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Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a median survival of 3-4 years after diagnosis. It is the most frequent form of a group of interstitial pneumonias of unknown etiology. Current available therapies prevent deterioration of lung function but no therapy has shown to improve survival. Periostin is a matricellular protein of the fasciclin 1 family. There is increased deposition of periostin in lung fibrotic tissues. Here we evaluated whether small interfering RNA or antisense oligonucleotide against periostin inhibits lung fibrosis by direct administration into the lung by intranasal route. Pulmonary fibrosis was induced with bleomycin and RNA therapeutics was administered during both acute and chronic phases of the disease. The levels of periostin and transforming growth factor-β1 in airway fluid and lung tissue, the deposition of collagen in lung tissue and the lung fibrosis score were significantly reduced in mice treated with siRNA and antisense against periostin compared to control mice. These findings suggest that direct administration of siRNA or antisense oligonucleotides against periostin into the lungs is a promising alternative therapeutic approach for the management of pulmonary fibrosis.

Objective: To investigate the effect of a novel topical wound-healing agent, low-concentration povidone-iodine ointment (LPIO) with a hydrophobic white petrolatum-rich base on skin-wound models in rats and rabbits., Method: The therapeutic efficacy of topically applied LPIO was compared to that of standard-concentration povidone-iodine ointment (SPIO) and non-treatment control, using a full-thickness skin-wound model in 24 hairless rats and a full-thickness skin-defect model in rabbit earlobes. The animals were kept under standardised conditions at the Central Research Laboratory of Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan). Therapeutic efficacy was evaluated based on macroscopic wound-size reduction, as well as histopathological and immuno-histochemical examinations., Results: LPIO enhanced wound healing in rat full-thickness skin ulcers, reducing wound size and inflammation, when compared with that in SPIO and non-treatment control. LPIO also markedly improved wound healing in rabbit earlobe ulcers by significantly improving re-epithelialisation, compared with that in SPIO., Conclusion: The results of this study suggest that LPIO is a useful topical therapy for ulcerative lesions.

Unlabelled: Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis., Summary: Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: Bronchial asthma is characterised by airway inflammation and remodelling with a decline of lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that play important roles in the pathogenesis of airway remodelling. Several clinical parameters are currently being used in routine clinical practice to assess outcome of therapy in asthma including frequency of rescue with short-acting β2-agonist and the asthma control test. In this study, we hypothesised that asthma control test is associated with circulating levels of fibrocytes in bronchial asthma., Methods: There were 20 patients with asthma and seven healthy controls. The number of CD45(+)Collagen I(+) circulating fibrocytes was assessed in the peripheral blood by flow cytometry., Results: The number of circulating fibrocytes was significantly increased in asthma patients with moderate and severe disease compared to controls, and it was inversely correlated with % forced expiratory volume in one second and % forced vital capacity (%FVC). The frequency of inhalation of short-acting β2 agonist and the asthma control test score was significantly and inversely correlated with the number of circulating fibrocytes., Conclusion: The results of this study showed that the number of circulating fibrocytes is inversely correlated with clinical asthma control parameters, further supporting the relevance of measuring circulating fibrocytes as a marker of clinical control in bronchial asthma., (Copyright © 2015 SEICAP. Published by Elsevier Espana. All rights reserved.)

Background: Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown., Objectives: This study investigated the role of PS in acute alcoholic hepatitis., Methods: A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol., Results: The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls., Conclusions: The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptors (PARs). However, the role of thrombin in the immune response is not clear., Objective: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma., Methods: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge., Results: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this., Conclusions: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Infectious complications frequently occur after major trauma, leading to increased morbidity and mortality. Thrombin-activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase in plasma, plays a dual role in regulating both coagulation and inflammation. Activated TAFI (TAFIa) has broad anti-inflammatory properties due to its inactivation of active inflammatory mediators (anaphylatoxins C3a and C5a, bradykinin, osteopontin)., Objectives: The purpose of this study was to determine if TAFI plays a role in the development of inflammatory complications after major trauma., Patients/methods: Upon arrival at the emergency department (ED), plasma levels of TAFI and TAFIa were measured in 26 multiple traumatized patients for 10 consecutive days. Systemic levels of inflammatory mediators, including interleukin-6 (IL-6), procalcitonin (PCT), C-reactive protein (CRP) and leukocytes were determined., Results: Fifteen patients developed pneumonia and/or sepsis (compl) and 11 had no complications (wo compl). Overall injury severity and age were comparable in both groups. Complications occurred approximately 5 days after trauma. IL-6 increased on day 5, whereas CRP, PCT and leukocytes started to increase on day 6 in the compl-group. Upon arrival at the ED and on days 1 and 4, TAFI levels were significantly lower in the compl-group compared to the wo compl-group (p=0.0215). Similarly, TAFIa was significantly lower on day 4 in the compl-group than in the wo compl-group (p=0.049)., Conclusions: This pilot study shows that TAFI levels are inversely correlated with inflammation-associated development of complications after major trauma., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Introduction: The thrombin-activatable fibrinolysis inhibitor (TAFI) is a potent inhibitor of fibrinolysis. However, the time course of TAFI and its activated form (TAFIa) following trauma, in particular in patients suffering trauma-induced coagulopathy, has been poorly examined., Methods: A total of 26 severely injured trauma patients were prospectively enrolled. TAFI and TAFIa levels were measured upon arrival and through hospital days one to 10. Trauma-induced coagulopathy was defined as elevated international normalized ratio (INR), and/or prolonged activated partial thromboplastin time (aPTT) and/or thrombocytopenia within one day of admission., Results: TAFIa and TAFI levels showed the largest decrease on days one and two, respectively, with a progressive increase thereafter. Overall, 11 patients developed coagulopathy. No statistically significant differences were found for TAFI levels between the two groups. For TAFIa, however, coagulopathic patients experienced significantly lower levels on admission and on days six to eight (all p<0.05). Statistically significant correlations were found between TAFIa level on admission and the amount of packed red blood cells (p=0.011; Spearman's correlation coefficient=-0.5) and fresh frozen plasma (p=0.044; Spearman's correlation coefficient=-0.405) transfused within the initial 24hours., Conclusion: Depletion of TAFIa may contribute to the development of trauma-induced coagulopathy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aims: Dehydroepiandrosterone exerts a protective effect against cardiovascular diseases. However, the relationship of dehydroepiandrosterone with the anticoagulant factor activated protein C, generated by the thrombin-thrombomodulin complex on vascular endothelial cells, remains unknown. This study aimed at studying the relationship between dehydroepiandrosterone and activated protein C generation in patients with Type 2 diabetes., Methods: Sixty-two male patients with Type 2 diabetes were enrolled in this study. Data obtained from 40 healthy male subjects were used as controls. The plasma levels of dehydroepiandrosterone, the activated protein C-protein C inhibitor complex, high-sensitivity C-reactive protein and monocyte chemoattractant protein-1 were measured by enzyme immunoassays. Carotid intima-media thickness was measured by ultrasonography., Results: The plasma levels of dehydroepiandrosterone (5.15 ± 2.81 vs. 3.76 ± 2.16 ng/ml; P < 0.005) and the activated protein C-protein C inhibitor complex (1.90 ± 1.07 vs. 1.02 ± 0.51 ng/ml; P < 0.001) were significantly lower in patients with diabetes than in normal subjects. Univariate analysis showed a significant correlation of the plasma level of dehydroepiandrosterone with that of the activated protein C-protein C inhibitor complex (r = 0.48, P < 0.001), high-sensitivity C-reactive protein (r = -0.30, P < 0.05) and with the mean intima-media thickness (r = -0.28, P < 0.05) in patients with diabetes. Stepwise multiple regression analysis showed that the plasma level of dehydroepiandrosterone is significantly correlated with the plasma levels of the activated protein C-protein C inhibitor complex (F = 18.06) and high-sensitivity C-reactive protein (F = 4.94). There was no correlation between the plasma levels of dehydroepiandrosterone and monocyte chemoattractant protein-1., Conclusions: These results suggest that lower circulating levels of dehydroepiandrosterone are associated with decreased activated protein C generation and higher intima-media thickness in patients with Type 2 diabetes., (© 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.)

