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Tumor necrosis factor-alpha (TNF-alpha) has two forms with apparently different biological activities: a membrane-associated form and a soluble form. TNF-alpha-converting enzyme (TACE) mediates a cleavage of membrane-associated TNF-alpha to induce its bioactive soluble form. We hypothesized that inhibition of TACE might prevent TNF-alpha-induced tissue injury while preserving the benefits of TNF-alpha. In this study, we evaluated the role of TACE in acute inflammation using an inhibitor of the enzyme in a rat model of lung transplantation. Inbred Lewis rats underwent left lung isotransplantation, and the donor lungs were kept in Euro-Collins solution with or without the inhibitor. After 6 hours of ischemia, the left lung was transplanted into the recipient rat and reperfused for 4 hours. Inhibition of TACE significantly attenuated endothelial and alveolar septal damage, as assessed by radiolabeled albumin leakage after transplantation. The inhibition also attenuated neutrophil accumulation in the alveolar space and other histopathologic findings, including intercellular adhesion molecule-1 expression. In addition, significantly lower levels of monocyte chemotactic protein-1, cytokine-induced neutrophil chemoattractant-1, high mobility group box-1, and soluble epithelial cadherin and decreased neutrophil elastase activity were observed in bronchoalveolar lavage fluid from the rats treated with the inhibitor. We conclude that TACE mediates a critical step in the development of post-transplantation lung injury.

Thrombin, a serine protease that plays a pivotal role in blood coagulation, wound healing, and angiogenesis, has also been implicated in the mitogenesis of various cell types. Previously, we showed that thrombin and the thrombin receptor agonist peptide (TRAP-14; SFLLRNPNDKYEPF) for protease-activated receptor 1 (PAR1) induce vascular endothelial growth factor (VEGF) secretion in PC-12 cells. In this study, we show that thrombin and TRAP-14 also stimulate VEGF secretion in the human NB-1 neuroblastoma cells. In these cells, we further show that thrombin-induced VEGF secretion was blocked by cycloheximide and actinomycin D, indicating that de novo protein synthesis is essential for this process. Reduced thrombin-induced VEGF secretion upon treatment with LY294002, calphostin C, or BAPTA, further suggests that the process is dependent on phosphatidyl-inositol-3-kinase, protein kinase C, and calcium. However, the complete loss of thrombin-induced VEGF production upon treatment with argatroban, a derivative of arginine and a potent anticoagulant/antithrombin agent, supports the notion that argatroban serves as a useful therapeutic tool for thrombin-associated pathologic conditions. Here, it appears that argatroban may be effective in controlling disorders linked to thrombin-induced VEGF production in neuronal cells.

The painful orogenital ulcerations of Behcet's disease are among the major symptoms of patients and are often intractable. We assessed the efficacy of granulocyte and monocyte adsorption apheresis therapy in two patients, a 21-year-old man with orogenital ulcerations and a 50-year-old woman with genital ulceration and abdominal pain. They underwent 5 and 8 granulocyte and monocyte adsorption apheresis treatments at 5-day intervals, respectively. The painful orogenital ulcerations of the man responded dramatically and the genital ulcer of the woman decreased in size and her abdominal pain was improved. Our results demonstrate that granulocyte and monocyte adsorption apheresis may be useful for treating orogenital ulcerations of Behcet's disease.

Impaired expression of the CD3 zeta chain in T cells associated with T cell anergy has been reported in cancer patients. However, few studies have investigated CD3 zeta expression in regional lymph node lymphocytes (LAL) in cancer patients. This study aims to confirm CD3 zeta expression levels by lymph node and peripheral blood lymphocytes (PBL) in gastric cancer patients and to discuss the clinical implications of intranodal or peripheral blood expression of CD3 zeta in gastric cancer.Twenty-two gastric cancer patients were enrolled. Macroscopically non-metastatic compartment 1 LNL (C1LNL), compartment 2 LNL (C2LNL) and PBL were surgically obtained. Two-color flow cytometry was then used to quantify the levels of CD3 zeta expression in C1LNL, C2LNL and PBL.Impaired CD3 zeta expression was confirmed in 9.5% of C1LNL, 8.9% of C2LNL and 4.2% of PBL. There was a significant difference in CD3 zeta expression levels between C1LNL and PBL (p0.01). CD3 zeta expression in PBL was significantly correlated with depth of invasion but not nodal involvement. Distinct differences between the respective lymph node compartments were not identified.Immunological paralysis following CD3 zeta impairment may occur more frequently in LNL than in PBL in gastric cancer. Identifying such patients during the perioperative period using flow cytometric methods will increase the efficacy of cytokine therapy aiming to normalize CD3 zeta expression levels.

