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An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. In the present study, we show evidence that supports a tight link between NO, Ca2+, protease and apoptosis in beta-cells. Three different NO donors, SNAP, NOR3 and NOC7, induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP at 200 microM increased cytosolic Ca2+ concentration ([Ca2+]i) and induced apoptosis. The SNAP-induced apoptosis was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. In conclusion, an excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized in pancreatic nerve fibers and islets and potently augments glucose-induced insulin secretion. The present study explored a possible extra-pancreatic action of PACAP. The specific PACAP receptor (PAC1 receptor) was expressed in the rat fat tissue and 3T3-LI adipocytes. PACAP-38 (10 nM) significantly enhanced insulin-induced 2-deoxyglucose uptake by 3T3-L1 adipocytes. Insulin-stimulated phosphatidylinositol 3-kinase activity was further increased by PACAP-38, whereas the tyrosine-phosphorylation of insulin receptor beta-subunit and insulin receptor substrate-1 was unaltered by PACAP-38. These results reveal that PACAP-38 enhances insulin-induced glucose uptake, an effect probably mediated by insulin-stimulated phosphatidyl-inositol 3-kinase, and that PACAP potentiates not only insulin secretion, but also insulin action in adipocytes.

Thrombin is a serine protease which is generated from its precursor prothrombin by the activation of the blood coagulation cascade. Thrombin converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a central role in thrombosis and hemostasis by regulating platelet aggregation and blood coagulation. Here, we show that thrombn enhanced vascular endothelial growth factor (VEGF) production in a dose- and time-dependent manner in the supernatant of cultured PC-12 cells, as determined by enzyme-linked immunosorbent assay (ELISA). Thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) exerted an effect similar to thrombin on VEGF production. Thrombin-induced VEGF production was significantly attenuated by recombinant human thrombomodulin (rTM) and its minimal functional domain E456. Furthermore, the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited thrombin-induced VEGF production. Thus, rTM and NAC apparently inhibited the effect of thrombin on VEGF production in neuronal cells.

IntroductionThe blood coagulation cascade is regulated by the luminal surface of the endothelial cell lining.1 Endothelial cells synthesize tissue factor pathway inhibitor (TFPI), which, in part, binds to the cell surface glycosaminoglycans and inhibits factors Xa, VIIa, and tissue factor.2 Endothelial cells also produce and exhibit thrombomodulin (TM) on their luminal surface.3 TM is a kind of thrombin receptor that forms a 1:1 complex with thrombin. In this complex, thrombin activates protein C (PC) more than 1,000-fold more than thrombin alone. TM then loses its procoagulant activities, which include fibrinogen clotting, activation of factors V and VIII, and platelet activation. Thus, TM converts thrombin from a procoagulant protease to an anticoagulant. Pathologic states, such as an endothelial injury or perturbation or continuous rapid coagulation cascade activation, overcomes the endothelial regulating activity, resulting in the development of intravascular coagulation and the induction of disseminated intravascular coagulation (DIC). Theoretically, then, supplementing soluble TM or activated PC (APC) to reconstitute the endothelial coagulation regulation system in the circulation and regulate pathologically-activated blood coagulation could be beneficial. In this chapter, application of soluble TM and APC in the treatment of DIC is reviewed.

OBJECTIVE—To clarify the mechanism of thrombin receptor mediated signal transduction and the induction of cytokines by thrombin stimulation in rheumatoid synovial fibroblasts. METHODS—Cytokines were measured by enzyme linked immunosorbent assay (ELISA) in the supernatants of cultured rheumatoid synovial fibroblasts stimulated by thrombin. To assess the mechanism of thrombin receptor mediated signal transduction in the rheumatoid synovial fibroblasts, electrophoretic mobility gel shift assay (EMSA), immunoglobulin κ-chloramphenicol acetyltransferase (CAT) assay, and immunostaining for NF-κB subunit molecule was performed. RESULTS—Thrombin stimulation activated the inducible transcription factor NF-κB, and then induced subsequent expressions of interleukin 6 (IL6) and granulocyte colony stimulating factor (G-CSF) in the cells. CONCLUSION—Thrombin receptor mediated signal transduction could induce the expressions of IL6 and G-CSF, and increase inflammatory events in the cavum articulare via NF-κB activation. Keywords: thrombin; NF-κB; cytokine; synovial cells

Crow-Fukase or POEMS syndrome of polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes is a rare multisystem disorder of obscure pathogenesis that is associated with microangiopathy, neovascularization, and accelerated vasopermeability. We examined the levels of the vascular endothelial growth factor/vascular permeability factor (VEGF) in the serum and cerebrospinal fluid (CSF) from 10 patients with this syndrome. Serum VEGF levels were about 15-30 times those in control subjects or patients with Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and other neurological disorders. The CSF VEGF levels, however, were similar to those found in GBS and CIDP. Elevated VEGF levels in the serum decreased in 7 patients with Crow-Fukase syndrome after conventional therapy. The principal isoform of VEGF in Crow-Fukase syndrome was VEGF165. Elevated VEGF was independent of M-protein. Our results suggest that the overproduction of VEGF is important in the pathogenesis of this disorder.

