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New strategy of IBD treatment using microbiota ○Takanori Kanai Department and Hepatology, Keio University School of Medicine,Tokyo,Japan

Both the incidence of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s diseases (CD), are rapidly increasing, not only in western countries, but also in developed Asian countries. In Japan, approximately 160,000 patients with UC and 40,000 with CD are currently registered by Japanese Health, Labour and Welfare Ministry. Although biologic agents targeting the immune system have been effective in patients with IBD, cessation of treatment leads to relapse in almost all patients, suggesting that immune dysregulation is an effect, not a cause, of IBD. Advances in next-generation gene sequencing technology have resulted in the identification of over 160 IBD-associated susceptibility genes within the past 10 years. These susceptibility genes, however, are unlikely to be the main cause of IBD, because in the past 50 years the numbers of IBD patients have increased 100-fold in Japan. It is more likely that dramatic changes in the social environment, especially dietary habits leading to the dysregulated composition of intestinal microbiota or dysbiosis, are fundamental causes of IBD. In Japan, upgrading of the water supply and sewerage systems, abuse of antibiotics, and especially dietary habits are now very similar to those in developed western countries. This review talk will provide current topics regarding new treatment using microbiota including probiotics.

MS1-1 AVMA Guidelines for the Euthanasia of Animals: 2013 Edition ○Makoto Suzuki Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

Article 40 of the Law for Humane Treatment and Management of Animals, mandates, ‟In case an animal must be destroyed, a method that minimizes pain and distress to the animal as much as possible shall be used.” The Guidelines for euthanasia of Animals state, ‟Administrator and operator should understand physiology and ecology, and should have appropriate euthanasia methods to minimize distress.” They further direct, ‟When euthanizing an animal, chemical or physical methods for euthanasia must render animals unconscious with minimal pain and distress, and they should stop cardiac or respiratory function.” However, these regulations include no description of euthanasia techniques, even though the Guidelines for Proper Conducts of Animal Experiments require detailed methods regarding final disposition of experimental animals. IACAC reviews euthanasia protocols from the point of scientific justification and humanness, while allowing the gathering of valid data. IACUC investigates whether animals are unconscious prior to death, whether euthanasia processes employed prevent unnecessary pain and distress, and whether they minimize risk to people and animals. ‟AVMA Guidelines for the Euthanasia of Animals: 2013” cover euthanasia methods for mammals, birds, reptiles, amphibians and finned fishes. Here I will introduce appropriate euthanasia methods for laboratory animals of different species or age.

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MS1-2 Anesthesia and analgesia for pain relief in laboratory animals ○Kazuto Yamashita Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen UNiversity, Ebetsu, Japan

Veterinary anesthesia is applied to various animals with different breeding purposes including animals living as a family member (companion animals), animals for the purpose of the food production (production animal), zoo animals, wild animals, and laboratory animals. The veterinary anesthesia is carried out for the purpose of sedation and immobilization, local anesthesia, general anesthesia, pain control, and euthanasia. In the companion animals, assuring high safety and pain relief are strongly demanded regardless of the productivity and economy. In laboratory animals, it is also urged to consider the principles of Replacement, Reduction and Refinement (the Three Rs) as the key strategies in order to achieve the goal of humane experimental techniques by the Act on Welfare and Management of Animals. In other words, veterinarians engaging animal experiments should have the ability to reduce the pain in the laboratory animals. Now, many anesthetics and analgesics have been approved for the animal use and make it possible to markedly raise quality of anesthesia and analgesia in Japan. In addition, the concepts of preventive analgesia, multimodal analgesia, and balanced anesthesia have been introduced and spread by enlightenment activities of the Japanese Society of Veterinary Anesthesia and Surgery and Japanese Society for Study of Animal Pain. In this lecture, I would like to focus on pain relief in anesthetized animals and to introduce strategies of pain control.