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation–induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature.

Summary Adult Still's disease is characterized by a high spiking fever, transient skin rash, and polyarthralgia. Joint pain is one of the major complaints and is often intractable. We assessed the efficacy of granulocyte and monocyte adsorption apheresis (GCAP) therapy for treating arthralgia in adult Still's disease. A 33-year-old woman with adult Still's disease who suffered from recalcitrant arthralgia resistant to systemic corticosteroids was treated with GCAP therapy. She underwent five GCAP treatments at 5-day intervals. Her joint pain responded dramatically to the GCAP therapy, suggesting that GCAP may be useful for treating adult Still's disease. We present a detailed description of the patient and this novel therapy.

To evaluate the contribution of hematologic factors and long-term inflammation to the development of myocardial infarction at a young age, we measured hematologic variables, including soluble fibrin and high-sensitivity C-reactive protein, in 90 patients who had myocardial infarction and 138 controls ≤45 years of age. Plasma levels of soluble fibrin and C-reactive protein were significantly higher in patients than in controls. On multivariate regression analysis, soluble fibrin was the strongest predictor of myocardial infarction at a young age.

Introduction Activated protein C (APC) is an important natural anticoagulant that is proteolytically generated from protein C (PC) by the modulation of thrombin activity in the presence of thrombomodulin on an endothelial surface. Recent studies have demonstrated that, beyond its anticoagulant acitivities, APC had anti-inflammatory and cytoprotective properties. The mechanisms underlying APC's anti-inflammatory effects remain unknown. Our goal was to elucidate and confirm these mechanisms. Methods We first examined the effect of APC on reactive oxygen species (ROS) and inflammatory cytokine production in murine macrophage-like RAW264.7 cells. We further examined the effect of APC on chemically induced lipid peroxidation and advanced glycation end-products (AGE) formation. Results and conclusions APC in the range of 10–50 μg/mL could reduce lipopolysaccharide (LPS)-induced ROS generation, nuclear factor κB (NF-κB) activation and resultant proinflammatory cytokine production. Additional cell-free experiments revealed that APC (10–50 μg/mL) had inhibitory effects on lipid peroxidation and AGE formation. These findings suggest that APC, via its intrinsic anti-oxidant properties, may, in settings of oxidant stress, exert important cytoprotective and anti-inflammatory effects that are distinct from its anticoagulant activity as an antioxidant protein. If that is true, APC may contribute to ROS-related chronic disorders including atherosclerosis and diabetes as well as acute shock conditions.

The association between biofilm formation and the accessory gene regulator (agr) types of methicillin-resistantStaphylococcus aureus(MRSA) strains in our hospital were investigated. The biofilm index and the incidence of MRSA strains carryingagr-2in the infection group (n= 91) were significantly higher than were those in the carrier group (n= 225), suggesting that biofilm formation andagrtype are associated with nosocomial MRSA infections.

Threatened premature labor (TPL) often results in preterm birth (PB). The aim of the present study was to evaluate the associations of periodontal and general health conditions with TPL and PB in relation to serum cytokine levels and the composition of subgingival plaque.Eighty-eight women were enrolled in the study. Systemic conditions were assessed, and subgingival plaque samples obtained for bacterial analysis. Periodontal examinations included assessments of plaque, gingivitis, clinical attachment level, probing depth, and bleeding on probing. Serum cytokine levels also were analyzed. Gestational age at delivery was recorded, and the mothers were divided into a TPL or non-TPL group, and into a non-TPL-TB (term birth), non-TPL-PB, TPL-TB, or TPL-PB group, accordingly.Forty subjects were classified as TPL and 18 as TPL-PB. There were significant differences between the TPL and non-TPL subjects in several of the systemic and periodontal parameters and serum cytokine levels. Significant differences were observed between the TPL-TB and TPL-PB groups in the percentage of Tannerella forsythensis (Tf, formerly Bacteroides forsythus), and the serum interleukin (IL)-8 and IL-1beta levels. Significant negative correlations between the gestational age at delivery and several periodontal parameters and serum IL-8 and IL-1beta levels, and significant positive correlations between periodontal status and serum IL-8 and IL-1beta levels, were observed.The TPL women revealed worsened periodontal conditions and elevated serum IL-8 and IL-1beta levels compared to the non-TPL women. The elevated levels of serum IL-8 and IL-1beta could have affected the maintenance of the proper uterine-fetus relationship, resulting in premature uterine contractions.

