Publications by authors named "Tomoaki Inoue"

31 Publications

Bilirubin is inversely related to diabetic peripheral neuropathy assessed by sural nerve conduction study.

J Diabetes Investig 2021 May 5. Epub 2021 May 5.

Fukuoka City Health Promotion Support Center, Fukuoka, Japan.

Aims/introduction: Diagnosis of diabetic peripheral neuropathy (DPN) depends on subjective findings, certain investigations for DPN risks have not been performed enough. Bilirubin protects against vascular complications by reducing oxidative stress in diabetes, but is not fully tested for DPN. This study aimed to evaluate sural nerve conduction impairments (SNCI) as an objective DPN marker and the contribution of bilirubin to SNCI.

Materials And Methods: Using DPN-Check , SNCI was defined as a decline of amplitude potential or conduction velocity below the normal limit in 150 inpatients with diabetes. The correlations between SNCI and conventional DPN diagnosis criteria, the incidence of diabetic retinopathy/nephropathy, biomarkers for atherosclerosis, cardiac function by ultrasonic cardiogram, and bilirubin were statistically tested, followed by the comparison of logistic regression models for SNCI to find confounders with bilirubin.

Results: The incidence of SNCI was 72.0%. The sensitivity and specificity of SNCI for DPN prediagnosis by simplified criteria were 54.6 and 90.5%, respectively, and similarly corresponded with diabetic retinopathy and nephropathy (sensitivity 57.4 and 50.0%, respectively). SNCI significantly related to diabetes duration, declined estimated glomerular filtration rate, albuminuria and total bilirubin. SNCI incidence was attenuated in the higher bilirubin tertiles (89.8/65.3/54.8%, P < 0.001). Bilirubin was an independent inverse risk factor for SNCI, even after adjustment by known risk factors for DPN and markers for microvascular complications.

Conclusions: SNCI is a comprehensive marker for diabetic complications. We first showed the independent inverse relationship between bilirubin and SNCI through the independent pathway with other complications, provably reducing oxidative stress, as previously reported.
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http://dx.doi.org/10.1111/jdi.13568DOI Listing
May 2021

Relationship between serum bilirubin levels, urinary biopyrrin levels, and retinopathy in patients with diabetes.

PLoS One 2021 11;16(2):e0243407. Epub 2021 Feb 11.

Fukuoka City Health Promotion Support Center, Fukuoka City Medical Association, Fukuoka City, Japan.

Objective: Previous reports have indicated that serum bilirubin levels may be associated with diabetic retinopathy. However, the detailed mechanism is not fully understood. In this study, we evaluated the relationship between the severity of diabetic retinopathy and various factors including bilirubin levels and factors influencing bilirubin metabolism.

Methods: The study participants consisted of 94 consecutive patients with diabetes mellitus admitted to Kyushu University Hospital from April 2011 to July 2012. The patients were classified into three groups: no retinopathy (NDR), simple retinopathy (SDR), and pre-proliferative or proliferative retinopathy (PDR). The relationship between the severity of retinopathy and various factors was evaluated using univariate and logistic regression analyses. In addition, multivariate regression analysis was performed to evaluate the significant determinants for bilirubin levels.

Results: In univariate analysis, a significant difference was found among NDR, SDR and PDR in bilirubin levels, duration of diabetes, systolic blood pressure, and macroalbuminuria. Logistic regression analysis showed that PDR was significantly associated with bilirubin levels, duration of diabetes, and systolic blood pressure (OR 0.737, 95% CI 0.570-0.952, P = 0.012; OR 1.085, 95% CI 1.024-1.149, P = 0.006; OR 1.036, 95% CI 1.011-1.062, P = 0.005, respectively). In turn, multivariate regression analysis showed that bilirubin levels were negatively associated with high-sensitivity C-reactive protein levels and PDR, but positively correlated with urinary biopyrrin levels, oxidized metabolites of bilirubin.

Conclusion: PDR was negatively associated with bilirubin levels. This negative association may be due to a decreased production of bilirubin rather than its increased consumption considering the positive association between bilirubin and biopyrrin levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877782PMC
February 2021

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals.

Toxicol In Vitro 2020 Aug 18;66:104832. Epub 2020 Mar 18.

Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan. Electronic address:

To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1β and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.
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http://dx.doi.org/10.1016/j.tiv.2020.104832DOI Listing
August 2020

The dipeptidyl peptidase-4 inhibitor, linagliptin, improves cognitive impairment in streptozotocin-induced diabetic mice by inhibiting oxidative stress and microglial activation.

PLoS One 2020 7;15(2):e0228750. Epub 2020 Feb 7.

Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan.

Objective: Accumulating epidemiological studies have demonstrated that diabetes is an important risk factor for dementia. However, the underlying pathological and molecular mechanisms, and effective treatment, have not been fully elucidated. Herein, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on diabetes-related cognitive impairment.

