インターロイキン-1β暴露下のラットの血管におけるエタノールによる誘導型一酸化窒素合成酵素抑制を介した弛緩反応の抑制

Nitric oxide produced by inducible nitric oxide synthase (iNOS) regulates sepsis-induced hypotension. During septic shock, interleukin (IL)-1β is synthesized in endothelial cells and smooth muscle cells by endotoxin. Ethanol (EtOH) suppresses endotoxin-induced hypotension. The present study aimed to elucidate the effect of EtOH on gradual relaxation and iNOS expression induced by IL-1β in isolated rat superior mesenteric arteries (SMAs). Exposure to IL-1β–induced contraction in SMA rings, followed by a gradual relaxation of phenylephrine precontracted tone. Contraction was abolished by indomethacin (IM), cycloheximide (Chx), and endothelium denudation. In contrast, the gradual relaxation was abolished by NOS inhibitors, Chx, endothelium denudation, and inhibited by EtOH (50 and 100 mM). However, IM had no effect on relaxation. Western blot analysis demonstrated that iNOS expression was induced by IL-1β and was inhibited by EtOH and endothelium denudation. Furthermore, messenger RNA expression of iNOS, but not endothelial NOS, was inhibited by EtOH. These data suggest that IL-1β–induced contraction is mediated by thromboxane A2, whereas IL-1β–induced relaxation occurs via NO derived from iNOS. The endothelium plays an important role in vasorelaxation. Taken together, EtOH inhibits IL-1β–mediated vasorelaxation by suppressing endothelium iNOS expression. This study provides the first evidence of EtOH -induced inhibition of IL-1β–mediated vasorelaxation.博士（医学）・甲第647号・平成28年3月15日© The Author(s)Copyright © 2016 by SAGE PublicationsThe definitive version is available at " http://dx.doi.org/10.1177/0960327115611944

Multiplex PCRを用いた簡便で感度の高い溺死診断法の開発

For diagnosing death due to drowning, the method of acid digestion of diatoms is widely used to detect plankton in the organs of the corpse. However, the method is limited by its　being complex, hazardous, time-consuming, and insufficiently sensitive. We therefore, developed a novel simple method to diagnose death due to drowning, and determined the location of drowning by detecting genes of representative bacteria in the environment. To procure all the information in one step, the multiplex PCR method was designed. For the diagnosis of drowning, the genes of upper respiratory indigenous bacteria, Streptococcus salivarius and Streptococcus sanguinis were used as indicators. For detection of the location of drowning, Aeromonas hydrophila and Microcystis aeruginosa were used as indicators of freshwater, and Vibrio harveyi as an indicator of seawater. A set of primers was designed for multiplex PCR. to amplify all the bacterial genes simultaneously. Using this method, 47 cases of drowning were examined, and the causes and locations of death were diagnosed.博士（医学）・乙第1428号・平成31年3月15

コンバラトキシンによる凝固亢進における単球由来組織因子陽性細胞外小胞の関与

Objectives: Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley (Convallaria majalis). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Methods: Blood samples were collected from healthy volunteers to measure plasma thrombin–antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. Results: CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. Conclusions: The findings of the present study have further clarified the procoagulant properties of CNT.本文は発行元が定める公開猶予期間終了後に公開

口腔内所見を用いた新たな年齢推定法

In forensic science, age estimation is an important step in identifying unidentified cadavers. As teeth are resistant to environmental degradation for long periods of time, they are often used to estimate age. Although there have been many reports of age estimation based on dental morphology, these methods tend to be subjective and cannot be used i n the case of edentulous jaws. In the present study, we developed a new method of age estimation from dental parameters (number of upper teeth [UT], lower teeth [LT], and prostheses [NP]; tooth attrition [TA]; and occlusal area [OA]) and the mandibular angle (MA) measured at proposed an equation for calculating the age. The results show that the mean error of this method is similar to that of previous methods, and even demonstrated improved accuracy in subjects aged >60 years. We also proposed an equation for age estimation from only the MA, and showed that we can perform age estimation even in edentulous cases using this equation. Because our method is superior in its simplicity, objectivity, and applicability when compared with previous methods, we believe our method wll prove useful for age estimation in a wide variety of cases.博士（医学）・甲611号・平成26年3月17