Granulocyte and monocyte adsorption apheresis (GCAP) is a new extracorporeal apheresis treatment modality that removes pathogenic granulocytes. Recently, we found that GCAP is useful for treating pyoderma gangrenosum and pustular psoriasis. We thought that this treatment may also be effective for treating other disorders attributable to activated granulocytes and studied the efficacy of GCAP in 4 patients with psoriatic arthritis. Treatment with GCAP resulted in remarkable clearing of joint pain, suggesting that GCAP is valuable for treating arthritis as well as skin disorders. We present a detailed description of these patients and this novel therapy.

We report a case of a 62-year-old man diagnosed as Crow-Fukase syndrome (POEMS syndrome), in which the serial platelet count and vascular endothelial growth factor (VEGF) concentration were determined before and during the state of disseminated intravascular coagulation (DIC). The serum VEGF concentration was noted to be gradually decreased prior to DIC, after which it abruptly decreased with a corresponding drop in platelet count upon the onset of DIC. The physiological effects of VEGF are viewed as one of the causative factors in DIC and its abrupt and excessive release may have caused the exacerbation of the patient's clinical symptoms.

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.

Summary. Background: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. Objectives: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. Methods: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. Results and conclusions: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-α, and TGF-β.

Acute hypotension, hypoxemia, cardiac arrhythmias, cardiac arrest, (or a combination of these), and sudden death are well-recognized complications of the cemented hip arthroplasty procedure. Collectively, these are known as the bone cement implantation syndrome (BCIS). The endogenous cannabinoids, anandamide (ANA) and 2-arachidonylglycerol (2-AG), are reported to be strong vasodilators and play a role in the hypotension associated with hemorrhagic and septic shock. In the present study, a potential role for the endogenous cannabinoids in influencing hemodynamic variables in BCIS was investigated. Thirty-five patients (35 hips) entered a prospective, randomized clinical trial. The patients were divided into two groups. Group 1 comprised 16 patients who had the component inserted using a conventional cementing technique, whereas group 2 consisted of 19 patients who had the femoral component inserted without cement. Blood samples were taken at six consecutive time points: before anesthesia, after reaming the femur, 2 min after insertion of stems with or without cement into the femur, and 10 min, 20, and 30 min after stem insertion. In group 1 (with cement), the mean levels of ANA and 2-AG significantly increased after stem insertion. In a comparison of each group after stem insertion, mean ANA and 2-AG levels in group 1 also significantly differed from those in group 2. By contrast, in group 2 (without cement) neither ANA nor 2-AG levels exhibited a significant increase or change at any point in time. In conclusion, we have shown for the first time that endogenous cannabinoids are candidates for lipid mediators of BCIS.