Method: Streptozotocin (STZ)-induced diabetic mice were treated with linagliptin (3 mg/kg/24 h) for 17 weeks. The radial arm water maze test was performed, followed by evaluation of oxidative stress using DNP-MRI and the expression of NAD(P)H oxidase components and proinflammatory cytokines and of microglial activity.

Results: Administration of linagliptin did not affect the plasma glucose and body weight of diabetic mice; however, it improved cognitive impairment. Additionally, linagliptin reduced oxidative stress and the mRNA expression of NAD(P)H oxidase component and TNF-α, and the number and body area of microglia, all of which were significantly increased in diabetic mice.

Conclusions: Linagliptin may have a beneficial effect on diabetes-related dementia by inhibiting oxidative stress and microglial activation, independently of glucose-lowering.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228750PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006898PMC
May 2020

Human-Induced Pluripotent Stem Cell-Derived Hepatocytes and their Culturing Methods to Maintain Liver Functions for Pharmacokinetics and Safety Evaluation of Pharmaceuticals.

Curr Pharm Biotechnol 2020 ;21(9):773-779

Consortium for Safety Assessment using Human iPS Cells (CSAHi), Japan.

Human hepatocytes are essential cell types for pharmacokinetics and the safety evaluation of pharmaceuticals. However, widely used primary hepatocytes with individual variations in liver function lose those functions rapidly in culture. Hepatic cell lines are convenient to use but have low liver functions. Human-Induced Pluripotent Stem (hiPS) cells can be expanded and potentially differentiated into any cell or tissue, including the liver. HiPS cell-derived Hepatocyte-Like Cells (hiPSHeps) are expected to be extensively used as consistent functional human hepatocytes. Many laboratories are investigating methods of using hiPS cells to differentiate hepatocytes, but the derived cells still have immature liver functions. In this paper, we describe the current uses and limitations of conventional hepatic cells, evaluating the suitability of hiPS-Heps to pharmacokinetics and the safety evaluation of pharmaceuticals, and discuss the potential future use of non-conventional non-monolayer culture methods to derive fully functional hiPS-Heps.
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http://dx.doi.org/10.2174/1389201021666200131123524DOI Listing
September 2020

Human induced pluripotent stem cell-derived mast cells useful for in vitro mast cell activation assay exhibiting phenotypes and morphological characteristics of human mast cells.

J Toxicol Sci 2019 ;44(11):789-797

Research Division, Chugai Pharmaceutical Co., Ltd.

Mast cells are key players in the inflammatory response with an important role in allergic reactions and are therefore useful for assessing the risk of anaphylaxis. However, they are difficult to isolate due to their low abundance and wide distribution. To overcome this, we generated and characterized mast cell-like cells derived from human induced pluripotent stem (hiPS) cells. These hiPS cell-derived mast cells (hiPS-MCs) were generated using recombinant human bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor 165 (VEGF), stem cell factor (SCF), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-9 (IL-9) in a StemPro-34 medium. The hiPS-MCs exhibited the morphological characteristics of human mast cells, expressing high affinity-IgE receptor (FcεRI) and mast cell markers such as tryptase, chymase, and CD117. In addition, FcεRI stimulation with agonistic anti-IgE functionally increased the expression of activation markers CD63 and CD203c, as well as the amount of released histamine. We think the hiPS-MCs generated in this study will be useful for assessing the pharmacology and toxicity of anti-allergy medicines.
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http://dx.doi.org/10.2131/jts.44.789DOI Listing
March 2020

Bilirubin reduces visceral obesity and insulin resistance by suppression of inflammatory cytokines.

PLoS One 2019 2;14(10):e0223302. Epub 2019 Oct 2.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Objective: Although previous studies have reported a negative relationship between serum bilirubin concentration and the development of diabetes mellitus (DM), the relationship between bilirubin and insulin resistance has not been thoroughly assessed. This study was designed to determine the relationships between bilirubin, body fat distribution, and adipose tissue inflammation in patients with type 2 DM and the effect of bilirubin in an obese animal model.

Method: Body fat distribution was measured using an abdominal dual bioelectrical impedance analyzer in patients with type 2 DM. We also measured glycemic control, lipid profile, serum bilirubin concentration and other clinical characteristics, and determined their relationships with body fat distribution. In the animal study, biliverdin (20 mg/kg daily) was orally administered to high-fat diet (HFD)-induced obese (DIO) mice for 2 weeks, after which intraperitoneal insulin tolerance testing was performed. Then, adipocyte area, adipocytokine expression, and macrophage polarization were evaluated in epididymal adipose tissues.

