Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells

ACE発現ヒトiPS細胞を用いたSARS-CoV-2感染の個人差再現と原因究明. 京都大学プレスリリース. 2021-04-19.Stem cells show gender differences in COVID-19 risk. 京都大学プレスリリース. 2021-04-19.Genetic differences are a primary reason for differences in the susceptibility and severity of COVID-19. As induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in SARS-CoV-2 infection in vitro. We found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected w SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, budding of viral particles, and production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We performed SARS-CoV-2 infection experiments on ACE2-iPS/ embryonic stem (ES) cells from eight individuals. Male iPS/ES cells were more capable of producing the virus compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro but also are a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences

鎖骨下動脈閉塞性動脈硬化症に対する血管内超音波評価を併用したステント留置術の長期治療成績

The objective of this study was to determine long-term outcomes after stent placement for subclavian artery (SA) obstructive lesions assisted by intraoperative intravascular ultrasound (IVUS). The study included 25 lesions in 24 patients who underwent stent placement assisted by intraoperative IVUS for subclavian artery stenosis or obstruction at our hospital between January 2003 and August 2010. Outcome was evaluated based on the results within 30 postoperative days (technical success rate, improvement in upper extremity ischemia, steal syndrome, left-right blood pressure difference, and perioperative complications) and the results after 30 postoperative days (incidence of vertebrobasilar artery territory infarction and restenosis). Stent placement and vessel dilatation were successful in all patients, without perioperative complications. Upper extremity ischemia, steal syndrome, and left-right blood pressure difference disappeared in all cases. During follow-up observation (6-96 months; median 51 months), no restenosis occurred at the stent placement site in any patient. In one case, four years after initial treatment, stenosis was noted proximal to the stent placement site. Satisfactory long-term as well as short-term outcomes were achieved after stent placement for SA obstructive lesions assisted by intraoperative IVUS evaluation.博士（医学）・乙第1362号・平成27年5月28日Copyright © 2014 by SAGE Publications. The definitive version is available at " http://dx.doi.org/10.15274/NRJ-2014-10023

不安定プラークを伴った頚動脈狭窄症に対するクローズセルステントを用いたステントインステント法による頚動脈ステント留置術

Purpose: To examine whether carotid artery stenting (CAS) of stenoses with unstable plaque using a closed-cell stent-in-stent technique prevents plaque protrusion. Materials and Methods: Between December 2014 and August 2018, 35 consecutive patients (mean age 75.8 years; 29 men) with carotid artery stenosis (20 symptomatic) and unstable plaque diagnosed by magnetic resonance imaging were prospectively analyzed. Mean diameter stenosis was 83.5%. All CAS procedures were performed with stent-in-stent placement of Carotid Wallstents using an embolic protection device and conservative postdilation. The technical success rate, incidence of plaque protrusion, ischemic stroke rate within 30 days, and new ipsilateral ischemic lesions on diffusion-weighted imaging (DWI) within 48 hours after CAS were prospectively assessed. Follow-up outcomes included the incidences of ipsilateral stroke and restenosis. Results: The technical success rate was 100%. No plaque protrusion or stroke occurred in any patient. New ischemic lesions were observed on DWI in 10 (29%) patients. During the mean 11.6-month follow-up, no ipsilateral strokes occurred. Two (6%) patients developed asymptomatic restenosis recorded as 53% lumen narrowing and occlusion, respectively. Conclusion: CAS using a closed-cell stent-in-stent technique for unstable plaque may be useful for preventing plaque protrusion and ischemic complications.博士（医学）・乙第1444号・令和元年12月5日This is a non-final version of an article published in final form in "https://doi.org/10.1177/1526602819847698

Application of susceptibility weighted imaging (SWI) for evaluation of draining veins of arteriovenous malformation: utility of magnitude images.

INTRODUCTION: The current study evaluated the signal characteristics of susceptibility weighted imaging (SWI) of arteriovenous malformation (AVM), especially for draining veins. For this purpose, we identified the draining veins of the AVM on angiography and evaluated the signal on magnitude image for SWI (SWI-mag) and minimum intensity projection image (SWI-minIP). METHODS: Subjects were 14 cases with angiographically proven AVM. SWI-mag, SWI-minIP, and time-of-flight (TOF) magnetic resonance angiography were acquired. For the draining veins of the AVM identified on angiography, we analyzed signal intensity on the images listed above, and classified it into hyperintensity (hyper), mixed intensity (mixed), hypointensity (hypo), and no visualization. RESULTS: On the analysis of 27 angiographically proven draining veins, 19 draining veins were classified as hyper, 3 as mixed, 0 as hypo, and 6 as no visualization on SWI-mag. On TOF images, 21 draining veins were classified as hyper, 2 as mixed, 0 as hypo, and 4 as no visualization, while 6 draining veins did not show hyperintensity on TOF, and SWI-mag visualized 3 of these 6 veins as hyper. CONCLUSION: SWI-mag depicted most draining veins of AVM as hyperintensity. We speculate that this is mainly due to the higher concentration of oxygenated hemoglobin (oxy-Hb) and inflow effect of the draining vein. SWI-mag seems to be useful in the analysis and follow-up for AVM as the signal on the image may reflect physiological status.博士（医学）・乙第1316号・平成25年7月22