Circulating level of α 2 -macroglobulin–β 2 -microglobulin complex in hemodialysis patients. Background The presence of α 2 -macroglobulin (α 2 M) in amyloid tissue from patients with dialysis-related amyloidosis (DRA) was demonstrated by Argiles et al in 1989. Thereafter, the formation of the complex of β 2 -microglobulin (β 2 m) with α 2 m was confirmed directly by in vitro study. In Alzheimer's disease, complex formation of amyloid β-peptide and α 2 M is considered to play an important role in the pathogenesis by modifying the degradation processes of amyloid protein. Thus, we hypothesized that the α 2 M-β 2 m complex is an important factor in the pathogenesis of DRA as well. Here, we measured the circulating levels of α 2 M-β 2 m complex in the maintenance hemodialysis patients and discussed about its clinical significance in DRA. Methods One hundred and thirty-seven hemodialysis patients and 11 prehemodialysis chronic renal failure (CRF) patients were included in this study. The affinity of purified α 2 M for β 2 m was confirmed by a highly sensitive 27MHz quartz crystal microbalance (QCM). The presence of circulating α 2 M-β 2 m complex was analyzed by immunoblotting analysis. Furthermore, the serum levels of α 2 M-β 2 m complex were measured by sandwich enzyme immunoassay. Results QCM analysis revealed the high affinity of α 2 M for β 2 m. The presence of circulating α 2 M-β 2 m complex was detected in two out of a total 11 prehemodialysis CRF patients and in 95 out of the total of 137 hemodialysis patients. None of the healthy subjects, however, were observed to present with any α 2 M-β 2 m complex. Serum levels of the α 2 M-β 2 m complex were correlated to the duration of hemodialysis ( P = 0.043). Serum levels of the α 2 M-β 2 m complex were significantly higher in patients with high DRA score than in patients with negative DRA score ( P = 0.018). Moreover, serum levels of the α 2 M-β 2 m complex showed significantly lower in the hemodiafiltration patients compared to the hemodialysis patients ( P = 0.002) and showed a strong correlation with DRA score in hemodialysis patients excluding 11 hemodiafiltration patients ( P = 0.0004). Conclusion This study is the first to demonstrate the presence of circulating α 2 M-β 2 m complex in hemodialysis patients. Furthermore, we observed the correlation between serum levels of α 2 M-β 2 m complex and clinical characteristics of DRA. Thus we concluded that a formation of an α 2 M-β 2 m complex may be implicated in DRA.

The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap-junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin-induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide-induced cell death was preceded by G0/G1 cell-cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stress-induced hepatocyte damage. In this context, methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor, or N-acetyl cysteine, an antioxidant, potently inhibited the anandamide-induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide-induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor.

High mobility group protein 1 (HMGB1) has been implicated in diverse cellular functions, including determination of nucleosomal structure and stability and binding of transcription factors to their cognate DNA sequences (1)(2)(3)(4). HMGB1 is also present in a membrane-associated form, termed amphoterin, that mediates neurite outgrowth (5). Amphoterin can interact with macrophage cell surface receptors for advanced glycation end products to enhance expression of tissue-type plasminogen activator (6). Recently, HMGB1 was identified as a late mediator of endotoxin lethality (7). Mice had increased serum HMGB1 concentrations after exposure to endotoxin, and sepsis patients who succumbed to infection also had increased serum HMGB1. It would therefore be useful to develop an easy and highly sensitive method to measure serum HMGB1. However, this study revealed that HMGB1 and HMGB2, with extremely high homology (81%) to HMGB1, coexist in the serum. We report an ELISA method we have developed that measures only HMGB1 without simultaneous determination of HMGB2. To prepare an anti-peptide monoclonal antibody reacting only with HMGB1, we selected a peptide sequence (peptide 1; GKGDPKKPRGK) with high antigenicity and different from that of HMGB2. The monoclonal anti-calf HMGB1 antibody was prepared against calf thymus-derived HMGB1, which was 98% homologous to human HMGB1. Each protein sample used for the analysis was prepared as follows. The peptides were purified and separated by HPLC. The peptide synthesized was added to maleimidobenzoyl- N -hydroxysuccinimide ester (Pierce Chemical Co.)-labeled keyhole limpet hemocyanin (Calbiochem) or maleimidobenzoyl- …

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.

Introduction: Hyperhomocysteinemia is a coronary risk factor, but its pathophysiologic mechanism remains unclear. Materials and methods: The importance of hyperhomocysteinemia in the pathogenesis of early myocardial infarction, was determined in case-control study of 127 men with a first early myocardial infarction ≤45 years and 150 age-matched male controls. We measured plasma concentrations of homocysteine, fibrinogen, antithrombin, tissue factor, tissue factor pathway inhibitor, tissue plasminogen activator, plasminogen activator inhibitor-I, plasminogen, α2-antiplasmin, lipoprotein(a), protein C, protein S, factor VII, and activated factor VII. Results: Homocysteine concentrations were higher in patients with early myocardial infarction than in controls (11.2±5.3 and 8.3±5.0 μmol/l, respectively, P

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.