MS1-3 Introduction to Medical Statistics ○Satoshi Miyata Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine

In medical science, data analysis by statistics is crucial to provide concrete evidence of association between some outcomes and correlating covariates. In this presentation of medical statistics, I will provide the fundamentals regarding 1) how to summarize data, 2) how to analyze data - interpretation of precondition and results -, and 3) how to use statistical models - revisit to basis -. 1) I will provide the essence on how to summarize data to describe basic properties of current data. To select an appropriate method to analyze data, it is important to understand the attribute of the data. 2) I will provide the essence about diagnosis of the model assumptions. Any statistical models require some mathematical preconditions, and it is necessary to double check if those assumptions are satisfied in the current data set. 3) I will revisit basic concepts of statistical analysis such as hypothesis testing and confidence interval. In this short course of medical statistics, I will not deal with mathematical details, but instead, I will help attendees understand basic procedure applied to any kinds of data analysis.

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MS2-1 Drug metabolism and pharmacokinetic research with humanized mice

○Hidetaka Kamimura Drug Development Solutions Division Sekisui Medical Co., Ltd., Tokyo, Japan

Chimeric mice with humanized livers (ChM) are especially a unique model which possesses human hepatocytes in their body and various applications have been reported recently. ChM exert their effectiveness in the prediction of clinical pharmacokinetics (PK) and human metabolism for the selection of final candidate compounds. Unfavorable human PK profiles may sometimes result in termination of the project. This presentation mainly focuses on a PK parameter, clearance (CL), which is a factor for removing drugs from the body and the most difficult to predict because of a wide range of variation among animal species and humans. Recently, a good correlation between CL of various drugs obtained in ChM and those in clinical studies was reported. However, they failed to predict human PK of some drugs which were susceptible to remnant mouse metabolism. Then we propose a novel method of removing the effect of mouse metabolism. On the other hand, if metabolites formed uniquely or disproportionately higher in humans are found in the process of new drug development, safety of the said metabolites are not considered to be warranted (the FDA Metabolites in Safety Testing (MIST) and subsequent ICH M3(2) guidance). Drug development may delay in some cases, if MIST concerned metabolites were found in the Phase I study. In this presentation, some examples for the prediction of human-specific metabolites and those which are common to animals but exhibit distinct PK properties in humans are introduced.

MS2-2 Drug discovery research for viral hepatitis using human hepatocyte chimeric mice ○Hikita Hayato, Tetsuo Takehara Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine

Chronic hepatitis B, which is caused by infection of hepatitis B virus; HBV, and chronic hepatitis C, which is caused by infection of hepatitis C virus; HCV, are ones of the most widely spread infectious diseases in the world. To conquer these HBV and HCV infectious disease, developing new therapies are desired. However, experiments using animal models were hampered by the fact that only human and chimpanzees can catch infection with HBV or HCV. Recently, humanized liver chimeric mice have developed, which makes it possible to infect mice with HBV and HCV. We examined new target or suitable therapy against HBV and HCV infection using humanized liver chimeric TK-NOG mice. We examined the effect of NTCP knockdown on HBV infection. Administration of siRNA against human NTCP using in vivo fectamin successfully reduced its expression level in human hepatocytes. It also reduced susceptibility to HBV infection, suggesting that NTCP would be a candidate of therapeutic target against HBV infection. Next, HCV-infected chimeric mice were treated with combinations among NA3A, NS5A and NS5B inhibitors. Drug resistant mutations were emerged when the treatment was failure. HCV with emerging drug resistant mutation by previous treatment was failed to eradiation by drug combinations including same class of resistant drug, suggesting that scrupulous attention is needed when re-treatment for patients with previous oral-treatment failure.