Background: Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by dominant T-helper (Th) 2 cytokine response. Bacillus Calmette-Guérin (BCG) has been used for preventing tuberculosis, and is regarded as a strong Th1 cytokine inducer. Antigen (Ag) 85B is a secretory protein present in Mycobacterium species that induces Th1 cytokine production., Objectives: We investigated the effects of combined vaccination of heat-killed BCG (hkBCG) and Mycobacterium kansasii Ag85B in an AD mouse model., Methods: For the AD model, keratin 14 promoter-derived caspase-1 overexpressing mice (KCASP1Tg) were used. The mice received a combination therapy of hkBCG at age 3 weeks and Ag85B twice weekly for 11 weeks from the 4th week; Ag85B monotherapy from the 4th week; hkBCG monotherapy at the 3rd week; or control saline. Areas of skin lesions, cytokine mRNA expression and serum interleukin (IL)-18 and immunoglobulin (Ig) E levels were analysed. Inducible Foxp3+  regulatory T cells (iTreg), IL-10-producing T cells (Tr1), and interferon (IFN)-γ/IL-4/IL-17-producing T cells were evaluated in the spleen., Results: Saline-treated mice and hkBCG monotherapy mice spontaneously developed severe dermatitis. However, combined therapy with hkBCG and Ag85B significantly suppressed the development of skin lesions and mast cell infiltrations. Elevations of the serum IgE and IL-18 levels were significantly suppressed with combined therapy. Mice treated with hkBCG and Ag85B had a normal number of iTreg in the spleen, and decreased number of both IL-4- and IL-17-producing CD4+ T cells. The effect of Ag85B monotherapy was limited., Conclusions: Combined vaccination with hkBCG and Ag85B decreases AD skin lesions by inducing regulatory T cells, suggesting that this vaccination is a potent and novel therapeutic strategy for AD., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Background: Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown., Objectives: We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy., Methods: Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month., Results: APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-β1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment., Conclusion: Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease., (© 2012 International Society on Thrombosis and Haemostasis.)

Background: Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein-E (ApoE) genotype has been associated with carcinogenesis., Objective: The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE-deficient mouse model., Methods: TAFI and ApoE double-knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild-type (wt) mice served as controls., Results: Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double-knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor-β1 were increased in TAFI/ApoE double-deficient mice treated with stz compared with the mice of the same genotype treated with saline., Conclusion: Apo-E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice., (© 2010 International Society on Thrombosis and Haemostasis.)

Background: 1,24-Dihydroxyvitamin D₃ (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D₃ inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells., Objectives: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells., Methods: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells., Results: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model., Conclusions: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels., (© 2010 The Authors. Journal Compilation © 2010 British Association of Dermatologists.)

Summary: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH., Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse., Methods and Results: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg(-1) monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice., Conclusions: TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation.

Objective: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse., Methods: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation., Results: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells., Conclusion: Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Background: Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions., Objective: To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX)., Methods: Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed., Results: Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-gamma., Conclusion: These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.

Background: C4b-binding protein (C4BP), a multimeric protein structurally composed of alpha chains (C4BPalpha) and a beta chain (C4BPbeta), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)-6. However, it is not fully understood how lipopolysaccharide (LPS) and IL-6 affect the plasma C4BP antigen level and C4BPalpha and C4BPbeta expression in hepatocytes., Objectives: To assess the effect of LPS and IL-6 on plasma C4BP, PS-C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro., Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg(-1)) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL-6 (10 microg kg(-1)) injection, and then gradually decreased up to 24 h after IL-6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPalpha and C4BPbeta in rat hepatocytes, and this effect was inhibited by NFkappaB and MEK/ERK inhibitors. IL-6 mediated increase in C4BPbeta expression in rat hepatocytes, which leads to increased plasma PS-C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT-3., Conclusion: LPS decreases both C4BPalpha and C4BPbeta expression via the NFkappaB and MEK/ERK pathways, whereas IL-6 specifically increases C4BPbeta expression via the STAT-3 pathway, causing an increase in plasma PS-C4BP complex, and thus decreasing the anticoagulant activity of PS.

There are no prospective comparison of the etiology and clinical outcome between hospital-acquired pneumonia (HAP) and nursing home-acquired pneumonia (NHAP) in non-intubated elderly. This study prospectively evaluated the etiology of HAP and NHAP in non-intubated elderly. A prospective cohort study was carried out in a rural region of Japan where the population over 65 years of age represents 30% of the population. A total of 108 patients were enrolled. There were 33 patients with HAP and 75 with NHAP. Etiologic diagnosis was established in 78.8% of HAP and in 72% of NHAP patients. The most frequent pathogens were Chlamydophila pneumoniae followed by Streptococcus pneumoniae, Staphylococcus aureus and Influenza virus. The frequency of Streptococcus pneumoniae and Influenza virus was significantly higher, whereas the frequency of Staphylococcus aureus and Enterobacteriaceae was significantly lower in NHAP compared to HAP. Performance and nutritional status were significantly worse in patients with HAP than in those with NHAP. Hospital mortality was significantly lower in patients with NHAP compared to those with HAP. This study demonstrated that C. pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus and Influenza virus are frequent causative agents of pneumonia in non-intubated elderly and that the responsible pathogens and clinical outcome differ between NHAP and HAP.