Results: In the clinical study, univariate analysis showed that a lower bilirubin concentration was significantly correlated with higher body mass index, waist circumference, triglyceride, uric acid, creatinine, visceral fat area and lower HDL-C. In multivariate analyses, bilirubin concentration significantly correlated with diastolic blood pressure, creatinine, and visceral fat area. However, there was no association between bilirubin concentration and subcutaneous fat area. In the animal study, DIO mice treated with biliverdin had smaller adipocytes than untreated DIO mice and biliverdin improved HFD-induced insulin resistance. Biliverdin treatment reversed the higher gene expression of Cd11c, encoding an M1 macrophage marker, and Tnfa, encoding the proinflammatory cytokine tumor necrosis factor-α, in the adipose tissues of DIO mice. These data suggest biliverdin administration alleviates insulin resistance by ameliorating inflammation and the dysregulation of adipocytokine expression in adipose tissues of DIO mice.

Conclusions: Bilirubin may protect against insulin resistance by ameliorating visceral obesity and adipose tissue inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223302PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774504PMC
March 2020

Collagen vitrigel promotes hepatocytic differentiation of induced pluripotent stem cells into functional hepatocyte-like cells.

Biol Open 2019 Jul 2;8(7). Epub 2019 Jul 2.

School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan

Differentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as , Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.
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http://dx.doi.org/10.1242/bio.042192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679405PMC
July 2019

Different players generate positive responses in two in vitro cytokine assay formats with aqueous and immobilized TGN1412 analog.

Biochem Biophys Res Commun 2018 07 24;502(1):91-97. Epub 2018 May 24.

Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513, Japan. Electronic address:

To detect potential risk of severe cytokine release syndrome, in vitro assay formats with human cells have been developed. The two major testing platforms are a combination of whole blood with aqueous-phase test articles (whole blood cytokine assay, WBCA) and peripheral blood mononuclear cells with solid-phase articles (PBMC assay). Significant induction of cytokines was seen in both assays after treatment with a widely used control agent, TGN1412 or its analog CD28SA, but the WBCA cytokine profile differed from what was expected from clinical experience. In the WBCA, potential risk of CD28SA was detected by elevation of IL-8 whereas IL-2, a key cytokine after stimulation of CD28, was not induced in approximately 40% of donor samples. Therefore, further mechanistic understanding of the different responses in the in vitro assay was needed. In this study of donor samples treated with CD28SA, we compared the induction of cytokines and identified the cytokine-producing cells in the two assays. IL-2 was markedly elevated in all the donors in the PBMC assay but only in 1 of 3 donors in the WBCA. IL-8, the most sensitive biomarker in the WBCA, was produced by monocytes and granulocytes. T cells, the most relevant player in the PBMC assay with CD28SA, did not contribute to the positive response seen in two donors in the WBCA, which suggests that different players caused the positive cytokine responses to CD28SA in the two assays.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.125DOI Listing
July 2018

Serum Bilirubin Concentration is Associated with Left Ventricular Remodeling in Patients with Type 2 Diabetes Mellitus: A Cohort Study.

Diabetes Ther 2018 Feb 15;9(1):331-338. Epub 2018 Jan 15.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Introduction: Previous studies have shown that serum bilirubin concentration is inversely associated with the risk of cardiovascular disease. The relationship between serum bilirubin concentration and left ventricular geometry, however, has not been investigated in patients with diabetes mellitus.

Methods: In this cohort study, 158 asymptomatic patients with type 2 diabetes mellitus without overt heart disease were enrolled. Left ventricular structure and function were assessed using echocardiography. Serum bilirubin concentration, glycemic control, lipid profile, and other clinical characteristics were evaluated, and their association with left ventricular geometry was determined. Patients with New York Heart Association Functional Classification greater than I, left ventricular ejection fraction less than 50%, history of coronary artery disease, severe valvulopathy, chronic atrial fibrillation, or creatinine clearance less than 30 ml/min, and those receiving insulin treatment, were excluded.

Results: Univariate analyses showed that relative wall thickness (RWT) was significantly correlated with diastolic blood pressure (P = 0.003), HbA1c (P = 0.024), total cholesterol (P = 0.043), urinary albumin (P = 0.023), and serum bilirubin concentration (P = 0.009). There was no association between left ventricular mass index and serum bilirubin concentration. Multivariate linear regression analysis showed that log RWT was positively correlated with diastolic blood pressure (P = 0.010) and that log RWT was inversely correlated with log bilirubin (P = 0.003). In addition, the patients with bilirubin less than 0.8 mg/dl had a higher prevalence of concentric left ventricular remodeling compared with those with bilirubin 0.8 mg/dl or more.

Conclusion: Our study shows that the serum bilirubin concentration may be associated with the progression of concentric left ventricular remodeling in patients with type 2 diabetes mellitus.
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http://dx.doi.org/10.1007/s13300-018-0368-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801254PMC
February 2018

Hyperinsulinemia and sulfonylurea use are independently associated with left ventricular diastolic dysfunction in patients with type 2 diabetes mellitus with suboptimal blood glucose control.

BMJ Open Diabetes Res Care 2016 18;4(1):e000223. Epub 2016 Aug 18.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan.