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MS2-3 Diverse human organ bud generation from iPSC ○Takanori Takebe1,2, Hideki Taniguchi1 1 2

Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA

In vitro organogenesis is now becoming a realistic goal of stem cell biology; however, one practical challenge is to develop a four-dimensional (4-D) stem cell culture system whereby multiple progenitors communicate in a spatiotemporal manner. By recapitulating multicellular communications involved in the budding of the rudimentary liver from the foregut, we recently showed that specified anterior visceral endodermal cells selforganized into 3-D iPSC-derived liver buds when co-cultivated with multiple stromal cell populations. By transplanting in vitro grown organ bud, we have demonstrated the vascularized and functional liver tissues in an immunodeficient animal with therapeutic potential (Nature, 2013). Furthermore, we also translated this approach to other systems by delineating the mechanisms guiding organ bud formation. Specifically, mesenchymal progenitors initiated organ bud formation within these heterotypic cell mixtures, which was dependent upon substrate matrix stiffness. Defining optimal mechanical properties of the substrate promoted formation of 3D, transplantable organ buds from tissues including kidney, pancreas and cartilage (J Clin Invest, 2014 & Cell Stem Cell, 2015). In this talk, I will summarize the state-of-art of these organ bud based approaches, and discuss their future potential applications.

MS3-1 Update of pre-clinical in vivo imaging systems ○Rikita Araki Bruker BioSpin, Yokohama, Japan

There are remarkable new technologies such as iPS cells in the molecular/cellular engineering. However, lots of efforts are required to apply these new technologies for the clinical use and animal experiments are indispensable in this bench-to-bedside process. In vivo imaging plays an important role in the pre-clinical research because it makes it possible to visualize the internal information of living animals without extracting organs. For example, when examining the effect of new drugs on disease model animals, in vivo imaging allows the researcher to follow each animal during the treatment and the number of animals used can be significantly reduced. There are several instruments/modalities in the in vivo imaging and each of them has pros/cons. For example, PET, SPECT and Optical imaging are quite useful to obtain the functional information, however, the resolution of the image is worse. On the other hand, with CT, high resolution 3D image can be easily acquired, but it is difficult to obtain functional information. Also, the contrast in the soft tissue is very poor in the CT image. So it becomes more and more important to scan animals with the multiple instruments and compensate the weakness of each instrument. In my talk, I will give the latest information of our pre-clinical in vivo imaging instruments including ultra-high field MRI, cryogen free PET-MR system, high-speed CT system and so on.

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MS3-2 MRI of neurological disease models in the marmoset ○Keigo Hikishima1, Yuji Komaki2,3, Hideyuki Okano3 1 Okinawa Institute of Science and Technology, Okinawa, Japan, 2Central Institute for Experimental Animals, Kawasaki, Japan, 3Keio University School of Medicine, Tokyo, Japan

Magnetic Resonance Imaging (MRI) is an imaging technique widely used in the clinical diagnosis. The advantages of MRI such as non-invasiveness and high soft-tissue contrast are attractive also to preclinical imaging study for investigation of structure and function of animal models. Through the recent advances of MRI such as ultra-high magnetic field strength (7 tesla ~) become mainstream for small experimental animals. The ultra-high magnetic field MRI enables us to obtain high enough image quality for advanced imaging of small experimental animals such as rat. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research. They are small enough to fit into the narrow bore of animal MRI system dedicated to rat, therefore the high spatial resolution MRI system is available also to marmoset. Using 7-tesla MRI, we have developed advanced neuroimaging technique such as brain volumetry, neuronal fiber tracking and functional activity mapping with brain atlas for marmoset. Here I will introduce such MR neuroimaging methods for the evaluation of neurological disease models in the marmoset.