Objective:To evaluate the usefulness of an assay using two polymerase chain reaction-based genotyping methods in the practical surveillance of methicillin-resistantStaphylococcus aureus(MRSA).Methods:Nosocomial infection and colonization were surveyed monthly in a university hospital in Japan for 20 months. Genotyping withmec-HVR is based on the size of themec-associated hypervariable region amplified by polymerase chain reaction. Toxin genotyping uses a multiplex polymerase chain reaction method to amplify eight staphylococcal toxin genes.Results:Eight hundred nine MRSA isolates were classified into 49 genotypes. We observed differing prevalences of genotypes for different hospital wards, and could rapidly demonstrate the similarity of genotype for outbreak isolates. The incidence of genotype D: SEC/TSST1 was significantly higher in isolates causing nosocomial infections (49.5%; 48 of 97) than in nasal isolates (31.4%; 54 of 172) (P= .004), suggesting that this genotype may represent the nosocomial strains.Conclusion:The combined use of these two genotyping methods resulted in improved discriminatory ability and should be further investigated.

To investigate the biological effect of cerivastatin on glioblastoma cells, we exposed them to various concentrations of cerivastatin. Cerivastatin exhibited dual effects on glioblastoma cells in a dose-dependent manner. Immunofluorescence microscopy showed disruption of actin stress fibers and focal adhesion plaques even at nanomolar concentrations. Matrigel assay demonstrated marked inhibition of glioblastoma cell invasion. Immunoblot analysis using a phosphospecific antibody against focal adhesion kinase (FAK) showed that inhibition of migration was associated with the down-regulation of tyrosine phosphorylation of FAK. Our data suggest that cerivastatin may be beneficial for combination therapy with conventional anti-cancer drugs by inhibiting the invasion of glioblastoma.

Recently, oscillometric devices have been developed that can measure blood pressure in the extremities and analyze pulse volume record. On the basis of the extremity pulse volume record, these devices can automatically determine three types of simply measured pulse wave velocity (PWV) (brachial PWV: heart to right upper arm; R-PWV: right upper arm-right ankle; and L-PWV: right upper arm-left ankle). The percent mean pulse volume record (%MPVR=the height that bisects the area of the pulse volume record/pulse pressure X100), a quantitative index of right brachial pulse volume record, can also be determined. To evaluate the usefulness of these new indices, we studied 1,067 consecutive subjects undergoing health checkups (648 men, 419 women; mean age, 50 +/- 9 years). In both sexes, age correlated positively with simply measured PWVs (men, brachial PWV: r=0.46, p

We report here that the traditional Chinese formulation, Chaihu-jia-Longgu-Muli-tang (CLM), significantly inhibited the increase in intimal thickening in rat carotid artery injured by balloon endothelial denudation, which mimics many aspects of restenosis after percutaneous coronary intervention (PCI) in humans. CLM, Saiko-ka-Ryukotsu-Borei-to in Japanese, is commonly prescribed for symptoms accompanying hypertension and atherosclerosis in Japanese kampo medical care. CLM administered orally 1 week before and 1, 4 and 8 weeks after balloon injury inhibited the increase in intimal area, intimal/medial ratio and stenosis ratio. To our knowledge, this is the first report demonstrating inhibitory effects of a traditional Chinese formulation on intimal thickening of carotid artery after balloon injury. It is worth noting that CLM maintained its inhibitory effect up to 8 weeks after balloon injury. The reduction in intimal thickening by CLM could have resulted from inhibition of intimal smooth muscle cell proliferation, which was assessed by immuno-histochemical analysis using monoclonal antibody against proliferating cell nuclear antigen. Therefore, CLM may be a favourable candidate for prevention of restenosis after PCI. Moreover CLM may have a therapeutic value in the prevention of atherosclerosis, because restenosis after PCI is considered to be an accelerated atherosclerosis.

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.

We encountered a patient with adult T-cell leukemia/lymphoma (ATL) which expressed neural cell adhesion molecule (NCAM). The tumor cells markedly infiltrated the central nervous system (CNS) during the course of the ATL. The patient died 20 months after disease onset, which was considered to be early in the course. During the invasion of the CNS, the surface phenotype of the peripheral blood ATL cells by flow cytometric analysis was CD2+, CD3+, CD4+, CD7-, CD8-, CD16-, NCAM (CD56)+, HLA-DR-. We speculate that the infiltration of ATL cells into the CNS was closely related to the expression of the NCAM in this patient.