Objective: Although diabetes mellitus is associated with an increased risk of heart failure with preserved ejection fraction, the underlying mechanisms leading to left ventricular diastolic dysfunction (LVDD) remain poorly understood. The study was designed to assess the risk factors for LVDD in patients with type 2 diabetes mellitus.

Research Design And Methods: The study cohort included 101 asymptomatic patients with type 2 diabetes mellitus without overt heart disease. Left ventricular diastolic function was estimated as the ratio of early diastolic velocity (E) from transmitral inflow to early diastolic velocity (e') of tissue Doppler at mitral annulus (E/e'). Parameters of glycemic control, plasma insulin concentration, treatment with antidiabetic drugs, lipid profile, and other clinical characteristics were evaluated, and their association with E/e' determined. Patients with New York Heart Association class >1, ejection fraction <50%, history of coronary artery disease, severe valvulopathy, chronic atrial fibrillation, or creatinine clearance <30 mL/min, as well as those receiving insulin treatment, were excluded.

Results: Univariate analysis showed that E/e' was significantly correlated with age (p<0.001), sex (p<0.001), duration of diabetes (p=0.002), systolic blood pressure (p=0.017), pulse pressure (p=0.010), fasting insulin concentration (p=0.025), and sulfonylurea use (p<0.001). Multivariate linear regression analysis showed that log E/e' was significantly and positively correlated with log age (p=0.034), female sex (p=0.019), log fasting insulin concentration (p=0.010), and sulfonylurea use (p=0.027).

Conclusions: Hyperinsulinemia and sulfonylurea use may be important in the development of LVDD in patients with type 2 diabetes mellitus.
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http://dx.doi.org/10.1136/bmjdrc-2016-000223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013397PMC
September 2016

Is an in vitro whole blood cytokine assay useful to detect the potential risk of severe infusion reaction of monoclonal antibody pharmaceuticals?

J Toxicol Sci 2016 ;41(4):523-31

Research Division, Chugai Pharmaceutical Co., Ltd.

After the life-threatening cytokine release syndrome (CRS) occurred in the clinical study of the anti-CD28 monoclonal antibody (mAb) TGN1412, in vitro cytokine release assays using human blood cells have been proposed for non-clinical evaluation of the potential risk of CRS. Two basic assay formats are frequently used: human peripheral blood mononuclear cells (PBMC) with immobilized mAbs, and whole blood with aqueous mAbs. However, the suitability of the whole blood cytokine assay (WBCA) has been questioned, because an unrealistically large sample size would be required to detect the potential risk of CRS induced by TGN1412, which has low sensitivity. We performed a WBCA using peripheral blood obtained from 68 healthy volunteers to compare two high risk mAbs, the TGN1412 analogue anti-CD28 superagonistic mAb (CD28SA) and the FcγR-mediated alemutuzumab, with a low risk mAb, panitumumab. Based on the cytokine measurements in this study, the sample size required to detect a statistically significant increase in cytokines with 90% power and 5% significance was determined to be n = 9 for CD28SA and n = 5 for alemtuzumab. The most sensitive marker was IL-8. The results suggest that WBCA is a practical test design that can warn of the potential risk of FcγR-mediated alemtuzumab and T-cell activating CD28SA but, because there was apparently a lower response to CD28SA, it cannot be used as a risk-ranking tool. WBCA is suggested to be a helpful tool for identifying potential FcγR-mediated hazards, but further mechanistic understanding of the response to CD28SA is necessary before applying it to T cell-stimulating mAbs.
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http://dx.doi.org/10.2131/jts.41.523DOI Listing
April 2017

Cardiac arrest due to massive hemorrhage from uterine adenomyosis with leiomyoma successfully treated with damage control resuscitation.

Acute Med Surg 2016 10 18;3(4):388-391. Epub 2016 Apr 18.

Advanced Medical Emergency and Critical Care Center Yamaguchi University Hospital Ube Japan.

Case: A 57-year-old woman was transferred to our emergency department by ambulance with cardiopulmonary arrest caused by massive genital bleeding. Cardiopulmonary resuscitation, including massive transfusion, was carried out and the return of spontaneous circulation was achieved. A giant uterine tumor was considered the source of the bleeding. Although hysterectomy was necessary to achieve definitive hemostasis, the patient was unable to tolerate the operation because of hemodynamic instability, acidosis, and coagulopathy. Therefore, we undertook vaginal gauze packing and uterine artery embolization to attain temporary hemostasis, which resulted in hemodynamic stabilization. Abdominal hysterectomy for definitive hemostasis was carried out 10 h after the embolization.

Outcome: The patient made a good post-surgical recovery without any complications.

Conclusion: In treating hemorrhagic shock due to uterine leiomyoma, damage-control resuscitation may be useful as a bridge prior to definitive hemostasis through hysterectomy.
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http://dx.doi.org/10.1002/ams2.198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667328PMC
October 2016

GLP-1 analog liraglutide protects against cardiac steatosis, oxidative stress and apoptosis in streptozotocin-induced diabetic rats.