MS3-3 Small animal in vivo imaging using high field MRI and functional contrast agents ○Ichio Aoki Japan Agency for Quantum and Radiological Science and Technology

The advent of functional contrast agents and nanoparticle drug delivery systems (nano-DDSs) is opening new pathways to understanding animal pathophysiology using high field MRI. Mn2+ can be used as a positive intracellular MRI contrast agent for small animal. Manganese-enhanced MRI (MEMRI) provides a unique opportunity to study neuronal activation and architecture. The underlying principle of MEMRI relies in the fact that Mn2+ behaves in a manner similar to Ca2+ in many biological systems. Extracellular Mn2+ can enter cells through NMDA receptors for glutamate and/or voltage-gated Ca2+ channels. Thus, Mn2+ can behave as a functional contrast agent depending on the cellular activity. In this presentation, I would like to summarize the recent progress of MEMRI and manganese-based contrast agents for small animal study both for neuroimaging and cancer research.Nanoparticles have also been investigated as possible MRI contrast agents. A nano-DDS is defined as the specific delivery of drugs to organs, tissues or cells using nanoparticles. Nano-carriers for DDSs can contain multiple functional elements, such as therapeutic drugs, MRI contrast agents, fluorescent or luminescent dyes, and radioisotopes, without serious changes to the particle kinetics/dynamics. The development of such multifunctional nano-DDSs and imaging has accordingly become a subject of widespread research. I will also summarize our recent research into nano-DDS-based contrast agents for cancer in small animal.

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MS4-1 Micro/nano device technology for medical applications ○Shoji Takeuchi IIS, U TOKYO

In this presentation, I am planning to talk about several MEMS/Microfluidic-based approaches for various medical applications. Fluorescent hydrogels hold great promise for in vivo continuous glucose monitoring with wireless transdermal transmission and long-lasting activity. We synthesized a highly-sensitive fluorescent monomer, and then fabricated injectable-sized fluorescent hydrogel beads and fibers. The fluorescent hydrogels transdermally glowed, and continuously responded to blood glucose concentration changes for up to 140 days, showing their potential application for long-term in vivo continuous glucose monitoring.Large-scale 3D tissue architectures that mimic microscopic tissue structures in vivo are very important for not only in tissue engineering but also drug development without animal experiments. We demonstrated a bottom-up tissue construction method using different types of cellular modules that serve as building blocks for thick and dense 3D tissues (eg. cell beads and cell fibers). As a practical application, for example, fiber encapsulating beta-cells is used for the implantation of diabetic mice, and succeeded in normalizing the blood glucose level.

MS4-2 in vivo mRNA delivery using polyplex nanomicelles ○Keiji Itaka The University of Tokyo, Tokyo, Japan

Administering mRNA is a promising approach for treating various diseases because mRNA has several advantages for providing therapeutic proteins or peptides to target tissues;for example,mRNA can be used for protein or peptide expression in their native forms at a target site.The sustained synthesis of proteins resulting from the delivered mRNA can facilitate the synchronization between the kinetics of signal receptor expression and bioactive factor availability.The combined use of two or more bioactive factors to provide better neurotrophic and neuroprotective effects is much easier than using recombinant proteins or peptides.Mostimportantly,the probability ofmRNA randomly integrating into the genome is negligible,thus avoiding the aberrant expression of oncogenes as a result of insertional mutagenesis.However,direct mRNA administration in vivo has been challenging due to two major issues of too unstable manner and its strong immunogenicity.Thus,using a suitable carrier is essential for effective mRNA administration.We recently established a novel carrier that was applicable for in vivomRNA delivery based on the self-assembly of polyethylene glycol(PEG)-polyamino acid block copolymer,i.e.polyplex nanomicelles,possessing a core-shell structure surrounded by a PEG outer layer,with the inner core of a functionalized polyamino acid.In this presentation,I will briefly introduce this system,and its applications for treating diseases using animal models.This system will be applicable for neurologic disorders,joint diseases,and other intractable diseases including regenerative medicine.