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen that specifically targets vascular endothelial cells. The objective of this study was to evaluate the role of VEGF in Kawasaki disease (KD), the most common cause of systemic vasculitis in childhood. Serum VEGF levels were measured by ELISA in 22 patients with KD, 22 febrile children with infection, and 19 healthy children. Samples from KD patients were divided into three groups: acute stage (n = 20), subacute stage (n = 13), and convalescent stage (n = 15). The results showed that KD patients in the acute and subacute stages had significantly higher levels of VEGF than did patients with infectious diseases and the healthy control subjects. When compared with the VEGF levels of patients with and without coronary artery lesions (CAL), significantly higher levels of VEGF were observed in the subacute stage in patients with CAL and in patients without CAL in the acute stage. Serial examination revealed that the serum VEGF levels in KD patients with CAL increased from a relatively low level in the acute stage to an extremely high level in the subacute stage. In contrast, patients without CAL were found to have extremely high levels of VEGF only in the acute stage of KD. In KD patients, the serum VEGF levels did not correlate with the inflammatory markers and clinical symptoms. Our results raise the possibility that VEGF is involved in the pathogenesis of KD, especially in the development of CAL. Further study is needed to clarify the biologic effect of VEGF on coronary arteries in KD.

Nε-(carboxymethyl)lysine in blood from maintenance hemodialysis patients may contribute to dialysis-related amyloidosis.BackgroundRecent studies demonstrated not only that advanced glycation end product could be found in amyloid tissue from patient with dialysis related amyloidosis, but also that amyloid β2-microglobulin was modified withnε-(carboxymethyl)lysine (CML). We wanted to determine if CML could be a biomarker in these patients.MethodsTo raise polyclonal anti-carboxymethyllysine antibody, human serum albumin was carboxymethylated by glyoxylic acid and was immunized to rabbits as antigen. Carboxymethyllysine-hemoglobin (CML-Hb) levels were measured by the dot blotting method using this antibody.ResultsThe levels of CML-Hb were 6.68 ± 3.10nmol CML/mg Hb in nondiabetic hemodialysis patients (N = 70), 6.39 ± 3.43nmol CML/mg Hb in diabetic hemodialysis patient (N = 21), and 3.13 ± 0.88nmol CML/mg Hb in 47 healthy volunteers. For clinical signs of dialysis-related amyloidosis, 70 nondiabetic hemodialysis patients were scored according Gejyo’s criteria. The CML-Hb levels in patients with a high amyloid score as well as a low amyloid score were significantly higher than in patients with negative amyloid score (8.89 ± 3.53nmol CML/mg Hb, 7.28 ± 2.32nmol CML/mg Hb vs. 5.11 ± 2.09nmol CML/mg Hb, P < 0.001, P < 0.05). Furthermore, the CML-Hb levels correlated significantly with serum values of the methylguanidine over creatinine ratio and hyaluronate.ConclusionsWe suggest that CML-Hb is increased in blood from patients on maintenance hemodialysis and is thus a potential biomarker of oxidative damage in these patients. Moreover, CML-modification of protein may play a pathogenic role in the development of dialysis related amyloidosis.

The effect of a novel recombinant soluble human thrombomodulin, ART-123, on protein C activation was investigated by measuring plasma prothrombinase activity in four healthy male volunteers. ART-123 at a dose of 0.3 mg was administered as a bolus intravenous injection for 1 minute. Plasma ART-123 concentration and prothrombinase activity were determined before and immediately, 24, and 48 hours after injection, and thromboelastography was recorded before and immediately and 24 hours after injection. The mean elimination half-life was 19.82 +/- 2.10 hours. Compared with pretreatment levels, ART-123 reduced prothrombinase activity by 44.2 +/- 11.7%, 52.1 +/- 10.8%, and 61.0 +/- 14.7%, respectively, immediately, 24, and 48 hours after injection, suggesting that ART-123 activated the protein C pathway. ART-123 did not affect thromboelastography values. There were no abnormal findings for objective signs or laboratory tests, including blood pressure, heart rate, electrocardiogram, body temperature, hematology, coagulation and hemostatic parameters, blood chemistry, and urinalysis. Based on these observations, ART-123 at a dose of 0.3 mg can activate the protein C pathway in healthy volunteers.

SummaryRecent studies have suggested that clot-bound thrombin plays an important role in thrombus growth. In this study, we examined the effects of recombinant human soluble thrombomodulin (rhsTM) on clot-induced coagulation. rhsTM enhanced the activation of protein C by clots, and attenuated clot-induced thrombin generation and fibrinopep-tide A (FPA) production in a dose-dependent manner. The inhibitory effect of rhsTM was abolished by anti-protein C antibody. The inhibitory effect of rhsTM on clot-induced thrombin generation continued for over 60 min after the addition of the clot, while an active site-directed thrombin inhibitor, argatroban, produced a more transient inhibition. rhsTM also inhibited the regrowth of the clot in 125I-fibrinogen-supplemented plasma. We also examined the effect of rhsTM by thromboelastography; rhsTM reduced the growth of the clot but had little effect on the time to begin clotting, while heparin and Fragmin (low molecular weight heparin) had effects opposite to those of rhsTM.These findings suggest that rhs-TM attenuates the growth of the clot by activating protein C and inhibiting further thrombin generation in the clot.