Atherosclerosis 2015 May 18;240(1):250-9. Epub 2015 Mar 18.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Objective: Accumulating evidence has implicated that GLP-1 may have a beneficial effect on cardiovascular but the mechanism is not fully understood. Here we show that GLP-1 analog, liraglutide, inhibits cardiac steatosis, oxidative stress and apoptosis in streptozotocin (STZ)-induced type 1 diabetic rats, via activation of AMPK-Sirt1 pathway.

Methods: Diabetic rats were treated with subcutaneous injections of liraglutide (0.3 mg/kg/12 h) for 4 weeks. Myocardial steatosis (detected by oil red O staining and myocardial triglyceride and diacylglycerol (DAG) contents assay), expression of protein kinase C (PKC), heart NAD(P)H oxidase activity, oxidative stress markers (8-hydroxy-2'-deoxyguanosine staining), apoptosis (TUNEL analysis) and genes that affect apoptosis and lipid metabolism were evaluated.

Results: Administration of liraglutide did not affect plasma glucose and insulin levels or body weights in STZ-induced diabetic rats, but normalized myocardial steatosis, expression of PKC, NAD(P)H oxidase activity, oxidative stress markers and apoptosis, all of which were significantly increased in diabetic hearts. Additionally, expression of genes mediating lipid uptake, synthesis and oxidation were increased in the diabetic hearts, and these increases were all reduced by liraglutide. In addition, liraglutide increased expression of Sirt1 and phosphorylated AMPK in the diabetic hearts.

Conclusions: Liraglutide may have a beneficial effect on cardiac steatosis, DAG-PKC-NAD(P)H pathway, oxidative stress and apoptosis via activation of AMPK-Sirt1 pathway, independently of a glucose-lowering effect.
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http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.026DOI Listing
May 2015

Maternal high-fat diet induces insulin resistance and deterioration of pancreatic β-cell function in adult offspring with sex differences in mice.

Am J Physiol Endocrinol Metab 2014 May 1;306(10):E1163-75. Epub 2014 Apr 1.

Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;

Intrauterine environment may influence the health of postnatal offspring. There have been many studies on the effects of maternal high-fat diet (HFD) on diabetes and glucose metabolism in offspring. Here, we investigated the effects in male and female offspring. C57/BL6J mice were bred and fed either control diet (CD) or HFD from conception to weaning, and offspring were fed CD or HFD from 6 to 20 wk. At 20 wk, maternal HFD induced glucose intolerance and insulin resistance in offspring. Additionally, liver triacylglycerol content, adipose tissue mass, and inflammation increased in maternal HFD. In contrast, extending previous observations, insulin secretion at glucose tolerance test, islet area, insulin content, and PDX-1 mRNA levels in isolated islets were lower in maternal HFD in males, whereas they were higher in females. Oxidative stress in islets increased in maternal HFD in males, whereas there were no differences in females. Plasma estradiol levels were lower in males than in females and decreased in offspring fed HFD and also decreased by maternal HFD, suggesting that females may be protected from insulin deficiency by inhibiting oxidative stress. In conclusion, maternal HFD induced insulin resistance and deterioration of pancreatic β-cell function, with marked sex differences in adult offspring accompanied by adipose tissue inflammation and liver steatosis. Additionally, our results demonstrate that potential mechanisms underlying sex differences in pancreatic β-cell function may be related partially to increases in oxidative stress in male islets and decreased plasma estradiol levels in males.
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http://dx.doi.org/10.1152/ajpendo.00688.2013DOI Listing
May 2014

Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

Atherosclerosis 2014 Jan 5;232(1):156-64. Epub 2013 Nov 5.

Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

Objective: Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress.

Methods: Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity.

Results: High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects.

Conclusions: Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications.
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http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.025DOI Listing
January 2014

Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice.

Biochem Biophys Res Commun 2013 Aug 22;438(1):224-9. Epub 2013 Jul 22.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2'-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.
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http://dx.doi.org/10.1016/j.bbrc.2013.07.063DOI Listing
August 2013

Plasma debris sputter resistant x-ray mirror.

Appl Opt 2013 Jun;52(16):3845-8

Laboratory of Advanced Science and Technology for Industry, University of Hyogo, Ako, Hyogo, Japan.

A diamond-like carbon (DLC) mirror, used as a grazing incident mirror in a plasma x-ray source, exhibits a high resistance to plasma debris sputtering. Good mirror reflectivity at a wavelength of 13.5 nm was confirmed using synchrotron radiation at the NewSUBARU facility. The erosion rate due to plasma debris sputtered at the incident debris angle of 20° was measured using a laser-produced Xe plasma source developed by the authors. The results indicate that the DLC film has a 5- and 15-fold higher sputtering resistance compared to films made of the traditional mirror materials Ru and Au, respectively. Because the DLC mirror retains a high sputtering resistance to Sn ions, it may be effective in Sn plasma source applications. We conclude that a grazing incident x-ray mirror coated with DLC can be of use as a plasma debris sputtering resistant mirror.
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http://dx.doi.org/10.1364/AO.52.003845DOI Listing
June 2013

Characteristics of a cylindrical collector mirror for laser-produced xenon plasma soft X-rays and improvement of mirror lifetime by buffer gas.