Background: We recently reported that human protein C inhibitor (PCI), a major inhibitor of activated protein C (APC), inhibits hepatocyte growth factor activator (HGFA) by forming HGFA-PCI complexes in vitro. In this study, we evaluated whether PCI regulates HGFA-mediated liver regeneration in a human PCI gene transgenic (hPCI-Tg) mouse model., Methods and Results: After partial hepatectomy in hPCI-Tg and wild-type (WT) mice, the degree of liver regeneration, protein and mRNA expression of HGFA, proHGF activation, plasma levels of PCI and HGFA-PCI complex, and other markers were evaluated in the remnant liver. We also evaluated the effect of anti-human PCI antibody on liver regeneration, which significantly decreased in hPCI-Tg mice compared to WT mice. HGFA mRNA levels in naive and remnant livers after hepatectomy were the same in both WT and hPCI-Tg mice; however, plasma HGFA levels and HGF activation in the liver were lower in hPCI-Tg than in WT mice. There was no difference in plasma levels of transaminases and inflammatory cytokines. However, sinusoidal congestion and bleeding were detected and the serum hyaluronic acid level was elevated in hPCI-Tg mice, indicating that human PCI aggravates sinusoidal injury by inhibiting the cytoprotective effect of APC. APC decreased thrombin-induced IL-6 production in isolated hepatic nonparenchymal cells (NPCs) in vitro. This impaired liver regeneration was reversed by anti-human PCI antibody treatment in vivo., Conclusion: PCI regulates liver regeneration after hepatectomy by forming an HGFA-PCI complex and aggravates hepatic NPC injury by inhibiting the cytoprotective effect of APC. Anti-PCI antibody treatment may be a novel therapy for improving liver regeneration.

Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) has been reported to affect wound healing and fibrotic processes, but its role in renal tubulointerstitial fibrosis remains unknown., Objective: To study its potential role, we compared TAFI-deficient and wild-type mice for the degree of renal fibrosis caused by unilateral ureteral obstruction (UUO)., Methods: The grade of tubulointerstitial fibrosis, the activity of plasmin, MMP-2 and MMP-9 were evaluated on days 4 and 9 after UUO., Results: The renal content of hydroxyproline and the activity of plasmin, MMP-2 and MMP-9 were significantly increased in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. These differences disappeared when animals with UUO from both groups were treated with the plasmin inhibitor tranexamic acid. The renal concentrations of fibrogenic cytokines were also significantly elevated in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice., Conclusion: The results of this study suggest that increased renal activity of plasmin in TAFI-deficient mice causes increased renal interstitial fibrosis in obstructive nephropathy.

Aims: Activated protein C (APC) is a key regulator of the clotting system and immune responses. We studied the relationship between the degree of atherosclerosis as measured by the intima-media thickness (IMT) of carotid artery and APC generation in Type 2 diabetic patients., Methods: Eighty-seven Type 2 diabetic patients and 35 control subjects participated. APC generation was assessed by the plasma APC-protein C inhibitor complex (APC-PCI) levels and the mean IMT of carotid artery was measured by ultrasonography. The plasma levels of the thrombin-anti-thromobin complex (TAT) and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunoassays., Results: Plasma TAT levels were significantly higher in diabetic patients [2.03 (1.12, 2.56) ng/ml, median (25th, 75th percentile)] compared with control subjects [0.85 (0.55, 2.08) ng/ml, P < 0.01]. Plasma APC-PCI levels were significantly lower in diabetic patients [0.93 (0.74, 1.22) ng/ml], than in control subjects [1.66 (1.25, 2.36) ng/ml, P < 0.001]. The mean IMT was significantly increased in diabetic patients (0.881 +/- 0.242 mm; mean +/- sd) compared with control subjects (0.669 +/- 0.140 mm; P < 0.01). Univariate analysis showed a significant and inverse correlation between plasma APC-PCI levels and mean IMT (r = -0.32, P < 0.005), and multivariate regression analysis confirmed the independent correlation (P < 0.05). Moreover, plasma APC-PCI levels significantly and inversely correlated with plasma PDGF levels in diabetic patients (r = -0.30, P < 0.01)., Conclusions: These results suggest that decreased APC generation is associated with vascular atherosclerotic changes in Type 2 diabetic patients.

Background: Hepatocyte growth factor (HGF) plays an important role in tissue repair and regeneration. HGF activator (HGFA), a factor XIIa-like serine protease, activates HGF precursor to HGF. The precursor of HGFA, proHGFA, is activated by thrombin generated at sites of tissue injury. It is known that protein C inhibitor (PCI), an inhibitor of activated protein C (APC), also inhibits thrombin-thrombomodulin (TM) complex., Objectives: In the present study we evaluated the effect of PCI on thrombin-catalyzed proHGFA activation in the presence of TM, and on HGFA activity., Results: PCI did not inhibit thrombin-TM-mediated proHGFA activation, but it directly inhibited activated HGFA by forming an enzyme inhibitor complex. The second-order rate constants (m(-1) min(-1)) of the reaction between HGFA and PCI in the presence or absence of heparin (10 U mL(-1)) were 4.3 x 10(6) and 4.0 x 10(6), respectively. The inhibition of HGFA by PCI resulted in a significant decrease of HGFA-catalyzed activation of HGF precursor. Exogenous HGFA added to normal human plasma formed a complex with plasma PCI, and this complex formation was competitively inhibited by APC in the presence of heparin, but very weakly in the absence of heparin. We also demonstrated using recombinant R362A-PCI that Arg362 residue of PCI is important for HGFA inhibition by PCI as judged from the three-dimensional structures constructed using docking models of PCI and HGFA or APC., Conclusion: These observations indicate that PCI is a potent inhibitor of activated HGFA, suggesting a novel function for PCI in the regulation of tissue repair and regeneration.

Tenascin-C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune-mediated hepatitis in wild-type (WT) and TNC-null (TNKO) mice. Eight-week-old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon-gamma, tumour necrosis factor-alpha, and interleukin-4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)-beta1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF-beta expression during progression of hepatitis to fibrosis., (Copyright (c) 2006 Pathological Society of Great Britain and Ireland.)

