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Advanced Clinical Research Center

1. Efficient gene transduction and integrationinto human blood cells with a new third-gen-eration lentiviral vector

YS Bai, Y Soda, et al.

As the difficulty of gene transduction of humanblood cells including hematopoietic stem cells is anobstacle to develop gene therapy targeting hemato-logical disorders, the development of new systems isstrongly desirable. Under these circumstances, somereports demonstrated promising results by usinglentiviral vectors, although more room for improve-ment has been left. In this study, we developed anovel third-generation self-inactivation (SIN) len-tiviral vector system based on HIV-1 to improve thetransduction efficiency and prevent vector-relatedtoxicity and production of replication-competentlentivirus. The VSV-G-pseudotyped HIV vector wasproduced by cotransfection of the vector and pack-aging constructs together with the VSV-G- andRev-expressing constructs into 293T cells. For com-parison, VSV-G-pseudotyped SIN MLV vector wasused. Transduction efficiencies were assessed by flu-orescence microscopy and flow cytometry, detectinghrGFP transgene expression. PCR for hrGFP wasalso performed to determine transduction efficien-cies of colony-forming cel ls derived fromhematopoietic cells. We examined integration of

Division of Molecular Therapy分子療法分野

Our laboratory is primarily concerned with the development of novel therapeutic op-tions against intractable hematological disorders including leukemia and lymphoma.In this year, our efforts based on molecular and cellular biology have produced thefollowing achievements which are clinically oriented.

Professor Shigetaka Asano, M.D., D.M.Sc.Associate Professor Arinobu Tojo, M.D., Ph.D.Lecturer Satoshi Takahashi, M.D.Lecturer Kaoru Uchimaru, M.D., Ph.D.Clinical Associate Jun Ooi, M.D.

教　授 医学博士　 浅　野　茂　隆助教授 医学博士　 東　條　有　伸講　師 医学博士　 高　橋　　　聡講　師 医学博士 内　丸　　　薫助　手 大　井　　　淳

these vectors in these cells by Alu-PCR method.Transduction efficiencies of leukemia cell lines withthe HIV and MLV vectors were almost 100% and lessthan 50%, respectively. The expression of the trans-gene persisted for eight weeks in cells transducedwith the HIV vector, but not with the MLV vector.Similar results were obtained in 11 kinds of primarycells obtained from leukemia or myeloma patients.In addition, resting peripheral blood lymphocytesand CD34-positive hematopoietic cells were success-fully transduced with the HIV vector, but not withthe MLV vector. Moreover, we confirmed integra-tions of vector genes in almost all colony-formingcells transduced with the HIV vector, but not withthe MLV vector. Our lentiviral vector system is con-sidered to be an excellent gene transduction systemfor human blood cells because of its high gene trans-duction and integration capability into hostchromosome.

2. Influence on gene transduction with a third-generat ion sin lentiviral vector: DNAmicroarray analysis of gene-transduced he-matopoietic progenitor cells

YS Bai, Y Soda, et al.

In the development of gene therapy targeting vari-ous kinds of disease including congenital enzyme

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deficiency, it is thought that gene transduction intohematopoietic stem cells is a very important method.A lot of reports have recently described that VSV-pseudotyped lentiviral vector is promising as a genetransduction method to hematopoietic stem cells be-cause of its excellent transduction efficiency andability of long-term gene expression. However, the ef-fect and safety of this vector system has not yet beenfully evaluated, despite improvements of the vectorsincluding the self-inactivation (SIN) of vectors toavoid the production of replication-competent virusand removal of cytotoxic accessory genes derivedfrom HIV. Therefore, we studied the influence of genetransduction with a third-generation SIN lentiviralvector on the gene expression of human hematopoiet-ic stem cells. CD34-positive cord blood cells(CD34+CBC) were obtained from cord blood usingmagnetic bead method, and transduced with thethird-generation VSV-pseudotyped SIN HIV vector[HIV(VSV)] at M. O. I. 20. Cells were cultured for fivedays in the presence of stem cell factor, granulocyte-colony stimulating factor, and thrombopoietin. Cellgrowth and viability was examined by trypan blue ex-clusion test and expression of surface antigen wasanalyzed by flow cytometry to access the cell differen-tiation. Gene expression in transduced cells wascompared with non-transduced cells by DNA mi-croarray analysis. The gene transduction efficienciesof cord blood CD34+CBC were 80%, and cell numberwas increased seven times for five days. In the trans-duced cells and the non-transduced cells, there wereno significant difference in cell number, viability, andexpression of surface antigens. DNA microarray anal-ysis demonstrated that there was no significantchange on the expression of 23,000 genes. OurHIV(VSV) vector did not influence on the gene ex-pression of CD34+CBC, and may be a useful and safevector system in clinical gene therapy.

3. Development of HIV (VSV) vectors targetingneutrophil disorders: study of promoter activ-ities in myeloid cells

XJ Li, Y Soda, et al.

Neutrophils are non-dividing effector cells of in-nate immunity. To treat neutrophil disorders,supplementation of defected gene in neutrophil isthought to be a promising approach. However, pre-vious trials using MLV vectors could not obtainsatisfactory results because of low transduction effi-ciency and low activity of promoters. Recently,VSV-pseudotyped HIV [HIV(VSV)] vectors was re-ported as a powerful tool for gene transduction intohuman blood cells, but the optimization of these vec-tor systems for mature granulocytes has been left. Inthis study, we determined the optimal internal pro-moter for the gene expression in myeloid cells. In thisstudy, we cultured CD34-positive cellsisolated from

umbilical cord blood (CD34+CBC) by two-step cul-ture method using 10ng/ml G-CSF, 5ng/ml SCF,5ng/ml GM-CSF and 5ng/ml IL-3. To determine themost efficient promoters for neutrophils, we con-structed six kinds of HIV(VSV) vectors that containinternal promoter such as CMV, PGK, EF-1α, CAG,MPO or defensin promoter. These vectors were inoc-ulated to CD34+CBC at an M. O. I. of 100, and theexpression levels of transgene in myeloid cells weredetermined by flow cytometry (FCM) to detect GFP.Microscopic observation and four color FCM detect-ing differentiaion antigens revealed that over 80% ofdifferentiated cells were neutrophils. On day 14 ofculture, GFP-positive rate of CD15-positive cellstransduced with CMV, PGK, EF-1α and CAG pro-moter-harboring vectors is 26. 0±5. 0%, 5. 7±2. 9%, 3.1±0. 6% and 3. 6±0. 6%, respectively. MPO and de-fensin promoter did not induce detectable GFPexpressions. Granule proteins, alkaline phosphotase,myeloperoxidase and esterase were comparably ex-pressed in transduced and non-transduced cells.Gene transduction with HIV vector did not affectedcell growth and function of differentiated cells, e. g.chemotaxis, phagocytosis, and the expression of my-loperoxidase and defensin. CMV promoter is themost suitable internal promoter in HIV (VSV) vectorfor gene modification of neutrophils and may be use-ful for gene therapy of neutrophil disoders.

4. Maxizyme, a novel ribozyme, induces celldeath of Philadelphia chromosome-positiveacute lymphoblastic leukemia (Ph+ ALL)

Y Soda, YS Bai, et al.

Patients with Ph+ ALL consisting about 30% ofALL have poor prognosis despite intensive treatmentincluding hematopoietic cell transplantation. We pre-viously demonstrated that an allostericallycontrollable novel ribozyme, designated as Maxizyme(Mz), induced cell death of chronic myelogenous leu-kemia (CML) cells expressing b2a2 type bcr-abltranscripts, and proposed that it might provide a newstrategy for future treatment of CML. Similar to CML,Ph+ ALL cells harbor e1a2 type bcr-abl fusion gene(e1a2) encoding the 190kD fusion protein (p190) and isinvolved in the pathogenesis of this disease. To devel-op a new therapeutic approach for Ph+ ALL, we havedesigned a Mz specifically cleaving e1a2 mRNA(e1a2Mz). We inserted Mz-coding gene downstreamof human transfer RNA (tRNAVal) gene having polIIIpromoter activity. This Mz expression unit was insert-ed in a third-generation self-inactivating lentiviralvector carrying a reporter gene of human CD2 toachieve high transduction and integration efficiencycomplied with strict safety. The virus particles wereproduced by cotransfection of the vectors and packag-ing constructs together with the VSV. G- andRev-expressing constructs into 293T cells. We trans-

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duced Mz into various types of cell lines includingPh+ ALL cells and primary cells from Ph+ ALL pa-tients with this lentiviral vector. In all types of cells,transduction efficiencies detected by flow cytometrywere almost 100%, and Mz expression was detectedby RT-PCR. In three of five Ph+ ALL cell lines, Mztransduction resulted in significant decrease of viabil-ity (about 20% on day 14), and increase of apoptoticcells. However, remaining two Ph+ ALL cell lines didnot show cell death similar to p190-negative K562 andNB4 cells. To elucidate the cause of the Mz-resistanceof Ph+ ALL cell lines, we measured the e1a2 mRNAlevel by quantitative RT-PCR. We observed decreasedlevel of e1a2 mRNA in all Ph+ ALL cells transducedwith Mz, however, the e1a2 mRNA level was higherin Mz-resistant cells than Mz-sensitive cells. In allthree primary Ph+ ALL cells tested, Mz inducedgrowth inhibition and cell death comparable to Mz-sensitive Ph+ ALL cell lines. Furthermore, Mz did notinfluence the growth and colony formation of normalCD34+ cord blood cells. In conclusion, e1a2Mz will bea useful tool for the gene therapy of Ph+ ALL. In vivostudy to see the effect of e1a2Mz on Ph+ ALL cells isunderway.

5. Development of a new targeting therapy forPhiladelphia chromosome-positive acute lym-phoblastic leukemia (Ph+ ALL): anti-CD19antibody-binding liposome (CD19-liposome)containing STI571 efficiently killed Ph+ ALLcells

M Harata, Y Soda, et al

Patients with Ph+ ALL have poor prognosis de-spite intensive treatments including hematopoieticcell transplantation. Recently, bcr-abl tyrosine kinaseinhibitor, STI571, proved to be a useful agent for Ph+ALL. However, most patients will soon acquire theresistance to STI571. To overcome this problem, highdose administration, which leads to high intracellu-lar concentration of STI571, is though to be apromising approach, although various toxicities willnot allow this approach. Therefore, dose escalationof STI571 only in target cells may resolve this prob-lem. Since almost all Ph+ ALL are derived fromprecursor B cells and express CD19 specifically onthe cell surface, targeting of Ph+ ALL cells usingCD19 monoclonal antibody is exploited. In thepresent study, we developed STI571 encapsuledanti-CD19 antibody binding liposomes for the pur-pose of developing a new cell targeting therapy ofPh+ ALL. The binding of anti-CD19 antibody to thesurface of liposomes was made via polyethylenegly-col (PEG). Transferrin (TF) binding PEG-liposomes(TF-liposome), PEG-liposomes and bare (DSPC/CH)liposomes were used as controls. Eight kinds of cells,such as OM9;22 Ph+ ALL, CML and APL cell lines,etc. were used. The efficacy of induction to the target

cells was examined by FCM and fluorescent micros-copy using calcein or DiI pigment encapsuledliposomes. The specificity of pigment induction viaCD19 was examined by a competitive inhibition ex-periment using anti-CD19 antibody. The effect ofSTI571 encapsuled liposomes(STI571-CD19-lipo-somes) was determined by observing viable cellnumber and survival rates in the time course. Wealso examined apoptosis of cells by Annexin-V assay.The induction efficiency by calcein encapsuled lipo-somes was almost 100% in all Ph+ ALL cell lines andwas significantly enhanced by CD19-liposomes com-pared with bare liposomes and PEG-liposomes. Thisenhancement was inhibited by the addition of ananti-CD19 antibody in a dose dependent manner. Incontrast, in CD19 non-expressing cells, the enhance-ment effect of CD19-liposomes was not observed.When STI571-CD19-liposomes were added, Ph+ALL and CD19-positive CML lymphoid crisis cellline, BV173, died in a shorter period compared withother STI571 containing liposomes or free STI571.This effect was observed proportionally to theSTI571 concentration in liposomes, but the amountof STI571 required to exert the effect was 0. 1µM orless in STI571-CD19-liposomes, being 1/10 or less offree STI571. A further study is now underway target-ing STI571 resistant cell lines, primary leukemia cellsfrom Ph+ ALL patients, and normal hematopoieticcells, in vitro and in animal model. STI571-CD19-li-posome was demonstrated to induce cell deathspecifically to Ph+ ALL cells. This new therapeuticapproach might be useful to obtain specific cell kill-ing effects for Ph+ ALL cells with much less sideeffects to other organs in vivo than administration offree STI571.

6. Isolation from phage-display libraries of threecandidate peptides which potentially targethematological malignancies

MH Chen, Y Soda, et al.

Selection of peptides which recognize specific celltypes is a promising strategy both in basic researchand clinical applications. Cell type-specific peptidescan be applied to delivery vehicles for small mole-cule compounds, nucleotides, genes as well asdiagnostic agents. They can also be useful as affinityreagents for cell purification and research tools forcell surface profiling. Recently, a number of cell ortissue-specific targeting peptides have been identi-fied by screening recombinant phage-displaylibraries in vitro and/or in vivo. Using the similarmethod, we isolated 3 distinct peptide-presentingphages which can bind to several human cancer celllines including myeloid/lymphoid leukemia celllines. We screened two types of phage-display ran-dom peptide libraries containing linear 12-merligands or disulfide constrained 7-mer ligands by the

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subtraction method. Daudi lymphoma cells were atfirst used to exclude non-specific binding and/orlymphoid specific binding of peptide-presenting ph-ages from libraries, and next AML-M2 derivedKasumi-1 cells were used to isolate peptides poten-tially targeting myeloid leukemia cells. Screening oflibraries was performed virtually according to thestandard protocol. Cells were incubated with 100 li-brary equivalent phages at 37oC for 1 hr. Afterextensive washing, cell-surface binding phages wererecovered by acid treatment of cells and neutralized.Internalized phages were prepared by lysing cells.After five rounds of screening procedures, we isolat-ed a single phage presenting 12-mer peptides(WAPPLFRSSLFY), which was specifically internal-ized by Kasumi-1 cells. In another selection, weidentified two Kasumi-1 cell-surface binding phagescontaining 12-mer inserts (GIQLANPPRLYG andLQAAYKGFARAG) after eighth rounds of screen-ing. Specific binding of the purified three phages toKasumi-1 cells at 4oC was confirmed by flowcytome-try using anti-M13 monoclonal antibody withphages containing random 12-mer inserts as control.We screened a panel of cancer cell lines to examinethe cell-type specificity of these three peptides. Weselected three 12-mer peptide sequences which candefinitely bind to several leukemia cell lines but notto normal lymphocytes. Further characterization ofthe peptides is ongoing.

7. Anti-NK cell treatment induces stable mixedchimerism in MHC-mismatched, nonmyeloab-lative bone marrow transplantation

SG Cho, Y Shuto, K Izawa et al.