Rev Sci Instrum 2012 Dec;83(12):123110

Laboratory of Advanced Science and Technology for Industry, University of Hyogo, 3-1-2 Kouto, Kamigori, Ako-gun, Hyogo 678-1205, Japan.

The focusing characteristics of a ruthenium-coated cylindrical mirror were investigated on the basis of its ability to collect and focus broadband 5-17-nm soft X-rays emitted from a laser-produced plasma. Based on the plasmas spectral intensity distribution and the reflectivity function of the mirror, we defined the optimum position of the integrated cylindrical mirror at which the X-ray energy flux transported and focused through the mirror was maximum. A minimum spot diameter of 22 mm at a distance of approximately 200 mm from a soft X-ray source was confirmed. The maximum intensity of the collected soft X-rays was 1.3 mJ/cm(2) at the center of the irradiation zone. Thus, the irradiation intensity was improved by approximately 27 times when compared to that of 47 μJ/cm(2) without the mirror. The debris sputtering rate on the reflection surface of the mirror can be reduced to 1/110 by argon gas at 11 Pa, while the attenuation rate of the soft X-rays due to absorption by the buffer gas can be suppressed to less than 10% at the focal point. The focusing property of the mirror is expected to be maintained for 3000 h or longer without significant degradation for a 100 W/320 pps laser shot if the ruthenium layer is thicker than 10 μm. These results suggest that a stand-alone broadband soft X-ray processing system can be realized by using laser-produced plasma soft X-rays.
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http://dx.doi.org/10.1063/1.4770328DOI Listing
December 2012

Chymase inhibition prevents myocardial fibrosis through the attenuation of NOX4-associated oxidative stress in diabetic hamsters.

J Diabetes Investig 2012 Aug;3(4):354-61

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences.

Unlabelled: Aims/Introduction:  Diabetic cardiomyopathy entails the cardiac injury induced by diabetes, independent of vascular disease or hypertension. Despite numerous experimental studies and clinical trials, the pathogenesis of diabetic cardiomyopathy remains elusive. Here, we report that chymase, an immediate angiotensin II (AngII)-forming enzyme in humans and hamsters, and NOX4-induced oxidative stress have pathogenic roles in myocardial fibrosis in diabetic hamsters.

Materials And Methods:   Expression of chymase was evaluated in the hearts of streptozotocin (STZ)-induced diabetic hamsters. The impact of chymase-specific inhibitors, TEI-E00548 and TEI-F00806, on myocardial fibrosis, and increased levels of intracardiac AngII, accumulation of 8-hydroxy-2'-deoxyguanosine (an oxidative stress marker in urine and heart tissue) and expression of heart NOX4 in diabetic hamsters were investigated.

Results:   Myocardial chymase expression was markedly upregulated in STZ hamsters in a glucose-dependent manner. A total of 8 weeks after STZ administration, the diabetic hamsters showed enhanced oxidative stress and NOX4 expression in the heart, in parallel with increased myocardial AngII production. Oral administration of chymase-specific inhibitors, TEI-F00806 and TEI-E00548, normalized heart AngII levels, and completely reversed NOX4-induced oxidative stress and myocardial fibrosis in STZ-induced diabetic hamsters, although they did not affect the activity of the systemic renin-angiotensin system or systolic blood pressure.

Conclusions:   Chymase inhibition might prevent oxidative stress and diabetic cardiomyopathy at an early stage by reducing local AngII production. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00202.x, 2012).
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http://dx.doi.org/10.1111/j.2040-1124.2012.00202.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019255PMC
August 2012

Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells.

J Pharmacol Sci 2011 25;117(3):149-59. Epub 2011 Oct 25.

Safety Assessment Department, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) hold great promise for development of in vitro research tools to assess cardiotoxicity, including QT prolongation. In the present study, we aimed to clarify the electrophysiological/pharmacological characteristics of hiPS-CMs using the patch-clamp technique. The hiPS cells were differentiated into beating cardiomyocytes by the embryoid body method. The expression of genes related to cardiac ion channels and differentiation markers in cardiomyocytes were detected by RT-PCR. Whole-cell patch-clamp recordings were performed using single hiPS-CMs dispersed from beating colonies. We confirmed voltage-dependence of major cardiac ion currents (I(Na), I(Ca), I(Kr), and I(Ks)) and pharmacological responses to ion-channel blockers. Action potential duration (APD) was prolonged by both I(Kr)/hERG and I(Ks) blockers, whereas it was shortened by an I(Ca) blocker, indicating that these ion current components contribute to action potential generation in hiPS-CMs. As for multiple ion channel blockers, terfenadine prolonged APD, but verapamil did not, results which were identical to clinically relevant pharmacological responses. These data suggest that patch-clamp assay using hiPS-CMs could be an accurate method of predicting the human cardiac responses to drug candidates. This study would be helpful in establishing an electrophysiological assay to assess the risk of drug-induced arrhythmia using hiPS-CMs.
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http://dx.doi.org/10.1254/jphs.11038fpDOI Listing
March 2012

Reduced expression of adipose triglyceride lipase enhances tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-kappaB.