In this study we demonstrate that prothrombin activates the cell proliferation of the lung adenocarcinoma A549 cells. The A549 cell expresses factor Xa-like prothrombinase activity on its surface and prothrombin was converted to thrombin on the cell surface. Furthermore, thrombin induced the activation of PKC, increased [Ca2+]i and potentiated MAP kinase activity through thrombin receptor. The mitogenic activity of prothrombin and the conversion to thrombin were completely abolished by the synthetic coagulation factor Xa inhibitor, DX9065a. These findings suggest that DX9065a is an effective agent for a therapeutic strategy against cancer itself and prothrombotic complications associated with malignancy.

血液学は医学の中でも,最も進歩の激しい領哉である.特に最近は診断.原因・病態,治療まで分子レベルでの進歩が著しい.ここではこの進歩著しい血液疾患の中で,血球系と凝固線溶,血小板系,さらにこれらを制御している内皮細胞系の検査の進歩を,特に実践的なものに的を絞った紹介した.

膠原病ではしばしば血栓,あるいは出血が見られる.これらは膠原病そのものの予後を大きく左右するのみか,薬剤が病巣局所にデリバリーされなくなり,基礎疾患である膠原病の難治化につながる.これらの血管合併症は,膠原病が血管炎をともなう疾患であるためと,膠原病によって血液凝固線溶系そのものが活性化,あるいは機能異常に陥っていることによって起こる.

Objective. To develop gene therapy for patients with human T cell leukemia virus type I (HTLV-I)-associated arthropathy (HAAP), we investigated the effects of ribozyme-mediated cleavage of HTLV-I tax/rex messenger RNA (mRNA) on synovial overgrowth. Methods. We introduced 2 hammerhead ribozymes targeted against HTLV-I tax/rex mRNA into synovial cells obtained from patients with HAAP and from patients with HTLV-I-negative rheumatoid arthritis (RA) and examined the ribozyme-mediated ablation of Tax expression. Using standard methods, we also determined the cells' ability to stop proliferating and to undergo apoptosis. Results. The ribozymes successfully cleaved tax/rex mRNA in HAAP patient synoviocytes. Both tax mRNA expression and Tax protein synthesis were inhibited significantly, resulting in inhibition of synovial cell growth and induction of apoptosis. In contrast, synovial cells from RA patients were not affected. Conclusion. In vitro results suggest that ribozyme-mediated gene therapy can inhibit the growth of HTLV-I-infected synovial cells, which is maintained by Tax protein, in HTLV-I-related diseases including HAAP.

We investigated the usefulness of ribozymes in inhibiting the expression of human T-cell leukemia virus type I (HTLV-I) gene. Two hammerhead ribozymes that were against HTLV-I rex (RR) and tax (TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitro cleavage analysis of run-off transcripts from tax/rex cDNA. Intracellular activities of the ribozymes were studied in HTLV-I tax cDNA-transfected rat embryonic fibroblasts (Rat/Tax cells), which expressed the Tax but not Rex. Ribozymes were delivered into cells using anionic or cationic liposomes fused with hemagglutinating virus of Japan (HVJ). Cellular uptake of ribozymes complexed with HVJ-cationic liposomes was 15-20 times higher cellular uptake than naked ribozymes, and 4-5 times higher than that of ribozymes complexed with HVJ-anionic liposomes. HVJ-cationic liposomes promoted accumulation of ribozymes in cytoplasm and accelerated transport to the nucleus. Tax protein levels were decreased about 95% and were five times lower when the same amount of TR was introduced into the cells using HVJ-cationic, rather than HVJ-anionic liposomes. Inactive ribozyme and tax antisense oligodeoxynucleotides reduced Tax expression by about 20%, whereas RR and tax sense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against tax mRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes.

We developed a novel assay using human thrombomodulin (TM), which detected overall abnormalities in the protein C anticoagulant pathway (PC pathway). This assay indicates the degree of inhibition of prothrombinase by TM, which is represented as the percentage of prothrombinase inhibition by 25 ng/ml of TM, termed PIP 25 (Prothrombinase Inhibition Percentage). We examined PIP25 in plasma samples from patients with systemic lupus erythematosus (SLE) with or without lupus anticoagulant (LA), patients with Behret's disease (BD), and patients with miscellaneous thrombotic vasculitis and compared these with the PIP25 of plasma samples from healthy volunteers in Japan. The PIP25s were significantly lower in SLE alone (35.5 ± 12.8%, P = 0.036) and SLE with LA (33.0 ± 13.3%, P = 0.030) and BD (33.3 ± 13.4%, P = 0.010) than those in healthy volunteers (43.5 ± 10.7%). There was no significance between healthy PIP25 and those with miscellaneous thrombotic vasculitis (44.2±8.4%, P = 0.823). These results suggest that the abnormalities of the protein C anticoagulant pathway were present in patients with SLE(LA) and BD.