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MS4-3 Development of Nanomachines Based on Polymeric Micelles Towards Future Medicine

○Nobuhiro Nishiyama1, Kazunori Kataoka2 1 2

Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan, Innovation Center of NanoMedicine (iCONM), Kawasaki, Japan

Recent advances in nanotechnology in the biomedical field have spurred the development of smart nanocarriers, which are integrated drug vehicles capable of (i) sensing targets inside the body and adapting their function based on these targets, (ii) modulating their properties and functions responding to internal or external stimuli, and (iii) operating at the target site. Polymeric micelles formed through the self-assembly of engineered block copolymers are promising nanocarriers, because the critical features of polymeric micelles, including the particle size, stability, and drug loading and release properties, can be modulated by engineering the constituent block copolymers. Several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Furthermore, polymeric micelles can be integrated with multiple functionalities such as targetability, environment-sensitivity and imaging functionality. We defined such smart nanocarries as a nanomachine. The combination systems of nanomachines and medical devices is also useful for super minimally invasive surgery. In my presentation, I will talk about recent achievements of smart polymeric micelles and useful animal models and methods to validate their functions in vivo.

MS5-1 Quality evaluation of pluripotent stem cells using developmental engineering ○Arata Honda Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki, Japan

Developmental engineering is a tool for obtaining the knowledge by making an artificial embryo. A developmental engineering technology, chimera production, is the mostly used to elucidate the gene function by gene targeting in mice. Mouse pluripotent stem cells (PSCs) can effectively contribute to form chimeras and can easily differentiate into germ cells by their naive pluripotency. On the other hand, human PSCs are called ‘prime;’ they have a poor ability to differentiate into germ cells and rarely contribute to form preimplantation embryos as chimeras. The potential to chimeric contribution of PSCs is one of the most critical indicators for their pluripotency. Previously, we have developed the methods to convert prime state PSC into naive-like state using rabbit ES cells and iPS cells as a translational research model. In this study, we converted Cynomolgus monkey ES cells into naive-like state. To verify their true naive pluripotency, naive-like Cynomolgus monkey ES cells were injected into mouse or rabbit preimplantation embryos. If successful, the naive conversion of Cynomolgus monkey pluripotent stem cells may lead to qualitative improvements and a method to assess naive-like conversion of human pluripotent stem cells. This should help accelerate future research on regenerative medicine.

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MS5-2 Techniques for creating genetically identical animals in the nonhuman primates ○Yusuke Sotomaru Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan

We are in search of methods to generate a genetically homogeneous non-human primate animal model of marmosets. Thus far, we have successfully produced cloned marmosets from fertilized eggs, including monozygotic twins, using a cloning technique in which 16-cell stage embryos are used as donors in nuclear transfer. However, we have not been able to produce cloned individuals from somatic cells or embryonic stem (ES) cells; these techniques clearly need to be improved. As an alternative to cloning techniques, we are also working on the application of an embryo-splitting technique. We have been successful so far in obtaining offspring from embryos split in quarters, and attempting to go even further. Furthermore, we are investigating improvements in the developmental potential of split embryos as well as the possibilities of removing restrictions on attained developmental stages and number of divisions by creating chimeras with tetraploid embryos. The confirmed effectiveness of these methods will hopefully enable them to be applied in the production of cloned individual marmosets from ES cells, using naive ES cells as donors.

MS5-3 Naive pluripotent stem cells ○Yasuhiro Takashima CiRA, Kyoto University, Kyoto, Japan

We are currently trying to establish germ line competent primate pluripotent stem cells (PSC) in order to generate primate genetically modified animals because germ line transmitted primate chimera animals by primate ES/iPS cells have not been reported yet.Recently, mouse PSC are separated by naive type and primed type. Naive mouse PSC correspond to epiblast in blastocysts and generate chimeric mice when they are injected into blastocysts. Furthermore, mouse naive mouse PSC can generate germ cells in vitro, on the other hand, mouse primed PSC cannot. Primate ES/iPS cells are thought to be primed type. Therefore primate ES/iPS cells might not have the ability to generate chimeric animals.Current human ES/iPS cells are also believed to be at a more advanced stage of development, as a primed state, than mouse ES/iPS cells. We have recently succeeded in generating human naive iPS cells, which are stem cells close to pre-implantation epiblasts similar as mouse ES/ iPS cells. We are challenging to apply this method for generating germ line competent primate PSC. I would like to discuss current state of primate PSC.

