National Defense Medical College Research Institute

Tokorozawa, Japan

National Defense Medical College Research Institute

Tokorozawa, Japan
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Miyazaki H.,National Defense Medical College | Nishiyama K.,National Defense Medical College Research Institute | Yamamoto J.,National Defense Medical College Research Institute
Journal of Leukocyte Biology | Year: 2013

Although mouse liver F4/80+ Kupffer cells consist of cytokine-producing CD11b+ cells and phagocytic CD68+ cells, an undefined CD11b- CD68- subset (30%) also exists. We herein demonstrate a more fundamental classification by adding CD32 (FcγRII), which covers most liver F4/80+ cells and the distinct functions of them. Among the F4/80+ cells, 50%, 40%, and 30% of cells were CD32+, CD68+, and CD11b+, respectively, and one-half of the CD68+ cells coexpressed CD32+. CD68+ and CD32+ cells, but not CD11b+ cells, expressed a phagocytosis-related CRIg. Gy (6) irradiation depleted liver CD11b+ cells and those in the spleen, bone marrow, and peripheral blood but not liver CD32/CD68+ cells. Transfer of bone marrow cells into the irradiated mice reconstituted liver CD11b+ cells. Conversely, clodronate pretreatment depleted only liver CD32/CD68+ cells but not liver CD11b+ cells and peripheral blood or spleen CD11b+ monocytes/macrophages. Moreover, the CD32+ cells might be precursors of CD68+ cells, as a large proportion of CD32+ cells expressed the c-kit (CD117), and CD34 and CD32+ cells acquired CD68 immediately after bacteria administration. CD32/CD68+ cells, but not CD11b+ cells, expressed resident macrophage-specific MerTK and CD64 (FcγRI). Challenge with Staphylococcus aureus or liver metastatic EL-4 tumor cells indicated that the CD6+8 subset is engaged in systemic bactericidal activity, whereas the CD11b+ subset is pivotal for liver antitumor immunity. Human liver CD14+ Kupffer cells could also be classified into three similar subsets. These results suggest that liver CD68+ Kupffer cells and CD11b+ Kupffer cells/macrophages are developmentally and functionally distinct subsets. J. Leukoc. Biol. 94: 1325-1336; 2013. © Society for Leukocyte Biology.


Saito A.,Waseda University | Miyazaki H.,National Defense Medical College Research Institute | Fujie T.,Waseda University | Ohtsubo S.,Waseda University | And 3 more authors.
Acta Biomaterialia | Year: 2012

Polymeric ultra-thin films (nanosheets) possess unique properties that make them suitable materials for various biomedical applications. In our previous study, we assessed the use of an antibiotic (tetracycline, TC)-loaded nanosheet (or "TC-nanosheet") for the treatment of gastrointestinal tissue defects. The nanosheet consisted of three functional layers: layer-by-layer nanosheet as a stable platform, TC as an antimicrobial agent with autofluorescence for tracing, and a poly(vinyl acetate) nanosheet to act as a protecting layer. The TC-nanosheet has high flexibility, adhesive strength and transparency. Here, we evaluated the effectiveness of the TC-nanosheet in preventing full thickness burn-wound infections. In an in vivo study, murine dorsal skin was injured by full-thickness burns and then infected with Pseudomonas aeruginosa (P. aeruginosa), a common bacterium causing burn-associated infections. The wound site was treated either with a TC-nanosheet, TC-unloaded nanosheet or left untreated. Wound management was facilitated by the high transparency of the TC-nanosheet. The TC-nanosheet significantly improved burn-wound infection by P. aeruginosa in mice. Indeed, all mice treated with the TC-nanosheet survived, whereas the other treatment groups displayed increased rates of mortality due to bacterial infection. According to histological analyses and viable bacterial counting in the liver (bacterial translocation), the TC-nanosheets were able to prevent not only the local inflammation but also systemic inflammation. We conclude that the TC-nanosheet can act as an effective treatment for full-thickness burn-wound infection. Hence, the TC-nanosheet is a promising therapeutic tool for burn-wound management in severely burn-injured patients. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


Maruhashi T.,Hiroshima University | Soga J.,Hiroshima University | Fujimura N.,Hiroshima University | Idei N.,Hiroshima University | And 12 more authors.
Circulation | Year: 2012