Background: Liver dysfunction caused by intrasinusoidal microthrombi is frequently observed in patients with cirrhosis after hepatectomy, but the mechanistic pathway remains unknown., Objective: In the present study, we evaluated the expression of protein S (PS) in hepatocytes and sinusoidal endothelial cells (SECs) from rats with dimethylnitrosoamine-induced cirrhosis before and after hepatectomy., Results: The plasma level of PS antigen was significantly decreased in cirrhotic rats as compared to control rats treated with vehicle. PS expression was significantly decreased in hepatocytes isolated from cirrhotic rats as compared to controls. In contrast, PS expression was significantly increased in SECs isolated from rats with cirrhosis as compared to controls. Interleukin-6 (IL-6) upregulated the expression of PS in hepatocytes, and tumor necrosis factor-alpha (TNF-alpha) decreased its expression in SECs from both cirrhotic and normal rats. The production of IL-6 and TNF-alpha by Kupffer cells and SECs was decreased in rats with cirrhosis as compared to controls. After hepatectomy, microthrombus formation was markedly enhanced in sinusoids from rats with cirrhosis, and the plasma levels of IL-6 and TNF-alpha were significantly increased in rats with cirrhosis as compared to controls. Furthermore, PS production in SECs was decreased, whereas that in hepatocytes was significantly increased in cirrhotic rats as compared to controls., Conclusions: These findings suggest that PS expression is differently regulated in hepatocytes and SECs of rats with cirrhosis before and after hepatectomy, that the expression of PS is regulated by locally released inflammatory cytokines, and that decreased expression of PS in SECs may cause liver microthrombus formation, which is frequently observed in patients with cirrhosis after hepatectomy.

Background: Protein C inhibitor (PCI) plays a role in multiple biological processes including fertilization, coagulation, fibrinolysis and kinin systems., Objectives: We hypothesized that PCI participates in the pathogenesis of pulmonary hypertension. To demonstrate this, we compared the development of pulmonary hypertension in mice overexpressing PCI in the lung with wild-type (WT) mice. Pulmonary hypertension was induced by s.c. injection of 600 mg kg-1 of monocrotaline weekly for 8 weeks., Results: Right ventricular arterial pressure was significantly increased in monocrotaline-treated WT mice compared with that in monocrotaline-treated transgenic mice. Bronchoalveolar lavage fluid (BALF) levels of thrombin-antithrombin complex, monocyte chemoattractant protein-1 and platelet-derived growth factor, and the plasma level of tumor necrosis factor-alpha were significantly increased in monocrotaline-treated WT mice as compared with monocrotaline-treated PCI transgenic mice. Increased level of PCI-thrombin complex was detected in BALF from monocrotaline-treated PCI transgenic mice as compared with saline-treated PCI transgenic mice., Conclusions: This study showed that increased expression of PCI in the lung is protective against monocrotaline-induced pulmonary hypertension, suggesting a potential beneficial effect of PCI for the therapy of this disease.

Background: The vitamin K-dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear., Objectives: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats., Results: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4-8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) transiently increased in plasma. IL-6 increased the protein expression of PS from hepatocytes, while TNF-alpha decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll-like receptor-4 (TLR-4) expression in both cells. Antirat CD14 and antirat TLR-4 antibodies inhibited LPS-induced NFkappaB activation, and a NFkappaB inhibitor suppressed LPS-induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS-induced decreased PS expression in both cells., Conclusions: These findings suggest that LPS-induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFkappaB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.

It has been previously demonstrated that activated protein C (APC) plays an important role in the inhibition of inflammation in the gastric mucosa from patients with Helicobacter pylori infection. However, the role of gastric epithelial cells in the anti-inflammatory activity of APC remains unknown. In the present study, we evaluated the anti-inflammatory activity of APC and the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in gastric epithelial cells. The gastric epithelial cell lines, MKN-1 and AGS, and gastric biopsy samples from patients with and without H. pylori infection were used in the experiments. Polymerase chain reaction showed that gastric epithelial cell lines express EPCR and TM. Flow cytometry analysis also showed EPCR expression in both cells. H. pylori infection significantly increased EPCR expression compared with non-infected cells. Similar results were observed in vivo when samples from patients with and without H. pylori infection were analyzed for EPCR protein expression. Significant TM activity was found on AGS and MKN-1 cells stimulated with LPS from Escherichia coli and VacA antigen. APC was able to significantly inhibit the secretion of MCP-1 and IL-1beta induced by H. pylori homogenate in AGS cells. APC also remarkably suppressed the mRNA expression and secretion of MCP-1 from AGS cells infected with H. pylori. These results demonstrated the expression of components of the protein C pathway on gastric epithelial cells and that APC may play a critical role in the protection against gastric mucosal inflammation.

Background: Several studies have shown that reflux esophagitis (RE) occurs after eradication of Helicobacter pylori. However, endoscopic findings do not allow prediction of the development of RE after successful treatment. In this study, we evaluated the relationship between the prevalence of RE after eradication therapy and the degree of hiatal hernia., Methods: The study comprised 148 patients who had undergone H. pylori eradication therapy over the past 5 years. The degree of RE and hiatal hernia was evaluated based on endoscopic findings. Hiatal hernia was graded according to Hill's gastroesophageal flap valve (GEFV; grades I-IV) classification. RE after eradication therapy was graded according to the Los Angeles classification system. H. pylori infection was confirmed in all patients by culture, urease test and histological examination of antral and fundic biopsy specimens., Results: Among 148 patients, there were 122 patients (82.4%) with successful and 26 (17.6%) with failed eradication therapy. RE was diagnosed in 25 (20.5%) out of 122 patients with successful therapy but only in 1 (3.8%) out of 26 patients with failed therapy (P < 0.05). After successful eradication, 25 patients had mild RE (12 with grade A, 13 with grade B). Among patients of the successful eradication group (n = 122), RE was diagnosed in 2 (5.3%) out of 38 patients without hiatal hernia and in 23 (27.4%) out of 84 patients with hiatal hernia (P = 0.0051). Furthermore, RE was diagnosed in 2 (5.3%) out of 38 patients with GEFV grade I, 13 (24.1%) out of 54 with grade II, 7 (30.4%) among 23 with grade III, and 3 (42.9%) out of 7 patients with grade IV. The pH level of gastric juice after eradication therapy was lower in the group with successful eradication than in the group with failed therapy regardless of the incidence and degree of RE., Conclusions: There is a high incidence of RE after successful H. pylori eradication therapy. This incidence of RE was closely associated with the presence and degree of hiatal hernia and with the decrease in gastric juice pH. These findings suggest that the presence of hiatal hernia together with increase in gastric acidity are important determinant factors for the development of RE after successful H. pylori eradication therapy.

