Kaname Nakatani

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta‐1 is expressed in a proportion of the skin. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and transforming growth factor‐β1 (TGF‐β1) are also secreted in the skin. We report here that Delta‐1, in concert with GM‐CSF and TGF‐β1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte‐associated antigen, CC chemokine receptor 6, E‐cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein‐1α (MIP‐1α). In response to CD40 ligand and tumor necrosis factor α, the cells acquire a mature phenotype of dendritic cells that is characterized by up‐regulation of human leukocyte antigen (HLA)‐ABC, HLA‐DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP‐1β and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta‐1, GM‐CSF, and TGF‐β1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand δ‐1 in human hematopoiesis.

Protein C activation in NIDDM patients

Summary Enhanced activation of the clotting system has been recently implicated in the pathogenesis of vascular complications in patients with diabetes mellitus. Abnormalities of the anticoagulant system may constitute a potential trigger factor for the haemostatic activation observed in diabetic subjects. The current study aimed to evaluate anticoagulant activity in diabetic patients by assessing the plasma levels of activated protein C-protein C inhibitor complex; and by measuring the anticoagulant response to exogenous thrombomodulin. This study comprised 61 patients (34 men, 27 women) with non-insulin-dependent diabetes mellitus (NIDDM) of whom 22 showed microalbuminuria and 39 normoalbuminuria. Data obtained in 31 non-obese and non-diabetic subjects were available for comparison. The plasma levels of fibrinogen (p < 0.02), prothrombin fragment 1 + 2 (p < 0.05), fibrin monomer (p < 0.0001), protein C antigen (p < 0.005), total protein S antigen (p < 0.02), soluble thrombomodulin (p < 0.005) and soluble E-selectin (p < 0.005) were significantly higher in diabetic patients than in healthy subjects. The plasma level of activated protein C-protein C inhibitor complex (7.4 ± 3.8 vs 3.0 ± 0.4 pmol/l) was significantly higher (p < 0.0001) and the anticoagulant response to exogenous thrombomodulin (23.4 ± 2.6 vs 35.3 ± 3.0 ng/ml) was markedly lower (p = 0.005) in all diabetic patients than in healthy subjects. Cases with microalbuminuria presented low plasma levels of activated protein C-protein C inhibitor complex (5.5 ± 0.6 vs 8.6 ± 0.7 pmol/l, p < 0.05) and significantly decreased values of the anticoagulant response to exogenous thrombomodulin (16.5 ± 2.9 vs 23.4 ± 2.6 %, p = 0.03) as compared to those with normoalbuminuria. The present study suggests that the hyper-coagulable state in NIDDM is associated with an increased activation of protein C but with a poor plasma reactivity to the anticoagulant effect of thrombomodulin. [Diabetologia (1996) 39: 1455–1461]

Troglitazone improves GLUT4 expression in adipose tissue in an animal model of obese type 2 diabetes mellitus

Troglitazone has been shown to improve peripheral insulin resistance in type 2 diabetic patients and animal models. We examined the effect of troglitazone on the expression of glucose transporter 4 (GLUT4) in muscle and adipose tissue from Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of obese type 2 diabetes mellitus. In addition, the effects of troglitazone on GLUT4 translocation and on glucose transport activity in adipocytes were also evaluated. Muscle and adipose tissues were isolated from 35-week-old male troglitazone-treated and untreated OLETF rats at a dose of 150 mg/kg per day for 14 days. In skeletal muscle, the protein and mRNA levels of GLUT4 were not significantly different between OLETF and control rats and they were not affected by troglitazone. On the other hand, GLUT4 protein and mRNA levels in adipose tissue from OLETF rats were significantly decreased (P<0.01) compared with control rats and they were significantly increased (1.5-fold, P<0.01) by troglitazone. Troglitazone had no major effect on GLUT4 translocation in adipocytes, but it significantly increased (1.4-fold, P<0.05) the basal and insulin-induced amounts of GLUT4 in plasma membrane (PM) in adipocytes from OLETF rats. Consistent with these results, the basal and insulin-induced glucose uptakes in adipocytes from troglitazone-treated OLETF rats were significantly increased (1.5-fold, P<0.05) compared with untreated OLETF rats. Our results suggest that troglitazone may exert beneficial effects on insulin resistance by increasing the expression of GLUT4 in adipose tissue.