Natural killer (NK) cells have been reported to beinvolved in resistance to engraftment of transplantedallogeneic stem cells. We investigated the effects ofNK cell depletion on engraftment and induction ofstable mixed chimerism after MHC-mismatched,nonmyeloablative bone marrow transplantation(NMT). Recipient mice (BALB/c, H-2kd) were inject-ed intraperitoneally with anti-asialoGM1 antibody(anti-NK Ab) on day -1. On day 0, they received TBIat a dose of 500 cGy, followed by intravenous infu-sion of 2 x 107 T-cell-depleted (TCD) bone marrow(BM) cells from allogeneic donors (C57BL/6, H-2kb).Early engraftment and chimerism were determinedby the relative ratio of peripheral blood (PB) lympho-cytes expressing either recipient or donor MHC classI molecules (FITC-H-2kb vs. PE-H-2kd) on day +21.Long-term engraftment and chimerism were evalu-ated by examination of PB and spleen byflowcytometry. All the recipients conditioned with asingle injection of anti-NK Ab and 500 cGy TBIshowed successful engraftment. In contrast, no re-cipients treated with TBI alone showed engraftment.Furthermore, a donor-dominant pattern of mixed

chimerism was observed in both PB and spleen.Spleen cells from recipients showing mixed chimer-ism revealed specific tolerance to both host anddonor strains, but were reactive to third-party cells(C3H/He). None of the reconstituted mice showedsigns of GVHD, and all survived up to day +330.These observations indicate that host NK cell deple-tion may be used to reduce the intensity of theconditioning regimen for engraftment of TCD grafts,and can contribute to the establishment of stablemixed chimerism in MHC-mismatched NMT.

8. Multi-lineage differentiation and lethal GVHD-like syndrome after transplantation into NOD/SCID mice of non-human primate commonmarmoset CD34+ cells

K Izawa, et al.

We have been trying to establish a non-human pri-mate model of preclinical studies such as genetherapy toward hematological disorders, using thesmall New World monkey, common marmoset (Cm)(Callithrix jacchus). However, handling with Cmstem/progenitor cells has been hampered by the lackof procedures for isolating those populations. Dis-crimination of stem/progenitor cells from matureblood cells usually depends on their surface expres-sion of CD34 antigens in human and rodents.Therefore, we developed for the first time anti-CmCD34 monoclonal antibody (MoAb) and tested cellpopulations isolated by this MoAb for their colony-forming capacity as well as repopulating activity inNOD/SCID mice. Cm cDNA encoding a human androdent CD34 homologue was cloned from bone mar-row (BM)-derived RNA. NIH3T3 cells wereengineered to express CM CD34 cDNA and immu-nized into BALB/c mice. The resulting MoAb wasused to isolate CD34+ cells in combination with amagnetic cell sorting system. Purified CD34+ cellswere subjected to colony-forming assays in methyl-cellulose and transplantation into sublethallyirradiated NOD/SCID mice. A series of murine Mo-A b s a g a i n s t h u m a n C D a n t i g e n s , w h i c hcross-reacted with corresponding Cm antigens, wereused to detect mature Cm blood cells in peripheralblood (PB) and BM of NOD/SCID mice. The openreading frame of Cm CD34 cDNA had 89% homolo-gy with the human sequences. One of the fiveavailable MoAbs, MA24 (IgM), recognized 1~2% ofBM mononuclear cells (MNCs), and purified CD34+

cells were approximately 11 to 30 fold enriched inCFU-GM content, compared with BM MNCs.Around one month after transplantation of up to 105

Cm CD34+ cells into NOD/SCID mice, CD2+, CD4+,CD8+, CD11b+ as well as CD14+ Cm cells could bedetected in PB and BM. Surprisingly, graft versushost disease (GVHD)-like syndrome including scle-roderma, weight loss or hunched posture developed

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Iijima Y, Okuda K, Tojo A, Tri NK, Setoyama M,Sakaki Y, Asano S, Tokunaga K, Kruh GD, andSato Y: Transformation of Ba. /F3 cells and Rat-1cells by ETV6/ARG. (2002) Oncogene 21:4374-4383

Duda, D. G., Sunamura, M., Lozonschi, L. ,Yokoyama, T., Yatsuoka, T., Horii, A., Tani, K.,Asano, S. , Nakamura, Y. and Matsuo, S.Overexpression of the p53-induced brain angio-genesis inhibitor 1 suppresses efficinetly tumorangiogenesis. Br. J Cancer 86:490-496, 2002.

Kawai,K.,Tani,K.,Yamashita,N.,Tomikawa,S.,Eriguchi,

Publications

M.,Fujime,M., Okumura,K., Kakizoe,T., Clift S.,Ando,D.,Mulligan, R., Yamauchi, A., Noguchi, M.,Asano, S., Akaza, H. Clinical course of an ad-vanced renal cell carcinoma patient treated withGM-CSF gene therapy (GVAX): The first experi-ence of cancer gene therapy in Japan. Int J Urol (inpress)

Ohata, J., Sakurai, J., Saito, K., Tani, K.,Asano, S.,Azuma, M. . Differential graft-versus-leukemia ef-fect by CD28 and CD40 costimulatory blockadeafter graft versus-host disease prophylaxis. ClinExp Immunol 129:61-68, 2002.

around 3 weeks after transplantation and thereafterworsened. Such a syndrome inevitably occurred inevery mouse which had Cm blood cells in PB. Dis-eased mice were sacrificed for histologicalexamination of various tissues, indicating massiveT(CD2+) cell infiltration into , liver, spleen. CmCD34+ cells isolated by MA24 demonstrate a repopu-

lating activity in a NOD/SCID transplantation mod-el. However, engraftment of Cm CD34+ cellscoincided with GVHD like-syndrome. In this xeno-geneic transplant model, the behavior of progeniesof Cm CD34+ is quite different from that of humanCD34+ cells which could not induce GVHD in thecomparable experimental conditions.

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Advanced Clinical Research Center

Division of Cellular Therapy細胞療法分野

Our major projects are (1) signal transduction of cytokine receptors, (2) identificationand characterization of novel cytokines, cytokine receptors, soluble factors, and tran-scription factors, (3) the roles of small GTPases and GAPs, (4) molecular mechanismof leukemogenesis, (5) identification of self-renewal factor for embryonic stem cells,(6) ontogeny of hematopoiesis, (7) characterization of hematopoietic stem cells, (8)molecular mechanism regulating hematopoiesis.

1. Isolation and characterization of new genes bya novel signal sequence trap method SST-REX

Yuichi Ikeda, Wing-Chun Bao, Toshihiko Oki,Takuya Sugiyama, Hidetoshi Kumagai1, SizhouFeng1, Kaori Tamitsu1, Yoshihiro Morikawa2,Hideaki Nakajima, Tetsuya Nosaka3, and ToshioKitamura: 1Takada Research Labs, Chugai Phar-maceutical Co., Ltd., 2Department of Neurobiologyand Anatomy, Wakayama Medical School of Medi-cine, 3Division of Hematopoietic Factors, TheInstitute of Medical Science, The University of To-kyo

Secreted and cell-surface proteins play essentialroles in cell-cell interaction. We have recently estab-lished a novel and efficient signal sequence trapmethod (SST-REX), in which cDNA fragments fusedto an extracellular deletion mutant of the constitu-t i v e l y a c t i v e M P L w e r e t r a n s d u c e d i n t oIL-3-dependent cells via retrovirus infection fol-lowed by the selection of factor-independent clones.Our method is quick and more accurate than the pre-viously published methods. In addition, type IImembrane proteins, which had never been isolatedby the previous SST methods, were also obtained byour SST-REX.

Several interesting genes have been isolated bythis method from various tissues including he-mopoiet ic s tem or progenitor ce l ls , AGM(aorta-gonad-mesonephros) cells, mast cells, andcardiocytes, and their functions are currently beinginvestigated.

2. Development of retrovirus vectors and packag-ing cell lines

Sumiyo Morita3, Fumi Shibata, Dan Wang, Toshi-hiko Oki, Tetsuo Kojima3, Yuko Koshino, HideakiNakajima, Tetsuya Nosaka3, Carol Stocking4, Wol-fam Ostertag4, Koichiro Tsuji and ToshioKitamura: 4Heinrich-Pette Institute, HamburgUniversity

We previously developed a MuLV-derived effi-cient retrovirual vector pMX that is suitable forlibrary construction. Combination of transient retro-virus packaging cell lines such as Bosc23 and thepMX vector produced high titer (106-107/ml) retrovi-ruses that gave 100% infection efficiency in NIH3T3cells, 10-100% infection efficiency in various he-mopoietic cell lines, and 1-20% in primary culturecells including T cells, monocytes, and mast cells.However, pMX did not work well in immature cells

教　授 医学博士　 北　村　俊　雄助教授 医学博士　 辻　　　浩一郎助　手 医学博士　 真　部　　　淳助　手 医学博士 中　島　秀　明

Professor Toshio Kitamura, M.D., D.M.Sc.Associate Professor Kohichiro Tsuji, M.D., D.M.Sc.Research Associate Atsushi Manabe, M.D., D.M.Sc.Research Associate Hideaki Nakajima, M.D., D.M.Sc.

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such as EC cells and ES cells. We have now devel-oped pMY and pMZ vectors that utilize PCMV's LTRand primer binding site, and can express GFP in ECcells and ES cells.

Recently, usefulness of transient packaging cellshas been recognized, however the titers of retrovi-ruses are rather unstable during culture. In order toestablish more stable packaging cell lines, we usedthe IRES sequence that allows simultaneous expres-sion of both gag-pol or env gene and drug resistancegene from one transcript. We used the strongest pro-moter EF-1α in making packaging constructs. Inaddition, to avoid inclusion of retrovirus sequencesas much as possible, we used only coding sequenceof gag-pol and env genes for the packaging constructs,which will not allow the formation of replication-competent retroviruses by recombination inpackaging cell lines. We established high-titer eco-tropic (PLAT-E) and amphotropic (PLAT-A)packaging cell lines where the EF-1α-gag-pol-IRES-p u r o r t o g e t h e r w i t h t h e c o r r e s p o n d i n gEF-1α-env-IRES-bsr were introduced into 293T cells.We have also established another new packaging cellline (PLAT-F) for efficient infection to human he-matopoietic stem cells by using env gene of felineendogenous retrovirus RD114, and the efficiency ofinfection of the viruses produced by PLAT-F, intohuman CD34 positive cells, is being investigated by along term reconstitution assay in SCID mice.

3. Analysis of the role of MgcRacGAP as a regula-tor of the small GTPase Rho family indifferentiation and cytokinesis

Toshiyuki Kawashima3, Yukinori Minoshima, Koi-chi Hirose3, Yukio Tonozuka, Takaya Satoh5,Yoshito Kajiro5, Hideaki Nakajima, Tetsuya Nosa-ka3, and Toshio Kitamura: 5Faculty of Bioscienceand Biotechnology, Tokyo Institute of Technology

In the search for key molecules that prevent mu-r ine M1 leukemic ce l l s f rom undergoingIL-6-induced differentiation into macrophages, weisolated an antisense cDNA that encodes full-lengthmouse MgcRacGAP through functional cloning. Inhuman HL-60 leukemic cells, overexpression of thehuman MgcRacGAP induced growth suppressionand macrophage differentiation. Analysis using themutants revealed that the GAP activity was dispens-able, but the myosin-l ike domain and thecysteine-rich domain were indispensable for growthsuppression and macrophage differentiation. Inter-estingly, MgcRacGAP localized to the nucleus ininterphase, accumulated to the mitotic spindle inmetaphase, and was condensed in the midbody dur-ing cytokinesis. Overexpression of an N-terminaldeletion mutant resulted in the production of multi-nucleated cells in HeLa cells. This mutant lost the

ability to localize in the mitotic spindle and mid-body. MgcRacGAP was also found to bind α-, β-, andγ-tubulins through its N-terminal myosin-like do-main. These findings indicate that MgcRacGAPdynamically moves during cell cycle progressionprobably through binding to tubulins and plays crit-ical roles in cytokinesis. Furthermore, using aGAP-inactive mutant, we have disclosed that theGAP activity of MgcRacGAP is required for cytoki-nesis, suggesting that inactivation of Rho familyGTPases may be required for normal progression ofcytokinesis. We have recently found that MgcRac-GAP is phosphorylated by some of the kinases thatare known to work in the midbody.

4. Identification of a small molecule which inhib-its leukemic cell growth caused by the internaltandem duplication mutations of Flt-3

Ken Murata3, Hidetoshi Kumagai1, Hideaki Naka-jima, Toshiyuki Kawashima3, Kaori Tamitsu1,Mariko Irie1, Shigetaka Asano6, Tetsuya Nosaka3,and Toshio Kitamura: 6Division of Molecular Ther-apy, Advanced Clinical Research Center, Instituteof Medical Science, The University of Tokyo

Internal tandem duplications of the juxtamem-brane region of the Flt-3 are found in about 20% ofthe human acute myeloid leukemia patients. Inscreening of the small compounds by the ability toselectively inhibit leukemic cell growth caused bysuch mutations of Flt-3, we have identified severalsmall chemical compounds. These molecules showstructural similarity to the tyrosine kinase inhibitor.One of the most effective molecules GTP14564 pref-erentially inhibited the growth of the Ba/F3 cellstransformed by the mutant Flt-3, thereby suppress-ing the tyrosine phosphorylation of STAT5, but notvery much in Ba/F3 cells driven by the Flt-3 ligand/wild type Flt-3. Forced expression of the dominantnegative STAT5A, but not treatment with the MEKinhibitors suppressed the mutant Flt-3-driven cellgrowth. On the other hand, the proliferative signalthrough the wild type Flt-3 was dependent on theactivation of MAP kinases. We also revealed that theN-terminal two tyrosine residues of the intracellulardomain of the mutant Flt-3 were responsible forSTAT5 activation and autonomous cell growth, butthe corresponding tyrosine residues of the intracellu-lar domain of the wild type Flt-3 was dispensable forcell growth.

5. STAT5 induces macrophage differentiation ofM1 leukemia cells through activation of IL-6production mediated by NF-κκκκκB p65

Toshiyuki Kawashima3, Ken Murata3, Yukio Tono-zuka, Yukinori Minoshima, Tetsuya Nosaka3, andToshio Kitamura

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U s i n g a c o n s t i t u t i v e l y a c t i v e S T A T 5 A(STAT5A1*6), we have shown that STAT5 inducesmacrophage differentiation of mouse leukemic M1cells through a distinct mechanism, autocrine pro-d u c t i o n o f I L - 6 . T h e s u p e r n a t a n t o fSTAT5A1*6-transduced cells contained sufficientconcentrations of IL-6 to induce macrophage differ-entiation of parental M1 cells, and STAT3 wasphosphorylated on their tyrosine residues in thesecells. Treatment of the cells with anti-IL-6 blockingantibodies profoundly inhibited the differentiation.We have also found that the STAT5A1*6 transacti-vated the IL-6 promoter, which was mediated by theenhanced binding of NF-κB p65 (RelA) to the pro-moter region of IL-6. These findings indicate thatSTAT5A cooperates with Rel/NF-κB to induce pro-duction of IL-6, thereby inducing macrophagedifferentiation of M1 cells in an autocrine manner. Insummary, we have shown a novel mechanism bywhich STAT5 induces its pleiotropic functions.

6. A novel secreted form of immune suppressorfactor with high homology to vacuolar ATPas-es identified by a forward genetic approach offunctional screening based on cell prolifera-tion

Edgardo E Tulin3, Hideaki Nakajima, N Onoda7, MMaeda7, M Hasegawa7, Atsushi Urano, Fumi Shi-bata, Tetsuya Nosaka3, H Nomura7, ShigetataAsano6, and Toshio Kitamura: 7Chugai ResearchInstitute for Molecular Medicine

In the search for stromal-derived growth factors,we have identified a novel secreted short form of im-mune suppressor factor (ISF) using a combination ofa genetic approach and retrovirus-mediated func-tional screening. This protein, which we termedShIF, was isolated based on its ability to support pro-liferation of a mutant clone S21, which wasestablished from Ba/F3 cells that are usually inter-leukin-3-dependent but became dependent on astroma cell line ST2 after chemical mutagenesis. ISF,a membrane protein harboring six transmembranedomains, was reported to have immunosuppressivefunctions. The coding region of ShIF started from thethird transmembrane domain of ISF. Biochemicalanalysis demonstrated that ShIF was expressed inboth the secreted and membrane-bound forms of 27-kDa protein, which was supposed to have an internalATG present in the third transmembrane domain ofISF as a start codon. In addition to the full-lengthform of ISF, a major protein with a molecular size of27 kDa was also expressed through the proteolyticprocess of ISF. ShIF resembles this naturally occur-ring short form of ISF (sISF). Deletion analysis of themajor domains of ISF cDNA revealed that ShIF is anactive functional domain of ISF with a capability to

support proliferation of S21 cells. Enforced expres-sion of ShIF in MS10 cells, bone marrow stroma cellsthat do not express endogenous ShIF or ISF, con-ferred on the cells an ability to support the growth ofS21 cells as well as bone marrow cells. Interestingly,ShIF shows a high sequence homology to the C-ter-minal part of a 95-kDa yeast vacuolar H (+)-ATPasesubunit, Vph1p (39%), and a 116-kDa proton pump(VPP1) (54%) of the rat and bovine synaptic vesicle.Therefore, it is possible that ShIF also acts as a protonpump and somehow prevents the cells from under-going apoptosis. We are currently examining theeffects of ISF and ShIF on the growth of hematopoie-tic progenitor cells from bone marrow, and alsotrying to identify the molecules that interact withthese factors.

