Shinichi Iwai

TNF-α Drives Human CD14+ Monocytes to Differentiate into CD70+ Dendritic Cells Evoking Th1 and Th17 Responses

Many mechanisms involving TNF-α, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-α to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14+ monocytes cultured in the presence of TNF-α and GM-CSF converted to CD14+ CD1alow adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-α, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (Mφ). The mature DC (CD83+ CD70+ HLA-DR high CD14low) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-γ and TNF-α, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-α added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the Mφ (CD14highCD70+CD83−HLA-DR−) produced large amounts of MMP-9 and TNF-α without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated Mφ. Therefore, additional stimulation of monocytes with TNF-α may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated Mφ-mediated innate immunity.

Alterations of Glucose-Dependent and -Independent Bladder Smooth Muscle Contraction in Spontaneously Hypertensive and Hyperlipidemic Rat

Alteration of bladder contractility was examined in the spontaneously hypertensive and hyperlipidemic rat (SHHR; age, 9 months; systolic blood pressure, >150 mm Hg; plasma cholesterol, >150 mg/dl). Carbachol (CCh) induced time- and dose-dependent contractions in Sprague-Dawley (age-matched control) rats and SHHR; however, maximal levels differed significantly (13.3 ± 2.2 and 5.4 ± 1.9 μN/mm2 following 10 μM CCh treatment, respectively; n = 5). This difference, which was maintained in calcium-replaced physiological salt solution (PSS), was suppressed by pretreatment with rho kinase inhibitor, 1 μM Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]; moreover, total activity of rho kinase was also reduced in SHHR bladder. Pretreatment of bladders under high-glucose (HG) conditions (22.2 mM glucose-contained PSS for 30 min) led to enhancement of CCh-induced contraction solely in control animals. Under HG conditions, both protein kinase C (PKC) activity and production of diacylglycerol (DG) derived from incorporated glucose declined in SHHR bladder; however, sustained elevation of plasma glucose level was not detected in SHHR. These results suggested that bladder contractility dysfunction in SHHR is attributable to alteration of rho kinase activity and the DG-PKC pathway. This dysfunction may occur prior to chronic hyperglycemia onset in progressive hypertension and hyperlipidemia.

Individual Difference in the Pharmacokinetics of a Drug, Pravastatin, in Healthy Subjects

The purpose of this investigation was to determine whether there were individual pharmacokinetic differences of a drug, pravastatin. Furthermore, the percentage of subjects who showed pharmacokinetic differences was determined. A single oral dose of pravastatin 10 mg was administered to 84 Japanese healthy male subjects. Serum concentrations of pravastatin were measured for 8 hours postdose. Area under the concentration‐time curve (AUC) and peak concentration (Cmax) were determined as primary evaluation parameters. An outlier was defined as follows:

Changes in mRNA Levels of Fibrinogen Subunit Polypeptides in Rats Defibrinogenated with Batroxobin

Batroxobin is a snake venom that is a thrombinlike enzyme used for clinical treatment. We analyzed hepatic mRNA levels for fibrinogen subunit polypeptides and prothrombin by reverse transcription-polymerase chain reaction as well as coagulation and fibrinolysis factors in plasma 1, 3, 5 and 24 hours after Batroxobin treatment (3 BU/100 g) in rats. The mRNA levels of alpha- and beta-chains of fibrinogen were significantly increased with decreases in plasma fibrinogen, alpha2-plasmin inhibitor, and plasminogen levels, while the mRNA levels for prothrombin remained unchanged. These results suggest that fibrinogen mRNA synthesis is regulated by plasma fibrinogen levels in Batroxobin-induced defibrinogenated rats.

Matrix metalloproteinase-9 in spontaneously hypertensive hyperlipidemic rats

The presence of hypertension and hyperlipidemia accelerates atherosclerosis and increases the risk of ocular disease. Since there were few rat models for atherosclerosis, spontaneously hypertensive rats (SHRs) and spontaneously hyperlipidemic rats (HLRs) were crossbred to obtain a new model: the spontaneously hypertensive hyperlipidemic rat (SHHR). Matrix metalloproteinases (MMPs) play an important role in ocular degeneration. The purpose of this study is to investigate changes in the MMP activities in vitreous and plasma as well as MMP expression in the retinas of SHHRs, which served as a model of vascular degeneration. We used 8-month-old Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats, SHRs, HLRs, and SHHRs. The MMP-2 and MMP-9 activities in plasma and vitreous were examined by zymography. The mRNA expression of MMP-2, MMP-9, and tissue inhibitor of metalloproteinases-3 (TIMP-3) in retina was examined by quantitative PCR. The localized expression of MMP-9 in the retinas was examined by immunostaining. The MMP-9 activity increased significantly in SHHRs compared with all other rats. MMP-9 was observed mainly at the superficial layer of the retina on immunostaining. The MMP-2, MMP-9, and TIMP-3 mRNA in retina was not significantly different in SHHRs as compared with all other rats. Increased MMP-9 activity in vitreous was influenced more intensely from plasma than retina because there was no change in MMP-9 expression in retina, and MMP-9 immunostaining was observed mainly at the surface of the retina, where blood vessels are present. In this study, the complications of hypertension and hyperlipidemia induced increased MMP-9 activity in vitreous and plasma. It is therefore suggested that MMP-9 may be involved in causing this result and in the development of retinal disease.