Decreased High–Molecular Weight Adiponectin–to–Total Adiponectin Ratio in Sera Is Associated With Insulin Resistance in Japanese Metabolically Obese, Normal-Weight Men With Normal Glucose Tolerance

Adiponectin is a key regulator of insulin resistance. Recent studies on multimer formation in human blood have demonstrated that high–molecular weight (HMW) adiponectin is the active form of the protein (1–3). Recently, Hara et al. (3) demonstrated that the ratio of HMW adiponectin to total adiponetin (HMWR) in systemic circulation is useful for the prediction of insulin resistance and metabolic syndrome. Metabolically obese, normal-weight (MONW) subjects (BMI <25 kg/m2) are characterized by excess visceral fat area (VFA; ≥100 cm2 by abdominal computed tomography scanning) and insulin resistance (4–6). In addition, we previously demonstrated that the …

The association of protein S Tokushima-K196E with a risk of deep vein thrombosis

Deep vein thrombosis (DVT) is a multifactorial disease caused by acquired risk factors such as a bed rest, surgery and malignancies. Although the factor V Leiden and the prothrombin-20210G>A mutation do not exist in Japanese populations, a mutation in protein S (PS) Tokushima (K196E) has been attracting attention in Japan. In this study, the genetic contribution of PS Tokushima (K196E) was evaluated in 60 Japanese patients with thrombosis in comparison to 234 healthy volunteers and 88 patients without thrombosis. Genes associated with the response to warfarin, cytochrome P450 2C9 (CYP2C9), vitamin K epoxide reductase complex subunit 1 (VKORC1), and γ-glutamyl carboxylase (GGCX) were also investigated simultaneously. PS Tokushima (K196E) was detected in 6 patients with thrombosis, in 3 without thrombosis and in 3 healthy volunteers, indicating that there is a high frequency of the PS Tokushima (K196E). There were no significant differences of CYP2C9, VKORC1 or GGCX between the patients with and without DVT. Therefore, PS Tokushima (K196E) is an important genetic risk factor for DVT in the Japanese population.

Elevated Plasma Levels of Fibrin Degradation Productsby Granulocyte-Derived Elastase in Patients with Disseminated Intravascular Coagulation

Plasma levels of granulocyte-derived elastase (GE-XDP), D-dimer, and soluble fibrin (SF) were examined in 177 patients with disseminated intravascular coagulation (DIC) of various etiologies. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with sepsis and solid cancer. The ratio of GE-XDP/D-dimer was significantly high in patients with trauma, burn, and sepsis, suggesting that fibrinolysis due to GE-XDP may be dominant in DIC. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with overt DIC and correlated with DIC score. Plasma levels of GE-XDP, but not SF, correlated significantly with D-dimer. Plasma levels of D-dimer, but not SF, correlated significantly with plasmin plasmin inhibitor complex (PPIC). Plasma levels of GEXDP and D-dimer, but not SF, were significantly high in nonsurvivors. Plasma levels of GE-XDP, but not SF, correlated significantly with sepsis-related organ failure assessment (SOFA) score. These results suggest that GE-XDP is a potentially useful marker for the diagnosis of overt-DIC and as a predictor of organ failure-related outcome.

The Membrane Proteinase 3 Expression on Neutrophils Was Downregulated After Treatment With Infliximab in Patients With Rheumatoid Arthritis

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-α therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-α antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFα, whereas it was significantly suppressed by plasma or α1-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

Multicenter evaluation of prototype real-time PCR assays for Epstein-Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients.

