Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis

Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses

Cannabinoids inhibit peptidoglycan-induced phosphorylation of NF-κB and cell growth in U87MG human malignant glioma cells

Nuclear factor (NF)-κB is the key transcription factor involved in the inflammatory responses, and its activation aggravates tumors. Peptidoglycan (PGN), a main cell wall component of Gram-positive bacteria, stimulates Toll-like receptor 2 (TLR-2) and activates a number of inflammatory pathways, including NF-κB. Cannabinoids have been reported to exert anti-inflammatory and antitumor effects. The mechanisms underlying these actions, however, are largely unknown. The purpose of this study was to investigate whether cannabinoids can suppress the PGN-induced activation of NF-κB and cell growth via cannabinoid receptors in U87MG human malignant glioma cells. PGN treatment induced the phosphorylation of NF-κB and cell proliferation in a concentration-dependent manner. The main endocannabinoid, 2-arachidonoylglycerol, prevented the PGN-induced phosphorylation of NF-κB, which was reversed by the CB1 cannabinoid receptor antagonist, AM281. The synthetic cannabinoid, WIN55,212-2, abolished the PGN-activated cell growth, and this effect was reversed by AM281. The preferential expression of CB1 rather than CB2 receptors in these cells was confirmed by reverse transcription-mediated polymerase chain reaction experiments and the observation that the WIN55,212-2-induced morphological changes were completely reversed by AM281 but not by the CB2 antagonist, AM630. Our finding that cannabinoids suppress the NF-κB inflammatory pathway and cell growth via CB1 receptors in glioma cells provides evidence for the therapeutic potential of targeting cannabinoid receptors for the treatment of inflammation-dependent tumor progression.Thesis of Ryosuke Echigo / 越後 亮介 博士論文 金沢大学医薬保健学総合研究科（保健学専攻

脳幹部転移性脳腫瘍に対する寡分割定位放射線治療の治療成績

Background: To assess the neuroimaging and clinical outcomes in patients with brainstem metastasis (BSM) treated with linac-based fractionated stereotactic radiotherapy (fSRT) with a micro-multileaf collimator. Methods: Between May 2007 and January 2017, 24 patients (15 male and 9 female) with BSM (25 lesions: midbrain, 10; pons, 13; and medulla oblongata, 2) were consecutively treated with linac-based fSRT. BSM originated from the lung (n = 18, 75.0%), colon (n = 3, 12.5%), and breast (n = 3, 12.5%). The median patient age was 67.0 (range: 42-80) years. Recursive partition analysis classified 2 patients as class I, 17 as class II, and 5 as class III. Overall survival was calculated using the Kaplan-Meier method. Results: Tumor volume ranged from 0.01 to 7.49 cm3 (median: 0.233 cm3), and patients were treated with a dose of 24-40 Gy in 7-13 fractions. The median OS was 9 months after fSRT (95% confidence interval 4.104-13.896). Large tumor volume, presence of brainstem-related symptoms, poor pretreatment Karnofsky performance status, and recursive partition analysis class III were significantly associated with low overall survival. Tumor volume decreased in 18 metastatic lesions, remained stable in 6, and increased in 1. No patient exhibited permanent radiation injury. Grade 2 nausea and vomiting according to the Common Terminology Criteria for Adverse Events 4.0 occurred in 1 patient who received corticosteroids. Conclusions: Linac-based fSRT with a micro-multileaf collimator delivered in the doses of 24-40 Gy in 7-13 fractions is a safe and effective local therapy for patients with BSM.博士（医学）・乙第1450号・令和2年3月16日Copyright © 2019 Elsevier Inc. All rights reserved

転移性脳腫瘍の倍量造影剤投与における、分割投与と単回投与の病変描出能の比較

PURPOSE: As stereotactic radiotherapy (SRT) becomes widespread, precise information including number, location, and margin of lesions is required when magnetic resonance (MR) imaging of brain metastasis is performed. We compare methods using 2 separate injections and a single injection for the administration of a double dose of contrast medium for contrastenhanced MR imaging. MATERIALS AND METHODS: We divided 40 patients with brain metastasis into 2 groups of 20 patients. Group A received 2 separate injections (0.2 + 0.2 mL/kg) of contrast medium (gadoteridol); Group B received a single injection of the same total dose (0.4 mL/kg). Group A underwent spin echo (SE) T1-weighted imaging (T1WI) and magnetization prepared rapid acquisition with gradient echo sequence (MPRAGE) after each injection, and Group B underwent the same MR studies at the same timing as Group A. We evaluated the number, signal-to-noise ratio (SNR), diameter, margin delineation, and volume of lesions and compared them between early and delayed studies by the 2 methods. RESULTS: The number of detected lesions was largest in delayed studies of MPRAGE in both groups. The SNR of the lesions was statistically lower in early studies of Group A than other studies. Delayed studies of Group B showed statistically better margin delineation than other studies on both SE-T1WI and MPRAGE studies. Diameter and enhanced volume were statistically significantly larger on delayed phase than early phase in both groups. CONCLUSION: Use of a single injection of double-dose contrast medium and longer delay time may improve margin delineation of lesions for the study of brain metastasis. Enhanced volume was larger on delayed phase, and it may influence selection of therapeutic strategy.博士（医学）・乙第1356号・平成27年3月16日Copyright © 2014 by Japanese Society for Magnetic Resonance in Medicine著作権は日本磁気共鳴医学会に帰属日本磁気共鳴医学会及び著者（共著者も含む）の許諾を得て登

ヒト細胞傷害性γδT細胞は膠芽腫細胞株を殺傷する：膠芽腫患者に対する免疫細胞治療の意義

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell–cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32 % U87MG, 15 % U138MG, 1 % A172, and 50 % K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.博士（医学）・甲第635号・平成27年5月28日© Springer Verlag. The definitive version is available at " http://dx.doi.org/10.1007/s11060-013-1258-4

Anti-NXP2 autoantibodies in adult patients with idiopathic inflammatory myopathies: Possible association with malignancy

Objectives: Myositis-specific autoantibodies (MSAs) are useful tools for identifying clinically homogeneous subsets and predicting prognosis of patients with idiopathic inflammatory myopathies (IIM) including polymyositis (PM) and dermatomyositis (DM). Recent studies have shown that anti-NXP2 antibody (Ab) is a major MSA in juvenile dermatomyositis (JDM). In this study the frequencies and clinical associations of anti-NXP2 Ab were evaluated in adult patients with IIM. Methods: Clinical data and serum samples were collected from 507 adult Japanese patients with IIM (445 with DM and 62 with PM). Eleven patients with JDM, 108 with systemic lupus erythematosus, 433 with systemic sclerosis and 124 with idiopathic pulmonary fibrosis were assessed as disease controls. Serum was examined for anti-NXP2 Ab by immunoprecipitation and western blotting using polyclonal anti-NXP2 Ab. Results: Seven patients (1.6%) with adult DM and one (1.6%) with adult PM were positive for anti-NXP2 Ab. Except for two patients with JDM, none of the disease controls were positive for this autoantibody. Among eight adult patients with IIM, three had internal malignancies within 3 years of diagnosis of IIM. Another patient with DM also had a metastatic cancer at the diagnosis. All of the carcinomas were at an advanced stage (stage IIIb-IV). Conclusions: While less common than in juvenile IIM, anti-NXP2 Ab was found in adult IIM. Anti-NXP2 Ab may be associated with adult IIM with malignancy