Background-Patients with Gilbert syndrome have mild unconjugated hyperbilirubinemia. It has been shown that bilirubin is an endogenous antioxidant. We evaluated the role of oxidative stress in endothelial function in patients with Gilbert syndrome under normal conditions without cardiovascular risk factors. Methods and Results-A total of 108 young men with Gilbert syndrome without cardiovascular risk factors and 108 age-matched healthy men (normal controls) were enrolled in this study. Serum concentrations of bilirubin were higher in patients with Gilbert syndrome than in control subjects (29.2±11.6 versus 9.4±2.7 μmol/L; P<0.001). Serum concentrations of malondialdehyde-modified low-density lipoprotein and urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG), as indices of oxidative stress, were lower in patients with Gilbert syndrome than in control subjects (61.8±24.5 versus 72.5±21.8 U/L, P=0.034; 7.8±2.4 versus 10.4±3.2 ng/mg creatinine, P=0.001, respectively). Flow-mediated vasodilation was greater in patients with Gilbert syndrome than in normal control subjects (7.2±2.2% versus 5.9±1.7%; P<0.001). Vascular responses to nitroglycerine were not significantly different between the 2 groups. Flow-mediated vasodilation correlated with serum concentration of bilirubin (r=0.44, P<0.001), malondialdehyde-modified low-density lipoprotein (r=-0.25, P=0.01), and urinary excretion of 8-OHdG (r=-0.27, P=0.004) in patients with Gilbert syndrome but not in control subjects. In addition, serum concentration of bilirubin correlated with malondialdehyde-modified low-density lipoprotein (r=-0.20, P=0.04) and 8-OHdG (r=-0.21, P=0.02) in patients with Gilbert syndrome but not in control subjects. Conclusions-Patients with Gilbert syndrome had low levels of oxidative stress associated with hyperbilirubinemia and enhancement of endothelium-dependent vasodilation. Clinical Trial Registration-URL: http://www.umin.ac.jp. Unique identifier: UMIN000003409. © 2012 American Heart Association, Inc.


Iba T.,Juntendo University | Saito D.,National Defense Medical College Research Institute | Wada H.,Mie University | Asakura H.,Kanazawa University
Thrombosis Research | Year: 2012

Introduction: Although supplementation with antithrombin (AT) concentrates has been widely accepted for the treatment of disseminated intravascular coagulation (DIC) in Japan, the effects and adverse effects have not been investigated. Materials and Methods: We conducted a nonrandomized multi-institutional survey. A total of 729 septic DIC patients with AT activity levels of 70% or lower, who had undergone AT substitution at either 1500 IU/day or 3000 IU/day for consecutive 3 days were analyzed. Of these, 650 and 79 patients had received 1500 IU/day (AT1500 group) and 3000 IU/day (AT3000 group), respectively. Results: Bleeding events were observed in 6.52% of patients (severe bleeding, 1.71%). A significant decrease in initial AT level (below 50%) was observed in 69.6% of patients in AT3000 group and 48.2% in AT1500 group, and this difference was significant (P < 0.01). A logistic-regression analysis conducted using age, gender, body weight, initial AT activity, and supplemented AT dose, revealed that higher initial AT activity (odds ratio (OR), 1.032; P < 0.001), AT dose of 3000 IU/day (OR, 1.912; P = 0.026), and age (OR, 0.985; P = 0.023) were significant factors for improved survival. Conclusion: The risk of severe bleeding is less than 2%, and concomitant administration of heparin did not increase the risk. The survival in AT1500 group was 65.2%, while that in AT3000 group was 74.7%. © 2012 Elsevier Ltd.


Kinoshita M.,National Defense Medical College | Uchida T.,National Defense Medical College | Sato A.,National Defense Medical College | Nakashima M.,National Defense Medical College | And 6 more authors.
Journal of Hepatology | Year: 2010