T1強調像での信号強度が出生後日数と負の相関性を示す新生児・乳児期の脳構造

Purpose: Although the neonatal and infantile brain typically shows sequential T1 shortening according to gestational age as a result of myelination, several structures do not follow this rule. We evaluated the relationship between the signal intensity of various structures in the neonatal and infantile brain on T1-weighted imaging (T1WI) and either postnatal or gestational age. Materials and Methods: We examined magnetic resonance images from 120 newborns and infants without any abnormalities in the central nervous system. Written informed consent was obtained from all parents and the institutional review board approved the study. Gestational age at examination ranged from 35 weeks, 3 days to 46 weeks, 6 days, and postnatal age ranged from 7 days to 127 days. Signal intensity on T1WI was evaluated on a scale from Grade 1 (indistinguishable from surrounding structures) to Grade 4 (higher than cortex and close to fat). We evaluated relationships between the T1 signal grades of various structures in the neonatal brain and postnatal or gestational age using Spearman’s correlation analysis. Results: Significant positive correlations were identified between T1 signal grade and gestational age in the pyramidal tract (P < 0.001). Conversely, significant negative correlations were evident between T1 signal grade and postnatal age (P < 0.001), in structures including the stria medullaris thalami, fornix cerebellar vermis, dentate nucleus and anterior pituitary gland. Conclusion: Significant negative correlations exist between signal intensity on T1WI and postnatal age in some structures of the neonatal and infantile brain. Some mechanisms other than myelination might play roles in the course of signal appearance.博士（医学）・乙第1405号・平成29年6月28日Copyright © 2017 by Japanese Society for Magnetic Resonance in Medicine : This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives International License(https://creativecommons.org/licenses/by-nc-nd/4.0/deed.ja)

Inhibition of Plasminogen Activator Inhibitor- 1 Attenuates Transforming Growth Factor- β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin.We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis

miR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells

マウス糞便を用いた大黄甘草湯中のセンノシドA腸内代謝とHPLC定量分析(発表論文抄録(2011))

Daiokanzoto (DKT, combination of rhubarb and glycyrrhiza), a Kampo medicine, is clinically effective for constipation. Sennoside A is well known to induce diarrhea. Sennoside A is a prodrug that is transformed into an active metabolite, rheinanthrone, by intestinal bacteria. In this study, we investigated the effects of glycyrrhiza on the activity of sennoside A metabolism in intestinal bacteria using mouse feces. A high-performance liquid chromatography (HPLC) method for the determination of sennoside A in incubation mixture of DKT with mouse feces was established. The retention time of sennoside A was 9.26±0.02 min with a TSKgel ODS-80TsQA column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile and detection at 265 nm. We found that the activity of sennoside A metabolism in intestinal bacteria was significantly accelerated when glycyrrhiza, liquiritin or liquiritin apioside coexisted with sennoside A, whereas that of glycyrrhizin was not altered. This method is applicable for determination of the activity of sennoside A metabolism by anaerobic incubation of DKT with mouse feces.Daiokanzoto (DKT, combination of rhubarb and glycyrrhiza), a Kampo medicine, is clinically effective for constipation. Sennoside A is well known to induce diarrhea. Sennoside A is a prodrug that is transformed into an active metabolite, rheinanthrone, by intestinal bacteria. In this study, we investigated the effects of glycyrrhiza on the activity of sennoside A metabolism in intestinal bacteria using mouse feces. A high-performance liquid chromatography (HPLC) method for the determination of sennoside A in incubation mixture of DKT with mouse feces was established. The retention time of sennoside A was 9.26±0.02 min with a TSKgel ODS-80TsQA column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile and detection at 265 nm. We found that the activity of sennoside A metabolism in intestinal bacteria was significantly accelerated when glycyrrhiza, liquiritin or liquiritin apioside coexisted with sennoside A, whereas that of glycyrrhizin was not altered. This method is applicable for determination of the activity of sennoside A metabolism by anaerobic incubation of DKT with mouse feces

Irradiation Accelerates Plaque Formation and Cellular Senescence in Flow‐Altered Carotid Arteries of Apolipoprotein E Knock‐Out Mice

[Background] Chronic inflammation through cellular senescence, known as the senescence‐associated secretory phenotype, is a mechanism of various organ diseases, including atherosclerosis. Particularly, ionizing radiation (IR) contributes to cellular senescence by causing DNA damage. Although previous clinical studies have demonstrated that radiotherapy causes atherosclerosis as a long‐term side effect, the detailed mechanism is unclear. This study was conducted to investigate the relationship between radiation‐induced atherosclerosis and senescence‐associated secretory phenotype in murine carotid arteries. [Methods and Results] Partial ligation of the left carotid artery branches in 9‐week‐old male apolipoprotein E‐deficient mice was performed to induce atherosclerosis. The mice received total body irradiation at a dose of 6 Gy using gamma rays at 2 weeks post operation. We compared the samples collected 4 weeks after IR with unirradiated control samples. The IR and control groups presented pathologically progressive lesions in 90.9% and 72.3% of mice, respectively. Plaque volume, macrophage accumulation, and phenotype switching of vascular smooth muscle cells were advanced in the IR group. Irradiated samples showed increased persistent DNA damage response (53BP1 [p53 binding protein 1]), upregulated cyclin‐dependent kinase inhibitors (p16INK4a and p21), and elevated inflammatory chemokines expression (monocyte chemotactic protein‐1, keratinocyte‐derived chemokine, and macrophage inflammatory protein 2). [Conclusions] IR promoted plaque growth in murine carotid arteries. Our findings support the possibility that senescence‐associated secretory phenotype aggravates atherogenesis in irradiated artery. This mice model might contribute to mechanism elucidation of radiation‐induced atherosclerosis