In humans, protein C inhibitor (PCI) is expressed in various tissues and present in many body fluids including plasma and seminal fluid. In rodents, PCI is expressed in reproductive organs only and is absent in plasma. In this study, we characterized the tissue expression and physiological role of PCI in novel human PCI gene transgenic (TG) mice. Northern blot and immunohistochemical analyses demonstrated that human PCI is expressed in liver hepatocytes, renal epithelial cells as well as heart, brain and reproductive organs of the TG mice. This PCI tissue distribution is similar to that found in humans. PCI in plasma of TG mice showed the same immunological and functional properties as human plasma PCI. Next, we evaluated the effect of PCI on coagulation, inflammation and tissue damage in lipopolysaccharide-treated TG mice. The results suggested that PCI efficiently inhibits not only the anticoagulant and anti-inflammatory activities of exogenously injected human activated protein C (APC) but also that of endogenously produced APC in mice with endotoxemia. These findings suggest that PCI exerts a procoagulant and proinflammatory effect by inhibiting APC. We believe our results also show how useful these TG mice may be for assessing the therapeutic effect of human APC in vivo and for evaluating the role of PCI in human physiological and pathological conditions.

Thrombin is a serine protease involved in many physiological functions and its receptor. the protease-activated receptor-1 (PAR-1), has a wide tissue distribution. We hypothesized that PAR-1 is expressed in gastric epithelial cells and that thrombin can modulate defence mechanisms through PAR-1. The rat gastric epithelial cell line (RMG1) and gastric biopsy specimens from gastritis patients were used in the study. Reverse transcriptase polymerase chain reaction analysis showed that the thrombin receptors PAR-1, PAR3 and PAR-4 are expressed by RGM1 gastric epithelial cell line. Immunohistochemical and electron microspcopic studies also showed PAR-1 expression in human gastric epithelial cells. Thrombin stimulated the secretion of mucin and prostaglandin E2 (PGE2) formation in RGM1 cells in a dose-dependent manner. PAR-1 agonist also stimulated PGE2 formation. In addition, thrombin significantly increases the expression of the PGE2 receptors EP2-R and EP4-R in RGM1 cells. In conclusion, the results of the present study showed for the first time that gastric epithelial cells express thrombin receptors and that these receptors may play a protective role in the gastric mucosa.

We investigated the relationship of the A/G variant of the tumor necrosis factor-alpha (TNF-alpha) gene promoter at position -308 with insulin resistance and abdominal fat distribution in type 2 diabetic patients in the Japanese population. The TNF-alpha polymorphism was evaluated by polymerase chain reaction-restriction fragment length polymorphism in 142 healthy volunteers and 132 type 2 diabetic patients. Insulin sensitivity was assessed by homeostasis model assessment (HOMA) index in healthy subjects and hyperinsulinemic euglycemic clamp in type 2 diabetic patients. Abdominal fat distribution was evaluated by computed tomography (CT) scanning in diabetic patients. The TNF-alpha polymorphism was detected in three healthy volunteers and three type 2 diabetic patients, all of them being heterozygotes. There was no significant difference in allele frequencies of the -308 polymorphism between healthy subjects (0.0106) and type 2 diabetic patients (0.0114). HOMA index was no significant difference between healthy subjects with and without polymorphism (1.09 +/- 0.03 vs. 1.02 +/- 0.05). Glucose infusion rate (GIR), an index of insulin sensitivity, was not significantly different between diabetic patients with and without TNF-alpha polymorphism (40.4 +/- 4.1 vs. 45.0 +/- 1.8 micromol/kg per min). Moreover, no remarkable effect of TNF-alpha polymorphism on abdominal fat distribution was observed in diabetic patients. These results suggest that A/G heterozygotes of the TNF-alpha gene promoter at position -308 play no major role in the pathogenesis of insulin resistance or abdominal fat distribution in Japanese type 2 diabetic patients.

The protein C (PC) pathway plays important roles in the regulation of the coagulation system and inflammatory response. This study evaluated the degree of PC activation in the airway of patients with bronchial asthma (BA), and the expression and regulation of PC and its receptor in airway epithelial cell lines. Thirteen BA patients and 8 healthy volunteers were enrolled in the study. BEAS-2B and A549 epithelial cell lines were used in experimental assays. Expression of anticoagulant factors was evaluated by RT-PCR and Western blotting. The activated protein C (APC)/thrombin (1.65 +/- 0.35 vs 3.34 +/- 0.59) and APC/PC (8.30 +/- 2.26 vs 24.41 +/- 9.88) ratios were significantly decreased and the concentrations of soluble thrombomodulin (TM) were significantly increased in induced sputum from BA patients compared with healthy subjects. Airway epithelial cells express PC, its receptor, and TM. PC antigen prepared from epithelial cells was significantly activated in the presence of thrombin. Thrombin increased the expression of PC antigen from lung epithelial cells. However, tumor necrosis factor-alpha, eotaxin, and RANTES (regulated on activation, normal T-cell expressed and secreted) decreased the expression of PC and its receptor in bronchial epithelial cells. Overall, these results showed for the first time that reduced activation of PC pathway occurs in the airway of BA patients and that TM, PC, and its receptor, are expressed by human airway epithelial cells. The expression of these PC pathway components was found to be downregulated by inflammatory cytokines. The decrease in PC activation may contribute to exacerbation of the inflammatory response in the airway of asthmatic patients.

We measured the plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) activity and antigen in patients with disseminated intravascular coagulation (DIC) to examine the relationship between hypofibrinolysis and the pathogenesis of DIC. TAFI activity and antigen levels in the plasma were both significantly low in patients with DIC. TAFI activity in plasma was correlated with TAFI antigen, indicating that activity and antigen correspond well. The decrease of TAFI activity in DIC may be due to enhanced consumption. Since the plasma thrombin-antithrombin III complex (TAT) level was found to be elevated in DIC, increase of thrombomodulin-thrombin complex generation is suggested in this state. TAFI activity and antigen levels were negatively correlated with TAT and D-dimer, suggesting that the plasma levels of TAFI are reduced by thrombin generation. Since TAFI was not correlated with fibrinogen, plasma-alpha(2)plasmin inhibitor complex (PPIC) and tissue type plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complex, TAFI might be a secondary modulator of fibrinolysis. The TAFI activity in plasma was significantly low in patients with infection and in those with organ failure, suggesting that TAFI may play an important role in the mechanism of organ failure in DIC-associated sepsis. In brief, TAFI may play an important role in the pathogenesis of DIC and organ failure.