Background & Aims: Liver Kupffer cells have been suggested to be heterogeneous macrophage lineage cells. We explored this possibility by classifying the mouse Kupffer cells into subpopulations and characterizing them by their phenotype and function. Methods: Liver mononuclear cells (MNCs) from C57BL/6 mice were isolated and their phenotypes and functions were analyzed. The effects of clodronate liposomes and gadolinium chloride (GdCl3) on Kupffer cells were also investigated. Results: Approximately 25% of liver MNCs were F4/80+ Kupffer cells. Of these, 46% were CD11b -CD68+, 22% were CD11b+CD68-, and 6% were CD11b+CD68+. CD68+ cells showed potent phagocytic activity and reactive oxygen species (ROS) production capacity after lipopolysaccharide (LPS) stimulation, whereas CD11b+ cells did not. CD11b+ cells showed a strong capacity for the production of cytokines (TNF and IL-12), which was much less prominent in CD68+ cells. At 24h after LPS or Escherichia coli injection into mice, the proportions of CD11b+CD68- and CD11b+CD68+ cells increased but that of CD11b-CD68+ cells decreased. The increase in CD11b+CD68+ cells appeared to be derived from the CD11b+CD68- subset. Although the CD11b+ cells augmented phagocytic activity after LPS injection, they did not increase ROS production, suggesting their weak lytic activity. Injection of clodronate or GdCl3 into mice depleted the CD68+ cells but increased CD11b+ cells proportionally because CD68+ cells may phagocytose these toxic reagents and undergo apoptosis. GdCl3-treated mice also consistently increased serum TNF after LPS challenge. Conclusions: Two F4/80+ Kupffer cell subsets may exist, a CD68+ subset with phagocytic activity and a CD11b+ subset with cytokine-producing capacity. © 2010 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver.


Terakawa M.,Keio University | Tsuda H.,National Defense Medical College | Ashida H.,National Defense Medical College Research Institute | Sato S.,National Defense Medical College Research Institute
Lasers in Surgery and Medicine | Year: 2010

Background and Objective: We previously delivered a therapeutic gene to skin grafts of rats by using photomechanical waves (PMWs), also called laser-induced stress waves (LISWs), with the objective of enhancing adhesion of grafted tissue. The objective of this study was to evaluate tissue alterations that are possibly caused by PMWs used for gene delivery on the basis of immunohistochemistry and electron microscopy. Materials and Methods: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532nm nanosecond laser pulses from a (of) Q-switched Nd:YAG laser. Tissue alterations were evaluated by histological analysis using hematoxylin and eosin (H&E) staining and immunohistochemical stainings, including anti-rat CD68 antibody staining to identify macrophages for detection of inflammation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining for assessment of apoptosis. Morphological changes of cell membranes and organelles were evaluated by transmission electron microscopy. Results: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm2 (42MPa in peak pressure), which is the optimum laser fluence (pressure) for therapeutic gene delivery to skin graft, showed no noticeable damage. At fluences higher than 1.8 J/cm2 (>51 MPa), fragmentation of nuclei was observed and the number of CD68-positive cells increased remarkably. No significant increases in the numbers of TUNEL-positive keratinocytes and fibroblasts were observed at 1.2 J/cm2. At fluences higher than 1.8 J/cm2, the averaged ratio of TUNELpositive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm2 caused neither damage to the cell membrane, nuclear membrane, or organelles. Conclusion: We observed no noticeable tissue alteration under the optimum laser irradiation conditions used for therapeutic gene delivery to a skin graft, demonstrating low invasiveness of our PMW-based gene transfection. © 2010 Wiley-Liss, Inc.


Takahashi Y.,National Defense Medical College Research Institute
Seishin shinkeigaku zasshi = Psychiatria et neurologia Japonica | Year: 2012

In 1996, the United Nations (UN) and the World Health Organization (WHO) published guidelines for developing suicide prevention measures at national level. In Japan, the Basic Act on Suicide Prevention was promulgated in June 2006 and a Comprehensive Suicide Measures Framework was formulated in June 2007, which announced that suicide prevention is an official concern and that entire societies should be engaged in such suicide prevention efforts. One of the fundamental principles moving forward is to place emphasis on coordination between medical and community models of care. The medical model is based on the notion that mental disorders that could lead to suicide should be diagnosed at earlier stages and addressed through appropriate psychiatric treatment. The community model, which concerns awareness activities that emphasize earlier detection and attentive social support, is also important. Implementing policies with a long-term perspective that maintain close linkages between these models is a critical strategy for suicide prevention. This presentation investigates the current conditions and issues around Japanese suicide prevention with reference to the UN/WHO guidelines for suicide prevention, and compares the situation to that in other countries.


