Minoru Miyake

Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions.

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-γ-producing Tim-3+ CD8+ T cells with enhanced granzyme B and perforin expression, although it induced CD4+ T cell apoptosis. It simultaneously increased the number of Tim-3+CD86+ mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8+ T cells with DCs from Gal-9-treated mice increased the number of IFN-γ producing cells and IFN-γ production. Depletion of Tim-3+ DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-γ-producing CD8+ T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8+ T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8+ T cells.

Electron paramagnetic resonance-guided normobaric hyperoxia treatment protects the brain by maintaining penumbral oxygenation in a rat model of transient focal cerebral ischemia.

Oxygen therapy for ischemic stroke remains controversial. Too much oxygen may lead to oxidative stress and free radical damage while too little oxygen will have minimal therapeutic effect. In vivo electron paramagnetic resonance (EPR) oximetry, which can measure localized interstitial partial oxygen (pO2), can monitor penumbral changes of pO2. Therefore, we used EPR to study the effects of oxygen therapy in a rat model of 90-mins middle cerebral artery occlusion (MCAO). We found that 95% normobaric O2 given during ischemia was able to maintain penumbral interstitial pO2 levels close to the preischemic value while it may cause a two-fold increase in penumbral pO2 level if given during reperfusion. Elevation of the penumbra pO2 to preischemic physiologic level during MCAO significantly reduced infarction volume, improved neurologic function, decreased the generation of reactive oxygen species (ROS), and reduced matrix metalloproteinase (MMP)-9 expression and caspase-8 cleavage in the penumbra tissue of rats brain treated with oxygen. These results suggest that maintaining penumbral oxygenation by normobaric oxygen treatment during ischemia lead to neuroprotection, which is further reflected by the decreased production of ROS, MMP-9, and caspase-8.

Generation of hydrogen peroxide during brief oxygen-glucose deprivation induces preconditioning neuronal protection in primary cultured neurons.

Although reactive oxygen species (ROS) have been implicated in ischemic preconditioning (IPC)‐induced neuronal protection, several key questions concerning ROS remain to be elucidated. The purpose of this study is to obtain direct evidence for the formation of specific ROS species generated by IPC, and to determine the specific species that is responsible for the observed neuronal protection. Primary cultured cortex neurons from rat embryos were preconditioned with 10 min of oxygen‐glucose deprivation (OGD), which increased the intracellular levels of superoxide and hydrogen peroxide. This preconditioning markedly induced neuronal protection against 2‐hr OGD stimuli. Preconditioning with exogenous ROS by the administration of xanthine/xanthine oxidase (X/XO), or hydrogen peroxide was also found to induce IPC‐like neuronal protection. Administration of hydrogen peroxide scavengers, such as catalase, glutathione, or the thiol reductant N‐(2‐mercaptopriopionyl)‐glycine, all reduced the increase in the intracellular hydrogen peroxide levels, which effectively eliminated IPC‐ or exogenous ROS‐induced neuronal protection. In contrast, administration of the membrane‐permeable superoxide dismutase mimic Mn(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin pentachloride was able to block the increase of intracellular superoxide levels during IPC, but did not abolish either IPC‐ or exogenous X/XO preconditioning‐induced neuronal protection. These findings strongly suggest that IPC enhances the generation of superoxide, which is then converted to hydrogen peroxide, and that hydrogen peroxide is likely the main trigger involved in the mechanism of IPC‐induced neuronal protection.

Normobaric hyperoxia inhibits NADPH oxidase‐mediated matrix metalloproteinase‐9 induction in cerebral microvessels in experimental stroke

Matrix metalloproteinase‐9 (MMP‐9) and NADPH oxidase contribute to blood–brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP‐9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase‐mediated MMP‐9 induction in transient focal cerebral ischemia. Male Sprague–Dawley rats (n = 69) were given NBO (95% O2) or normoxia (21% O2) during 90‐min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP‐9 and NADPH oxidase. BBB damage was non‐invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91phox and MMP‐9 expression were up‐regulated in ischemic hemispheric microvessels after 90‐min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP‐9 increase, indicating a causal link between NADPH oxidase‐derived superoxide and MMP‐9 induction. NBO treatment inhibited gp91phox expression, NADPH oxidase activity, and MMP‐9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91phox containing NADPH oxidase plays an important role in MMP‐9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP‐9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF‐1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF‐1‐regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF‐1 on cellular redox status in SH‐SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF‐1α expression by HIF‐1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non‐transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF‐1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF‐1 regulated not only glucose transporter‐1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA‐transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF‐1 protects cells from hypoxia and ischemia mediated injuries.