フォン・ヴィレブランド因子の機能を調節することで、マウスの急性腎虚血再灌流障害を緩和できる

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and may become fatal. In addition to its hemostatic functions, von Willebrand factor (VWF) is known to play a role in cross-talk between inflammation and thrombosis. We hypothesized that VWF may be involved in the pathophysiology of AKI, major causes of which include insufficient renal circulation or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the role of VWF in AKI using a mouse model of acute ischemia-reperfusion (I/R) kidney injury. We analyzed renal function and blood flow in VWF-gene deleted (knock-out; KO) mice. The functional regulation of VWF by ADAMTS13 or a function-blocking anti-VWF antibody was also evaluated in this pathological condition. Greater renal blood flow and lower serum creatinine were observed after reperfusion in VWF-KO mice compared with wild-type (WT) mice. Histological analysis also revealed a significantly lower degree of tubular damage and neutrophil infiltration in kidney tissues of VWF-KO mice. Both human recombinant ADAMTS13 and a function-blocking anti-VWF antibody significantly improved renal blood flow, renal function and histological findings in WT mice. Our results indicate that VWF plays a role in the pathogenesis of AKI. Proper functional regulation of VWF may improve the microcirculation and vessel function in the kidney, suggesting a novel therapeutic option against AKI.博士（医学）・甲第744号・令和2年3月16日© The Author(s) 2019. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Repression of factor VIII inhibitor development with apoptotic factor VIII-expressing embryonic stem cells

Development of factor VIII (fVIII)-neutralizing antibodies, called inhibitors, is a challenging problem in the management of hemophilia A patients. We explored the possibility of pretreatment with apoptotic fVIII-expressing embryonic stem (ES) cells to prevent the development of fVIII inhibitors. Murine ES cells integrated with the human F8 gene were differentiated into embryoid bodies, dissociated to a single cell suspension, subjected to hypo-osmotic shock to induce apoptosis, and intraperitoneally injected into hemophilia A mice. Inhibitors were induced by periodic intraperitoneal injections of recombinant human fVIII (rhfVIII). In the groups in which intraperitoneal injections of rhfVIII began at 1-3 weeks after pretreatment, the titers of inhibitors were significantly lower after the third administration of rhfVIII compared with that in the control group in which apoptotic Ainv18 ES cells (without the human F8 gene) were used for pretreatment, and continued to show lower levels until the sixth administration of rhfVIII. These results suggest that pretreatment with apoptotic hfVIII-expressing ES cells might be promising for the prevention of fVIII inhibitor development in hemophilia A patients

Endothelium-Independent Relaxation of Vascular Smooth Muscle Induced by Persimmon-Derived Polyphenol Phytocomplex in Rats

The vasorelaxant effect of polyphenols is well known, and the mortality rate due to coronary artery disease is low in people who consume polyphenol-containing foods. We aimed to elucidate the mechanism by which polyphenols derived from persimmon juice (PJ) and persimmon leaves (PLs) induce vasorelaxation and suppress vasocontraction in the superior mesenteric arteries isolated from male Sprague Dawley rats. Vasocontraction was induced with 1 µM phenylephrine, and polyphenol-induced vasorelaxation was expressed as a percentage of the previous tone induced by phenylephrine. PJ powder (100 mg/L) induced higher levels of vasorelaxation (mean ± standard error of the mean, 88.6% ± 4.4%) than PLs powder (1 g/L; 72.0% ± 10.8%). Nitric oxide pathway inhibitors (NG-nitro-L-arginine methyl ester + carboxy-PTIO) did not affect persimmon-derived polyphenol-induced vasorelaxation, whereas potassium chloride, tetraethylammonium, and potassium-channel inhibitors did. Vasorelaxation was endothelium independent with both extracts. Phenylephrine-induced vasocontraction was suppressed by pretreatment with PJ and PLs powder, even when inositol triphosphate-mediated Ca2+ release and extracellular Ca2+ influx were inhibited. These results suggest that persimmon-derived polyphenol phytocomplex cause vasorelaxation and inhibit vasocontraction through hyperpolarization of smooth muscle cells. Persimmon-derived polyphenols may be able to prevent cardiovascular diseases caused by abnormal contraction of blood vessels