7. Molecular mechanism of stem cell self renewalon bone marrow stroma

Hideaki Nakajima, Yuko Koshino, Yuseok Moonand Toshio Kitamura

Hematopoietic stem cells (HSC) keep self-renew-ing in the bone marrow in order to supportcontinuous blood cell production. These processesare thought to occur in the bone marrow niche, a spe-cial microenvironment created by stromal cells.HSC-stromal cell interaction is thought to provideunknown signals to keep HSC in immature state andmakes them undergo extensive self-renewal. How-ever, molecular mechanism of these processes ispoorly understood. We are trying to address thisquestion by following approaches. 1) Identify cellsurface molecules that are expressed on stromal cellsand important for HSC self-renewal by utilizing avariety of technologies (i. e. signal sequence trap,mRNA subtraction) and analyze their function in vit-ro and in vivo. 2) Identify genes that are induced inHSC by contacting with stromal cells. These genesare strong candidates that are involved in the self-renew processes evoked by stromal cell contact. Weare now characterizing two novel molecules that arespeculated to be important for these processes.

8. Role of co-repressors in STAT5-dependenttranscription

Hideaki Nakajima, Paul K. Brindle8, Makoto Han-da9, and James N. Ihle8: 8Department ofBiochemistry, St Jude Children's Research Hospi-tal, 9Blood Center, Keio University School ofMedicine

STAT5 is a latent transcription factor activated bya variety of cytokines including IL-3, GM-CSF anderythropoietin. To gain more insight into the molecu-lar mechanism how STAT5 regulates the variety ofcytokine responses, we set out to explore the proteins

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that interact with STAT5 in vivo. We employed yeasttwo-hybrid screening to look for STAT5-interactingmolecules, and identified silencing mediator for ret-inoid and thyroid hormone receptor (SMRT) as apotential binding partner. SMRT interacted withboth STAT5A and 5B, and the association was detect-ed both in vitro and in vivo. Interestingly, SMRTstrongly repressed STAT5-dependent transcriptionboth on heterologous and native promoters in re-porter assays. To further clarify the physiologicalrole of this interaction, we created a stable cell lineoverexpressing SMRT. In clear contrast to parentalcell line, expression of STAT5 target genes in this cellline was not sustained and was quickly suppressedwithin 2 hours after normal initial phase of induc-tion. Conversely, histone deacetylase inhibitor,Trichostatin A effectively enhanced and prolongedinduction of STAT5-target genes in a parental cellline. Extensive mutational and binding analysis re-vealed that this interaction was mediated throughN-terminal coiled-coil region of STAT5. Surprising-ly, previously identified point mutation in thecoiled-coil region that makes STAT5 hyperactive,disrupted Stat5-SMRT interaction, suggesting over-all transcriptional activity of STAT5 is determined bythe balance of coactivators and corepressors boundto it. In addition, above data predicts that naturallyoccurring dominant negative mutant of STAT5 lack-ing carboxyl-transactivation domain interacts onlywith SMRT, but not with CBP, suggesting that it actsas dominant negative by actively repressing targetgenes through SMRT. Together, this study illumi-nates the potential role of SMRT in negativeregulation of STAT5-dependent transcription, andreveals a novel crosstalk between nuclear receptorand JAK-STAT signaling pathways.

9. The role of CCAAT/ enhancer-binding protein εεεεεin normal hematopoiesis and leukemogenesis

Hideaki Nakajima, Naohide Watanabe9, MakotoHanda9, Yasuo Ikeda10, James N Ihle8 ScottKogan11, Grant McArthur12 and Toshio Kitamura:10Division of Hematology, Department of InternalMedicine, Keio University School of Medicine,11Comprehensive Cancer Center, University of Cal-ifornia, San Francisco, 12Division of Hematology/Medical Oncology, Peter MacCallum Cancer Insti-tute,

Granulocyte colony-stimulating factor (G-CSF) isa major cytokine that regulates proliferation and dif-ferentiation of myeloid cells , although theunderlying mechanisms by which G-CSF controlsmyeloid differentiation are largely unknown. Differ-entiation of hematopoietic cells is regulated bylineage-specific transcription factors, and gene-tar-geting studies previously revealed the critical rolesof CCAAT/enhancer-binding protein C/EBPα and

C/EBPε, respectively, in the early and mid-late stag-es of granulocyte differentiation. The expression ofC/EBPε in 32Dcl3 cells and FDCP1 cells expressingmutant G-CSF receptors was examined and it wasfound that G-CSF up-regulates C/EBPε. The signalfor this expression required the region containing thefirst tyrosine residue of G-CSF receptor. Dominant-n e g a t i v e S T A T 3 b l o c k e d G - C S F - i n d u c e dgranulocytic differentiation in 32D cells but did notblock induction of C/EBPε, indicating that these pro-teins work in different pathways. It was also foundthat overexpression of C/EBPε greatly facilitatedgranulocytic differentiation by G-CSF and, surpris-ingly, that expression of C/EBPε alone was sufficientto make cells differentiate into morphologically andfunctionally mature granulocytes. Overexpression ofc-myc inhibits differentiation of hematopoietic cells,but the molecular mechanisms of this inhibition arenot fully understood. In 32Dcl3 cells overexpressingc-myc that do not differentiate by means of G-CSF,induction of C/EBPε is completely abrogated. Ectop-ic expression of C/EBPε in these cells inducedfeatures of differentiation, including changes in nu-clear morphologic character is t ics and theappearance of granules. The data show that C/EBPεconstitutes a rate-limiting step in G-CSF-regulatedgranulocyte differentiation and that c-myc antago-nizes G-CSF-induced myeloid differentiation, atleast partly by suppressing induction of C/EBPε.

Acute promyelocytic leukemia is characterized bythe balanced taranslocation t(15;17), which generatesPML-RARα fusion protein. This fusion protein isthought to affect key differentiation pathway of nor-mal myeloid development, one of which is C/EBPε.We employed PML-RARα transgenic mouse modelto show that restoration of C/EBPε expression canrevert leukemic phenotype of these mice. These ob-servations reveal that C/EBPε is a critical target ofPML-RARα and suggest that targeted modulation ofC/EBP activities could provide a new approach totherapy of AML

10. Identification of factor(s) supporting self-re-newal of primate embryonic stem cells

Takuya Sugiyama3, Atsushi Urano, Tetsuya Nosa-ka3, Hideaki Nakajima and Toshio Kitamura

Dissection of molecular nature of embryonic stem(ES) cells may promote our understanding of cellularpluripotency and inner cell mass (ICM) develop-ment, and also can assist ES-based t issueengineering. Both mouse and human ES cells requirefeeder layer cells to retain the undifferentiated state.Whereas mouse ES cells were reported to remain un-differentiated without feeder cells in the presence ofleukemia inhibitory factor (LIF), primate ES cellswithout feeder cells do differentiate even in the pres-ence of LIF. Our goal is to identify the feeder-derived

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factor(s) supporting undifferentiated state andgrowth of the primate ES cells, through cDNA ex-pression cloning. This project is in collaboration withDrs. Suemori and Nakatsuji at Kyoto University.

11. Whole embryo culture (WEC) analysis on he-matopoietic cell development

Daisuke Sugiyama, Feng Ma, Kohichiro Tsuji

Although precursors with the potential to gener-ate definitive hematopoietic stem cells (HSC) appearindependently in yolk sac (YS) and intraembryonicparaaortic splanchnopleura (P-Sp) as mentionedabove, it remains unanswered whether both early YSand P-Sp contribute to definitive hematopoiesis intheir circumstances in vivo. To address this issue, wedeveloped an embryo-grafting system using WEC. Inthis system, whole embryos from 8. 25 days post-coi-tum (dpc) , a t ime before the formation ofomphalomesenteric artery which connect betweenYS and embryo proper, could be cultured to 11. 0 dpcafter its formation. We first investigated whether he-matopoietic cell development of embryos in WECcan compare with normal one. We isolated and dis-pased AGM region from the embryos after WEC, andperformed fetal thymus organ culture (FTOC) assayand co-culture with OP9 stromal cells to detect T andB lymphoid and hematopoietic potentials. After 2. 75days of WEC, lymphohematopoietic progenitors ex-isted in AGM region of the cultured embryo as wellas normal 11. 0 dpc embryo. We then made YS-YSchimera embryos at 8. 25 dpc. When the chimera em-bryos were cultured in WEC, vigorous blood flowwas formed within the YS graft. The developed sys-tem may provide a useful tool for analysis ofhematopoieitc cell development , especially its ori-gin.

12. Erythropoiesis from acetylated low-densityprotein (Ac-LDL)-incorporating endothelialcells into circulation at pre-liver stage.

Daisuke Sugiyama, Kohichiro Tsuji

Erythropoiesis is characterized by two distinctwaves of production during mouse embryogenesis: aprimitive one originating from YS and a definitiveone produced from both the YS and the embryoproper. How this last wave is generated remains un-clear. We put forward the hypothesis that erythroidcells could be generated by endothelial cells (ECs). Toinvestigate this problem, we have designed a methodto label ECs at 10 dpc. This label associates two tech-niques: an intracardiac inoculation that allowsmolecules to be delivered into the blood stream, fol-lowed by a WEC period. DiI-conjugated acetylatedlow-density lipoprotein (Ac-LDL-DiI) was used to

specifically tag ECs from the inside. One hour afterinoculation, DiI staining was found along the entireendothelial tree. Flow cytometric analysis revealedthat DiI+ cells were CD31+, CD34+ and CD45-, an an-tigen make-up characteristic for the endotheliallineage. Twelve hours after inoculation, 43% of DiI+

circulating cells belonged to the erythroid lineage.These cells expressed Ter 119 and displayed an adultglobin chain arrangement, thus belonged to the de-finitive lineage as confirmed in erythroid colonyformation. The rest of the cells likely represent com-mitted white blood cells or multi-potent progenitorsas revealed by a mix-colony formation. Beyond the29-somite stage, the proportion of the DiI+ erythroidcells gradually decreased. These results demon-strate, for the first time in the mouse embryo, thegeneration of hematopoietic cells from an endothe-lial intermediate, using an in vitro tracing. We thusprovide evidence for a release of these cells are ableto colonize the fetal liver and generate definitiveerythrocytes in vivo.

13. Development of human lymphohematopoie-sis defined by CD34 and CD81 expressions.

Feng Ma, Kohichiro Tsuji

Cord blood (CB) CD34+ cells expressed CD81, amember of transmembrane 4 superfamily, and wereclassified into three subpopulations based on theirexpression levels: CD34+CD81+, CD34LowCD81+ andCD34+CD81High cells. We then examined the lym-phohematopoietic activity of each subpopulation,using suspension and clonogenic cultures for he-matopoietic potential, coculture with MS-5 cells for Bcell potential, NOD/SCID mouse fetal thymus organculture for T cell potential, coculture with NOD/SCID mouse fetal liver-derived stromal cells for nat-ural killer (NK) cell and mast cell potentials, andxenotransplantation into NOD/SCID mice for long-term repopulating ability. CD34+CD81+ cellsrepresented a heterogeneous population that had alllymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34LowCD81+

cells were more enriched in erythroid, megakaryo-cytic and NK lineage potentials but already lost Tand B potentials and long-term repopulating ability.CD34+CD81High fraction had depleted of most lym-phohematopoietic potential except for NK cell andmast cells potentials. Thus, along the differentiationcascade from CD34+CD81+ lymphohematopoieticstem cells, an up-regulation of CD81 or a down-regu-l a t i o n o f C D 3 4 m e a n s t h e c h a n g e o flymphohematopoietic property. CD81 may serve asa marker to define developmental stages of lympho-hematopoietic stem cells.

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14. Reconstitution of human hematopoiesis inNOD/SCID mice by clonal cells expanded fromsingle CD34+CD38- cells expressing Flk2/Flt3

Yasuhiro Ebihara, Atsushi Manabe, Mika Wada,Kohichiro Tsuji,

In the present study, we examined the expressionof Flk2/Flk3, a tyrosine kinase receptor, on humanCB CD34+ hematopoietic progenitor/stem cells. Inflow cytometric analysis, Flk2/Flt3 was expressed onfour fifths of CD34+ cells and their immature sub-populations, CD34+CD33- and CD34+CD38- cells.Methycellulose clonal culture of sorted Lin-

CD34+Flk2/Flt3+ and Lin-CD34+Flk2/Flt3- cellsshowed that most of myelocytic progenitors ex-pressed Flk2/Flt3, but erythroid and hematopoieticmultipotential progenitors were shared by both frac-tions. When 1 x 104 Lin-CD34+Flk2/Flt3- cells weretransplanted into four NOD/SCID mice, no recipi-ents possessed human CD45+ cells in bone marrow11 to 12 weeks after the transplantation. By contrast,all of four recipients transplanted with 1 x 104 Lin-

CD34+Flk2/Flt3+ cells showed a successfulengraftment. Furthermore, clonal cells expandedfrom single Lin-CD34+CD38-Flk2/Flt3+ cells in theculture with Flk2/Flt3 ligand (FL), stem cell factor(SCF), thrombopoietin (TPO), and a complex of IL-6/soluble IL-6 receptor (IL-6/sIL-6R) were individual-ly transplanted into NOD/SCID mice. Twenty to 21weeks after the transplantation, 3 of 10 clones har-vested at day 7 of culture, and 3 of 6 clones at day 14could reconstitute human hematopoiesis in recipientmarrow. These results demonstrated that Flk2/Flt3was expressed on a wide variety of human hemato-poietic cells including long term-repopulatinghematopoietic stem cells.

15. TEK expression and hematopoietic and an-giogenic potentials in cord blood CD34+ cells.

Mika Wada, Yasuhiro Ebihara, Feng Ma, MiyukiIto, Kohichiro Tsuji

Tunica interna endothelial cell kinase (TEK) is ex-pressed on commonly expressed on bothhematopoietic and endothelial cells, and plays somecrucial roles in mouse development. In human, how-ever, little has been known about the hematopoieticand angiogenic ability of TEK-expressing cells in CBcells, which originate from human fetus period. Wethen compared the hematopoietic and angiogenicability between CB CD34+TEK+ and CD34+TEK-

cells, using clonogenic assay and xenotransplanta-tion into NOD/SCID mice. The result showed that

colony-forming cells and cells capable of repopulat-ing in NOD/SCID mice were present in bothCD34+TEK+ and CD34+TEK- cells, and their hemato-poietic activities were similar. By contrast, thepotential to differentiate into endothelial cells in vivowas greater in the former. All the seven NOD/SCIDmice engrafted with CD34+TEK+ cells had humanCD31 and VE-cadherin-expressing endothelial cellsin vessels of ischemic musles and/or human CD31,KDR and ecNOS-expressing endothelial cells in liversinusoidal cells, while such endothelial cells were de-tected in only three of the seven recipients engraftedwith CD34+TEK- cells. The present result has impor-tant impl ica t ions in ce l lu lar therapy forhematopoietic disorders and vascular diseases usingCB cells.