Hydroxyl radical formation is greater in striatal core than in penumbra in a rat model of ischemic stroke

Although hydroxyl radical (•OH) formation has been implicated in the pathophysiological changes of ischemic stroke, •OH production in the core and penumbra regions is not clear. It is extremely important to distinguish penumbra from ischemic core in focal cerebral ischemia studies, because the penumbra contains viable tissue, which can be salvaged by appropriate treatment. This study evaluated •OH production in both core and penumbra regions of ischemic striatum during ischemia and reperfusion. Microdialysis probes were placed in striatal tissue of rats subjected to the middle cerebral artery occlusion model of ischemic stroke. The •OH‐trapping agent 4‐hydroxybenzoic acid (4‐HBA) was administered by both i.v. and probe infusion. Dialysate levels of the 4‐HBA oxidation products, 3,4‐dihydroxybenzoic acid (3,4‐DHBA), were determined by HPLC‐ECD. After microdialysis probe delivery of 4‐HBA, •OH production was significantly increased in the striatal core during both ischemia and reperfusion. Penumbra •OH production increased only during reperfusion. Alterations of 3,4‐DHBA concentration in dialysate following i.v. 4‐HBA administration were likely related to alterations in tissue blood flow. The findings were confirmed by a greater oxidation of dihydroethidium in the ischemic core than in the penumbra as determined by fluorescent microscopy. The findings of •OH production in ischemic striatum are the opposite of those reported for ischemic cortex and suggest critical regional variations in •OH production that may have significant clinical implications in the treatment of ischemic stroke.

Galectin-9 expands immunosuppressive macrophages to ameliorate T-cell-mediated lung inflammation.

Galectin‐9 (Gal‐9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T‐cell‐mediated autoimmune models. However, it remains unclear if Gal‐9 plays a suppressive role for T‐cell function in non‐autoimmune disease models. We assessed the effects of Gal‐9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal‐9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii‐induced lung inflammation, as the levels of IL‐1, IL‐6, IFN‐γ, and IL‐17 were significantly reduced in the BALF of Gal‐9‐treated mice. Moreover, co‐culture of anti‐CD3‐stimulated CD4 T cells with BALF cells harvested from Gal‐9‐treated mice on day 1 resulted in diminished CD4 T‐cell proliferation and decreased levels of IFN‐γ and IL‐17. CD11b+Ly‐6ChighF4/80+ BALF Mϕ expanded by Gal‐9 were responsible for the suppression. We further found in vitro that Gal‐9, only in the presence of T. asahii, expands CD11b+Ly‐6ChighF4/80+ cells from BM cells, and the cells suppress T‐cell proliferation and IFN‐γ and IL‐17 production. The present results indicate that Gal‐9 expands immunosuppressive CD11b+Ly‐6Chigh Mϕ to ameliorate Th1/Th17 cell‐mediated hypersensitivity pneumonitis.

Galectin-9 expands unique macrophages exhibiting plasmacytoid dendritic cell-like phenotypes that activate NK cells in tumor-bearing mice

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C(+)CD11b(+)F4/80(+) macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.

Florid cemento-osseous dysplasia. Report of a case.

A case of florid cemento-osseous dysplasia in a 16-year-old Japanese boy is presented. The lesion was unusually large and affected all four quadrants. Progressive increase in the bulk of the lesion was seen.

Acetoxymethoxycarbonyl nitroxides as electron paramagnetic resonance proimaging agents to measure O2 levels in mouse brain: a pharmacokinetic and pharmacodynamic study.

Measurement of O2 concentration and distribution in brain is essential to understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that can potentially map O2 concentration in the brain. In a previous study, we demonstrated that, after intraperitoneal administration of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (1) to mice, this nitroxide crossed the blood-brain barrier into brain tissue where, after hydrolysis, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) was liberated and entrapped. This pilot study suggested that nitroxide 1 is a proimaging agent that can deliver nitroxide 2 to brain tissue, where O2 levels can be estimated. In the present study, we conducted a series of pharmacokinetic and pharmacodynamic experiments designed to assess the uptake of structurally disparate nitroxides into brain tissue and retention, after hydrolysis, of the anions of the corresponding nitroxide acids. From these findings, nitroxide 1 and trans-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (5) meet the requirement as EPR proimaging agents for mapping O2 distribution in the brain following stroke.