To get insight into the involvement of tumor necrosis factor-alpha (TNF-alpha) and Fas (CD95) ligand in apoptosis (programmed cell death) of monocyte/macrophages in HIV-1-infected individuals, various T cell and myelomonocytic cell lines, including the HIV-1-infected clones OM-10.1 and U1 cells, were cultured in the presence of either TNF-alpha alone, anti-Fas agonist monoclonal antibody (Fas-mAb) alone, or their combinations. TNF-alpha moderately decreased the viability of myelomonocytic cell lines in a dose-dependent fashion (1-100 ng/ml). Unlike HIV-1-infected T cell lines, the viability of OM-10.1 and U1 cells was not affected by the treatment with Fas-mAb alone at concentrations up to 1,000 ng/ml. However, the viability of OM-10.1 cells further decreased with increasing concentrations of Fas-mAb when exposed simultaneously to TNF-alpha, suggesting that TNF-alpha sensitizes the cells to Fas-mAb-induced cell death. FACScan analysis and DNA gel electrophoresis revealed that the cell death was due to apoptosis. Such an effect of Fas-mAb was not identified in U1 cells. TNF-alpha but not Fas-mAb activated latent HIV-1 in OM-10.1 and U1 cells. Although all myelomonocytic cell lines expressed Fas on their cell surface, TNF-alpha significantly up-regulated the expression of Fas in only OM-10.1 cells. These results indicate that, unlike T cells, HIV-1-infected myelomonocytic cells are generally resistant to the Fas-mediated apoptosis. However, they would become sensitive to the apoptosis if the expression of Fas could be up-regulated by TNF-alpha or other factors.

Thrombomodulin (TM) is a glycoprotein that was originally identified on vascular endothelium and characterized as a natural endothelial anticoagulant. We reported previously that TM was also expressed at cell-cell boundaries of squamous epithelium, except in the basal layer cells and upper granular layer cells. The expression pattern of TM in the squamous cells implies that this molecule might be associated with keratinocyte differentiation, as well as being a potent anticoagulant. In the present study we examined TM expression immunohistochemically in biopsy specimens from 65 patients with primary oral squamous cell carcinoma (OSCC), and compared the results with the biological behavior of the carcinomas. The patients with intense TM expression in the carcinoma showed a significantly lower frequency of lymph node metastasis and significantly more favorable survival than those with negative TM expression. The TM expression was a better marker than the other prognostic factors, such as differentiation degree, tumor size and invasion mode. When TM expression was compared between the primary and metastatic lesions in the 36 patients who had lymph node metastasis, 14 (39%) showed decreased TM expression, 19 (53%) showed no change, and 3 (8%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P < 0.05). These results indicate that reduced expression of TM is associated with metastasis of carcinoma cells. The reduction of TM expression seems to play an important role in the metastatic process of OSCC, and to be related with a poor outcome for the patients.

Poly(dimethylsiloxane) (PDMS) and aromatic polyamide (aramid) multiblock copolymer (PAS) membranes containing ≥55 wt % of PDMS were prepared. Their tensile strength, morphology, and oxygen permeation property were investigated. The observed high tensile strength of PAS with 55 wt % of PDMS indicates the presence of PDMS-aramid co-continuous phases with lamellar structures; furthermore, the microphase-separated structures of PAS membranes were observed by means of transmission electron microscopy. The overall oxygen permeation resistance of a conventional silicone rubber showed typical dependence on stirrer speed, which was derived from the macroscopic relationship between the membrane-liquid interfacial resistance and the stirrer speed. However, the overall oxygen permeation resistances of the PAS membranes were found not to simply depend on stirrer speed. Combining with the oxygen permeability of PAS in the case of a gas-membrane-gas system, the interface resistances of the membranes were evaluated. The interface resistances of the PAS membranes with the two-phase nature were more susceptible to the hydrodynamic parameter than that of the silicone rubber and became lower than that of the silicone rubber at higher stirrer speeds. The low interface resistance together with the high tensile strength of the PAS membranes enables us to provide highly oxygen permeable membranes in practical applications with a membrane-liquid interface. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 64: 1153–1159, 1997

We investigated the pathophysiology of nerve hyperexcitability in a patient with Isaacs' syndrome, who had typical clinical and electromyographic features and responded to plasma exchange. Immunoblotting and immunohistochemistry studies showed that antibodies from this patient reacted with the lysate of a neuronal cell line (PC12). In Western blots, constituents of the patient's serum, particularly immunoglobulin M, reacted with proteins of approximately 50 and 18 kDa, whereas the control serum did not. A cross-linking study with alpha-dendrotoxin (7 kDa) showed a 57 kDa protein-peptide complex. Immunohistochemistry showed that the patient's serum reacted with PC12 cells and human intramuscular nerve axons. Our findings indicate that in Isaac's syndrome nerve hyperexcitability is the result of the immunological involvement of the voltage-dependent potassium channels located along the distal motor nerve or at the nerve terminal.

トロンビンが各種細胞に働く際の受容体がトロンビン受容体(thrombin receptor, TR)である. TRは膜を7回貫通したG蛋白共役型で,トロンビンはTRの細胞外N末端側を限定分解する.すると新たにむき出しになったN末端がアゴニストとして働くというユニークな構造をしている. TRはトロンビンによる血小板凝集,血管内皮細胞活性化,血管平滑筋細胞の遊走・増殖などに関わり,血栓や種々の血管病変に関係するものと考えられる.

