高力価VA RNA欠失アデノウイルスベクターの新規作製法の開発と応用

学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 藤堂 具紀, 東京大学教授 小池 和彦, 東京大学教授 川口 寧, 東京大学准教授 立川 愛, 東京大学教授 真鍋 俊也University of Tokyo(東京大学

The role of proboscis of the malaria vector mosquito Anopheles stephensi in host-seeking behavior

<p>Abstract</p> <p>Background</p> <p>The proboscis is an essential head appendage in insects that processes gustatory code during food intake, particularly useful considering that blood-sucking arthropods routinely reach vessels under the host skin using this proboscis as a probe.</p> <p>Results</p> <p>Here, using an automated device able to quantify CO₂-activated thermo (35°C)-sensing behavior of the malaria vector <it>Anopheles stephensi</it>, we uncovered that the protruding proboscis of mosquitoes contributes unexpectedly to host identification from a distance. Ablation experiments indicated that not only antennae and maxillary palps, but also proboscis were required for the identification of pseudo-thermo targets. Furthermore, the function of the proboscis during this behavior can be segregated from CO₂detection required to evoke mosquito activation, suggesting that the proboscis of mosquitoes divide the proboscis into a "thermo-antenna" in addition to a "thermo-probe".</p> <p>Conclusions</p> <p>Our findings support an emerging view with a possible role of proboscis as important equipment during host-seeking, and give us an insight into how these appendages likely evolved from a common origin in order to function as antenna organs.</p

Application of complement component 4d immunohistochemistry to ABO-compatible and ABO-incompatible liver transplantation.

Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti-donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO-C grafts and 15 of the 29 patients (52%) with ABO-I grafts showed C4d positivity. In the ABO-C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor-specific anti-human leukocyte antigen DR antibodies (HLA-DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single-antigen bead assay (P = 0.04). Conversely, the presence of HLA-DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2-year follow-up, neither C4d positivity nor HLA-DR DSAs were related to graft loss. Among ABO-I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d-positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I grafts, and they were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO-C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO-I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated

Distinct Osteogenic Potentials of BMP-2 and FGF-2 in Extramedullary and Medullary Microenvironments

Bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) have been regarded as the major cytokines promoting bone formation, however, several studies have reported unexpected results with failure of bone formation or bone resorption of these growth factors. In this study, BMP-2 and FGF-2 adsorbed into atellocollagen sponges were transplanted into bone defects in the bone marrow-scarce calvaria (extramedullary environment) and bone marrow-abundant femur (medullary environment) for analysis of their in vivo effects not only on osteoblasts, osteoclasts but also on bone marrow cells. The results showed that BMP-2 induced high bone formation in the bone marrow-scarce calvaria, but induced bone resorption in the bone marrow-abundant femurs. On the other hand, FGF-2 showed opposite effects compared to those of BMP-2. Analysis of cellular dynamics revealed numerous osteoblasts and osteoclasts present in the newly-formed bone induced by BMP-2 in calvaria, but none were seen in either control or FGF-2-transplanted groups. On the other hand, in the femur, numerous osteoclasts were observed in the vicinity of the BMP-2 pellet, while a great number of osteoblasts were seen near the FGF-2 pellets or in the control group. Of note, FCM analysis showed that both BMP-2 and FGF-2 administrated in the femur did not significantly affect the hematopoietic cell population, indicating a relatively safe application of the two growth factors. Together, these results indicate that BMP-2 could be suitable for application in extramedullary bone regeneration, whereas FGF-2 could be suitable for application in medullary bone regeneration

Microrna-9-5p-CDX2 axis: A useful prognostic biomarker for patients with stage II/III colorectal cancer

A lack of caudal-type homeobox transcription factor 2 (CDX2) protein expression has been proposed as a prognostic biomarker for colorectal cancer (CRC). However, the relationship between CDX2 levels and the survival of patients with stage II/III CRC along with the relationship between microRNAs (miRs) and CDX2 expression are unclear. Tissue samples were collected from patients with stage II/III CRC surgically treated at Kyoto University Hospital. CDX2 expression was semi-quantitatively evaluated by immunohistochemistry (IHC). The prognostic impacts of CDX2 expression on overall survival (OS) and relapse-free survival (RFS) were evaluated by multivariable statistical analysis. The expression of miRs regulating CDX2 expression and their prognostic impacts were analyzed using The Cancer Genome Atlas Program for CRC (TCGA-CRC). Eleven of 174 CRC tissues lacked CDX2 expression. The five-year OS and RFS rates of patients with CDX2-negative CRC were significantly lower than those of CDX2-positive patients. Multivariate analysis of clinicopathological features revealed that CDX2-negative status is an independent marker of poor prognosis in stage II/III CRC. miR-9-5p was shown to regulate CDX2 expression. TCGA-CRC analysis showed that high miR-9-5p expression was significantly associated with poor patient prognosis in stage II/III CRC. In conclusion, CDX2, the post-transcriptional target of microRNA-9-5p, is a useful prognostic biomarker in patients with stage II/III CRC

Speech corpora in NINJAL, Japan demonstration of corpus concordance systems : Chunagon and Kotonoha

National Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsNational Institute for Japanese Language and LinguisticsThe National Institute for Japanese Language and Linguistics, Japan (NINJAL, Japan) provides a demonstration site in the LPSS 2019 conference. This manuscript presents an overview of the demonstration of three corpora: Corpus of Spontaneous Japanese, Corpus of Everyday Japanese Conversation, and Corpus of Japanese Dialects.NINJAL also demonstrates two concordance systems. The first is "Chunagon (中納言)" which is a morpheme based concordance system that was made publicly available in 2011. The second is the currently developing system "Kotonoha" released in 2018 that enables query of multiple corpora in terms of register type and period

Translocator protein imaging with 18F-FEDAC-positron emission tomography in rabbit atherosclerosis and its presence in human coronary vulnerable plaques

Background and aims: This study aimed to investigate whether N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), a probe for translocator protein (TSPO), can visualize atherosclerotic lesions in rabbits and whether TSPO is localized in human coronary plaques.Methods: 18F-FEDAC-PET of a rabbit model of atherosclerosis induced by a 0.5% cholesterol diet and ballooninjury of the left carotid artery (n = 7) was performed eight weeks after the injury. The autoradiography intensity of 18F-FEDAC in carotid artery tissue sections was measured, and TSPO expression was evaluated immunohistochemically.TSPO expression was examined in human coronary arteries obtained from autopsy cases (n = 16), and in human coronary plaques (n = 12) aspirated from patients with acute myocardial infarction (AMI).Results: 18F-FEDAC-PET visualized the atherosclerotic lesions in rabbits as high-uptake areas, and the standard uptake value was higher in injured arteries (0.574 ± 0.24) than in uninjured arteries (0.277 ± 0.13, p < 0.05) or myocardium (0.189 ± 0.07, p < 0.05). Immunostaining showed more macrophages and more TSPO expression in atherosclerotic lesions than in uninjured arteries. TSPO was localized in macrophages, and arterial autoradiography intensity was positively correlated with macrophage concentration (r = 0.64) and TSPO (r = 0.67). TSPO expression in human coronary arteries was higher in AMI cases than in non-cardiac death, or in the vulnerable plaques than in early or stable lesions, respectively. TSPO was localized in macrophages in all aspirated coronary plaques with thrombi.Conclusions: 18F-FEDAC-PET can visualize atherosclerotic lesions, and TSPO-expression may be a marker of highrisk coronary plaques

Functional tooth number and mortality

Aim: Previous studies on the association between intraoral conditions and mortality in community-dwelling older individuals reported that fewer present teeth (PT) are significant risk factors for mortality. However, how the number of PT relative to the number of functional teeth (FT), including both present and rehabilitated teeth, influences mortality has not been investigated fully. This study examined the impact of the number of FT on mortality among community-dwelling Japanese older adults. Methods: This study was a retrospective, observational and population-based follow-up study, which examined 1188 older individuals who participated in an annual geriatric health examination from 2009 to 2015. The average follow-up period was 1697.0 ± 774.5 days. The primary outcome was all-cause mortality at follow-up. The numbers of PT and FT of each participant were counted during an oral examination. In addition, demographics, clinical variables, blood nutrient markers, physical functions and perceived masticatory function were measured. Results: Kaplan–Meier analysis, followed by a log-rank test, revealed that fewer PT (P < 0.001) and FT (P = 0.002) were significantly associated with a reduced survival rate. Cox's proportional hazard analysis indicated that the number of FT, but not the number of PT, was a significant independent mortality risk factor after adjusting for demographics, clinical variables, nutrient markers and physical functioning (P = 0.036, hazard ratio: 2.089). Conclusions: Current results suggest that the number of FT more strongly predicts all-cause mortality than the number of PT among community-dwelling older adults. Further studies are necessary to consider the confounding of socioeconomic status and disability status

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a ‘double-unit’ AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact ‘excisional-expression’ unit consisting of a target cDNA ‘upstream’ of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the ‘double-unit’ AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity

Number of functional teeth more strongly predicts all‐cause mortality than number of present teeth in Japanese older adults

Aim Previous studies on the association between intraoral conditions and mortality in community‐dwelling older individuals reported that fewer present teeth (PT) are significant risk factors for mortality. However, how the number of PT relative to the number of functional teeth (FT), including both present and rehabilitated teeth, influences mortality has not been investigated fully. This study examined the impact of the number of FT on mortality among community‐dwelling Japanese older adults. Methods This study was a retrospective, observational and population‐based follow‐up study, which examined 1188 older individuals who participated in an annual geriatric health examination from 2009 to 2015. The average follow‐up period was 1697.0 ± 774.5 days. The primary outcome was all‐cause mortality at follow‐up. The numbers of PT and FT of each participant were counted during an oral examination. In addition, demographics, clinical variables, blood nutrient markers, physical functions and perceived masticatory function were measured. Results Kaplan–Meier analysis, followed by a log‐rank test, revealed that fewer PT (P  Conclusions Current results suggest that the number of FT more strongly predicts all‐cause mortality than the number of PT among community‐dwelling older adults. Further studies are necessary to consider the confounding of socioeconomic status and disability status