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MS6-1 Innovation of Bacterial Identification tests ○Nobuhito Hayashimoto ICLAS Monitoring Center, Central Institute for Experimental Animals, Kawasaki, Japan

In the field of laboratory animal science, microbiological tests are used mainly for health monitoring, spot tests, and quarantine tests. To improve rapidity and accuracy is an everlasting problem in microbiological tests. Among microbiological tests, bacterial culture tests are useful tests that can approach to bacterial characteristics from various aspects. However bacterial culture tests are time-consuming because it depends on the growth speed of bacterial isolates. Recently, Matrix Assisted Laser Desorption/ Ionization-Time of Flight Mass Spectrometer, MALDI-TOF MS, begins to be used for bacterial identification as a new test. In the bacterial identification using MALDI-TOF MS, bacterial isolates are ionized by laser radiation, then mass spectrums pattern that consist of ionized proteins in bacterial isolates is compared with all data in the device. Large merits of the bacterial identification using MALDI-TOF MS are simple operation and reduction of test times. In this symposium, the outline of bacterial identification using MALDI-TOF MS will be explained as well as introduction of actual figure of bacterial identification test using the device.

MS6-2 Innovation of Serology Test ○Shigeri Maruyama Charles River Laboratories Japan, Inc., Ibaraki, Japan

Serology test is a major microbiological monitoring method which tests the history of infection of animals using the presence of specific antibodies in blood of them. This method does not detect microbes itself. It tests antibodies which are produced after the entry of microbes into host animals. Consequently, it is beneficial to testing inapparent infections and to other cases in which microbes are not easily detected. In addition, because it only deals with antibodies, it reduces the risk of spread of pathogenic microbes. Testing methods using antibodies has been widely accepted and microbiological monitoring, including the use of ELIZA, is one of them. Recently, Multiplexed fluorometric immunoassay (MFI) has been developed. Compare to ELIZA, which only measure a single analyze, MFI permits multiplexing up to 100 different assays from a tiny amount of serums. As technology advanced, new methods and tools for detecting infection has been developed and available. According to the development of laboratory equipment and innovations, the way of microbiological monitoring diverges into many branches, but detection of antibodies through serological reaction is one of the outstanding monitoring methods. In this symposium, we will be introducing the development of antibody detection methods which use serologic reaction and be explaining characteristics of those respectively. We also would like to describe our effort to develop easy blood sampling methods.

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MS6-3 Toward the innovations of microbiological monitoring ○Fumio Ike Experimental Animal Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan

Fine animal production by commercial breeders, highly manufactured caging systems, well-established rederivation procedures, and reliable transport network; all of these support excellent and reproducible animal experiments especially on mice. Knowledge of molecular genetics enhanced creation of genetically modified (GM) organisms and has been combined with ‟mouse science”, which is welcomed by mouse community and still induces increasing number of GM mice generation. Concept of ‟3Rs”, however, has reduced mouse usage in a single experiment. This means animal facilities handle a lot of mouse strains with limited numbers and thus microbiological monitoring of experimental mice is changing its meaning from mass to individual. RIKEN BioResource Center has been working to prepare high-quality mice resources by trustworthy genetic and microbial tests. We think there exist three key points to keep good microbiological conditions; establishment of clean colony (management and microbiological tests of breeding colony which will be used to prepare recipient and/or foster mothers, and microbiological tests of recipient and/or foster mothers themselves), good housing condition keeping (environmental microbiological tests) and microbiological monitoring of live mice colonies (dirty-bedding sentinel tests and complement tests). In this symposium, I will talk about the development of Multiplex Microfluidic Chip for individual serology tests using small amount of serum and new sentinel ideas that would help detecting weak pathogens.

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Mini Symposium.

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