16. Impaired neutrophil maturation in the truncat-ed mouse granulocyte colony-stimulatingfactor (G-CSF) receptor-transgenic mice.

Tetsuo Mitsui, Sumiko Watanabe13, Sachiyo Hana-da, Yasuhiro Ebihara, Kohichiro Tsuji: 13Divisionof Molecular and Developmental Biology, IMSUT

Severe congenital neutropenia (SCN) is a hemato-poietic disorder characterized by neutropenia inperipheral blood and maturation arrest of neutrophilprecursors in bone marrow. Patients with SCN mayevolve to have myelodysplastic syndrome or acutemyelocytic leukemia. In approximately 20% of SCNcases, a truncation mutation is found in the cytoplas-mic region of the G-CSF receptor (G-CSFR). We thengenerated mice carrying murine wild type G-CSFRand its mutants equivalent to truncations at aminoacids 718 and 731 in human G-CSFR, those were re-ported to be related to leukemic transformation ofSCN. Although numbers of peripheral white bloodcells, red blood cells and platelets had no differenceamong mutant and wild type G-CSFR transgenic(Tg) mice, both of the mutant receptor Tg mice hadone third of peripheral neutrophil cell counts com-pared to wild type receptor Tg mice. The mutantreceptor Tg mice also showed impaired resistance tothe infection with Staphylococcus aureus. Moreover,bone marrow of these Tg mice had an increased per-centage of immature myeloid cells, a feature of SCN.This maturation arrest was also observed in in vitrocultures of bone marrow cells of truncated G-CSFRTg mice under G-CSF stimulation. In addition, clonalculture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSFin myeloid progenitors. Our Tg mice may be usefulin the analysis of the role of truncated G-CSFR inSCN pathobiology.

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Kitamura, T., Kawashima, T., Minoshima, Y.,Tonozuka, Y., Hirose, K., and Nosaka, T. Role ofMgcRacGAP/CYK4 as a regulator of the smallGTPase Rho family in cytokinesis and cell differ-entiation. Cell Structure and Function, 26:645-651, 2002.

Tulin, E. E., Onoda, N., Hasegawa, M., Nosaka, T.,Nomura, H. and Kitamura, T. Genetic approachand phenotype-based complementation screeningfor identification of stromal cell-derived proteinsinvolved in cell proliferation. Exp. Cell Res.272:23-31, 2002.

Tulin, E. E., Onoda, N., Hasegawa, M., Nomura, H.and Kitamura, T. Inhibition of human endothelialcell proliferation by ShIF, a vacuolar H+-ATPAse-like protein. Oncogene 21:844-848, 2002.

Sai, X., Kawamura, Y., Kokame, K., Yamaguchi, H.,Shiraishi, H., Suzuki, R., Suzuki, T., Kawaichi, M.,Miyata, T., Kitamura, T., De Strooper, B.,Yanagisawa, K., and Komano, H. Endoplasmicreticulum stress-inducible protein, Herp, en-hances presenilin-mediated generation of amyloidbeta-protein. J. Biol. Chem. 277:12915-12920, 2002.

Nosaka, T. and Kitamura, T. Pim-1 expression is suf-ficient to induce cytokine independence in murinhematopoietic cells, but is dispensable for BCR-ABL-mediated transformation. Exp. Hematol.30:697-702, 2002.

Nakamura, T., Ouchida, R., Kodama, T., Kawashima,T., Watanabe, S., Morimoto, C., Kitamura, T., andTanaka, H. Cytokine receptor common β subunit-mediated STAT5 activation confers NFκB activa-tion in murine pro B cell line Ba/F3 cells. J. Biol.Chem. 277:6254-6265, 2002.

Perez, O. D., Kinoshita, S., Hitoshi, Y., Payan, D. G.,Kitamura, T., Nolan, G. P. and Lorens, J.Acitivation of the PKB/AKT pathway by ICAM-2.Immunity 16:51-65, 2002.

Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M.,Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A.,Kimoto, M., and Miyake, K. Essential role of MD-2in LPS responsiveness and TLR4 distribution. Nat.Immunol. 3:667-672, 2002.

Gugasyan, R., Quilici, C., I, S. T., Grail, D., Verhagen,A. M., Roberts, A., Kitamura, T., Dunn, A. R., andLock, P. Dok-related protein negatively regulatesT cell development ts RasGTPase activating pro-tein and Nck docking sites. J. Cell Biol. 158:115-125, 2002.

Stoeclin, G., Colombi, M., Raineri, I., Leuenberger, S.,Mallaun, M., Schmidlin, M., Gross, B., Lu, M.,Kitamura, T., and Moroni, C. Functional cloning ofBRF-1, a regulator of ARE-dependent mRNAturnover. EMBO J. 21:4709-4718, 2002.

Ezoe, S., Matsumira, I., Ishihara, K., Minegishi, N.,Takahashi , S . , Machii , T . , Kitamura, T. ,

Yamamoto, M., Enver, T., and Kanakura, Y.GATA-2 affects cytokine-dependent growth of he-matopoietic cells through accumulation ofp21WAF1 and p27Kip1 proteins. Blood 100:3512-3520, 2002.

Komano, H., Kawamura, Y., Shiraishi, H., Sai, X.,Suzuki, R., Kawaichi, M., Kitamura, T., andYanagisawa, K. A new functional screening sys-tem for the identification of DNA encoding a regu-lator of γ-cleavage. J. Biol. Chem. 277:39627-39633,2002.

Yujiri, T., Nawata, R., Takahashi, T., Sato, Y.,Tanizawa, Y., Kitamura, T., and Oka, Y. MEK ki-nase 1 interacts with focal adhesion kinase andregulates insulin receptor substrate-1 expression.J. Biol. Chem. in press.

Bao, Y-C., Tsuruga, H., Hirai, M., Kitamura, T., andKumagai, H. Identification of a human cDNA se-quence which encodes a novel membrane-associ-ated protein containing a zinc metalloproteasemotif. DNA Research in press.

Piekorz RP, Hoffmeyer A, Duntsch CD, McKay C,Nakajima H, Sexl V, Snyder L, Rehg J, Ihle JN. Thecentrosomal protein TACC3 is essential for he-matopoietic stem cell function and genetically in-terfaces with p53-regulated apoptosis. EMBO J.21:653-64, 2002

Carpino N, Kobayashi R, Zang H, Takahashi Y, JouST, Feng J, Nakajima H, Ihle JN. Identification,cDNA cloning, and targeted deletion of p70, anovel, ubiquitously expressed SH3 domain-con-taining protein. Mol Cell Biol. 22:7491-500, 2002.

Truong BT, Lee YJ, Lodie TA, Park DJ, Perrotti D,Watanabe N, Koeffler HP, Nakajima H, Tenen DG,Kogan SC. CCAAT/enhancer binding proteins re-press the leukemic phenotype of acute myeloidleukemia. Blood. in press.

Ueno H, Sakita-Ishikawa M, Morikawa Y, Nakano T,Kitamura T, Saito M: A stromal cell-derived mem-brane protein that supports hematopoietic stemcells. Nature Immunology, in press.

Minoshima Y, Kawashima T, Hirose K, Tonozuka Y,Kawajiri A, Bao Y-C, Deng X, Tatsuka M,Narumiya S, May Jr. SW, Nosaka T, Semba K,Inoue T, Satoh T, Inagaki M, Kitamura T: Phos-phorylation of Mgc Rac GAP by Aurora B Kinaseleads to induction of latent GAP activity towardRho A during mitosis; identification of a Rho GAPindispensable for the completion of cytokinesis.Developmental Cell, in press.

Ito M, Kobayashi K, Suzue K, Kawahata M, Hioki K,Ueyama Y, Koyanagi Y, Sugiyama K, Tsuji K,Hiramatsu H, Heike T, Nakahata T: NOD/SCID/gcnull mouse: A novel excellent recipient mousefor engraftment of human cells. Blood 100: 3175-3182, 2002.

Publications

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Ebihara Y, Wada M, Ueda T, Manabe A, Tanaka R,Mugishima H, Ito M, Mugishima H, Asano S,Nakahata T, Tsuji K: Reconstitution of human he-matopoiesis in NOD/SCID mice by clonal cellsexpanded from single CD34+CD38- cells express-ing Flk2/Flt3. Br J Haematol 119: 525-534, 2002.

Tsuji K, Ueda T: Protocol for cytokine mediated ex-pansion of human NOD/SCID-repopulating cells.Methods in Molecular Medicine-Cytokines andColony Stimulating Factors. edited by Korholz Dand Kiess W (The Human Press, Totowa), 387-395,2002.

Mitsui T, Watanabe S, Hanada S, Ebihara Y, Sato T,Nakahata T, Tsuji K: Impaired neutrophil matura-tion in the truncated mG-CSF receptor-transgenicmice. Blood, in press.

Sugiyama D, Hirose I, Arai K, Tsuji K: Erythropoiesisfrom acetyl LDL-incorporating endothelial cells

into circulation at pre-liver stage. Blood, in press.Wada M, Ebihara Y, Ma F, Yagasaki H, Ito M,

Mugishima H, Takahashi T, Tsuji K: TEK expres-sion and hematopoietic and angiogenic potentialsin cord blood CD34+ cells. Int J Hematol, in press.

Tsuji K, Ma F, Wang D: Development of humanlymphohematopoiesis defined by CD34 and CD81expressions. Leukemia & Lymphoma, in press.

北村俊雄（2002）レトロウイルスを利用したサイトカインシグナル伝達機構の解析　臨床免疫 37:72-77.

中島秀明、北村俊雄（2002）受容体／シグナル伝達　造血幹細胞　基礎から遺伝子治療、再生医療へ（中外医学社）p53-65.

殿塚行雄、北村俊雄（2002）Thymic Stromal Lymphopoietin（TSLP）臨床免疫 37:376-380.

北村俊雄（2002）サイトカイン受容体概論　サイトカインがわかる（宮島篤　編　羊土社）p20-29.

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Advanced Clinical Research Center

Division of Infectious Diseases感染症分野

1. Analysis of human immunodeficiency virustype 1 Nef, focusing on the proline-rich do-main using a Sendai virus vector.

Takeshi Yamada et al.

Human immunodeficiency virus type 1 (HIV-1)Nef protein plays a role in the down-regulation ofhuman-leukocyte antigen class I (HLA-I) moleculeson the surface of T cells, which is likely to be relatedwith its evasion from cytotoxic T lymphocytes(CTLs). To investigate in detail such a function ofNef, we have designed Sendai viruses (SeVs) to high-ly and readily express recombinant proteins in thefloating cells, for example, human T-lymphocytes.All of the generated recombinant SeVs achieved highlevels of gene expression in CEM cells by 24 hours ofculture after infection at a multiplicity ofinfection(MOI) of 10. By flow cytometric analysis, itwas confirmed that HLA-class I and CD4 moleculeson CEM cells were efficiently down-regulated byNef. We applied this new system using recombinantSeVs to analyze the proline-rich domain (amino acid69-78)of Nef by site-directed mutagenesis. As a re-sult, we elucidated that the amino acid proline atposition 78(Pro-78) was the most crucial amino acidin the downregulation of HLA-class I. However, theconversion of Pro-78 into alanine did not alter thedown-regulation of CD4 molecules. These resultssuggest that a single amino acid, Pro-78 in Nef distin-guishes the down-regulation of HLA-class I fromCD4.

2. Immunogene therapy for AIDS by presenting aCTL-epitope peptide with Sendai virus vec-tors.

Ai Kawana-Tachikawa et al.

We study a Sendai virus (SeV) vector system forexpression of major histocompatibility complex(MHC) class I/peptide complexes, which may be aneligible therapeutic molecules in immunogene thera-py. We cloned the extracellular domain of a humanMHC class I heavy chain, HLA-A*2402, and humanbeta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virustype 1 (HIV-1) cytotoxic T-lymphocyte (CTL)epitopes (e-beta2m) in separate SeV vectors. Whenwe coinfected nonhuman mammalian cells with theSeVs, naturally folded human MHC class I/peptidecomplexes were secreted in the culture supernatants.Biotin binding peptide sequences on the C terminusof the heavy chain were used to tetramerize the com-plexes. These tetramers made in the SeV systemrecognized specific CD8-positive T cells in peripher-al blood mononuclear cells of HIV-1-positivepatients with a specificity and sensitivity similar tothose of MHC class I tetramers made in an Escheri-chia coli system. Solo infection of e-beta2m/SeVproduced soluble e-beta2m in the culture superna-tant, and cells pulsed with the soluble protein wererecognized by specific CTLs. Furthermore, whencells were infected with e-beta2m/SeV, these cellswere recognized by the specific CTLs more efficient-

Main subjects of the Division of Infectious Diseases are human immunodeficiencyvirus (HIV) infection and related disorders

教　授 医学博士　 岩　本　愛　吉助教授 医学博士　 北　村　義　浩助　手 医学博士　 高　橋　　　孝助　手 医学博士　 小田原　　　隆

Professor Aikichi Iwamoto, M.D., D.M.Sc.Associate Professor Yoshihiro Kitamura, M.D., Ph.D.Research Associate Takashi Takahashi, M.D., D.M.Sc.Research Associate Takashi Odawara, M.D., Ph.D.

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ly than the protein pulse per se. SeV is nonpathogen-ic for humans, can transduce foreign genes intonondividing cells, and may be useful for immuno-therapy to enhance antigen-specific immuneresponses. Our system can be used not only to detectbut also to stimulate antigen-specific cellular im-mune responses.

3. Dihydrofolate Reductase Gene Polymor-phisms in Pneumocystis carinii f. sp. hominisin Japan

Takashi Takahashi et al.

Pneumocystis carinii f. sp. hominis (P. carinii) is animportant causative pathogen of morbidity and mor-tality in immunocompromised patients with humanimmunodeficiency virus type-1 infection, hemato-logical malignancies, organ transplantation state orconnective tissue diseases. We examined polymor-phisms in the dihydrofolate reductase (DHFR) geneof Pneumocystis carinii isolates from 27 patientswith P. carinii pneumonia in Japan. Four substitutionsites with two synonymous and two non-synony-mous changes were found. Two synonymoussubstitutions at nucleotide positions 540 and 312were identified in one and 13 patients, respectively.Two amino acid substitutions (Ala67Val, Cysl66Tyr)were found in two different patients. No linkage ofamino acid substitutions in DHFR to those in dihy-dropteroate synthase was observed. The twopatients whose isolates showed non-synonymousDHFR mutations were not exposed to DHFR inhibi-tors before they developed PCP and were treatedsuccessfully with co-trimoxazole.

4. Polymorphisms and haplotypes of the humangene and their association with the clinicalcourses of HIV-positive Japanese patients.

Noriko Kobayashi and Hitomi Taguchi-Nakamuraet al.