In this study, we examined changes in the plasma levels of total plasminogen activator inhibitor-I (PAI-I) and tissue-type plasminogen activator (tPA)/PAI-I complex in patients with disseminated intravascular coagulation (DIC) and in those with thrombotic thrombocytopenic purpura (TTP) to investigate the fibrinolytic function and its relation to organ failure. The plasma levels of total PAI-1 and tPA/PAI-I complex were significantly higher in patients with DIC, pre-DIC, and TTP than in those with non-DIC. The plasma levels of thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex (PPIC), D-dimer, thrombomodulin (TM), total PAI-I, and tPA/PAI-I complex were significantly higher in patients with organ failure than in those without organ failure. The plasma levels of total PAI-I and tPA/PAI-I complex were markedly increased in patients with acute leukemia. The plasma levels of total PAI-I, but not those of tPA/PAI-I complex, were significantly increased in patients with sepsis or with solid cancer. In all cases, total PAI-I or tPA/PAI-I complex was not significantly correlated with any hemostatic marker. Measurement of total PAI-I and tPA/PAI-I complex may be useful in the diagnosis of DIC.

Background and Aim: Low-grade fasting hyperbilirubinemia is a common observation in healthy subjects (HS), whereas high-grade fasting hyperbilirubinemia is believed to be a characteristic finding of Gilbert's syndrome. This study was undertaken to assess the role of mutation in bilirubin UDP- glycosyltransferase gene (UGT1A1) on fasting hyperbilirubinemia., Methods: Analysis of UGT1A1 and a caloric restriction test (400 kcal for 24 h) were performed in 56 healthy subjects (25 males, 31 females), and 28 patients with Gilbert's syndrome (18 males, 10 females). There were 29 healthy subjects with no mutation in UGT1A1, and 27 healthy subjects and 26 Gilbert's syndrome patients with mutations in the coding and/or promoter (TATA box) regions of UGT1A1., Results: The mean increment of serum bilirubin (DeltaSB) was 7.6 micromol/L [corrected] (males) and 4.1 micromol/L (females) in subjects with no UGT1A1 mutation. Subjects with mutation in UGT1A1 showed higher levels of DeltaSB than individuals without mutation. Among healthy subjects, gender difference in DeltaSB values was observed only in individuals with the wild type of UGT1A1, but not in those with mutations in this gene., Conclusion: The results of the present study suggest that UGT1A1 mutation has a role in the development of high-grade fasting hyperbilirubinemia after caloric restriction.

It is well recognized that activation of the coagulation system plays an important role in bleomycin (BLM)-induced lung injury and fibrosis. The protein C (PC) pathway is an important regulator of the coagulation system. In this study, we evaluated the bronchoalveolar lavage fluid (BALF) concentration of activated PC (APC) and the therapeutic effect of the intratracheal administration of APC on BLM-induced lung fibrosis in mice. APC levels in BALF were significantly lower in BLM-treated animals than in the saline-treated group. Fibrotic changes were progressive in mice treated with BLM and intratracheal instillation of vehicle (BLM/Veh) after 14 and 21 d of BLM infusion. Compared with the BLM/Veh group, histologic findings on Days 14 and 21 in mice treated with BLM and intratracheal instillation of APC (BLM/APC) showed less fibrotic lesions in the subpleural and central areas of the lung. The mean Aschcroft's fibrosis score in the BLM/Veh group was significantly (p < 0.05) higher than in the BLM/APC group. The lung hydroxyproline content on Day 21 was significantly higher (p < 0.05) in the BLM/Veh group (1.78 +/- 0.07 micromol/lung weight) than in the BLM/APC (1.30 +/- 0.06 micromol/lung weight) group. The ratio of plasminogen activator activity to thrombin level in BALF was significantly increased in the BLM/APC group compared with the BLM/ Veh group on Day 21. The expression of tumor necrosis factor-alpha and interleukin-1beta was significantly decreased in the lungs of the BLM/APC group compared with the BLM/Veh group on Day 14 after BLM infusion. These results showed that intratracheal APC administration inhibits the development of lung fibrosis in BLM-induced lung injury, giving further support to the important role that the PC pathway plays in the mechanism of lung fibrosis.

Inhalation of nitric oxide (NO) is useful for the treatment of patients with pulmonary hypertension. However, the potential toxicity of inhaled NO is still unclear. Coagulation activation plays an important role in lung injury. We assessed the effect of low- and high-dose inhaled NO on the coagulation system in the intraalveolar space of mice. The animals were assigned to five groups (n = 6): [RA] group, mice exposed to fresh air alone; [RA+2 ppm NO] group, fresh air and 2 ppm NO; [RA+40 ppm NO] group, fresh air and 40 ppm NO; [RA+2 ppm NO+O(2)] group, fresh air, 2 ppm NO and O(2); and [RA+40 ppm NO+O(2)] group, fresh air, 40 ppm NO and O(2). Each group was treated for 3 wk. Lung specimens of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups showed significant nitrotyrosine immunoreactivity. BALF concentrations of total protein, thrombin and soluble tissue factor were significantly increased in mice of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups compared with [RA] group. However, BALF concentrations of total protein, thrombin, and soluble tissue factor were not significantly increased in mice of [RA+2 ppm NO] and [RA+2 ppm NO+O(2)] groups compared with [RA] group. Lung tissue factor mRNA expression was higher in the high-dose NO group than in the low-dose NO group. NO donor increased significantly tissue factor activity on alveolar epithelial cells. This study has shown for the first time that long-term inhalation of high, but not low, concentration of NO may activate the clotting system by increasing the lung expression of tissue factor.