SummaryTo evaluate the association between haemostatic parameters and increased risk of myocardial infarction (MI) at a young age, we measured fibrinogen, factor VII, antithrombin III, protein C, protein S, tissue factor (TF), free form tissue factor pathway inhibitor (TFPI), plasminogen, 2-antiplasmin, tissue plasminogen activator (tPA), plasminogen activator inhibitor-I (PAI-I), and lipoprotein (a) in 140 young men with MI before age 45 and 150 age-matched healthy men. TF, TF/TFPI ratio, PAI-I, PAI-I/tPA ratio, plasminogen, and lipoprotein (a) in young MI patients were all significantly higher than controls, while TFPI, antithrombin III, and tPA were significantly lower (P

Rationale Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated. Objectives To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH. Methods Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested. Results HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats. Conclusions Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.

Background/Aim: Carboxymethyllysine is one of the common advanced glycation end products in vivo. In a previous report, we were the first to describe the increase of circulating carboxymethyllysine-hemoglobin (CML-Hb) levels in hemodialysis patients, particularly in patients with dialysis-related amyloidosis (DRA). The aim of this study was to investigate the predictive value of CML-Hb in the progression of DRA using computed tomography images of 2-year or 3-year follow-up periods of patients with amyloid bone cysts at the hip joint. Methods: Circulating CML-Hb levels were measured as previously reported, and computed tomography scanning was conducted from 1996 to 1998 or 1999 in 57 hemodialysis patients whose original renal disease had been confirmed to be nondiabetic. Patients who showed a new growth of cysts or a growth rate of 30% or more were classified as progressive cases, while the other patients were classified as nonprogressive cases. Results: (1) The circulating CML-Hb levels showed a strong correlation with the DRA score by multiple regression analysis, and (2) patients with progression of amyloid cysts showed a significantly higher circulating CML-Hb level than patients without progression. Conclusion: Based upon these results, we conclude that measurement of circulating CML-Hb levels has a potential value in both judgment of the clinical state and prediction of progression of DRA in hemodialysis patients.

By electron microscopy, we examined the skeletal muscle from two patients with Ullrich's disease. One patient had a deletion in the collagen VI alpha 2 gene. The muscle biopsy specimens showed no collagen VI immunoreaction, while the expression of collagen IV was increased. Collagen VI is a microfibrillar protein in the extracellular matrix with cell adhesive properties, and collagen IV is a principal component of the basal lamina. Electron microscopy revealed unevenness, extension, and folding of the muscle plasma membrane, and showed thickening and overproduction of the basal lamina. The data show that type VI collagen is certainly one of the important extracellular matrix components maintaining the structural integrity of skeletal muscle, and a defect of the collagen VI protein causes abnormalities of the muscle plasma membrane, dystrophic muscle changes, and up-regulation of collagen IV.

Toll-like receptors 2 and 4 (TLR2 and TLR4) have been found to transduce signals of peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively, for NF-κB activation. However, little is known about the expression and regulation of the TLR2 gene in monocytes/macrophages in response to the two typical bacterial products. We show in the present study that both PGN and a high concentration of LPS increase TLR2 gene expression in macrophage-like cells, 1α,25-dihydroxyvitamin D3-differentiated human HL60 and mouse RAW264.7 cells, and human monocytes in a dose- and time-dependent manner. Actinomycin D and pyrrolidine dithiocarbamate inhibition of gene transcription and NF-κB activation, respectively, blocks LPS- and PGN-elevated TLR2 mRNA in monocytic cells. The LPS-induced increase in TLR2 mRNA in monocytic cells is abolished by polymyxin B pretreatment and is observed in peripheral blood mononuclear cells from pigs subjected to endotoxic shock. Further, high concentrations of LPS and synthetic lipid A increase TLR2 mRNA expression in peritoneal macrophages from both TLR4-deficient C3H/HeJ mice and normal C3H/HeN mice, a process that constitutes induction of TLR4-independent TLR2 expression. These findings demonstrate that TLR2 gene expression is upregulated in macrophage responses to PGN and to high concentrations of LPS in vitro and in vivo and correlates with NF-κB activation.