Effect of Lipid-Hydroperoxide-Induced Oxidative Stress on Vitamin E, Ascorbate and Glutathione in the Rabbit Retina

Background: It is possible that oxidative stress causes several retinal diseases. However, the natural biogenic role of antioxidants in the retina is not clear. Purpose: This study investigates the change in concentration of vitamin E (VE), ascorbate and glutathione (GSH) in the retina following vitreous injection of 600 µg 18:2 linoleic acid hydroperoxide (LHP) in male New Zealand rabbits. Method: LHP was injected above the retinal surface. The animals were sacrificed and the eyes enucleated before LHP injection, 1, 3, 6, 12, 24 h and 4 and 7 days after LHP injection. Retinas were removed, VE and ascorbate measured by HPLC, and GSH determined by a fluorometric method. Results: The concentration of VE in the retina decreased from pretreatment levels of 154.6 ± 29.7 nmol/g wet weight (n = 7) and was lowest at 6 h (61.1 ± 18.1 nmol/g wet weight, n = 4, p < 0.05), then increased gradually, returning slowly to pre-LHP levels by 7 days. The concentration of ascorbate in control retinas decreased at 6 h from pretreatment levels of 7.33 ± 0.93 µmol/g wet weight (n = 7) to 2.74 ± 0.16 µmol/g wet weight (n = 4, p < 0.05) and returned to pretreatment levels rapidly by 24 h after injection. The concentration of GSH in retinas decreased from baseline levels of 109.53 ± 8.19 µg/g wet weight (n = 9), was lowest at 12 h (72.40 ± 11.17 µg/g wet weight, n = 5, p < 0.05) and returned to pretreatment levels by 7 days. Conclusion: The results suggest that intravitreous LHP injection is a contributor to oxidative stress in the rabbit retina by causing a reduction in antioxidant capacity.

A new method for measuring osteoclast formation by electrical impedance.

Osteoclasts are important target cells for osteoporosis treatment. Recently, a real-time cell analysis (RTCA) system was developed to observe cell morphology and adhesion; however, the use of RTCA to study osteoclastogenesis has not been reported. Here, we investigated whether osteoclast formation could be monitored in real-time using RTCA. The cell index determined via electrical impedance using RTCA, and the number of osteoclasts exhibited a significant positive correlation. RTCA was useful for determining the effect of (-)-epigallocatechin-3-gallate on the inhibition of bone resorption. We established a new method of measuring osteoclast formation in real-time using RTCA.

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis

AIMS In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases. MAIN METHODS Four-month-old male Sprague-Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR. KEY FINDINGS In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR. SIGNIFICANCE This study suggested that these four genes might be important in early atherosclerosis development.

Appointment of New Section Editors

Pinar Aydin O’Dwyer, Professor of Ophthalmology and neuroophthalmologist, is a member of the Advisory Committee and Assessment Committee and Head of the Ethics Subcommittee of the International Council of Ophthalmology. She serves as Executive Board Member and Treasurer of the Turkish Board of Ophthalmology and General Secretary of the Postgraduate and Continuing Education Committee. Since 2000 she has been Program Secretary of the Scientific Section for Neuroophthalmology of the European Association for Vision and Eye Research (EVER); since 2002 she has been a Regional Representative on the EVER Board and is currently the treasurer of EVER. She has held conferences, and organized and given courses in America, Europe, Asia and Africa. She has authored approximately 150 scientific publications in peer-reviewed ophthalmology journals and edited seven books in ophthalmology. Since 2004 she has served on the Editorial Board of Ophthalmic Research . Ophthalmic Research is pleased to announce the appointment of new section editors for the field of Neuroophthalmology and Glaucoma. In addition, a new section – Clinical Retina – is introduced to manage the growing number of submissions in this field. Section editors have important duties and are responsible for guiding a submission through the peer review process. They are assigned to a particular article and are responsible for selecting reviewers for that submission, communicating with the authors and reviewers throughout the process, making a decision in favor of acceptance or rejection, and finally managing the successful papers through the editing process. Drs. Aydin (Neuroophthalmology) and Wiedemann (Clinical Retina) have already served as board members of Ophthalmic Research . Dr. Orgül (Glaucoma) begins his tenure on the board this year. We hope to continue the success of the journal and to provide useful information to the medical and scientific communities. At the same time, I personally would also like to thank Drs. Tsukahara and van den Berg for their time and commitment as section editors for many years. Uwe Pleyer, Editor-in-Chief Published online: December 11, 2006

Vitamin E-Coated Dialyzer Inhibits Oxidative Stress

Aim: To examine the effects of vitamin E-coated dialyzer on oxidative stress in vitro. Methods: A dialyzer with a synthetic polymer membrane (APS-11SA) and vitamin E-coated dialyzer (VPS-11SA) were connected to a blood tubing line, and U937 cells were circulated in the device. The circulating fluid was collected at 1, 2, 5, 10, 25, and 50 cycles, which are estimated numbers of passes through the dialyzer. Intracellular reactive oxygen species (ROS) production, malondialdehyde (MDA), and Cu/Zn-superoxide dismutase (SOD) were quantified. Results: Intracellular ROS production was increased in the first cycle by APS-11SA and was decreased throughout the experiment by VPS-11SA. Intracellular ROS production in the VPS-11SA device was lower, and MDA levels were decreased. MDA levels were lower during VPS-11SA processing than during APS-11SA processing. Cu/Zn-SOD levels remained unchanged. Conclusion: Our results highlight anti-oxidative-stress effects of a vitamin E-coated dialyzer.