Quantitative PCR is becoming widespread for diagnosing and monitoring post‐transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home‐brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post‐transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV‐positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤4.15 for EBV among three different sites and ≤3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home‐brew assay were high (EBV, 0.73–0.83, median = 0.78; CMV, 0.54–0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter‐laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.

Effect of Genetic Polymorphism of CYP3A5 and CYP2C19 and Concomitant Use of Voriconazole on Blood Tacrolimus Concentration in Patients Receiving Hematopoietic Stem Cell Transplantation.

Background: Blood tacrolimus (TAC) concentration delivered via intravenous administration is known to be influenced by genetic polymorphism of CYP3A5 and interaction with triazole antifungal agents. However, interindividual variability of blood TAC concentration is as of yet still difficult to predict during the early stages of hematopoietic stem cell transplantation (HSCT). This study was conducted to assess the wide variability of blood TAC concentrations because of the hepatic metabolic activities of CYP3A and CYP2C19 in HSCT recipients. Methods: This study is a single-institute prospective study that includes 21 adult patients who underwent HSCT and received 24 hours continuous intravenous administration of TAC at the Mie University Hospital between January 2009 and March 2014. After HSCT, the changes in blood TAC concentration/dose (C/D) ratio and TAC dose reduction from initial dose were investigated. Results: Significant differences between HSCT recipients with CYP3A5*1 allele and CYP3A5*3/*3 genotype were observed with respect to the median TAC C/D ratio on day 14 (563 versus 742 ng/mL per mg/kg, P < 0.01) and day 21 (672 versus 777 ng/mL per mg/kg, P < 0.05) after HSCT. Concomitant administration of voriconazole (VRCZ), but not of lansoprazole, was found to significantly increase the median TAC C/D ratio on day 14 (557 versus 723 ng/mL per mg/kg, P < 0.01). Possession of the CYP3A5*3/*3 genotype (day 14: odds ratio, 32.2; day 21: odds ratio, 33.0; P < 0.05) and concomitant administration of VRCZ (day 14: odds ratio, 37.8; P < 0.05) were found to be independent risk factors, which significantly contributed to an increased TAC C/D ratio. In HSCT recipients with CYP3A5*3/*3 genotype (78.0%), the median TAC dose ratio (day 21/day −1) was significantly lower compared with HSCT recipients with the CYP3A5*1 allele (94.1%), whereas VRCZ administration itself had no significant influence. Interestingly, in HSCT recipients with CYP2C19*1/*1, we found that the influence of VRCZ on the TAC dose ratio (85.7%) was relatively mild, even in a recipient with CYP3A5*3/*3. Conclusions: In HSCT recipients, the variability of intravenous TAC concentration in the blood could be explained in part by the genetic variation of CYP3A5. The study results also strongly imply that the magnitude of hepatic interaction between TAC and VRCZ is affected by the genetic polymorphism of both CYP3A5 and CYP2C19 genes.

Thrombomodulin accelerates activated protein C production and inhibits thrombin generation in the plasma of disseminated intravascular coagulation patients

Thrombomodulin (TM) has been under development as a medicine for disseminated intravascular coagulation (DIC), and is expected to exhibit strong anticoagulant activity by inhibiting thrombin generation via the acceleration of protein C activation. In the present study, we examined the pharmacological action of TM in plasma obtained from DIC patients. TM was found to inhibit thrombin generation and accelerate activated protein C (APC) production at 0.3–30 TM units/ml in plasma obtained from DIC patients irrespective of their underlying disorders. In addition, there was a positive correlation between the inhibition of thrombin generation and the amount of APC produced. Thrombin generation was inhibited by over 50% when the plasma level of APC was increased by more than 0.2 μg/ml. These results indicate that TM inhibits thrombin generation in plasma obtained from DIC patients by accelerating APC production. Moreover, the results imply that the thrombin generation test may be a good method to speculate the efficacy of TM on every patient before the administration of TM.