The human liver-specific intercellular adhesionmolecule 3 (ICAM-3) -grabbing nonintegrin protein(CD209L, L-SIGN, DC-SIGNR) is a type II membraneprotein homologous to the dendritic cell (DC)-specificcounterpart (CD209, DC-SIGN) and is expressed inliver and lymph nodes but not in DCs. Reportedly,CD209 is required for activation of resting T cellsthrough its binding to ICAM-3, which stabilizes the

DC/T-cell contact zone. CD209L may have a similarfunction. CD209L as well as CD209 were shown tocapture human immunodeficiency virus type I (HIV)virions by binding HIV gp120 with carbohydrate rec-ognition domains (CDRs) and to transmit them to Tlymphocytes. Therefore, the genetic polymorphismsof CD209L and CD209 may affect HIV pathogenesis.Studies to test such a hypothesis have not been con-ducted, so far, at least on CD209L. The human CD209Lgene consists of 8 exons and spans about 6. 5 kb onchromosome 19. Exon 4 encodes the neck domain andexons 5-7 CRDs. IMSUT/JST (http://snp. ims. u-to-kyo. ac. jp) has discovered 5 single-nucleotidepolymorphisms (SNPs; one in intron 4, one in exon 5,and three in intron 5) in the CD209L gene in a Japanesepopulation (IMS-JST025120 to IMS-JST025124).Soilleux et al. described a variable-number-of-tan-dem-repeats (VNTR) of a 69 bp-sequence in exon 4;this VNTR results in 23 aa repeats in the neck domainof CD209L. We typed the 5 SNPs in DNA samplesfrom 59 HIV-infected patients. We also typed theVNTR polymorphism in the same samples. Among 44HIV-infected patients, whose CD4+ cells were count-ed on more than three occasions before the start ofanti-retroviral treatment, (5 untreated, and 54 treated),the A allele in intron 5 at IMS-JST025124 was associat-ed with the higher number of lowest CD4+ cell countsduring untreated periods (statistical significance: A/A vs. G/G and G/A, P = 0. 0069 by Mann-Whitney's UTest).

We also look for association of frequency and ex-tent of adverse effects by anti-retroviral drugs withpolymorphisms of gene involved in lipid metabo-lism and drug transporters.

5. Dynamics of HIV variations in HIV-infected Jap-anese patients

Tae Furutuki and Noriaki Hosoya et al.

Deciphering the dynamics of HIV variation willbring deeper understanding of the mechanisms thatgenerate viral diversity. Because HLA-A24 is themost common HLA genotype among Japanese, weare investigating HLA-A24-restriction in cytotoxic T-cell (CTL) response with this aim. From clinicalisolates we are cloning and sequencing part of nef,env, and pol regions that encode CTL epitopes re-stricted by HLA-A24. We are likely to be findingsome evidence of immunological pressure involvedin viral evolution in human individuals.

Ae, K., N. Kobayashi, R. Sakuma, T. Ogata, H.Kuroda, N. Kawaguchi, K. Shinomiya, and Y.Kitamura. 2002. Chromatin remodeling factor en-

coded by ini1 induces G1 arrest and apoptosis inini1-deficient cells. Oncogene 21:3112-20.

Endo, T., T. Takahashi, M. Suzuki, F. Minamoto, M.

Publications

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and A. Iwamoto. 2001. Acute parvovirus B19 in-fection during anti-retroviral therapy. J InfectChemother 7:110-2.

Takahashi, T., T. Endo, T. Nakamura, H. Sakashitat,K. Kimurat, K. Ohnishit, Y. Kitamura, and A.Iwamoto. 2002. Dihydrofolate reductase genepolymorphisms in Pneumocystis carinii f. sp.hominis in Japan. J Med Microbiol 51:510-5.

Takahashi, T., M. Goto, T. Endo, T. Nakamura, N.Y u s a , N . S a t o , a n d A . I w a m o t o . 2 0 0 2 .P n e u m o c y s t i s c a r i n i i c a r r i a g e i nimmunocompromised patients with and withouthuman immunodeficiency virus infection. J MedMicrobiol 51:611-4.

Takahashi, T., A. Hitani, H. Yamada, T. Nakamura,and A. Iwamoto. 2001. Desenitization tof l u c o n a z o l e i n a n A I D S p a t i e n t . A n nPharmacother 35:642-3.

Tobiume, M., M. Takahoko, T. Yamada, M. Tatsumi,A. Iwamoto, and M. Matsuda. 2002. Inefficient en-hancement of viral infect ivi ty and CD4downregulation by human immunodeficiency vi-rus type 1 Nef from Japanese long-termnonprogressors. J Virol 76:5959-65.

Watanabe, N., M. Tomizawa, A. Tachikawa-Kawana,M. Goto, A. Ajisawa, T. Nakamura, and A.Iwamoto. 2001. Quantitative and qualitative ab-normalities in HIV-1-specific T cells. Aids 15:711-5.

Xin, X., K. Nakamura, H. Liu, E. E. Nakayama, M.Goto, Y. Nagai, Y. Kitamura, T. Shioda, and A.Iwamoto. 2001. Novel polymorphisms in humanmacrophage inflammatory protein-1 alpha (MIP-1alpha) gene. Genes Immun 2:156-8.

Yamada, H., H. Kotaki, T. Nakamura, and A.Iwamoto. 2001. Simultaneous determination ofthe HIV protease inh ib i tors ind inav i r ,amprenavir, saquinavir, ritonavir and nelfinavirin human plasma by high-performance liquidchromatography. J Chromatogr B Biomed SciAppl 755:85-9.

Yamada, T., N. Kaji, T. Odawara, J. Chiba, A.Iwamoto, and Y. Kitamura. 2003. Proline 78 IsCrucial for Human Immunodeficiency Virus Type1 Nef To Down-Regulate Class I Human Leuko-cyte Antigen. J Virol 77:1589-1594.

Yamamoto, Y., T. Takasaki, K. Yamada, M. Kimura,K. Washizaki, K. Yoshikawa, A. Hitani, T.Nakamura, and A. Iwamoto. 2002. Acute dissemi-nated encephalomyelitis following dengue fever. JInfect Chemother 8:175-7.

Goto, K. Okuzumi, N. Oyaizu, T. Nakamura, andA. Iwamoto. 2001. Mycobacterium haemophiluminfection in a Japanese patient with AIDS. J InfectChemother 7:186-90.

Ikegawa, M., J. Yuan, K. Matsumoto, S. Herrmann,A. Iwamoto, T. Nakamura, S. Matsushita, T.Kimura, T. Honjo, and K. Tashiro. 2001. Elevatedplasma stromal cell-derived factor 1 protein levelin the progression of HIV type 1 infection/AIDS.AIDS Res Hum Retroviruses 17:587-95.

Kawana-Tachikawa, A., M. Tomizawa, J. Nunoya, T.Shioda, A. Kato, E. E. Nakayama, T. Nakamura, Y.Nagai, and A. Iwamoto. 2002. An efficient and ver-satile mammalian viral vector system for majorhistocompatibility complex class I/peptide com-plexes. J Virol 76:11982-8.

Kobayashi, N., H. T. Nakamura, M. Goto, T.Nakamura, K. Nakamura, W. Sugiura, A.Iwamoto, and Y. Kitamura. 2002. Polymorphismsand haplotypes of the CD209L gene and their as-sociation with the clinical courses of HIV-positiveJapanese patients. Jpn J Infect Dis 55:131-3.

Koibuchi, T., A. Hitani, T. Nakamura, N. Nojiri, K.Nakajima, T. Jyuji, and A. Iwamoto. 2001. Pre-dominance of genotype A HBV in an HBV-HIV-1dually positive population compared with anHIV-1-negative counterpart in Japan. J Med Virol64:435-40.

Nakamura, H., T. Nakamura, M. Suzuki, F.Minamoto, N. Oyaizu, T. Shiba, M. Miyaji, and A.Iwamoto. 2002. Disseminated coccidioidomycosiswith intra- and paravertebral abscesses. J InfectChemother 8:178-81.

Nakayama, E. E., L. Meyer, A. Iwamoto, A. Persoz, Y.Nagai, C. Rouzioux, J. F. Delfraissy, P. Debre, D.McIlroy, I. Theodorou, and T. Shioda. 2002. Pro-tective effect of interleukin-4 -589T polymorphismon human immunodeficiency virus type 1 diseaseprogression: relationship with virus load. J InfectDis 185:1183-6.

Ohmine, T., T. Katsube, Y. Tsuzaki, M. Kazui, N.Kobayashi, T. Komai, M. Hagihara, T. Nishigaki,A. Iwamoto, T. Kimura, H. Kashiwase, and M.Yamashita. 2002. Anti-HIV-1 activities and phar-m a c o k i n e t i c s o f n e w a r y l p i p e r a z i n y lfluoroquinolones. Bioorg Med Chem Lett 12:739-42.

Sakuragi, J., A. Iwamoto, and T. Shioda. 2002. Disso-ciation of genome dimerization from packagingfunctions and virion maturation of human immu-nodeficiency virus type 1. J Virol 76:959-67.

Taguchi, H., T. Takahashi, M. Goto, T. Nakamura,

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Advanced Clinical Research Center

Division of Bioengineering臓器細胞工学分野

1. Development of innovative cancer therapy

a. Critical Involvement of Nitric Oxide Produc-tion in the Establishment of Tumor SpecificCellular Immunity Induced with Dendritic CellsActivated with Streptococcal Preparation OK-432.

Yong-kook Kim, Naoya Ichikawa, Hideaki Tahara

Dendritic cells (DCs) are potent professional anti-gen presenting cells and play a critical role in cellularimmunity. We have previously demonstrated thatspecific antitumor immune response can be inducedwith intratumoral (i. t.) injection of bone marrow-de-rived DCs genetically engineered with interleukin(IL)-12 genes. In this study, we examined whetherDCs stimulated with OK-432 could be used in thesame manner. In vivo administration of recombinantIL-12 causes NK cells and T cells to secrete IFN-γ andenhances the cytolytic functions against tumor cells.These cytokines, however, induce abundant secre-t ion of n i t r i c ox ide (NO) f rom ac t ivatedmacrophages, and appear to suppress cellular im-munity in murine system. OK-432, a penicillininactivated and lyophilized preparation of low-viru-lence strain (i. e. Su) of Streptococcus pyogenes, hasbeen clinically used as a potent biological modifiersfor treating cancer patients and is known to inducemultiple cytokines including IFN-γ and IL-12 in vitro.In tumor models of C57BL/6 mice inoculated withMCA205 (fibrosarcoma) or MC38 (colon adenocarci-

noma) intradermally, i. t. injection of bone marrow-derived DCs with or without OK-432 showedmarginal antitumor effects on day 7 established tu-mors. However, significant antitumor effects wereobserved when the tumor bearing mice were treatedwith the combination of DCs, OK-432, and N-nitro-L-arginine methyl ester (L-NAME), which inhibitsinducible nitric oxide synthase (iNOS)-mediated NOproduction. Furthermore, tumor specific and potentcytotoxic T lymphocytes (CTLs) could be obtainedfrom the splenocytes of the mice treated with thecombined therapy. In contrast, the tumors treatedwith DCs, OK-432, and N-nitro-D-arginine methylester (D-NAME), which is an inactive optical isomerof L-NAME and has no inhibitory effects on iNOS,showed only marginal effects at the levels similar tothose of mice treated with DCs with or without OK-432 alone. These results suggest that tumor specificcellular immunity in our murine models could bestrongly suppressed by NO, which is abundantly se-creted by macrophages and DCs stimulated withOK-432. Thus, i. t. injection of DCs and OK-432 couldbe a promisingly cancer immunotherapy if NO pro-duction is properly regulated.

b. Immunomonitoring for cancer vaccine usingHLA- tetramer

Toshiyuki Baba, Takuya Tsunoda, Hiroyuki Mush-iake, Hideaki Tahara

The HLA tetramer is a powerful tool to quantita-

Our department has two major goals in basic research; 1) Development of innovativecancer therapy using immunologic approaches and gene therapy strategies, and 2)Mechanistic study on transplantation immunology to further develop clinical trans-plantation.

教　授 医学博士　 田　原　秀　晃講　師 医学博士　 角　田　卓　也助　手 医学博士 安　藤　裕　一

Professor　 Hideaki Tahara, M.D., D.M.Sc.Lecturer Takuya Tsunoda, M.D., D.M.Sc.Research Associate Yuichi Ando, M.D., D.M.Sc.

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tively evaluate the CTLs specific to antigen peptidepresented on particular HLA. Although HLA-A24 isthe most common MHC class I allele in Japanese,A24 tetramer assay has not been established yet. Weestablished CTL lines using CE3 that was nonamerpeptide encoded in CEA and CMVpp65 A24 peptide.We also prepared A24/CE3 and A24/CMV tetram-ers, and succeeded in evaluations the frequency ofCE3 and CMV peptide specific CTL using A24/CE3and A24/CMV tetramer sufficiently. Still more, weevaluated the frequency of antigen specific CTLs inthe PBMC from the melanoma patients treated withgp100 peptide vaccination.

c. Penicillin-killed Streptococcus pyogenes (OK-432) promote dendritic cell maturation neitherthrough TLR2 nor 4 but through beta 2 integrin

Saori Nakahara, Takuya Tsunoda, ToshiyukiBaba, Hideaki Tahara.

Dendritic cells (DC) are potent antigen presentingcells which has recently been used for cancer immu-notherapy using epitope peptides derived fromtumor rejection antigens. Accumulating results ofthe clinical trial of such strategy suggest that matura-tion of the DCs applied is one of the key factorswhich influence the outcome of the vaccination. Ithas been suggested that DCs need to have “mature”phenotype which is capable of inducing cytotoxic Tcells (CTL) efficiently. The characteristics of the ma-ture DCs (mDCs) include high expression of MHCand co-stimulatory molecules and the production ofIL-12. In this study, we examined the effects of peni-cillin-killed Streptococcus pyogenes (OK-432,clinical grade in Japan) on DC maturation. Further-more, we also examined the potency of OK-432stimulated DCs on the induction of CTLs specific tothe epitope peptide.

Peripheral blood mononuclear cells (PBMC) wereobtained from healthy donors, selected by the adher-ence, and cultured in AIM-V medium supplementedwith 1000U/ml of GM-CSF and 1000U/ml of IL-4 for5-7 days. Phenotypic analysis on them showed thatmore than 90% of prepared cells showed the immu-nophenotype consistent with immature DC (iDC).These iDC were divided into 4 groups and culturedfurther in AIM-V containing following agents; A.AIM-V alone, B. TNF-α (100 ng/ml), C. LPS (100 ng/ml), D. OK-432 (10µg/ml)(OK-DC). After 72 hours,cells were harvested and surface phenotypes and cy-tokine production using FACS and ELISArespectively. DCs in groups B, C, and D showed sig-nificantly higher CD83 expression (B, 85. 9%; C, 84.7%; D, 61. 0%) when compared with control, ( A,3.82%). Furthermore, DCs in group D showed signif-icantly higher production of IL-12 (40. 7 ± 3. 1ng/ml) and IFN-γ (1976. 8 ± 272. 6pg/ml) when com-pared with those of other groups. These results

indicate that OK-432 could promote the maturationof iDC to produce significant amount of Th1 type cy-tokines. To examine the influence of the OK-432 onthe induction of peptide specific CTLs, CE3 (HLA-A*2402 restricted 9 mer peptide derived fromCarcinoembryonic antigen, TYACFVSNL) was usedfor inducing peptide specific CTLs. The 51chromium-releasing assay and the tetramer assay of the CD8+ Tcells showed that highest cytotoxic activity and high-est CTL frequency were induced with OK-DCstimulation. Furthermore, we investigated the signal-ing pathway of OK-432 using the TLR indicator celllines and the blocking antibodies. These resultsshowed that OK-432 does not use either TLR2 orTLR4, but the β2 integrin for was the stimulation.These results strongly suggest that OK-432 could be auseful agent for peptide-based cancer vaccine usingDCs.

d. Development of novel chemoimmunotherapyusing S-1 and Lentinan

Hiroyuki Mushiake, Takuya Tsunoda, HideakiTahara

Cancer chemotherapy has limitations that it is dif-ficult to obtain survival benefit, even if the tumorregression was accomplished temporarily. Combineusage of biological response modifier with anti-can-cer drug, so called “chemoimmunotherapy”, hasbeen paid attention to these effects and benefits.However, little is known about the mechanisms. Thisinvestigation was conducted to clarify the mecha-nisms of synergistic effect of β-glucan, Lentinan, anda novel oral anti-cancer drug, S-1, in cancer cachexicmouse model. On the hypothesis that a β-glucan,Lentinan will be able to enhance the phagocyte effi-ciency of dendritic cells, we have been trying to breakthe peripheral T cell tolerance toward tumor self anti-gen, CEA, expressed by MC-38 stably transducedwith CEA in C57BL/6J mice transgenic for CEA.

e. In vivo electroporation of human FLT3-Ligandplasmid DNA induce anti-tumor specific immu-nity by mobilization of dendritic cells in situ.