Ethanol attenuates vasorelaxation via inhibition of inducible nitric oxide synthase in rat artery exposed to interleukin-1β.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) regulates sepsis-induced hypotension. During septic shock, interleukin (IL)-1β is synthesized in endothelial cells and smooth muscle cells by endotoxin. Ethanol (EtOH) suppresses endotoxin-induced hypotension. The present study aimed to elucidate the effect of EtOH on gradual relaxation and iNOS expression induced by IL-1β in isolated rat superior mesenteric arteries (SMAs). Exposure to IL-1β–induced contraction in SMA rings, followed by a gradual relaxation of phenylephrine precontracted tone. Contraction was abolished by indomethacin (IM), cycloheximide (Chx), and endothelium denudation. In contrast, the gradual relaxation was abolished by NOS inhibitors, Chx, endothelium denudation, and inhibited by EtOH (50 and 100 mM). However, IM had no effect on relaxation. Western blot analysis demonstrated that iNOS expression was induced by IL-1β and was inhibited by EtOH and endothelium denudation. Furthermore, messenger RNA expression of iNOS, but not endothelial NOS, was inhibited by EtOH. These data suggest that IL-1β–induced contraction is mediated by thromboxane A2, whereas IL-1β–induced relaxation occurs via NO derived from iNOS. The endothelium plays an important role in vasorelaxation. Taken together, EtOH inhibits IL-1β–mediated vasorelaxation by suppressing endothelium iNOS expression. This study provides the first evidence of EtOH -induced inhibition of IL-1β–mediated vasorelaxation.博士（医学）・甲第647号・平成28年3月15日© The Author(s)Copyright © 2016 by SAGE PublicationsThe definitive version is available at " http://dx.doi.org/10.1177/0960327115611944 "identifier:Human & experimental toxicology [Epub ahead of print] pii: 0960327115611944(2015 Oct 22)identifier:09603271identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3184identifier:Human & experimental toxicology, Epub ahead of print: pii: 096032711561194

Therapeutic approaches for treating hemophilia A using embryonic stem cells

Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future. Keywords: Cell therapy, Embryonic stem cell, Hemophilia A, Mouse mode

A Sensitive and Time-Saving Method for the Diagnosis of Drowning by Multiplex PCR.

For diagnosing death due to drowning, the method of acid digestion of diatoms is widely used to detect plankton in the organs of the corpse. However, the method is limited by its　being complex, hazardous, time-consuming, and insufficiently sensitive. We therefore, developed a novel simple method to diagnose death due to drowning, and determined the location of drowning by detecting genes of representative bacteria in the environment. To procure all the information in one step, the multiplex PCR method was designed. For the diagnosis of drowning, the genes of upper respiratory indigenous bacteria, Streptococcus salivarius and Streptococcus sanguinis were used as indicators. For detection of the location of drowning, Aeromonas hydrophila and Microcystis aeruginosa were used as indicators of freshwater, and Vibrio harveyi as an indicator of seawater. A set of primers was designed for multiplex PCR. to amplify all the bacterial genes simultaneously. Using this method, 47 cases of drowning were examined, and the causes and locations of death were diagnosed.博士（医学）・乙第1428号・平成31年3月15日identifier:Journal of Nara Medical Association Vol.69 No.4,5,6 p.77-85 (2018.12)identifier:13450069identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3560identifier:Journal of Nara Medical Association, 69(4,5,6): 77-8