Introduction Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. Methods Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. Results Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. Conclusions Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Thrombin-mediated activation of human platelets involves the G-protein-coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4 is thought to modulate platelet activation and subsequent procoagulant reactions. However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully elucidated, particularly under flow conditions.A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600s(-1)) and large arteries and small veins (240s(-1)).At a shear rate of 600s(-1), SCH79797 (10μM) efficiently reduced fibrin-rich platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P0.001). The inhibitory activity of SCH79797 was diminished at 240s(-1). YD-3 (20μM) had no significant effect at either shear rate. The antithrombotic effects of SCH79797 were significantly augmented when combined with aspirin and AR-C66096 (P2Y12 antagonist), but not with YD-3. In contrast, no significant inhibition of tissue factor-induced clot formation under static conditions was observed in blood treated with SCH79797 and YD-3, although thrombin generation in platelet-rich plasma was weakly delayed by these antagonists.Our results suggest that the antithrombotic activities of PAR1 and/or PAR4 antagonism is influenced by shear conditions as well as by combined platelet inhibition with aspirin and a P2Y12-antagonist.

Stroke is a major cause of mortality and disability worldwide. The main cause of stroke is atherosclerosis, and the most common risk factor for atherosclerosis is hypertension. Therefore, antihypertensive treatments are recommended for the prevention of stroke. Three angiotensin receptor blockers (ARBs), telmisartan, irbesartan and candesartan, inhibit the expression of the receptor for advanced glycation end-products (RAGE), which is one of the pleiotropic effects of these drugs. High mobility group box 1 (HMGB1) is the ligand of RAGE, and has been recently identified as a lethal mediator of severe sepsis. HMGB1 is an intracellular protein, which acts as an inflammatory cytokine when released into the extracellular milieu. Extracellular HMGB1 causes multiple organ failure and contributes to the pathogenesis of hypertension, hyperlipidemia, diabetes mellitus, atherosclerosis, thrombosis, and stroke. This is the first review of the literature evaluating the potential of three ARBs for the HMGB1-RAGE axis on stroke therapy, including prevention and acute treatment. This review covers clinical and experimental studies conducted between 1976 and 2013. We propose that ARBs, which inhibit the HMGB1/RAGE axis, may offer a novel option for prevention and acute treatment of stroke. However, additional clinical studies are necessary to verify the efficacy of ARBs.

Background: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from the systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. Materials and Methods: In this study, we produced a new adsorbent material by chemically treating polystyrene fiber. We first determined whether the adsorbent material efficiently adsorbed HMGB1 in vitro using a bovine HMGB1 solution and a plasma sample from a swine model of acute liver failure. We then constructed a column by embedding fabric sheets of the newly developed fibers into a cartridge and tested the ability of the column to reduce plasma HMGB1 levels during a 4-hour extracorporeal hemoperfusion in a swine model of acute liver failure. Results: The in vitro adsorption test of the new fiber showed high performance for HMGB1 adsorption (96% adsorption in the bovine HMGB1 solution and 94% in the acute liver failure swine plasma, 2 h incubation at 37°C; p < 0.05 vs. incubation with no adsorbent). In the in vivo study, the ratio of the HMGB1 concentration at the outlet versus the inlet of the column was significantly lower in swine hemoperfused with the newly developed column (53 and 61% at the beginning and end of perfusion, respectively) than in those animals hemoperfused with the control column (94 and 93% at the beginning and end of perfusion, respectively; p < 0.05). Moreover, the normalized plasma level of HMGB1 was significantly lower during perfusion with the new column than with the control column (p < 0.05 at 1, 2, and 3 h after initiation of perfusion). Conclusion: These data suggest that the newly developed column has the potential to effectively adsorb HMGB1 during hemoperfusion in swine.

Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells to express the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.

Swine are becoming increasingly attractive as animal models for clinical research and the recently developed Microminipig (MMPig) has emerged as a possible experimental animal model. In this study, we demonstrated age-dependent changes in hematological parameters and coagulation activity in healthy MMPigs (58 male and 67 females, aged 0-34 months), and investigated white thrombus formation (WTF) using an in vitro microchip flow-chamber system (four males and four females, aged 22-23 months). There was no clear sex or age-dependent differences in any hematological parameters. While activated partial thromboplastin time (APTT) was shorter than prothrombin time (PT), with APTT:PT of 0.88:1, microchip flow-chamber system analysis showed that WTF time was shorter than that in humans, suggesting a possible thrombotic tendency in the MMPig. These results could be useful to life science researchers in the use of the MMPig as an experimental model animal for thrombus formation.