Takuya Takayama, Kazuya Shimao, Hideaki Tahara

We have established the genetically modified DCto regulate the immune response. We have also fo-cused on Flt3-Ligand, a recently reported cytokine, isa stimulator for proliferation and differentiation ofDC not only in vitro but also in vivo. In this study, weevaluated the effects of Flt3-Ligand on DC mobiliza-tion, proliferation, maturation and immune responseusing in vivo electroporation (IVE). After Flt3-Ligandtransfection using IVE, significantly high level ofFlt3-Ligand was detected in the serum during 10days after IVE. The frequency of DC both in spleen

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and bone marrow significantly was increased afterFlt3-Ligand IVE when compared with those of con-trol group. In mouse tumor model, Flt3-Ligand IVEinduced anti-tumor effects that were associated withproliferation and mobilization of DC. These resultsimplied that Flt3-Ligand gene transfer using IVEcould utilize to the clinical application for cancergene therapy.

f. Vaccination of SLC gene-Modified tumor cellsinduce anti-tumor immunity modulating den-dritic cell’s functions in situ

Kazuya Shimao, Takuya Takayama, Hideaki Tahara

Secondary lymphoid –tissue chemokine (SLC),which is a member of CC chemokine, promotes themigration of mature dendritic cell (DC) expressedCCR7. In this study, we are going to examine the effi-cacy of SLC on DC mobilization in vivo andanti-tumor immunity by SLC gene modified tumorvaccination.

2. Mechanistic study on transplantation immu-nology

a. The promotion of Donor engraftment with Non-lethal irradiation, G-CSF and Tolerogenic DCin allergenic BMT.

Kaname Yamamoto, Yoshifumi beck, Yuichi Ando,Hideaki Tahara

We analyzed the effect of G-CSF and vitamin D3on maturation and character of DC in vitro mice BMculture with GM-CSF and IL-4. The addition of G-CSF (10 - 100ng/mL) in BM-DC culture inducedexpansion of the percentage and the number ofCD11c+ CD86_cells with G-CSF receptor. It is report-ed that G-CSF polarize to Th-2 in helper T balance. Sowe evaluate the effect of pre/post G-CSF injectioninto major mismatched BMT recipient. G-CSF injec-tion enhanced donor engraftment after BMT withsub-lethal irradiation (700R), but not with non-lethalirradiation (500-600R). It is unclear that how G-CSFpromote the donor engraftment. Next we demon-strated that the addition of vitamin D3 (10-100nM) inBM-DC culture suppressed the expression of costim-ulatory molecule (CD86, CD40) on CD11c+ cells andDC derived with vitamin D3 had no stimulatory ac-tivity on MLR with allo-splenocytes. Furthermorewe separated CD11c ＋ CD86_cells from vitamin D3derived DC by MACS. But pre-injection of Macs sep-arated this tolerogenic DC into allo-BMT recipienthad no effect of the promotion of donor engraftment.We examine the effect of injection of vitamin D3 intoallo-BMT recipient.

b. Evaluation of in vivo tolerogenicity of geneti-cally modified recipient dendritic cells(syngeneic DC) pulsed with immunogenicpeptides (allopeptides) derived from donorHLA molecule in HLA class I transgenicmouse.

Sumito Tamura, Yoshifumi beck, Yuichi Ando,Hideaki Tahara

Foreign tissue allografts are rejected because thehistocompatibility antigens they express stimulate aresponse from the host’s immune system. The pre-dominant cell type involved in this response is the Tlymphocyte. Before T cell causes rejection, naive al-loreactive T cell must be activated by antigenpresenting cells (APCs) that bear both allogeneicMHC molecule and co-stimulatory signals. DC, aprofessional APC, plays a critical role in this initia-t ion and modulation of immune response.Regulating the activity of DC is, in theory, an idealapproach to establish donor specific tolerance. Ourgoal is to establish a state of alloantigen specific tol-erance by manipulating the interaction of DC andreactive T cell.

T cells of C3H. B51 recognize HLA-B*3501 mole-cules expressed on C3H. B35 as allo-MHC class Iantigens and rejects vascularized C3H. B35 grafts bycellular mechanism. (Ando and Beck et al., Trans-plantation 68, 904-908, 1999.) HLA-B*3501 derivedpeptide induces long-term heart graft survival inC3H. B35 into C3H. B51 cardiac transplantationmodel (Ando and Beck et al., Transplantation 68,904-908, 1999) by intra-thymic injection. (Transplantproceedings, 30, 3890-3891, 1998). As explained inthe last annual report, we initially planned to clarifythe tolerogenic capability of syngeneic DC involvedwith allo-MHC molecules based on this HLA TGMheart transplant model. The attempt, however, wasfaced by difficulties in creating an adequate allo-pep-tide pulsed syngeneic bone marrow DC withallogeneic effect for further in vitro assay plannedprior to in vivo application. In order to circumventthe stagnation, we have decided to alter our ap-proach. We have decided to study the mechanism oftolerance in this model, hoping that this approachwill give us the “lead” to the establishment of pe-ripheral allo-specific tolerance.

Results obtained so far suggest that B35 allo-pep-tide seems to be involved with class II rather thanclass I in inducing tolerance. T cell obtained from tol-erant recipients showed both proliferation and CTLgeneration following stimulation by alloantigen, astate so called ”split tolerance”. This means that sim-ple deletion based on MHC-allo-peptide molecularmimicry theory is unlikely as the cause of graft spe-cific tolerance. On the other hand, adoptive transferof bulk splenocytes obtained from sygeneic donorsthat received intra-thymic administration of allo-

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Publication

peptide resulted in prolongation of graft survival,suggesting that regulatory T cell may be involved intolerance induction. We are currently planning to

Adachi O, Nakano A, Sato O, Kawamoto S, TaharaH, Toyoda N, Yamato E, Matsumori A, TabayashiK, and Miyazaki J. Gene transfer of Fc-fusioncytokine by in vivo electroporation: application togene therapy for viral myocarditis. Gene Ther 9:577-583, 2002.

Takayama T, Kaneko K, Morelli AE, Li W, Tahara H,and Thomson AW. Retroviral delivery of trans-forming growth factor-β1 to myeloid dendriticcells; inhibition of T cell priming ability and influ-ence on allograft survival. Transplantation 74:112-119, 2002.

Goto H, Osaki T, Nishino K, Tachibana I, Takeda Y,Yoneda T, Funakoshi T, Kimura H, Hayashi S andTahara H. Construction and Analysis of New Vec-tor Systems with Improved Interleukin-18 Secre-tion in Xenogeneic Human Tumor Model: JImmunother 25 Suppl 1:S35-41, 2002.

Tanaka F, Hashimoto W, Robbins PD, Lotze MT andTahara H. Therapeutic and specific antitumor im-munity induced by co-administration of immaturedendritic cells and adenoviral vector expressingbiologically active IL-18. Gene Ther 9:1480-6, 2002.

Hashimoto W, Tanaka F, Robbins PD, Taniguch M,Okamura H, Lotze MT and Tahara H. NK but not

NKT cells play a necessary role to promote an in-nate antitumor response induced by IL-18. Int JCancer (In press)

Mikihito Nakamori, Makoto Iwahashi, KentaroUeda, Takuya Tsunoda, Hiroshi Terasawa,Hirofumi Hamada and Hiroki Yamaue: Dose ofadenoviral vectors expressing interleukin-2 playan important role in combined gene therapy withcytosine deaminase/5-fluorocytosine: PreclinicalConsideration. Jpn. J. Cancer Res. 93; 706-715,2002.

Yasuhiro Koh, Takuya Tsunoda, Makoto Iwahashi,Hiroki Yamaue, Kiwao Ishimoto, HiroshiTanimura, Hisao Fukumoto, Takashi Nakamura,Yasuaki Tatsumi, Mikiko Shimizu, NagahiroSaijo, Kazuto Nishio: Decreased expression of α2,8sialyltransferase and increased expression of β1,4N-acetylgalactosaminyltransferase in gastrointes-tinal cancers. Exp. Biol. Med 227(3); 196-200, 2002

Takuya Tsunoda, Kenji Matsuda, Hiroki Yamaue.Enhancement of cytotoxic T lymphocytes re-sponses in patients with gastrointestinal malig-nancies following vaccination with CEA peptidespulsed dendritic cells. J Immunother. 25, S13,2002.

study the role of regulatory cells in the HLA. TGMmodel.

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Advanced Clinical Research Center

Division of Clinical Immunology免疫病態分野

1. Crk-associated substrate lymphocyte type(Cas-L) project

Satoshi Iwata, Hiroshi Kobayashi, Akiko Souta-K u r i b a r a , T a k a h i r o S a s a k i , R i k a k oMiyake-Nishijima, Seiji Kobayashi, MasahikoUchiyama, Mamoru Nori, Osamu Hosono, HiroshiKawasaki, Hirotoshi Tanaka, and Chikao Morimo-to

β1 integrins play crucial roles in a variety of cellprocesses such as adhesion, migration, proliferation,and differentiation of lymphocytes. Previously weshowed that co-immobilized ligand or anti-β1 inte-grin mAbs with a submitogenic dose of anti-CD3mAb induced a marked increase of IL-2 secretionand proliferative response of T cells, indicating therole of integrin/ligand binding in T cell activation.Furthermore, we showed that the ligation of β1 inte-grins induces protein tyrosine phosphorylation ofpp125FAK (focal adhesion kinase), paxillin, andpp105 in H9 cells as well as peripheral T cells. Pp105was first described in our laboratory as a protein that

is predominantly tyrosine phosphorylated by the li-gation of β1 integrins in H9 cells. Recently, we havedemonstrated that pp105 is a hematopoietic variantof p130Cas (Crk-associated substrate), and thus des-ignated Cas-L (Crk-associated substrate lymphocytetype).

Cas family proteins (p130Cas, Cas-L/HEF-1, Efs/Sin) have common structural features. From N-ter-minal end, they have SH3 domain, SD (substratedomain) that is a cluster of YXXP motifs, serine-richdomain, YDYVHL motif, Helix-loop-helix domain,and coiled-coil domain. Based on this structuralanalysis, we showed that 1) FAK binds SH3 domainof Cas-L, 2) Crk, Nck, and SHP-2 bind YXXP motifsin SD, and 3) Src family PTKs (Fyn, Lck, and Src)bind YDYVHL motif, respectively. Furthermore, wehave recently identified a new Cas-L binding pro-tein, and designated it as MICAL (MoleculeInteracting with Cas-L). MICAL binds SH3 domainof Cas-L through its proline rich sequence, and bindvimentin, through its C-terminal portion, suggestingits role in cytoskeletal reorganization.

Our present projects aim at 1) identifying novel

Our long term goal is to define the molecular basis for the mechanisms of the immuneabnormalities observed in various immune-mediated disorders such as autoimmuedisease as well as to cure patients suffering from the above immune-mediated disor-ders. To accomplish this goal, we have focused on defining the structure and functionof cell surface and intracellular molecules expressed in human T cells and other cellsand on understanding how the immune regulatory system works in normal and dis-ease conditions. Our study will provide new insights into understanding the precisemolecular mechanisms that underlie immune abnormalities found in various autoim-mune diseases as well as other immune-mediated disorders and will lead to thedevelopment of rational therapy for the manipulation of the abnormalities found insuch diseases.

教　授 医学博士　 森　本　幾　夫助教授 医学博士　 田　中　廣　壽

Professor Chikao Morimoto, M.D., D.M.Sc.Associate Professor Hirotoshi Tanaka, M.D., D.M.Sc.

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Cas-L binding proteins by Two-Hybrid screening, 2)investigating the biological significance of those pro-tein-protein interactions in vitro, and 3) evaluatingthe clinical relevance of those interactions in a vari-ety of disorders, including inflammatory diseasesand malignancies.

a. Cloning and characterization of binding molecules forCas-L by Two-Hybrid screening

Although our previous analysis revealed that Cas-L plays a crucial role in T cell costimulation andmigration through the engagement of β1 integrinsand TCR/CD3, exact biological roles of their Cas-Lin these processes has not fully understood. To gainan insight in this issue, we have screened several mil-lions of cDNA clones from a HTLV-I (humanT-lymphotropic virus type I) transformed human Tcell line (SLB-I) and human fetal brain by Two-Hy-brid assay in search for Cas-L binding molecules. Asa result, we have successfully isolated some unchar-acterized clones as well as the clones for knownproteins. Two cDNAs for FAK were among thoseclones, which have been already reported as Cas-Lbinding proteins. Interestingly, we identified twocDNAs for HTLV-I tax and one for Smad7. GFP-taxand GFP-Smad7 expressed in 293T cells show co-pre-cipitation with Cas-L. These results were furtherconfirmed by full length cDNAs for tax and Smad7subcloned in mammalian expression plasmid.

Tax was originally identified as cis-activator ofLTR-mediated HTLV-I replication. It has been re-ported that Tax transactivate a variety of host genes,especially downstream of NF-κB.

Smad7 is an endogenous inhibitor of TGF-β sig-naling pathway. Golemis et al. have recentlyreported that Smad 3 is also one of the binding part-ners of Cas-L/HEF1, which is involved in TGF-βsignaling pathway as a positive regulator (Co-Smads). Since Cas-L binds both positive andnegative regulator of TGF-β signaling pathway, weevaluate overall biological outcome of Cas-L-Smadsinteraction.

At this moment, we focus on these clones as puta-tive Cas-L binding proteins.

b. Induction of Cas-L by HTLV-I tax; Role of Cas-L in ATL(adult T cell leukemia)

Since Cas-L is overexpressed in various HTLV-I-infected T cell lines, we evaluated the effect of p40taxprotein on expression and phosphorylation of Cas-Lusing JPX-9 cells in which tax is induced by additionof CdCl2. Surprisingly, tyrosine phosphorylation aswell as the expression of Cas-L was markedly en-hanced through the induction of tax protein in JPX-9cells. Furthermore, these cells showed markedly en-hanced motile behavior on FN-coated Transwell™insert, confirming that Cas-L play an important role

in cell migration in those cells.In the clinical setting, we have shown that leuke-

mic cells from ATL patients express significantlyhigher amount of Cas-L compared to the healthy in-dividuals. Furthermore, we have shown thattyrosine phosphorylation of Cas-L is spontaneouslyelevated in the case with ATL patients, suggestingthat extremely invasive nature of ATL cells may beattributed to the enhanced expression and phospho-rylation of Cas-L.

c. Role of Cas-L in pathogenesis of arthritis in HTLV-I taxtransgenic mice and human rheumatoid arthritis

It has been reported that the expression of β1 inte-grins and their l igands are elevated in theinflammatory sites in rheumatoid arthritis (RA), sug-gesting that β1 integrins play an important role intriggering and maintaining the inflammatory re-sponse of such diseases.