To investigate the relationship between peripheral blood levels of agouti-related protein (AGRP) and various parameters of obesity, we measured the plasma level of AGRP in 15 obese and 15 nonobese men and evaluated its relationship with body mass index (BMI), body fat weight, and visceral, sc, and total fat areas measured by computed tomography, fasting insulin levels, glucose infusion rate during an euglycemic hyperinsulinemic clamp study, serum leptin, and plasma alpha-MSH. Obese men had significantly higher plasma concentrations of AGRP than nonobese men (P < 0.01). Univariate analysis showed that the plasma levels of AGRP are proportionally correlated with BMI, body fat weight, and sc fat area in obese men (BMI: r = 0.732, P < 0.01; body fat weight: r = 0.603, P < 0.02; sc fat area: r = 0.668, P < 0.01) and in all men (BMI: r = 0.839, P < 0.0001; body fat weight: r = 0.818, P < 0.0001; sc fat area: r = 0.728, P < 0.0001). In all men, the plasma levels of AGRP were significantly correlated with the visceral fat area (r = 0.478, P < 0.01), total fat area (r = 0.655, P < 0.0001), fasting insulin level (r = 0.488, P < 0.01), glucose infusion rate (r = -0.564, P < 0.01), serum level of leptin (r = 0.661, P < 0.0001), and the plasma level of alpha-MSH (r = 0.556, P < 0.01). In all subjects, multiple regression analysis showed that the plasma levels of AGRP are significantly (F = 15.522, r = 0.801, P < 0.03) correlated with the plasma levels of alpha-MSH, independently from the total fat area. However, the correlation between plasma levels of AGRP and serum levels of leptin was found to be dependent on the total fat area. In brief, these findings showed that the circulating levels of AGRP are increased in obese men and that they are correlated with various parameters of obesity. Although correlation does not prove causation, the results of this study suggest that peripheral AGRP may play a role in the pathogenesis of obesity.

Objective: To investigate the usefulness of the homeostasis model assessment as an index of insulin resistance (HOMA-IR) for evaluating the clinical course of patients with type 2 diabetes., Research Design and Methods: The usefulness of HOMA-IR and its relationship with insulin resistance assessed by the hyperinsulinemic-euglycemic clamp study (clamp IR) were evaluated in 55 Japanese patients with type 2 diabetes before and after treatment. The patients were subjected to diet (approximately 1,440-1,720 kcal/day) and exercise therapy (walking 10,000 steps daily) for 6 weeks during their hospitalization., Results: Univariate regression analysis disclosed a significant correlation between log-transformed HOMA-IR and log-transformed clamp IR before (r = -0.613, P < 0.0001) and after ( = -0.734, P < 0.0001) treatment. Neither the slopes (-0.71 +/- 0.12 vs. -0.79 +/- 0.09, F = 0.25, P = 0.61) nor the intercepts (y-intercept = 1.67 vs. 1.70, x-intercept = 2.36 vs. 2.15, F = 0.02, P = 0.88) of the regression lines between HOMA-IR and clamp IR were significantly different before and after treatment. There was a significant correlation between the decrease in log-transformed HOMA-IR and the increase in clamp IR during treatment (r = -0.617, P < 0.0001)., Conclusions: HOMA-IR may constitute a useful method not only for diagnosing insulin resistance, but also for follow-up during the treatment of patients with type 2 diabetes.

Several hemostatic and vascular endothelial cell markers were measured in 39 patients with thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) and in 20 healthy volunteers to examine the relationship between the occurrence of hemostatic abnormality or vascular endothelial cell injury and patient outcome. The plasma levels of von Willebrand factor, tissue plasminogen activator (TPA), plasminogen activator inhibitor (PAI-1), and the TPA-PAI-1 complex were significantly increased in TTP/HUS patients; however, the levels of these markers were not significantly different between TTP/HUS patients who survived and those who died, suggesting that these markers might not be directly related to outcome. The plasma levels of soluble granule membrane protein (GMP)-140 were significantly higher in TTP/HUS patients than in healthy volunteers, suggesting that platelets and vascular endothelial cells are activated or injured in TTP/HUS. There was no significant difference in GMP-140 levels between TTP/HUS patients with good and poor prognoses; this may be owing to the release of GMP-140 from platelets. The plasma thrombomodulin (TM) levels in TTP/HUS patients were significantly higher than in healthy volunteers; the plasma TM levels were significantly higher in patients who died than in patients who survived. These findings showed that TM levels reflect the outcome and that the outcome of TTP/HUS depends on the presence vascular endothelial cell injury. The plasma protein C and antithrombin levels were markedly reduced in TTP/HUS patients who died compared with those who survived. These findings suggest that reduced plasma antithrombin and protein C may be useful markers of systemic vascular endothelial injury. In conclusion, the results of this study showed that the outcome of TTP/HUS is related to vascular endothelial cell injury and that plasma TM, antithrombin, and protein C levels may be useful markers of systemic vascular endothelial cell injury.

The patient was a 32-year-old obese woman with a history of type 2 diabetes and hypertension for 6 years. Although she was treated with antihypertensive agents and intensive insulin therapy, her hyperglycemia was difficult to control. She wanted to have a baby but pregnancy was not recommended because her diabetes was under poor control and the use of antihypertensive medication. To achieve good control of obesity, diabetes and hypertension, she was admitted to our clinical department for weight reduction using very low calorie diet (VLCD). During VLCD she had a 19.8 kg reduction in body weight and her blood glucose and blood pressure were in good control without the use of drugs. Five months later, she became pregnant after the fourth trial of intrauterine insemination (IUI) and gave birth to a female baby under insulin therapy. This is the first report that showed the usefulness of VLCD for prepregnant control of glucose metabolism and blood pressure in an obese hypertensive patient with type 2 diabetes mellitus.

Plasma levels of tissue factor pathway inhibitor (TFPI)-activated factor Xa (FXa) complex were measured in patients with disseminated intravascular coagulation (DIC), pre-DIC, and DIC. Plasma levels of plasmin-plasmin inhibitor complex (PPIC), D-dimer, and soluble fibrin monomer (SFM) were significantly higher in patients with DIC than in those with pre-DIC or non-DIC; the levels of these hemostatic markers were significantly higher in patients with pre-DIC than in those with non-DIC. Plasma levels of thrombin-antithrombin complex (TAT) were significantly higher in patients with DIC or pre-DIC than in those with non-DIC. Plasma levels of tissue factor (TF), total TFPI, free TFPI, and TFPI-Xa complex were significantly higher in patients with DIC than in those with non-DIC. Plasma levels of TFPI-Xa complex were significantly increased in patients with pre-DIC as compared to those with non-DIC; however, plasma free TFPI levels were significantly decreased in patients with pre-DIC as compared to those with non-DIC. These findings suggest that free TFPI might be consumed in the pre-DIC state, thereby confirming the activation of the extrinsic pathway. Plasma levels of TFPI-Xa complex were significantly correlated with TF, free TFPI, and total TFPI. Increased plasma TFPI-Xa complex levels might be useful for the diagnosis of DIC or pre-DIC, particularly that occurring by activation of the extrinsic pathway of blood coagulation.