The mulberry plant (Morus alba L.) contains abundant anthocyanins (ANCs), which are natural antioxidants. The aim of this study was to determine the ANC composition of Thai Morus alba L. fruits and to assess the effect of an ANC extract on blood glucose and insulin levels in male leptin receptor-deficient Zucker diabetic fatty (ZDF) rats. The major components of the ANC extract were identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry. ZDF and lean rats were treated with 125 or 250 mg ANCs/kg body weight, or 1% carboxymethylcellulose (CMC) twice daily for 5 weeks. Neither ANC dose had an effect on body weight. Following 5 weeks of treatment, glucose levels were observed to increase from 105.5±8.7 to 396.25±21 mg/dl (P

Poly(dimethylsiloxane) (PDMS) and aromatic polyamide (aramid) multiblock copolymers (PASs) ranging from 26 wt % to 75 wt% in PDMS content were prepared and cast into transparent, ductile, and elastomeric films from N,N′-dimethylacetamide solutions. The gas permeation properties and dynamic thermomechanical properties of the PAS films were investigated. It was found that the PASs containing < 75 wt % of PDMS had two-phase morphologies due to the great difference between the solubility parameters of the two components, in spite of the relatively low molecular weight of each segment. PASs containing ≥ 35 wt % of PDMS showed the PDMS continuous phase and interfacial mixing occurred clearly between the two phases at the higher PDMS contents. PAS containing ≥ 53 wt % of PDMS showed high enough gas permeability compared with conventional silicone rubbers. The gas permeation properties can be well predicted by the PDMS contribution to the continuous phase rather than by the modulus behaviors alone. © 1996 John Wiley & Sons, Inc.

Disiloxane–aromatic polyamide(aramid) multiblock copolymers(2SiPASs) were synthesized using 1,3-bis(3-aminopropyl)-1,1,3,3-tetramethyldisiloxane(BATS) as an analog of aramidsilicone resin consisting of aromatic polyamide and poly(dimethylsiloxane) (PDMS). 2SiPASs afford a transparent and toughened plastic film. The surface properties of 2SiPAS were investigated by X-ray photoelectron spectroscopy (xps) and static contact angle measurement. The results of surface analysis suggested that BATS content of the 2SiPAS surface increased with increasing BATS content in bulk. The interaction between the platelets and the 2SiPAS surface was found to be very weak when the BATS content reached 26 wt % in bulk. © 1996 John Wiley & Sons, Inc.

Multiblock copolymers consisting of aromatic polyamide(aramid) and poly(dimethylsiloxane) (PDMS) aramid-silicone resins (PASs) were synthesized by low temperature solution polycondensation, and PAS films were prepared by casting from an N,N'-dimethylacetamide solution. In this study, we investigated bovine serum albumin (BSA) adsorption, L929 cell adhesion, and tissue reaction on the surface of PAS in order to clarify the interaction between PAS and biomolecules. It was found that the amount of adsorbed biomolecules on PAS was extremely low in contrast with those on aramid and nylon films, and it was comparable to SILASTIC® 500-1 film. This suppression of adsorption of biomolecules onto PAS seemed to be due to the low surface free energy of the outermost surface of PAS, where PDMS block was condensed.

The regulatory mechanisms of microcirculation might differ in the liver from other organs, because macrophages are resident in the hepatic sinusoids and sinusoidal endothelial cells are unique in shape and function. Thrombomodulin expression in endothelial cells and tissue factor activity in isolated macrophages were studied in the liver and lung of rats. In normal rats, the thrombomodulin expression was minimal in hepatic sinusoids, but prominent in pulmonary capillaries, while the tissue factor activity in the presence of endotoxin was higher in pulmonary macrophages than in Kupffer cells, although the levels in the absence of endotoxin were comparable in both cells. The tissue factor activity in hepatic macrophages was increased after priming of the cells with Corynebacterium parvum or after induction of liver necrosis or cirrhosis with carbon tetrachloride. In the necrotic or cirrhotic liver, increased thrombomodulin expression was seen along capillaries extending in necrotic areas and regenerating nodules, but this increase was minimal in the Corynebacterium parvum-treated rat liver. Blood coagulation equilibrium in microcirculation regulated by endothelial cells and macrophages may differ between the liver and lung. Such equilibrium in the liver may vary depending on pathological status.

Human thrombomodulin (hTM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Focusing on the establishment of the practical evaluation of hTM immobilized materials, the activity of immobilized hTM was evaluated by in vivo and ex vivo blood contacting tests. As the basis for immobilization, regenerated cellulose films and hollow fibers were used. For the in vivo test, hTM immobilized cellular hollow fibers were implanted into dog blood vessels. Using hTM immobilized cellulose hollow fibers, a small scale dialyzer was assembled and its antithrombogenic activity was studied using human blood. As a result, it was revealed that the immobilized hTM still has co-enzymatic activity for activation of Protein C and anticoagulant activity. The coagulation time of the human blood passed through the hTM immobilized small dialyzer was effectively prolonged. It is expected that hTM immobilized cellulose should be a useful antithrombogenic biomaterial.