The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined. Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E. coli-type lipid A (LA-15-PP) enhanced VEGF mRNA expression. LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level. Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.

Human thrombomodulin (TM) was for the first time immobilized on glass beads by the reaction between the carboxyl group of TM and the amino group of glass beads using water-soluble carbodiimide. Immobilized human TM exhibited both anticoagulant activity and inhibition of platelet aggregation of human blood.

We recently had the opportunity to study a 25-year-old male with both Ehlers-Danlos syndrome (EDS) and von Recklinghausen neurofibromatosis (VRNF). We describe the clinical manifestations of the case and discuss the probable pathomechanism of the combination of the two syndromes, with a review of the literature. As recent literature suggests that both syndromes are linked to chromosome 17, we conclude that their combination is not coincidental, but genetically linked.

Aberrant angiogenesis is associated with lesion formation in chronic periodontitis. However, little is known about the mediators that contribute to angiogenesis or about therapeutic agents that control the production of the mediators. Roxithromycin (RXM), which is a new 14-member macrolide antibiotic, has a wide antibacterial spectrum against oral pathogens and an immunomodulatory effect. In the present study, we examined the effects of RXM on tumor necrosis factor (TNF)-alpha-induced vascular endothelial growth factor (VEGF) in human periodontal ligament (HPDL) cells. In addition, the effect of RXM on VEGF expression in HPDL cells was examined.HPDL cells were plated at 5 x 10(5) cells/ml in 150 cm2 cell culture dishes. The confluent-stage cells were pretreated with or without 10 microg/ml of RXM or other antibiotics in 1% FBS-containing alpha-MEM for 24 hours, followed by simultaneous treatment with 10 ng/ml of TNF-alpha and 10 microg/ml of these antibiotics. After incubation for various periods, the culture supernatants and sediments were collected and analyzed by ELISA, Northern blot, and gel shift assays.VEGF mRNA and its protein were constitutively expressed in HPDL cells, and the level of expression was markedly enhanced by stimulation with TNF-alpha. RXM strongly inhibited the expression of VEGF mRNA and the production of VEGF. Furthermore, RXM suppressed activation of transcription factors AP-1 and SP-1, which were critical factors in VEGF transcription, in TNF-alpha-stimulated HPDL cells.These results indicate that TNF-alpha, one of the proinflammatory cytokines implicated in the pathogenesis of periodontitis, induces excess induction of VEGF in HPDL, which may account for increased angiogenesis in periodontitis lesions. Interestingly, the antibiotic roxithromycin inhibits TNF-mediated VEGF induction, suggesting its possible therapeutic utility in periodontitis and other chronic inflammatory conditions involving VEGF induction.

Remnant-like particles, which have been recognized to be atherogenic derivatives of chylomicrons and very low density lipoproteins, can be measured using a new assay kit. The purpose of the present study was to investigate the association of remnant-like particles with the coagulation system that has an important role in the pathogenesis of myocardial infarction. We assayed blood levels of total cholesterol, triglyceride, HDL-cholesterol, apolipoproteins, remnant-like particles-cholesterol, remnant-like particles-triglyceride, fibrinogen, factor VII antigen, activated factor VII, and tissue factor in 111 patients with a history of myocardial infarction and 128 control subjects. In simple regression analysis, plasma levels of remnant-like particles-cholesterol and remnant-like particles-triglyceride showed a significant positive correlation with the levels of activated factor VII (r=0.319, p

In this study, we attempted to detect altered gene expressions in the cells that had adhered to various surfaces using the differential display method. Thioglycollate-elicited peritoneal exudate cells (PEC) and mouse fibroblast (L929) cells were cultured on the polymer films. After a predetermined time, the total RNA was isolated from cells and the differential mRNA expressions were evaluated by RT-PCR method. As a result, in the differential display of amplified cDNA from PEC, the different patterns of cDNA fragments among the samples were obtained. This indicates that there were many different mRNA expressions depending on the polymer surfaces. The use of differential method was proven to be useful for studying cell-polymer interaction.

