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JSPS Asian Core Program 1st annual seminar abstract 1-1 Prof. Kenzaburo Tani Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector. Ryo Kurita 1, Erika Sasaki 2, Tomoko Yokoo 1, Kashiya Takasugi1, Yan Dong4, Hideyuki Imoto1, Youko Suehiro1, Yoshimiti Tachikawa1,Takashi Hiroyama 3, Yukoh Nakazaki 1, Kiyoko Izawa 4, Hajime Ishii 2, Yoshikuni Tanioka 2, Kisaburo Hanazawa 5, Yuan Son Bai 4, Yasushi Soda 4 and Kenzaburo Tani 1. 1 Molecular Genetics, Bioregulation, Fukuoka, Fukuoka, Japan, 812-8582; 2 Division of Animal Experimentation, Central Institute of Experimental Animals, Kawasaki, Kanagawa, Japan, 216-0001; 3 Cell Engineering Division, Riken Bio Resource Center, Cell Bank, Tukuba, Ibaragi, Japan, 305-0074; 4 Division of Molecular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Tokyo, Japan, 108-8639 and 5 Department of Urology, Juntendo University School of Medicine, Tokyo, Japan. We recently demonstrated that non-human primates of common marmosets (CM) are suitable for the preclinical studies of hematopoietic stem cell therapy and established CM ES cell lines (Sasaki, E. et al., Stem Cells 2005). In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods. The results demonstrated the feasibility of gene modified ES cell therapy. First, we tested various in vitro culture conditions including embryoid body formation or co-culturing with stromal cells to induce hematopoietic cells, but the hematopoietic activities were rather low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that the culture conditions were lacking essential factor for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors which were constructed to express hematopoietic genes such as marmoset tal1/scl, gata1, gata2, Lh2 and human hoxB4 genes under the EF1 promoter and transduced them into CM ES cells. Only when tal1/scl gene was transduced, hematopoietic induction from CM ES cells was dramatically stimulated and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were observed by immunochemical and morphological analyses. JSPS Asian Core Program 1st annual seminar abstract 1-2 Prof. Kenzaburo Tani Furthermore, FACS analysis and RT-PCR results showed that CM CD34, one of the hematopoietic stem cell markers, was expressed in the tal1/scl overexpressed-EB cells. These results were similarly obtained from experiments using three independent CM ES lines. After the xenotransplantation of ES-derived cells into immunodeficient mice, CM CD45+ cells and immature erythroid cells were observed in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation both in vitro and in vivo. Above results suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Determination of both long-term hematopoietic reconstitution capacity and safer intracellular delivery of tal1/scl gene products are under investigation for the purpose of developing new hematopoietic stem cell therapy. JSPS Asian Core Program 1st annual seminar abstract 2 Dr. Kennosuke Karube The pathology of adult T-cell lymphoma/leukemia Kennosuke Karube, Koichi Ohshima Department of Pathology, Kurume University Adult T cell lymphoma/leukemia (ATLL) is caused by human T cell lymphotropic virus type I (HTLV-1) and characterized by aggressive clinical course and poor prognosis. ATLL is prevalent in areas endemic for HTLV-1, including Kyushu, which locates southwestern Japan, and the Caribbean basin. Until now, many studies about HTLV-1 virus and the mechanism of infection progressed, but the prognosis of ATLL is still poor and the oncogenic mechanism of ATLL has not been known completely. In this seminar, we would like to introduce our study analyzing the morphological, epidemiological, genetical, and immunohistochemical features of ATLL. (1) The incidence of ATLL in Japan (2) The morphological feature of ATLL (3) The immunohistochemical feature of ATLL (4) The association of ATLL lymphoma cells and CD4+CD25+ regulatory T cells: FoxP3 expression in ATLL and its effect on the clinical feature JSPS Asian Core Program 1st annual seminar abstract 3 Prof. Naotaka Hamasaki Japanese Thrombophilia Protein S and Protein C gene mutations in Japanese deep vein thrombosis patients. Naotaka Hamasaki, M.D., Ph.D. Faculty of Pharmaceutical Science Nagasaki International University Coagulation factor V Leiden has not been detected in Japanese patients suffering from thrombosis. Hitherto the constitutional background of Japanese thrombotic patients has never been systematically examined. We have performed a systematic investigation to determine pathogenesis for deep vein thrombosis in Japanese population. Routine coagulation and fibrinolysis tests were performed to determine the activities of protein S, protein C, antithrombin, plasminogen and fibrinogen. Gene analysis was performed in thrombotic patients having the low activities of these factors. Our study indicates that the frequency (19/85=0.22) of mutations of protein S gene in the Japanese patients was 5-10 times higher than that of mutations of protein S gene in Caucasian patients, and the frequency (8/85=0.09) of mutations of protein C gene was almost three times higher than that of Caucasian patients. Protein S reinforces the activated protein C activity and cooperatively regulates the coagulation activity with the activated protein