Fujie T.,Waseda University | Saito A.,Waseda University | Kinoshita M.,National Defense Medical College | Miyazaki H.,National Defense Medical College Research Institute | And 3 more authors.
Biomaterials | Year: 2010

An ultra-thin polymer film (nanosheet) composed of polysaccharides (i.e., polysaccharide nanosheet) provides sufficient adhesiveness, flexibility and robustness to act as an effective wound dressing. We have recently demonstrated the sealing effect of a nanosheet on a murine cecal puncture. Nevertheless, a small percentage of bacteria penetrated the nanosheet because of its ultra-thin structure. Here, we have developed an antibiotic-loaded nanosheet to inhibit bacterial penetration and investigated its therapeutic efficacy using a model of a murine cecal puncture. Tetracycline (TC) was sandwiched between a poly(vinylacetate) (PVAc) layer and the polysaccharide nanosheet (named PVAc-TC-nanosheet). Under physiological conditions, TC was released from the nanosheet for 6 h. Microscopic observation between the interface of the PVAc-TC-nanosheet and bacteria demonstrated how its potent anti-microbial effect was achieved. In vivo studies show that overlapping therapy with the PVAc-TC-nanosheet (thickness: 177 nm) significantly increases mouse survival rate after cecal puncture as well as suppressing an increase in the intraperitoneal bacterial count and leukocyte count. © 2010 Elsevier Ltd.


Sato S.,National Defense Medical College Research Institute | Kawauchi S.,National Defense Medical College Research Institute | Okuda W.,Tokyo University of Agriculture and Technology | Nishidate I.,Tokyo University of Agriculture and Technology | And 2 more authors.
PLoS ONE | Year: 2014

Despite many efforts, the pathophysiology and mechanism of blast-induced traumatic brain injury (bTBI) have not yet been elucidated, partially due to the difficulty of real-time diagnosis and extremely complex factors determining the outcome. In this study, we topically applied a laser-induced shock wave (LISW) to the rat brain through the skull, for which real-time measurements of optical diffuse reflectance and electroencephalogram (EEG) were performed. Even under conditions showing no clear changes in systemic physiological parameters, the brain showed a drastic light scattering change accompanied by EEG suppression, which indicated the occurrence of spreading depression, long-lasting hypoxemia and signal change indicating mitochondrial energy impairment. Under the standard LISW conditions examined, hemorrhage and contusion were not apparent in the cortex. To investigate events associated with spreading depression, measurement of direct current (DC) potential, light scattering imaging and stereomicroscopic observation of blood vessels were also conducted for the brain. After LISW application, we observed a distinct negative shift in the DC potential, which temporally coincided with the transit of a light scattering wave, showing the occurrence of spreading depolarization and concomitant change in light scattering. Blood vessels in the brain surface initially showed vasodilatation for 3-4 min, which was followed by long-lasting vasoconstriction, corresponding to hypoxemia. Computer simulation based on the inverse Monte Carlo method showed that hemoglobin oxygen saturation declined to as low as ∼35% in the long-term hypoxemic phase. Overall, we found that topical application of a shock wave to the brain caused spreading depolarization/depression and prolonged severe hypoxemia-oligemia, which might lead to pathological conditions in the brain. Although further study is needed, our findings suggest that spreading depolarization/depression is one of the key events determining the outcome in bTBI. Furthermore, a rat exposed to an LISW(s) can be a reliable laboratory animal model for blast injury research. © 2014 Sato et al.


Sato S.,National Defense Medical College Research Institute | Ando T.,Keio University | Obara M.,Keio University
Optics Letters | Year: 2011

We developed an optical-fiber-based photomechanical gene transfer system for endoscopic or catheter-based application. A fiber tip with a laser-absorbing film covered with a transparent plastic disk for plasma confinement was attached to a quartz fiber; the film was irradiated with nanosecond laser pulses transmitted through the fiber to generate photomechanical waves (PMWs). Characteristics of PMWs emitted from the fiber tip were examined to confirm the necessary conditions for gene transfer. We then attempted to transfer reporter genes to the rat skin as a test tissue in vivo with the fiber system, and the results showed significantly high protein levels and spatially selective pinpoint gene expressions in the tissue. © 2011 Optical Society of America.

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