J Biol Chem 2011 Sep 2;286(37):32045-53. Epub 2011 Aug 2.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.
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http://dx.doi.org/10.1074/jbc.M111.285650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173212PMC
September 2011

In vitro gene expression analysis of hepatotoxic drugs in rat primary hepatocytes.

J Appl Toxicol 2008 Mar;28(2):227-36

Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.

The study examined the feasibility of screening for hepatotoxicity by an in vitro gene expression analysis using rat primary hepatocytes and Affymetrix Rat Toxicology U34 arrays. Hepatocytes were exposed for 6 or 24 h to eight drugs, with different mechanisms of hepatotoxicity, at one third of the cytotoxic concentration TC50, i.e. acetaminophen, cyclophosphamide, clofibrate, chlorpromazine, lithocholic acid, cisplatin, diclofenac and disulfiram. The types of transcriptional changes observed in this study were generally consistent with previously reported in vivo data, although there were some differences. In hierarchical cluster analysis, drugs formed clusters depending on their mode of toxicity against cells. The number of transcripts affected by the cholestatic hepatotoxicants (lithocholic acid and chlorpromazine) or the drugs that rarely cause of hepatotoxicity (cisplatin, diclofenac and disulfiram) were limited compared with the other drugs (acetaminophen, clobifibrate and cyclophosphamide), where they did not induce transcriptional changes apparently related to toxicity. It is concluded that in vitro gene expression analysis of hepatocytes using microarray is a useful tool for evaluating the toxicological profile of drugs and in screening for the direct toxicity of drugs against hepatocytes.
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http://dx.doi.org/10.1002/jat.1328DOI Listing
March 2008

In vitro gene expression analysis of nephrotoxic drugs in rat primary renal cortical tubular cells.

J Appl Toxicol 2008 Mar;28(2):237-48

Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan.

Rat primary renal cortical tubular cells were exposed to seven test substances, some with, and some without, known direct renal tubular cell toxicity. Cells were exposed to the substances at either one-third or one-tenth of the TC50 for cytotoxicity for 6 h or 24 h, so as not to induce cytotoxicity but to cause some transcriptional changes. Transcriptional profiles were investigated by using the Affymetrix Rat Toxicology U34 arrays, containing probes for more than 850 genes and ESTs. Four direct toxicants, cisplatin (CDDP), its less nephrotoxic analogue carboplatin (CBDCA), cephaloridine and gentamicin, were grouped together in a hierarchical clustering. In addition, the four direct toxicants affected more than 32 transcripts at their subcytotoxic concentrations at either 6 h or 24 h exposure. On the other hand, diclofenac, cyclosporine A and zinc, which are not considered to be directly toxic to tubules, affected less than 12 transcripts. Decreased Map3k12 and increased Hmox1 were commonly observed among the four direct toxicants, which appeared to be responses to cellular damage. Two platinum complexes, CDDP and CBDCA, induced similar changes, regardless of exposure duration or concentration. The types of transcriptional changes observed in this study were consistent with previously reported in vivo data, although there were some differences. These observations suggest that an in vitro gene expression analysis approach using GeneChip is feasible for screening for direct tubular toxicity of drugs and may help to clarify the underlying mechanisms of tubular toxicity.
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http://dx.doi.org/10.1002/jat.1329DOI Listing
March 2008

Protonation and methylation of an Anderson-type polyoxoanion [IMo6O24]5-.

Inorg Chem 2007 Feb 26;46(4):1464-70. Epub 2007 Jan 26.

Department of Chemistry, Kwansei Gakuin University, Sanda 669-1337, Japan.