Mutations in p53, a tumor suppressor gene, are one of the most common genetic lesions of human cancers. The relationship between pS3 gene mutation and ultraviolet (UV) light has been demonstrated in skin cancers of sun-exposed sites. In this study, genomic DNA from 12 skin cancers was screened for mutations in exons 5 to 9 of this gene using the polymerase chain reaction — single strand conformation polymorphism (PCR-SSCP) analysis followed by DNA sequencing. DNA samples were obtained from 8 basal cell carcinomas (BCCs): 1 from an organoid nevus, 1 from a patient with basal cell nevus syndrome, 1 from a patient with xeroderma pigmentosum, and 1 from a recurrent and 4 from primary sporadic lesions on actinic damaged skin, and from 4 squamous cell carcinomas (SCCs): 1 from a burn scar, 1 from a patient with epidermodysplasia verruciformis, and 2 from actinic keratosis. Mutation of the p53 gene was detected in only 1 case of SCC which had arisen from actinic keratosis. The mutation occurred at codon 159 in exon S with a GCC to CCC base-pair substitution resulting in an amino acid change of alanine to proline. This mutation does not correspond to results of UV mutagenesis studies reported in the literature. Our findings imply that, although p53 gene mutation and UV exposure play an important role in the carcinogenesis of some skin cancers, they are not crucial, especially in skin cancers that develop from underlying skin disorders.

It is known that adult T-cell leukemia (ATL) cells undergo spontaneous apoptosis in vitro as a results of expression of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. However, the molecular mechanism of the apoptosis remained to be elucidated. We examined the expression of Fas-Ag, Bcl-2 and Tax in ATL cells prior to culture and following culture. ATL cells from some patients showed a increased level of Fas-Ag and a decreased Ievel of Bcl-2 compared to peripheral Iymphocytes from healthy individuals. Culture of ATL cells resulted in apoptosis. However, the apoptosis was confined to cells associated with a high level of Tax and a decreased level of Bcl-2. In addition, we found that apoptosis induced by HTLV-I Tax results in destruction of the HTLV-I genome itself. These results suggested that decreased Bcl-2 may be important in Tax-induced apoptosis.

To examine the presence of CD34-positive cells and intranuclear factors in acute coronary thrombi, we compared thrombi in patients with type 2 diabetes mellitus (DM, n = 21) and without DM (n = 29). Immunohistochemical staining revealed the constitutive presence of platelets, fibrin, erythrocytes, neutrophils, extracellular high-mobility group box 1 (HMGB-1), and histone H3 in all thrombi. There were significantly more oval or flat CD34-positive cells and significantly larger HMGB-1-positive areas in the thrombi from patients with DM. The flat CD34-positive cells expressed ecto-nucleoside triphosphate diphosphohydrolase (a platelet aggregation inhibitor). The number of CD34-positive cells was negatively correlated with the serum levels of glucose and hemoglobin A1c, whereas the HMGB-1-positive area was positively correlated with the levels of serum glucose. The paucity of CD34-positive cells and the high levels of HMGB-1 expression in acute coronary thrombi from patients with type 2 DM could facilitate thrombus formation.

A lot of fruits contain nutrient substances that can prevent, cure or suppress various diseases. They are nature’s true medicines, and diets rich in fruits are consistently associated with a decreased risk of cancer and other chronic diseases. Apricot “Prunus armeniaca” is the fruit of a rosaceous tree (rosaceae), which is produced in most parts of the world (Gur, 1985). The name Prunus armeniaca is thought to be a misnomer based upon the long-held view that apricots initially originated in Armenia. It is known that apricots originated in the Far East, most likely in the Himalayas, the Northern and Western regions of China from where they spread to Armenia and Russia (Gu, 1979; Gulcan, 1988). Apricot is found semi-wild and wild in the northern hills of China and in a broad belt across the hills, mountains, and plateaus of Central Asia as far as the Caucasus Mountains. The first record of the domestication of apricots is an account of their cultivation in China, about 4000 years ago. It is likely that tribe people of Central Asia established traditional rights to harvest their parts of the apricot forests for millennia before this time.

Thrombomodulin (TM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, the immobilization of hTM was investigated in detail using surface modified polymers. As the basis of immobilization, poly(acrylic acid) (PAAc) surface-grafted poly(ethylene) (PAAc-g-PE) film was used with the expectation of increasing the immobilization amount of hTM. The effect of the immobilization reaction on the hTM activities, and the comparison of the activities of the immobilized hTM with the free hTM, were studied.

To investigate how the number of mast cells is controlled, we studied a murine mastocytoma cell line. Based on electron microscopic observation of nuclear condensation and electrophoretic evidence with DNA fragmentation, these mastocytoma wells were shown to undergo apoptosis. This apoptosis was dependent on the concentrations of serum and L-arginine and was enhanced by TNF-alpha. We confirmed that apoptosis was mediated by nitric oxide (NO) synthase; inducible NO synthase (iNOS) mRNA was strongly expressed in apoptotic cells, while an inhibitor of NOS, NG-monomethyl-L-arginine, and dexamethasone prevented apoptosis in addition to inhibiting iNOS mRNA expression. Our results suggest that iNOS expression is very important in regulating the proliferation of mast cells under pathological conditions.

Human thrombomodulin (hTM), which is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant, was immobilized on to various substrates by two immobilization methods. As the substrates of immobilization, poly(acrylic acid) surface grafted poly(ethylene) (PAAc-g-PE) film, poly(vinylamine) surface grafted poly(ethylene) film, and PAAc surface grafted nylon were used. For immobilization, simultaneous preactivation methods were used. The effect of the immobilization reaction on hTM activities, the comparison of the activities of immobilized hTM with those of free hTM, and the effect of the thrombin incorporation on antithrombogenic activity were studied. The hTM immobilized onto PAAc-g-PE by preactivation showed the highest antithrombogenic activity. The thrombin incorporation affected protein C activation activity but not the fibrinogen clotting time. hTM immobilized nylon showed greater antithrombogenicity in vitro.