The authors evaluated the hemostatic abnormalities occurring in the postoperative period of eight patients with malignant tumors and compared them with those occurring in the postoperative period of eight patients with benign tumors. Two of the patients with malignant tumor presented pulmonary embolism after operation. Plasma fibrinogen and fibrin degradation product levels in patients with malignant tumors were already high before operation and further increased significantly after operation. The plasma levels of D-dimer, thrombin-antithrombin complex, and free-tissue factor pathway inhibitor were increased in both groups after operation, but they were higher in patients with malignant tumors than in patients with benign tumors. The plasma levels of protein C and antithrombin were significantly decreased in both groups after operation. but they were significantly lower in patients with malignant tumors than in those with benign tumors. The decreased activity of protein C or antithrombin may be not only a risk factor of thrombotic disease, such as pulmonary embolism, but also the cause of thrombosis. In patients with malignant tumors, the operation time was significantly longer than that in patients with benign tumors. This long operative period might cause vascular endothelial cell injury which is reflected by the plasma levels of free-tissue factor pathway inhibitor, antithrombin, and protein C.

Activation of the coagulation system in the alveolar space plays an important role in the pathogenesis of interstitial lung disease (ILD) and pulmonary fibrosis. The protein C (PC) pathway is the main modulator of coagulation activation. This study evaluated whether dysfunction of the PC pathway is associated with increased collagen synthesis in the intraalveolar space of patients with ILD. This study comprised 22 patients with ILD; of these, five had idiopathic pulmonary fibrosis (IPF), nine had sarcoidosis-associated ILD, and eight had collagen vascular disease-associated ILD (CVD-ILD). Thrombin-antithrombin complex (TAT) was measured as a marker of coagulation activation. As markers of the PC pathway activity, the concentration of activated PC-PC inhibitor (APC-PCI) complex and the APC-PCI/PC ratio were measured and, as a marker of collagen synthesis, the concentration of aminoterminal propeptide of type III procollagen (PIIINP) was measured in bronchoalveolar lavage fluid (BALF) of ILD patients. TAT was significantly increased in BALF from ILD patients as compared to control subjects. The concentrations of PIIINP were significantly elevated in patients with ILD as compared to healthy subjects. In contrast, the concentration of APC-PCI and the values of APC-PCI/PC ratio were significantly decreased in BALF from patients with ILD. BALF concentration of PIIINP was significantly and inversely correlated with the concentration of APC-PCI and with the APC-PCI/PC ratio. These findings suggest that dysfunction of the protein C pathway may have important physiopathologic implications in the development of pulmonary fibrosis in ILD.

Plasma levels of activated protein C (APC)-protein C inhibitor (PCI) were significantly increased in patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), acute myocardial infarction (AMI), pulmonary embolism (PE), or deep vein thrombosis (DVT) and in patients undergoing hemodialysis (HD). Plasma levels of APC-alpha(1)-antitrypsin (AT) complex were significantly increased in patients with DIC and in those with TTP. Plasma levels of PCI were significantly decreased in patients with DIC, non-DIC, or TTP and in those undergoing HD. In the pre-DIC stage, the plasma levels of APC-PCI complex were significantly increased but not those of APC-alpha(1)-AT complex. These data suggest that measurements of APC-PCI complex and APC-alpha(1)-AT complex may be useful for the diagnosis of DIC. After treatment of DIC, the plasma levels of APC-PCI complex and APC-alpha(1)-AT complex were significantly decreased, but not those of PCI. Plasma levels of thrombin-antithrombin complex (TAT), plasmin-alpha(2)-plasmin complex (PPIC), D-dimer, and soluble fibrin monomer (SFM) were markedly increased in patients with DIC or pre-DIC and were moderately increased in patients with non-DIC, TTP, AMI, PE, or DVT and in those undergoing HD. The receiving operating characteristic (ROC) analysis showed that SFM and the APC-PCT complex are useful markers for diagnosis of DIC. The specificity of plasma TAT and PPIC levels was low. The positive rate of APC-PCI complex was higher than 90% with DIC, TTP, AMI, PE, and it was higher than 60% with DVT and HD. Since the APC-PCI complex was elevated not only in patients with venous thrombosis but also in those with arterial thrombosis, components of the protein C pathway might be useful markers for the diagnosis of arterial thrombosis., (Copyright 2000 Wiley-Liss, Inc.)

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.

This study evaluated hemostatic data in 28 patients with newly diagnosed acute promyelocytic leukemia (APL) and 15 patients with relapsed APL. Activated partial thromboplastin time and prothrombin time were prolonged at initial onset of APL. Plasma level of fibrinogen was significantly decreased in patients with initial disease of APL, but it was not decreased significantly during the relapse of APL. Plasma fibrin and fibrinogen degradation products levels were significantly increased and platelet counts significantly decreased in both groups. Plasma levels of antiplasmin significantly decreased at initial onset but not during relapse. Plasma levels of antithrombin were within normal range in patients with initial disease but significantly decreased in those with relapse. Plasma levels of D-dimer, soluble fibrin monomer (sFM), plasmin-plasmin inhibitor complex (PPIC), and thrombin antithrombin complex (TAT) levels were significantly high in both groups. Plasma levels of PPIC, sFM, and D-dimer were significantly higher at initial onset of APL than during relapse. However, there was no significant difference in DIC score between patients with initial onset and those with relapse; plasma levels of tissue factor (TF) significantly increased in both groups, but they were significantly higher at initial onset of APL than during relapse. TF and tissue type plasminogen activator (t-PA) antigen levels in leukemic cell lysate were significantly increased in both groups, and they were significantly lower during relapse than at initial onset. Hemostatic abnormalities occurring in patients with relapsed APL might be the result of the decrease of TF and t-PA in leukemic cells. These findings suggest that DIC in APL patients with relapse might not be caused only by TF and t-PA and thus should be treated with different therapy from patients with initial onset of APL., (Copyright 2000 Wiley-Liss, Inc.)

We evaluated the effect of troglitazone (given orally 400 mg/day) on glucose intolerance and on the plasma levels of tumour necrosis factor-alpha (TNF-alpha) in 12 obese patients with type 2 diabetes for 12 weeks. Troglitazone significantly decreased fasting plasma glucose, serum C-peptide, serum insulin and HbA1c levels. Plasma levels of TNF-alpha were significantly reduced by troglitazone administered for 8 and 12 weeks. Troglitazone administration significantly improved insulin resistance, but did not affect pancreatic beta-cell function as evaluated by the homeostasis model assessment (HOMA). In the present study, we reported for the first time that troglitazone administration significantly reduces plasma levels of TNF-alpha in obese patients with type 2 diabetes.