Shoji Furusako

Effects of Aryl Hydrocarbon Receptor Signaling on the Modulation of Th1/Th2 Balance

An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor γ-chain complex

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Presepsin (sCD14-ST): development and evaluation of one-step ELISA with a new standard that is similar to the form of presepsin in septic patients

The current international consensus guidelines for the management of sepsis and septic shock emphasize the need to establish a rapid method to diagnose sepsis (1). Currently, procalcitonin (PCT) is used as a marker to diagnose sepsis or severe sepsis (2). However, a meta-analysis of PCT showed that this marker has poor diagnostic utility for differentiating sepsis from other non-infectious causes. Thus, better biomarkers of sepsis are needed (3). As a result, it is still a challenge to rapidly and accurately diagnose sepsis and severe sepsis. We previously identified a sepsis-specific novel form of CD14, the soluble CD14 subtype (sCD14-ST: renamed presepsin). To measure presepsin specifically, we developed a two-step sandwich enzyme-linked immunosorbent assay (ELISA), and demonstrated that presepsin was elevated specifically in septic patients (4). Receiver operating characteristic (ROC) curve analysis of presepsin revealed that presepsin is a more specific and sensitive marker for sepsis compared with conventional markers of sepsis, including interleukin-6 (IL-6) and PCT (5). Although the first generation two-step ELISA was sufficient to evaluate presepsin as a diagnostic marker for sepsis, this assay was not convenient and lacked the rapidity and precision that is required for routine presepsin tests in intensive care units. In this letter, we describe the development and evaluation of a one-step ELISA that uses recombinant presepsin as a new standard. The two-step ELISA measured plasma presepsin levels within 4 h, and concentrations were extrapolated based on recombinant CD14 (S286C) antigen (approx. 40 kDa) as the standard. However, an analysis of native presepsin in septic patients revealed that this protein consists of the N-terminal 13 kDa fragment of the CD14 protein wwhen examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditionsx (6). Based on these data, we prepared recombinant human pre-

Nrf2 deficiency leads to behavioral, neurochemical and transcriptional changes in mice

Nrf2 is a transcription factor that regulates the antioxidant and detoxification enzyme genes and provides defense against oxidative and electrophilic stresses in various tissues. In brain, while neuroprotective functions of Nrf2 have been well documented, Nrf2 contribution to the brain function remains to be elucidated. To address this issue, we investigated whether Nrf2 deficiency affects psychological behaviors, neurotransmitter systems and gene expressions in mice. We conducted four behavioral tests, social interaction, open‐field, rotarod and forced swimming tests and found that Nrf2 knockout mice exhibited reduced immobility in the forced swimming test. Neurochemical analyses revealed that the dopamine and serotonin metabolites increased in the brains of Nrf2 knockout mice. We also present a catalog of genes whose expression is Nrf2‐dependent in brain under unstressed conditions, which includes a number of xenobiotic‐metabolizing enzyme genes. These results thus support our contention that Nrf2 regulates its target genes in brain under unstressed conditions and loss of Nrf2 affects various brain functions.

Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase

1. Polyamine oxidase was purified from the soluble fraction of porcine liver by more than 70,000-fold to electrophoretic homogeneity using N8-acetylspermidine-Sepharose 4B affinity chromatography. 2. The molecular weight and isoelectric point of this enzyme were 62,000 and pH 4.5, respectively. 3. Optimal pH for the catalytic activity was close to 10.0. 4. The enzyme activity was enhanced by 5 mM dithiothreitol or 5 mM benzaldehyde. 5. Preferential substrates for this cytoplasmic PAO were N1-acetylspermine, N1-acetylspermidine and spermine. 6. Spermidine was not virtually the substrate for this enzyme. 7. The present results suggested the physiological roles of cytoplasmic PAO, being coupled with the reaction of spermidine/spermine N1-acetyltransferase, in recycling the cellular polyamines to putrescine.

The new sepsis marker, sCD14-ST, induction mechanism in the rabbit sepsis models

Soluble CD14 subtype (sCD14-ST) is a fragment of CD14 and is markedly increased in sepsis patients. We developed a new immunoassay to detect sCD14-ST and evaluated the efficacy of this marker for diagnosis of sepsis. For developing the strategies of sCD14-ST as a sepsis diagnostic marker, the induction mechanism must be known.

The novel Nrf2 inducer TFM-735 ameliorates experimental autoimmune encephalomyelitis in mice

ABSTRACT The transcription factor NF‐E2‐related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms against oxidative stress. Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system, is characterized by progressive demyelination and neurodegeneration induced by inflammation and oxidative stress. The induction of Nrf2 signaling has been shown to inhibit disease development and progression in the experimental autoimmune encephalomyelitis (EAE) model of MS in mice. In the present study, we performed a high‐throughput screening assay using a chimeric construct of the N‐terminal portion of Nrf2 fused to LacZ. Using this approach, we identified the novel Nrf2 inducer TFM‐735. Using human primary cell profiling systems, we found that TFM‐735 inhibited T cell proliferation and exerted immuno‐modulatory effects by inhibiting the production of IL‐6 and IL‐17. TFM‐735 also inhibited IL‐17 secretion from human peripheral blood mononuclear cells stimulated with anti‐CD3 and anti‐CD28. In EAE mice treated with TFM‐735, the expression of the Nrf2 target gene Nqo1 increased in the brain and spleen, disease severity was ameliorated, and plasma IL‐17 levels decreased. Furthermore, TFM‐735 inhibited luciferase activity in Wim‐6 transgenic EAE mice expressing the human interleukin 6‐luciferase (hIL6‐BAC‐Luc) reporter. Therefore, these findings indicate that TFM‐735 is a potent Nrf2 inducer that inhibits inflammatory cytokine production and disease progression in mice with EAE and that TFM‐735 is a promising therapeutic agent for MS.

Intestine-Targeted DGAT1 Inhibition Improves Obesity and Insulin Resistance without Skin Aberrations in Mice

Objective Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice. Design and Methods We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500), reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO) mice. Results The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice. Conclusions Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance.

Properties of soluble glycoprotein VI, a potential platelet activation biomarker

Abstract Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.

Effect of trapidil on effector functions of monocytes related to atherosclerotic plaque.

The infiltration and activation of inflammatory cells play an important role in the formation and stability of coronary atherosclerotic plaque in patients with acute coronary syndrome. In this study, we evaluated the effect of trapidil, an anti-platelet agent, on atheroma-related functions of human T cells and monocytes. Trapidil and anti-CD154 (CD40 ligand) antibody inhibited the increase of procoagulant activity in the mixed lymphocyte reaction; trapidil also suppressed the induction of tissue factor, monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 in the mixed lymphocyte reaction. Trapidil did not alter CD154 expression on isolated T cells, but it diminished CD40 expression on isolated monocytes and human monocytic leukemia THP-1 cells stimulated with interferon-gamma. Moreover, trapidil reduced MCP-1 production of isolated monocytes and THP-1 cells stimulated with interferon-gamma plus CD154-transfected cells. This effect was not seen with other tested anti-platelet agents and coronary vasodilators. In conclusion, trapidil directly acts on monocytes/macrophages to lower their susceptibility to CD154 on T cells.