C. Since factor V Leiden shows resistance to the protein S/protein C system, coagulation regulation by the protein S/protein C system profoundly fails in individuals having factor V Leiden, yielding the same phenotypes as those with reduced protein S/protein C anticoagulation activity. It is interesting to note that the frequency of factor V Leiden (20-50%) in Caucasian deep vein thrombosis patients is similar to that (30%) of heterozygous gene mutations of the protein S/protein C anticoagulation system in Japanese deep vein thrombosis patients. Key Words: Deep vein thrombosis; protein S gene mutation; protein C gene mutation; factor V Leiden; Japanese thrombophilia. JSPS Asian Core Program 1st annual seminar abstract 4 Prof. Ampaiwan Chuansumrit THROMBOEMBOLISM IN THAI CHILDREN Ampaiwan Chuansumrit, M.D., Nongnuch Sirachainan, M.D. Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Objective: To analyze the etiology, management and outcome of Thai pediatric patients with thromboembolism. Methods: A retrospective study of patients admitted at the Department of Pediatric, Ramathibodi Hospital from 1987 to 2003, was conducted. Results: Total 108 patients with thromboembolisms were enrolled in the study. The sites of thromboembolism were in the venous sinus or arteries of the central nervous system 33%; the skin with necrosis, 20%; deep vein of the extremities, 16%; the toes and fingers with gangrene, 14%; and other sites such as the heart and lugs, 17%. Most of the patients had triggering conditions (54%) including endothelial injury, stasis of blood and alteration of coagulation factors. Factor V Leiden and prothrombin 20210 were not found among the studied patients; however, low levels of antithrombin III, protein C or protein S were found in 39% (22/57) of the patients. Most of these were transient except for three who were diagnosed with homozygous (n=3) and heterozygous (n=4) protein C deficiency. The management included administration of standard heparin or low molecular weight heparin, if not contraindicated replacement of fresh frozen plasma 10 ml/kg twice a day and treatment of triggering and underlying conditions. The fatality rate was 14%. Subsequent episodes of thromboembolism were found in 13 patients who did not receive appropriate management. Conclusion: Comprehensive investigation and specific treatment for pediatric patients are emphasized in order to prevent recurring thromboembolic episodes. JSPS Asian Core Program 1st annual seminar abstract 5 Assoc.Prof. Shuji Shimizu High-quality interactive telecommunication system using super-fast Internet connections and its application to hematology Kyushu University, Fukuoka, Japan Shuji Shimizu, Naoki Nakashima, Nobuhiro Torata, Koji Okamura, Koichiro Muta, Masao Tanaka Amid rapid change in medical knowledge and patient care, education and training for younger generation is of utmost importance beyond geographic borders. International telecommunication system via super-fast broadband Internet, which was established between Japan and Korea in 2003 and can transmit moving images without any loss of quality, was reported to be a powerful tool for this purpose. The aim of our study was to expand our advanced system to whole range of Asia-Pacific regions and to widen our application fields over rapidly growing optic fiber network. Kyushu University Hospital in Japan was newly linked to over 20 medical institutions or meeting venues in China, Taiwan, Thailand, Vietnam, Singapore, and Australia through domestic and international research and development networks. A digital video transport system (DVTS), which can transform digital video signals directly to Internet protocol with the bandwidth of 30 Mbps per line, and cipher security programs to protect patient’s privacy were used. Of the 60 international teleconferences conducted during three years, real-time demonstrations and interactive teleconferences with recorded videos were included. Not only two stations were connected pier-to-pier, but also multiple stations were connected without any conversion to analog signals. The medical fields involved were surgery, endoscopy, hematology, medical informatics, nursing, health care, etc. The network remained stable, and the time delay between stations was restricted to 0.3 – 1.0 sec. We have established a high-quality telecommunication system in wide area of Asia-Pacific, and this is the first time to apply high-speed Internet technology to medicine in such a big international scale. Because it is not only high-quality but also economical and easily accessible, we believe this system will promote worldwide expansion of efficient medical education and active academic exchanges. JSPS Asian Core Program 1st annual seminar abstract 6 Dr. Nopphol Pausawasdi Telemedicine: Siriraj’s education and research network Nopphol Pausawasdi, PhD. Faculty of Medicine Siriraj Hospital, Mahidol University The Faculty of Medicine Siriraj Hospital has three major missions which are (a) provides medical education (b) produces medical research and (c) provides medical care. To accomplish those missions effectively, the faculty has consider advanced technology which would enhance teaching and learning activities, research projects and collaborations, and medical care skills and knowledge. Telemedicine is a