We have shown that Cas-L plays a crucial role in Tcell costimulation and migration through the en-gagement of β1 integrins and TCR/CD3 complex. Toinvestigate the role of Cas-L in pathophysiology ofRA, we utilized HTLV-I tax transgenic mice, sincethey develop RA-like disease. We compared litter-mate control mice (Ct), tax transgenic mice withoutarthritis (Ntg) and those with arthritis (Atg) at 4-14weeks after birth. Transendothelial migration assayrevealed that migratory activity of spleen cells fromAtg and Ntg mice was much higher than that of Ctmice. Biochemical studies using the lysate of spleencells showed that Cas-L protein and its spontaneoustyrosine phosphorylation were increased in both Atgand Ntg mice compared to Ct mice. Interestingly, theextent of elevation in Cas-L protein and its phospho-rylation level was higher in Atg mice than that of Ntgmice, which paralleled with the migratory capabilityof spleen cells. To define the kinases that phosphory-lated Cas-L, we analyzed Src family PTKs (Fyn andLck) and β1 integrin-associated PTKs (FAK andPyk2). Subsequently we found that spontaneous ty-rosine phosphorylation of Fyn and Lck wereobserved in spleen and lymph node of Atg mice,whereas FAK and Pyk2 were only marginally phos-phorylated. Immunohistochemical analysis showeda large number of Cas-L positive lymphocytes andleukocytes migrating into the affected joints withRA. These results strongly suggest that increased ex-pression as well as tyrosine-phosphorylation ofCas-L protein is involved in the development of ar-thritis in HTLV-I tax transgenic mice.

Finally, we investigated the human materials frompatients with rheumatoid arthritis, and have shownthat tyrosine phosphorylation of Cas-L are markedlyenhanced in SFMC (synovial fluid mononuclear cells)from RA patients. These cells exhibited increased ca-pability of transendothelial migration compared toPBMC from RA patients and healthy volunteers. Since

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β1 integrins play a key role in triggering and main-taining inflammation of RA, the above resultsstrongly suggest that Cas-L protein appears to play animportant role in the pathophysiology of RA.

2. Structural basis for CD26 mediated T cell co-stimulation and function in normal anddisease conditions.

Chikao Morimoto, Kei Ohnuma, Masahiko Uchiya-ma, Hiroshi Kobayashi, Satoshi Iwata, OsamuHosono, Hiroshi Kawasaki, and HirotoshiTanaka(in collaboration with Nam H Dang, MDAnderson Cancer Center, USA, and with RafaelFranco, University of Barcelona, Spain).

CD26 is a 110-kDa cell surface glycoprotein thatposseses dipeptidyl peptidase IV(DPPIV) (EC.3.4.14.5) activity in its extracellular domain and a pri-mary marker of activated T cells. In the resting state,CD26 is preferentially expressed on a subset of CD4memory T cells where they account for the majority ofIL-2 secretory capabilities and help for B cell Ig pro-duction and are the primary responders to recallantigen such as tetanus toxoid. CD26 is also capable ofproviding a potent costimulatory or “second” signalwhich can augment other activation pathways lead-ing to proliferation, cytokine production and effectorfunctions. The mechanism of costimulation remainsunclear since the cytoplasmic domain consists of only6 amino acid and lacks a phosphorylation site, leadingto the conclusion that CD26 interacts with other cellsurface molecules. We have already shown that CD26may interact with CD45RO which modulates TcR/CD3 activity through its intracellular tyrosine phos-phatase domain. Recently, we have detected anotherCD26 binding protein, the mannose-6-phosphate/in-sulin-like growth factor II receptor (M6P/IGFIIR) asbeing critical for this interaction for CD26 mediated Tcell costimulation in addition to adenosine deaminase(ADA). More recently, we have shown that CD26 lo-calizes into lipid rafts,and targeting of CD26 to rafts isnecessary for signaling events through CD26. Impor-tantly, aggregation of CD26 by anti-CD26 mAbcrosslinking also causes coaggregation of CD45 intorafts. In addition, we have demonstrated that recom-binant soluble CD26 (sCD26) has an enhancing effecton T cell proliferation in the presence of the recall anti-gen, tetanus toxoid. This enhancement resulted in anincrease in the surface expression of the costimulatorymolecule CD86 on monocytes following uptake ofsCD26.

Currently we are focusing on the molecular basisfor CD26 mediated T cell activation signaling andenhancement of memory T cell response by sCD26.Furthermore , we are focusing on the translationalresearch of utilization of anti-CD26 mAb as well assoluble CD26 for malignant tumors, immune-medi-ated disorders and immune deficiency diseases.

a. Presence of CD26/DPPIV is associated with enhancedtopoisomerase II alpha expression and increasedsensitivity to drug-induced G2-M arrest.

CD26 is a 110kD surface-bound ectopeptidasewith dipeptidyl peptidase (DPPIV) activity that hasmultiple functions, including a role in T cell activa-tion and we demonstrated that the expression ofCD26 and its DPPIV activity enhanced the sensitivityof CD26 Jurkat transfectants to doxorubicin-inducedcell cycle arrest at the G2-M checkpoint. We extendour previous findings by showing that surface ex-pression of CD26/DPPIV enhanced sensitivity ofCD26 Jurkat transfectants to G2-M arrest mediatedby the antineplastic agent etoposide. Similar to thecase found with doxorubicin, the increased sensitivi-ty to etoposide-induced G2-M arrest was associatedwith disruption of cell cycle-related events, includ-i n g h y p e r p h o r y l a t i o n o f p 3 4 c d c 2 k i n a s e ,prophorylation of cdc25C and alterration in cyclinΒ1 expression. CD26/DPPIV-associated enhance-ment of doxorubicin and etopioside-induced G2-Marrest was also ovserved in serum-free media. Con-sistent with the fact that CD26 Jurkat transfectantsdisplayed greater sensitivity to the topoisomerase II-targeted drugs doxorubicin and etoposide, as well assuggesting a potential mechanism for this enhancedsusceptibility, our findings strongly suggested thatCD26/DPPIV surface expression was associatedwith increased topoisomerase II alpha levels.

b. G1/S cell cycle arrest provoked in human T cells byantibody to CD26

The expression of CD26 is enhanced after activa-tion of T cells, while it is preferentially expressed ona subset of CD4+ memory T cells in the resting state.We demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibitshuman T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cellclones by inducing G1/S arrest, which is associatedwith enhancement of p21Cip1 expression. This effectdepends on the DPPIV enzyme activity of the CD26molecule. Moreover, we show that expression ofp21Cip1 after treatment with the anti-CD26 mAb 1F7appears to be induced through activation of extracel-lular signal-regulated kinase (ERK) pathway. Thesedata thus suggest that anti-CD26 treatment mayhave potential use in the clinical setting involvingactivated T cell dysregulation, including autoim-mune disorders and graft-vs.-host disease.

c. Reduction of serum soluble CD26/DPPIV enzyme ac-tivity and its correlation with disease activity in SLE

The soluble form of CD26 is present in serum andrecombinant soluble CD26 (rsCD26) can enhance invitro antigen-specific T cell responses. To determine

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the role of soluble CD26 (sCD26) in the pathophysi-ology of patients with systemic lupus erythematosus(SLE), we measured levels of sCD26 and its specificDPPIV activity in serum. Serum sCD26 levels andDPPIV activity were measured by sandwich ELISAin 53 patients with SLE and 54 healthy controls. Se-rum sCD26 was identified by immunoprecipitationand immunoblot analysis. Expression of CD26 on Tcells was analyzed by flow cytometry. Serum levelsof sCD26 and its specific DPPIV activity were signif-icantly decreased in SLE and were inverselycorrelated with SLE disease activity index score, butnot with clinical variables or clinical subsets of SLE.Close correlation between sCD26/DPPIV and dis-ease activity was observed in the longitudinal study.Our results showed that serum levels of sCD26 maybe involved in the pathophysiology of SLE, and ap-pear to be useful as a new disease activity marker forSLE.

d. Soluble CD26/DPPIV enhances transendothelial mi-gration via its interaction with M6P/IGFIIR

In addition to its membrane form of CD26, CD26exists in plasma as a soluble form (sCD26), which isthe extracellular domain of the molecule thought tobe cleaved from the cell surface. We demonstratethat sCD26 mediates enhanced transendothelial Tcell migration, an effect that requires its intrinsic DP-PIV enzyme activity. We also show that sCD26directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor(M6P/IGFIIR) on the endothelial cell surface acts asa receptor for sCD26. Our findings therefore suggestthat sCD26 influences T cell migration through itsinteraction with M6P/IGFIIR.

e. Regulation of epithelial and lymphocyte cell adhesionby ADA-CD26 interaction

The extra-enzymic function of cell-surface adenos-ine deaminase (ADA), an enzyme mainly localizedin the cytosol but also found on the cell surface ofmonocytes, B cells and T cells, has lately been thesubject of numerous studies. Cell-surface ADA isable to transduce co-stimulatory signals in T cells viaits interaction with CD26, an integral membrane pro-tein that acts as ADA-binding protein. We attemptedto explore whether ADA-CD26 interaction plays arole in the adhesion of lymphocyte cells to humanepithelial cells. To meet this aim, different lympho-cyte cell lines (Jurkat and CEM T) expressingendogenous, or overexpressing human, CD26 pro-tein were tested in adhesion assays to monolayers ofcolon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA.Interestingly, the adhesion of Jurkat and CEM T cellsto a monolayer of Caco-2 cells was greatly dependenton CD26. An increase by 50% in the cell-to-cell adhe-

sion was found in cells containing higher levels ofCD26. Incubation with an anti-CD26 antibody raisedagainst the ADA-binding site or with exogenousADA resulted in a significant reduction (50-70%) ofT-cell adhesion to monolayers of epithelial cells. Therole of ADA-CD26 interaction in the lymphocyte-ep-ithelial cell adhesion appears to be mediated byCD26 molecules that are not interacting with endog-enous ADA (ADA-free CD26), since SKW6.4 (B cells)that express more cell-surface ADA showed loweradhesion than T cells. Adhesion stimulated by CD26and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interactionin cell-to-cell adhesion was confirmed further in inte-grin activation assays. FACS analysis revealed ahigher expression of activated integrins on T celllines in the presence of increasing amounts of exoge-nous ADA. Taken together, these results suggest thatthe ADA-CD26 interaction on the cell surface has arole in lymphocyte-epithelial cell adhesion.

3. Therapeutically targeting transcription factors

Hirotoshi Tanaka, Yuichi Makino, NoritadaYoshikawa, Noriaki Shimizu, Tsunenori Kodama,Rika Ouchida, Hiroshi Nakamura, Tetsuya Hisada,Chikao Morimoto (Division of Clinical Immunolo-gy), Hiroshi Handa (TokyoInstitute of Technology),Masatoshi Kusuhara, Fumitaka Ohsuzu (NationalDefence Medical College) (in colaboration withLorenz Poellinger Lab, Karolinska Institute, Swe-den)

We are interested in the mechanism of eukaryoticgene expression and development of novel therapyand/or drug which target transcriptional machiner-ies. For this purpose, our recent work is mainlyfocused on conditional regulation of transcriptionfactors including the glucocorticoid receptor and hy-poxia-inducible factor-1α.

a. Glucocorticoid receptor project

Glucocorticoid hormones are effective in control-ling inflammation, but the mechanisms that conferthis action are largely unknown. It has been shownthat both positive and negative regulation of geneexpression are necessary for this process. The geneswhose activity is negatively modulated by glucocor-ticoids in the inflammatory process code for severalcytokines, adhesion molecules. Most of them do notcarry a classical binding site for regulation by theglucocorticoid receptor (GR), but have instead regu-latory sequences for transcription factors such asAP-1 or NF-κB. Considering various severe side ef-fects of glucocorticoids, it may be pharmacologicallyimportant to dissociate these negative regulatoryfunction of the GR from induction of metabolic en-

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zymes, gene expression of which has been shown tobe positively regulated by the GR. We propose that acertain class of compounds (surprisingly, some ofthem are non-steridal chemicals) may dissociatetransactivation and transrepression function of theGR and offer opportunities for the design of suchcompounds that could function more effectively asantiinflammatory drugs. In this line, we are develop-ing the strategy for identification of noveltherapeutic bullets.

(i) Redox Regulation of the Glucocorticoid ReceptorRedox regulation is currently considered as a

mode of signal transduction for coordinated regula-tion of a variety of cellular processes. Transcriptionalregulation of gene expression is also influenced bycellular redox state, most possibly through the oxi-do-reductive modification of transcription factors.The GR belongs to a nuclear receptor superfamilyand acts as a ligand-dependent transcription factor.We demonstrated that the GR function is regulatedvia redox-dependent mechanisms at multiple levels.Moreover, it is suggested that redox regulation of thereceptor function is one of dynamic cellular respons-es to environmental stimuli and plays an importantrole in orchestrated crosstalk between central andperipheral stress responses.

(ii) Development of Dissociating Ligand for the Glucocor-ticoid Receptor

The GR function could be differencially regulatedby ligands. We have recently shown that not onlysynthetic glucocorticoids but also certain bile acidscould differentially modulate GR function. More-over, the effects of those compounds are indicated tobe ascrived to the ligand binding domain of the re-ceptor. In this line, we are going to isolate thedissociating ligand that preferencially promotestransrepression function of the GR.

On the other hand, receptor specificity is anotherimportant aspect of novel GR regulator. In this line,we have shown that cortivazol is extremely specificfor GR and does not bind to MR. We are studying themolecular basis for this receptor specificity of theligand using cortivazol as a model.

(iii) Molecular biology of a novel protein HEXIM1We have recently cloned the cDNA encoding a

novel protein HEXIM1, expression of which is in-duced by treatment of vascular smooth muscle cellswith a differentiation inducer hexamethylane bisace-tamide. We showed that HEXIM1 is a nuclearprotein and represses NF-κB-dependent transcrip-tion. Since NF-κB plays a pathological role in smoothmuscle cell proliferation, our study will not only un-veil pathogenesis of but also contribute to therapy ofatherosclerotic vascular disorders.

(iv) Hypoxia-inducible Factor (HIF)-1a projectHIF-1α is essential for not only angiogenesis but

also development of certain organs. In this line, mo-lecular biology of HIF-1α will provide us possibleadvantage to characterize and manupilate such pro-cesses.

b. Transcriptional Network Controlling Angiogenesis inHealth and Diseases

Angiogenesis is regulated by a combination ofvarious factors including transcription factors. Re-cently, we have isolated cDNA encoding the novelprotein IPAS which can squelch HIF-1a. Its tissue-specific expression argues the physiological role oftranscriptional network for orchestrated regulationof angiogenesis. We are currently studying the mo-lecular mechanism of the interaction between HIF-1aand IPAS. This negative regulator may also thera-peutically applicable for treating a number ofangiogenic disorders including cancer, diabetic ret-inopathy, and rheumatoid arthritis. Moreover, wehave recently shown that IPAS is a splice variant ofHIF-3a, and its mRNA expression is enhanced underhypoxic conditions. This conditional regulation ofsplicing is our current interest.

On the other hand, we have recently identifiedthat HIF-1α function is regulated in a various fashionin certain physiological settings, which may be im-portant for homeostatic control of tissue function. Inthis line, we are now identifying the molecular mech-anism for such regulation of HIF-1α.

4. Immunobiology and clinical applications ofchemokines and their receptors

Hiroshi Kawasaki, and Chikao Morimoto (in col-laborat ion wi th Katsuaki Sato , TakamiMatsuyama, and Kouichi Hirai)

We have been pursuing the structure and func-tional analysis of human chemokine•chemokinereceptor system in order to clearly address their rolesin innate and acquired immune system. Since thediscovery of chemokine receptors as the HIV co-re-ceptors, this area of immune mediators have drawntremendous attention. We are in the process of thera-peutically apply antibodies to chemokine receptorsin a fully humanized fashion. Major target moleculesat present are CCR-1, CCR-3 and IL-12.

An abortive ligand-induced activation of CCR1-mediated downstream signaling event and adeficiency of CCR5 expression are associated withthe hyporesponsiveness of human naive CD4+ Tcells to CCL3 and CCL5.