This study demonstrated that intravenous infusion of recombinant human soluble thrombomodulin (rhs-TM) could inhibit disseminated intravascular coagulation (DIC) caused by 4 hr infusion of tissue factor (TF) in rats. Extended infusion of TF reduced fibrinogen and platelet counts and elevated serum FDP level. Pretreatment and coinfusion of rhs-TM could block changes of these DIC-parameters without prolongation of APTT. Heparin, which is a potent anti-DIC drug, could also inhibit these changes with extra prolongation of APTT and PT. Thus, these results suggest thrombomodulin prevent DIC less bleeding tendency than heparin.

Ultrathin films of poly(dimethylsiloxane)-polyamide copolymers (PAS) with different silicone contents were prepared by the methods of water casting and spin coating. The surface properties of the films were investigated in detail using contact angle measurements and X-ray photoelectron spectroscopy (XPS). The water-cast films were thin enough to be applicable as laminate films for an artificial lung or an air-separator for oxygen-enrichted air. Uniform and ultrathin films were obtained by the spin coating method. XPS and contact angle measurements suggested that poly(dimethylsiloxane) segments were condensed at the outermost surface of spin-coated PAS films. XPS measurements also revealed that the surface properties of PAS were affected by the molding method, especially the solvent evaporation conditions. Ultradunne Filme aus Polydimethylsiloxan(PDMS)-Polyamid-Copolymeren (PAS) mit unterschiedlichen Silikonanteilen wurden durch Filmgiesen auf Wasser und Rotationsbeschichten hergestellt. Die Oberflacheneigenschaften der Filme wurden mittels Kontaktwinkelmessungen und Rontgenphotoelektronenspektroskopie (XPS) untersucht. Die auf Wasser gegossenen Filme waren dunn genug, um als Laminatfilme in kunstlichen Lungen oder als Trennmembranen zur Sauerstoffanreicherung von Luft Anwendung finden zu konnen. Gleichmasig dicke und ultradunne Filme wurden durch Rotationsbeschichten erhalten. Aus den XPS- und Kontaktwinkelmessungen kann geschlossen werden, das sich die PDMS-Segmente an der Oberflache der Filme anreichern. Aus den XPS-Messungen geht auserdem hervor, das die Oberflacheneigenschaften von PAS von der Herstellungsmethode, insbesondere von den Bedingungen der Losemittelverdampfung abhangen.

SummaryWe reported that recombinant human soluble thrombomodulin (rhs- TM) is effective for disseminated intravascular coagulation (DIC) in vivo, in mice and rats. In the present work, we investigated the effects of decreased plasma antithrombin III (ATIII) levels on anticoagulant effects of rhs-TM, as compared to findings with heparin, of which effect is lowered by the decreased plasma ATIII levels in patients with DIC. Rat plasma ATIII levels decreased when we mixed plasma with anti-rat ATIII antibody and the potential of heparin to prolong APTT or PT was markedly diminished. The potential of rhs-TM to prolong APTT and PT was not affected. In rats injected with anti-rat ATIII antibody, plasma ATIII levels decreased immediately. When the rats were infused with tissue factor (TF), DIC was induced. At doses of rhs-TM and heparin which were equally effective at inhibiting the decrease in platelet count and fibrinogen level in cuntiol rats treated with TF, only rhs-TM remained effective in preventing DIC in rats with reduced ATIII levels. Heparin was not effective, when administered to these rats with reduced ATIII levels. Therefore, rhs-TM effectively inhibits coagulation independent of ATIII levels, in contrast to heparin, which depends on the ATIII level.

Aramid-silicone resins (PASs) consisting of aromatic polyamide (aramid) and poly(dimethyl-siloxane) (PDMS) segments were synthesized by low temperature solution polycondensation. For the evaluation of blood compatibility in vitro, two kinds of experiments were carried out. One was the thromboxane B2(TXB2) release test from platelets attaching to PAS and Biomer. The other was the observation of the platelet adhesion on the surfaces of PAS by scanning electron microscopy (SEM). The results indicated that PAS was bio-inert in vitro. The surface chemical composition of PAS films was investigated by means of electron probe micro analysis (EPMA), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle measurements. The relationship between blood compatibility and surface composition of PAS is discussed.

We describe three human T lymphotropic virus type I (HTLV-I) carriers, who are characterized by a dissociation between the clinical features including scarce cognitive impairments and peculiar hyperintensities in the deep and subcortical white matter detected by T2-weighted magnetic resonance imaging (MRI) scans. Unlike spotty MRI findings which are encountered in clinically asymptomatic elderly individuals and are previously described in some HTLV-I carriers, the MRI changes in our patients were diffuse and extensive. Known central nervous system disorders accompanying a cerebral white matter involvement were failed to be diagnosed. These findings support a possibility that extensive lesions in the supratentorial white matter are associated with HTLV-I infection with minimal symptoms, and suggest that MRI scans may detect occult diffuse inflammatory changes associated with the virus infection in these patients.