technology that helps the faculty to establish collaborations with other medical institutions over the world which allows us to create medical education and research network. In 2004, the faculty initially utilized telemedicine in a Siriraj annual medical conference to allow medical staffs who were working in Siriraj hospital and other private hospitals in Bangkok to see presentations from the conference venue as well as to be able to communicate with speakers. In the same year, the faculty began to provide medical education to medical students who studied in Ratchaburi hospital which is located in Ratchaburi province. Moreover, we also collaborated with a group of doctors from Kyushu University Hospital to conduct a telemedicine session from the Faculty of Medicine Siriraj Hospital to Kyushu University Hospital. Since then the Faculty of Medicine Siriraj Hospital set up its telemedicine internet network connection with great supports from many national and international organizations including Mahidol University, Uninet, Nectec, and Kyushu University Hospital. Currently the faculty is able to utilize telemedicine technology in everyday routine working basis and other special events. We collaborate with many local and global medical institutions including Ramathobodi Hospital, Ratchaburi Hospital, Samitivet Hospital, Bangkok General Hospital, and those of APAN medical working group such as Kyushu University Hospital and Seoul National University Bundang Hospital in order to develop education and research network among doctors and medical staffs around the world. The Faculty of Medicine Siriraj Hospital will continue to expand its network to other medical institutions to enrich medical information exchange which would help developing medical education and research of the faculty and the others. JSPS Asian Core Program 1st annual seminar abstract 7-1 Prof. Shinji Nakao Specific antibodies to moesin, a membrane-cytoskeleton linker protein, in the serum of patients with aplastic anemia: their incidence and possible role in the pathophysiology of bone marrow failure. Shinji Nakao, Hiroyuki Takamatsu. Cellular Transplantation Biology, Kanazawa University Graduate School of Medical Science Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. pathophysiology of AA. It is as yet unclear whether these auto-Abs play some roles in the We recently demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34 + cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients’ sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 g/ml of control IgG or anti-moesin Abs derived from an AA patient’s serum, TNF- production from THP-1 cells stimulated by anti-moesin Abs was 1.9-2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF- production. These results indicate that anti-moesin Abs may be JSPS Asian Core Program 1st annual seminar abstract 7-2 Prof. Shinji Nakao involved in the suppression of hematopoiesis of AA patients by stimulating TNF- production from monocytes. JSPS Asian Core Program 1st annual seminar abstract 8 Dr. Yukimi Sakoda Pathophysiology and treatment of chronic GVHD after allogeneic hematopoietic stem cell transplantation Sakoda Yukimi Department of Medicine and Biosystemic Science, Kyushu University Chronic graft-versus-host disease (GVHD) is the most common cause for poor long-term outcomes after allogeneic bone marrow transplantation (BMT). Unfortunately, despite success in the prevention of acute GVHD by the introduction of cyclosporine, the incidence of chronic GVHD has not decreased. However, the pathophysiology of chronic GVHD still remains poorly understood. Recent animal studies suggested that donor CD4+ T cells play a critical role in the induction of chronic GVHD, thus T cell-depletion (TCD) could reduce chronic GVHD as well as acute GVHD. However, the first randomized study comparing TCD-BMT with T cell replete BMT found that TCD reduces rates of acute GVHD, but not chronic GVHD, thus suggesting that thymic-dependent T cells play a role in mediating chronic GVHD. We tested the hypothesis that the impaired thymic function of the recipients will permit the emergence of pathogenic T cells that cause chronic GVHD using chimeric mice where thymic negative selection was impaired. Lethally irradiated C3H/HeN (H-2k) recipients were reconstituted with TCD bone marrow cells from MHC class II-deficient B6 (H-2b) mice. These mice developed diseases that showed all of the clinical and histopathological features of human chronic GVHD. Thymectomy prevented chronic GVHD, thus confirming the causal association of the thymus. We also found that CD4+ T cells isolated from chronic GVHD mice were primarily donor-reactive and adoptive transfer of these CD4+ T cells caused chronic GVHD in recipient mice in the presence of donor-derived antigen presenting cells. Our results demonstrate for the first time that T cells which escape from negative thymic selection could cause chronic GVHD. These results also suggest that self reactivity of donor T cells play a role in this chronic GVHD and improvement in the thymic function may have a potential to decrease chronic GVHD. A recent study from another group showed donor B cells play a role in chronic GVHD. We therefore conduct a phase II trial of anti-B cell therapy with rituximab in steroid refractory chronic GVHD.