We report a marked difference in concentration of vascular endothelial growth factor (VEGF) between serum and plasma in patients with Crow-Fukase syndrome (CFS). The serum/plasma VEGF levels in 4 CFS patients were 8,634/152, 5,203/176, 3,724/127, and 868/13 pg/ml, respectively. We also showed that platelets were a major source of this VEGF and that VEGF was released during platelet aggregation by physiological stimulation. It is suggested that in CFS, local VEGF concentration is markedly elevated by aggregation of platelets containing excessive VEGF and their adhesion to vascular walls, resulting in excessive physiological activities of VEGF. Our findings provide important information for developing more effective therapeutic trials.

We demonstrated that leptin showed effects on both platelets and endothelial cells through its functional receptor. These effects are the vector to inducing thrombotic tendency. Leptin concentrations used in our experiments correspond to that of leptin in the circulation of obese individuals. Thus we suggest that increased leptin may act as a risk factor for thrombosis in obese individuals.

Castleman's disease is a lymphoproliferative disorder of unknown etiology characterized by enlarged hyperplastic lymph nodes with marked vascular proliferation. To evaluate the possible involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of Castleman's disease, we studied VEGF expression in sera and lymph nodes from four patients with either the plasma-cell type or mixed type of Castleman's disease. Clinically, one patient had the multicentric type and the others the localized type. The VEGF levels of the sera and the supernatants of the cultured lymph nodes were higher than those of normal controls. VEGF was strongly expressed in plasma cells in the interfollicular region of the lymph nodes, but rarely in normal lymph nodes. The disregulated IL-6 gene expression is considered to be a primary event that could be related to the etiology of this disease. Recently, Kaposi's sarcoma virus/human herpes virus 8 (KSHV/HHV-8) has been reported to be associated with a subset of the multicentric type of Castleman's disease, and a viral homologue of IL-6 (vIL-6) encoded by KSHV/HHV-8 has been shown to induce VEGF expression. Human IL-6 produced in the affected lymph nodes of Castleman's disease may induce paracrine VEGF-production by plasma cells and vascular proliferation in the lymph node. The confirmation of the role of VEGF in the pathogenesis of Castleman's disease may provide a therapeutic strategy.

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt are essential for the fibroblast transformation induced by many viral products. Tax, encoded by human T-cell leukemia virus type I (HTLV-I), has been demonstrated to induce the transformation of rat fibroblast Rat-1 cell through NF-kappaB activation. By stable transfection of Rat-1 cells with expressing constructs of Tax and its mutant M47, which is defective in HTLV-I LTR transactivation, we selected their transformed clones, which have characteristics of NF-kappaB activation and colony formation beyond the cell monolayer (a malignant phenotype). However, these two characteristics in the transformed clones of Tax and M47 disappear after these cells have been treated with wortmannin, a specific inhibitor of PI3K. Further, increased activity of the PI3K/Akt is observed in the transformed clones of Tax and M47 as compared to the clones of empty vector Neo and the M148, which is defective in NF-kappaB activation and cell transformation. Increased activity of PI5K is present in the transformed clones of both Tax and M47 and in the M148 clone as compared to that in the Neo cell. It is known that the efficiency of Tax-induced cell transformation is not high; a minority of Tax-expressing clones show transformation, although the majority of Tax-expressing clones show activated NF-kappaB. A Tax-expressing, nontransformed clone after transfection with an active form of the catalytic subunit of PI3K, p110alpha, becomes transformed. Consistent with these results, a Tax highly-expressing human T-cell line MT2 exhibits both higher polyphosphoinositide turnover and higher activities of PI3K and PI5K than those of Jurkat or MT1 and HTLV-I-negative and a Tax-unexpressing cell line, respectively. These results demonstrate that the activation of the PI3K/Akt signaling pathway, excepting for the NF-kappaB, is also required for the cell transformation induced by Tax.

The expression of thrombomodulin (TM) and caveolin-3 on biopsied muscle fibers from 17 patients with various neuromuscular diseases was studied immunocytochemically. We also compared the expression of TM and caveolin-3 with that of desmin, which is a marker for muscle fiber regeneration. Most TM-positive muscle fibers strongly expressed caveolin-3 and desmin. Our results suggest that TM is expressed and caveolin-3 is upregulated during the process of muscle fiber regeneration.