A series of protonated and methylated Anderson-type molybdoperiodates as well as the unprotonated [IMo6O24]5- have been synthesized and structurally characterized as tetra-n-butylammonium salts: [(n-C4H9)4N]5[IMo6O24] [monoclinic, space group C2/c, a = 33.6101(3) A, b = 15.2575(1) A, c = 24.0294(2) A, beta = 126.9569(3) degrees , Z = 4], [(n-C4H9)4N]4[IMo6O23(OH)] [monoclinic, space group P21/c, a = 9.5587(1) A, b = 24.1364(2) A, c = 18.2788(2) A, beta = 90.1562(5) degrees , Z = 2], [(n-C4H9)4N]3[IMo6O22(OH)2].2DMF [monoclinic, space group P21/a, a = 17.6105(4) A, b = 15.5432(5) A, c = 29.3316(9) A, beta = 91.475(3) degrees , Z = 4], [(n-C4H9)4N]4[IMo6O23(OMe)].3H2O [orthorhombic, space group Pbca, a = 17.0679(4) A, b = 25.6998(6) A, c = 20.7428(4) A, Z = 4], [(n-C4H9)4N]3[IMo6O22(OMe)2] [monoclinic, space group P21/n, a = 10.4009(1) A, b = 14.6658(3) A, c = 23.5395(4) A, beta = 100.324(1) degrees , Z = 2]. In all of these compounds, the [IMo6O24]5- anion is protonated or methylated selectively at O atoms shared by two Mo atoms. The results have also revealed that the protonated Anderson-type molybdoperiodates readily react with methanol in a very selective manner, while the unprotonated [IMo6O24]5- anion does not react with methanol under similar conditions.
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http://dx.doi.org/10.1021/ic061881zDOI Listing
February 2007

Removal of p-alkylphenols from aqueous solutions by combined use of mushroom tyrosinase and chitosan beads.

Biosci Biotechnol Biochem 2006 Oct 7;70(10):2467-75. Epub 2006 Oct 7.

Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, Japan.

Enzymatic removal of p-alkylphenols from aqueous solutions was investigated through the two-step approach, the quinone conversion of p-alkylphenols with mushroom tyrosinase (EC 1.14.18.1) and the subsequent adsorption of quinone derivatives enzymatically generated on chitosan beads at pH 7.0 and 45 degrees C as the optimum conditions. This technique is quite effective for removal of various p-alkylphenols from an aqueous solution. The % removal values of 97-100% were obtained for p-n-alkylphenols with carbon chain lengths of 5 to 9. In addition, removal of other p-alkylphenols was enhanced by increasing either the tyrosinase concentration or the amount of added chitosan beads, and their % removal values reached >93 except for 4-tert-pentylphenol. This technique was also applicable to remove 4-n-octylphenol (4NOP) and 4-n-nonylphenol (4NNP) as suspected endocrine disrupting chemicals. The reaction of quinone derivatives enzymatically generated with the chitosan's amino groups was confirmed by the appearance of peaks for UV-visible spectrum measurements of the chitosan films incubated in the p-alkylphenol and tyrosinase mixture solutions. In addition, 4-tert-pentylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide.
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http://dx.doi.org/10.1271/bbb.60205DOI Listing
October 2006

Differentiation and maintenance of mast cells from CD34(+) human cord blood cells.

Exp Hematol 2006 Mar;34(3):320-9

Pharmaceutical Research Department III and Preclinical Research Department II, Chugai Pharmaceutical Co., Ltd, Kamakura, Japan.

Objective: Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells.

Materials And Methods: Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy.

Results: CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro.

Conclusion: Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.
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http://dx.doi.org/10.1016/j.exphem.2005.12.007DOI Listing
March 2006

In vitro and in vivo assays for high-molecular-weight pegylated muteins of granulocyte colony-stimulating factor.

Methods Mol Biol 2004 ;249:167-76

Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.

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http://dx.doi.org/10.1385/1-59259-667-3:167DOI Listing
February 2004

Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

Environ Health Perspect 2003 Aug;111(11):1411-20

Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan.

Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1241634PMC
http://dx.doi.org/10.1289/ehp.6164DOI Listing
August 2003

Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines.

J Toxicol Sci 2002 Aug;27(3):229-37

Department of Pharmacology, Dalian Medical University, 465 Zhongshan Road, Dalian, 116027, P. R. China.

We investigated and compared the cytotoxicity of 16 reference compounds in four in vitro systems: primary cultured rat hepatocytes, hepatoma HepG2 cell line, non-hepatic HeLa and Balb/c 3T3 cell lines. After 24 hr of exposure to the test compounds, the water-soluble tetrazolium salts WST-1 assay was used as an endpoint to evaluate cytotoxicity. Acetaminophen, diclofenac sodium cyclophosphamide and disulfiram displayed from 2 to more than 10 times higher IC50 values in three cell lines than in rat primary cultured hepatocytes. The cytotoxic effects of aspirin, amiodarone, clorfibiric acid, chlorpromazine, erythomycin, lithocholic acid, cisplatin and quinidine in rat hepatocytes were similar or 2 times stronger than those observed in cell lines. Ketoconazole resulted in the lowest IC50 value in the HeLa cell line. The data suggested that the compounds which are known to be metabolism-mediated liver toxicants have a differential hepatotoxicity in vitro and that primary cultured rat hepatocytes could represent a valuable tool for both screening and study of the effects of bio-transformation on the cytotoxicity of new chemical entities and xenobiotics in vitro.
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http://dx.doi.org/10.2131/jts.27.229DOI Listing
August 2002