Human memory CD4(+) T cells respond better toinflammatory CCLs/CC chemokines, CCL3 and

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CCL5, than naive CD4(+) T cells. We analyzed theregulatory mechanism underlying this difference.Memory and naive CD4(+) T cells expressed similar-ly high levels of CCR1; however, CCR5 was onlyexpressed in memory CD4(+) T cells at low levels.Experiments using mAbs to block chemokine recep-tors revealed that CCR1 functioned as a majorreceptor for the binding of CCL5 in memory and na-ive CD4(+) T cells as well as the ligand-inducedchemotaxis in memory CD4(+) T cells. Stimulation ofmemory CD4(+) T cells with CCL5 activated proteintyrosine kinase-dependent cascades, which were sig-nificantly blocked by anti-CCR1 mAb, whereas thisstimulation failed to induce these events in naiveCD4(+) T cells. Intracellular expressions of regulatorof G protein signaling 3 and 4 were only detected innaive CD4(+) T cells. Pretreatment of cell membranefractions from memory and naive CD4(+) T cellswith GTP-gamma S inhibited CCL5 binding, indicat-ing the involvement of G proteins in the interactionof CCL5 and its receptor(s). In contrast, CCL5 en-hanced the GTP binding to G(i alpha) and G(q alpha)in memory CD4(+) T cells, but not in naive CD4(+) Tcells. Thus, a failure of the ligand-induced activationof CCR1-mediated downstream signaling event aswell as a deficiency of CCR5 expression may be in-volved in the hyporesponsiveness of naive CD4(+) Tcells to CCL3 and CCL5.

CC-chemokine receptor 3: a possible target intreatment of allergy-related corneal ulcer.

To determine the suppressive effects of antibodies(Abs) against CC-chemokine receptor (CCR)-1 andCCR-3 on eosinophil chemotaxis induced by culturesupernatant from corneal keratocytes and by tearsfrom severely allergic patients with corneal ulcer.Primary cultures of human corneal keratocytes wereincubated with interleukin (IL)-4 (33.3 ng/mL) andtumor necrosis factor (TNF)-alpha (33.3 ng/mL) for48 hours. In tear samples collected from five severelyallergic patients and three nonallergic control sub-jects, eosinophils were immunostained for CCR.Next, eosinophils purified from peripheral bloodwere preincubated with or without anti-CCR-1 andanti-CCR-3 Abs before a Boyden chamber assay wasconducted. Recombinant human (rh) eotaxin, rh-reg-

ulated on activation normal T-cell expressed and se-creted (rh-RANTES), culture supernatant fromhuman corneal keratocytes, and tear samples wereused as chemoattractants. Eosinophils in tears fromallergic patients expressed CCR-1 and -3 on their sur-faces. Anti-CCR-1 and -3 Abs each inhibitedeosinophil chemotaxis induced by rh-RANTES.Anti-CCR-3 Ab (but not anti-CCR-1 Ab) also inhibit-ed eosinophil chemotaxis induced by rh-eotaxin.Anti-CCR-1 and -3 Abs, respectively, inhibited up to75.2% and 94.6% of eosinophil chemotaxis inducedby culture supernatant, as well as 27.8% and 74.5% ofchemotaxis induced by tear samples. CONCLU-SIONS: Anti-CCR-1 and -3 Abs inhibited eosinophilchemotaxis induced by culture supernatant fromcorneal keratocytes and tear samples from severelyallergic patients. Anti-CCR-3 Ab was more effectivethan anti-CCR-1 Ab. Inhibition of CCR-3 on eosino-phils may be a treatment for corneal ulcer in patientswith ocular allergy.

Langerhans Cell-mediated Transferred Antigen-Loaded Dendritic Cells Initiate T Cell Activation

Evidence obtained from experiments on animalsuggests that epidermal Langerhans cells (LCs) anddermal dendritic cells (DCs) play a crucial role in ep-icutaneous immune responses. However, themechanism underlying the initiation of the epicuta-neous immune response in humans remains obscure.To clarify the mechanism responsible for the initia-tion of an Ag-specific immune response in epidermisin the human system, we examined the role of theinterplay between DCs and LCs, both derived fromhuman peripheral blood (PB) monocytes, in vitro.DCs exhibited more potent expressions of the MHCproduct and costimulatory molecules than LCs. LCswere less effective for the internalization of exoge-nous Ag, and the activation of allogeneic andautologous Ag-specific T cells than DCs. DCs andLCs expressed different in chemokine receptor rep-ertoire and responsiveness. LCs can transferunprocessed Ag to DCs via cell to cell contact, andthese trans-Ag-loaded DCs induced an Ag-specific Tcell response. Thus, cross-priming between DCs andLCs is crucial for the initiation of epicutaneous im-mune responses.

Publication

Ouchida, R., Kusuhara, M., Shimizu, N., Hisada, T.,Makino, Y., Morimoto, C., Handa, H., Ohsuzu, F.,Tanaka, H. Suppression of NF-κB-dependent geneexpression by a hexamethylene bisacetamide-in-ducible nuclear protein HEXIM1 in human vascu-lar smooth muscle cells. Genes to Cells. 2003, inpress.

Sato, K., Kawasaki, H., Morimoto, C., Matsuyama, T.Langerhans Cell-mediated Transferred Antigen-

Loaded Dendritic Cells Initiate T Cell Activation. JImmunol 2003, in press.

Aytac, U., Sato, K., Ohnuma, K., Mills, GB.,Cabnillas, F., Morimoto C., Dang, NH. Presemceof CD26/DPPIV is associated with enhancedtopoisomerase II alpha expression and increasedsensitivity to drug-induced G2-M arrest. Br. J.Cancrer. 2003, in press.

Watanabe, S., Murakami, T., Nakamura, T.,

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Morimoto, C., Arai, K. Human GM-CSF inducesHIV-1 LTR by multiple signalling pathways.Biochimie. 84:633-642,2002.

Ohnuma, K., Ishii, T., Iwata, S., Hosono, O.,Kawasaki, H., Uchiyama, M., Tanaka, H.,Yamochi, T., Dang, NH., Morimoto, C. G1/S cellcycle arrest provoked in human T cells by anti-body to CD26. Immunology. 107:325-333,2002.

Dang, NH., Morimoto, C. CD26: an expanding role ini m m u n e r e g u l a t i o n a n d c a n c e r . H i s t o lHistopathol. 17:1213-1226,2002.

Hase, H., Kanno, Y., Kojima, H., Morimoto, C.,Okumura, K., Kobata, T. CD27 and CD40 inhibitp53-independent mitochondrial pathways inapoptosis of B cells induced by B cell receptor liga-tion. J Biol Chem. 277:46950-46958,2002.

Kobayashi, H., Hosono, O., Mimori, T., Kawasaki,H., Dang, NH., Tanaka, H,. Morimoto C. Reduc-tion of serum soluble CD26/dipeptidyl peptidaseIV enzyme activity and its correlation with diseaseactivity in systemic lupus erythematosus. JRheumatol. 29:1858-1866,2002.

Ikushima, H., Munakata, Y., Iwata, S., Ohnuma, K.,Kobayashi, S., Dang, NH., Morimoto, C. SolubleCD26/dipeptidyl peptidase IV enhancestransendothelial migration via its interaction withmannose 6-phosphate/insulin-like growth factorII receptor. Cell Immunol. 215:106-110,2002.

Hisakawa, N., Tanaka, H., Hosono, O., Nishijima, R.,Ohashi, Y., Saito, S., Nishiya, K., Hashimoto, K.,Morimoto, C. Aberrant responsiveness toRANTES in synovial fluid T cells from patientswith rheumatoid arthritis. J Rheumatol. 29:1124-1134, 2002.

Sato, K., Kawasaki, H., Morimoto, C., Yamashima,N., Matsuyama, T. An abortive ligand-induced ac-tivation of CCR1-mediated downstream signal-ing event and a deficiency of CCR5 expression areassociated with the hyporesponsiveness of humannaive CD4+ T cells to CCL3 and CCL5. J Immunol.168:6263-6272,2002.

Iwata, S., Kobayashi, H., Miyake-Nishijima, R.,Sasaki, T., Souta-Kuribara, A., Nori, M., Hosono,O., Kawasaki, H., Tanaka, H., Morimoto, C. Dis-tinctive signaling pathways through CD82 andbeta1 integrins in human T cells. Eur J Immunol.32:1328-1337,2002.

Suzuki, T., Nakamoto, T., Ogawa, S., Seo, S.,Matsumura, T., Tachibana, K., Morimoto, C.,Hirai, H. MICAL, a novel CasL interacting mol-ecule, associates with vimentin. J Biol Chem.277:14933-14941,2002.

Gines, S., Marino, M., Mallol, J., Canela, EI.,Morimoto, C., Callebaut, C., Hovanessian, A.,Casado, V., Lluis, C., Franco, R. Regulation of epi-thelial and lymphocyte cell adhesion by adenosine

deaminase-CD26 interaction. Biochem J. 361:203-209,2002.

Yoshikawa, N., Makino, Y., Okamoto, K., Morimoto,C., Makino, I., Tanaka, H. Distinct interaction ofcortivazol with the ligand binding domain confersglucocorticoid receptor specificity: cortivazol is aspecific ligand for the glucocorticoid receptor.J Biol Chem. 277:5529-5540,2002.

Nakamura, T., Ouchida, R., Kodama, T., Kawashima,T., Makino, Y., Yoshikawa, N., Watanabe, S.,Morimoto, C., Kitamura, T., Tanaka, H. Cytokinereceptor common beta subunit-mediated STAT5activation confers NF-kappa B activation in mu-rine proB cell line Ba/F3 cells. J Biol Chem.277:6254-6265,2002.

Makino, Y., Kanopka, A., Wilson, WJ., Tanaka, H.,Poellinger, L., IPAS is an hypoxia-inducible splic-ing variant of the HIF-3α locus. J. Biol. Chem.277:32405-32408, 2002

Nishi, T., Shimizu, N., Hiramoto, H., Sato, I.,Yamaguchi, Y., Hasegawa, M., Aizawa, S.,Tanaka, H., Kataoka, K., Watanabe, H., Handa, H.Spatial redox regulation of a critical cysteine resi-due of NF-κB in vivo.J. Biol. Chem. 277:44548-44556, 2002

Jögi, A., Ora, I., Nilsson, H., Lindeheim, Å., Makino,Y., Poellinger, L., Axelson, H., Påhlman ,S. Hy-poxia alters gene expression in human neuroblas-toma cells toward an immature and neural crest-like phenotype. Proc. Natl. Acad. Sci. U.S.A. 99:7021-7026.2002

Makino, Y., Okamoto, K., Tanaka, H. Thioredoxinand redox regulation of the nuclear receptor. InMethods in Molecular Biology. Edited byArmstrong D. (Humana press), pp171-181, 2002.

Kataoka, S., Kudo, A., Hirano, H., Kawakami, H.,Kawano, T., Higashihara, E., Tanaka, H., Delarue,F., Sraer, J-D., Mune, T., Krozowski, ZS., Yan ,K.11β-Hydroxysteroid Dehydrogenase Type 2 Is Ex-pressed in the Human Kidney Glomerulus.J Clin Endocrinol Metab. 87: 877-882, 2002

Itoh, M., Adachi, M., Yasui, H., Takekawa, M.,Tanaka, H., Imai, K. Nuclear export of glucocorti-coid receptor is enhanced by c-Jun N-terminal ki-nase-mediated phosphorylation. Mol Endocrinol. 16:2382-2392, 2002

Hiramoto, M., Shimizu, N., Nishi, T., Shima, D.,Aizawa, S., Tanaka, H., Hatakeyama, M.,Kawaguchi, H., Handa, H. High-performance af-finity beads for identifying anti-NF-κB drug re-ceptors. Methods Enzymol. 353:81-88, 2002

Fukagawa, K., Okada, N., Fujishima, H., Nakajima,T., Tsubota, K., Takano, Y., Kawasaki, H., Saito,H., Hirai, K. CC-chemokine receptor 3: a possibletarget in treatment of allergy-related corneal ulcer.Invest Ophthalmol Vis Sci. 43:58-62,2002.

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Advanced Clinical Research Center

1. Research and development of informationnetwork system for Translational Researchunder the concept of Evidence Based Medi-cine

National cost for Japanese health now amounts upto 3000 billion yen per year. Medical accidents andinsufficient or surplus medical cares cause serioussocial problems. These facts will strongly requiretranslational research which means application ofgenome science to clinical medicine. For promotionof translational research, EBM(Evidence Based Med-icine) is the most necessary method for assurance ofclinical protocol. For realizing true EBM, it is neces-sary to research and develop the most appropriatedgenome science based clinical databases and theirprocessing system.

Basic clinical database should be consist of the setof correct descriptions of clinical actions. An clinicalaction means 5w1h(when, where, who, whom, why,and how) and its quantitative result from the logicalatomism point of view. These clinical databases,which are processed by the appropriate statistical

method including data mining technology, revealthe most appropriate clinical protocol.

The research hospital has introduced clinical or-der entry system. New clinical information systemsare to be introduced and EBM-oriented sever com-puter system is going to operate in the background.

Because the Research Hospital has many experi-ences in blood and immunity disease, our EBMdatabase will be expected to give us much useful in-formation about the mechanism of these diseases.We started the construction pathological knowledgedatabase including genome, protein, and their rela-tion, i. e. pathway database for pathologicalapplication of genome science.

2. Research and development of experimentalsystem for cell therapy

Pattern recognition and control technologies ofhuman cell and microscopic cell organs are very im-portant for genomic sc iences and c l inicalapplications. New clinical protocol should be testedin human cell experimental system before its clinical

The purpose of the Division of Medical Data Processing Network System for the Re-search Hospital is to research and develop advanced system engineering methodologyand computer technology suitable for the 21-th century type research hospital, espe-cially Translational Research, which is performed as application of genome science toclinical medicineÅD Our main research objectives are as follows;- Construction of clinical knowledge database system for evidenced based medicine,- Computer technologies suitable for genome based medicine,- Data processing methodologies of advanced medical instrument , and- Pattern recognition methodologies and control technologies of living human cells.

Professor Tetsuo Shimizu 教　授 　 清　水　哲　男

Division of Medical Data ProcessingNetwork Systemゲノム医療情報ネットワーク分野

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application. We are researching to develop automat-ic cDNA(RNA, or protein) mechanical injectionmachine to human cell nucleus. This machine is ex-pected also to be useful to inject variable drugs inhuman cells.

At first the computational pattern recognitionmethod of human cell organs must be developed. Weare successful to recognize the fluorescence patternin cell organ over about 90% efficiency. This resultwill be applied to the automatic cDNA injection ma-chine combined with flow metric equipment, whichwill process over 100000 human cells in a day andwill be applied to cell therapy for many kinds ofblood or immunity disease.

We also started to introduce a cell observation sys-tem, which will enables us to monitor living humancells and track them continuously. The instrumentwill be expected to give us much pathological knowl-edge concerning living cell dynamism.

3. Research and development of bioinformaticssimulation system to explain blood systemdifferentiation and immunity mechanism

Many research projects started to study genomicdisease in Japan. For the clinical application of these

results, systematic studies are necessary how to inte-grate the genomic database and clinical data as wellas new computer simulation algorithm. We are go-ing to simulate blood differentiation and immunitymechanism.

At first, genomic database for blood differentia-tion pathway should be arranged. The next step is tocollect concerning protein pathway database. At last,the differentiation process and immunity mecha-nism will be simulated by bioinformatics technologyunder development in this Institute. The results willbe verified by automatic human cell processing sys-tem to be applied to the clinical protocol. In thisintegrated system consisting of mutually intercon-nected in- vivo, in- vitro, and in- silico simulation,many kind of blood and immunity disease will beprevented. This system will be hoped to contributemuch to national health programs of Japan.

For the first case, we started hematological bioin-formatics simulation of erythrocyte maturation.Using appropriate genome and protein interactivepathway data including many interactions betweennucleus and mitochondria, we obtained good simu-lation result which will be expected to give usexplanation of the pathological processes concerninghemoglobin accumulation in the erythrocyte.

清水哲男：医科学のためのシステム論，放射線科学， vol. 45No. 1, 13-23, 2002

Publication