RESEARCH ACTIVITIES

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Research in this division is directed toward understanding the complex interactionsthat occur between pathogenic bacteria and their human hosts at very early stageof bacterial infectious processes. Our special interest is focused upon the molecu-lar pathogenicity of enteropathogenic bacteria, such as Shigella, Helicobacter py-lori, enteropathogenic E. coli and enterohemorrhagic E. coli. We are also search-ing for effective methods to protect or regulate bacterial infection by using knowl-edge accumulated.

1. Escape of Intracellular Shigella from Auto-phagy.

Michinaga Ogawa, Tamotsu Yoshimori1, Toshi-hiko Suzuki, Hiroshi Sagara, Noboru Mi-zushima2 and Chihiro Sasakawa: 1National In-stitute of Genetics, 2Tokyo Metropolitan Insti-tute of Medical Science

Autophagy is an intracellular bulk degrada-tion system in which cytoplasmic macro mole-cules or organelles are directed to the ly-sosomes. Shigella and Listeria are able to disruptphagocytic vacuole of macrophage and epithe-lial cells and escape into the cytoplasm. It im-plies that invaded bacteria in cytoplasm can betarget for autophagy, and that cytoplasmic bac-teria can exert some function to evade auto-phagy. we investigated the possibility that Shig-ella could be entrapped by autophagosomes.MDCK expressing GFP-LC3 (a marker of auto-phagosomes) were infected with Shigella flexneriwild type or icsB mutant and the colocalizationof bacterium and GFP-LC3 was observed.Analysis by confocal laser microscopy, thin-

sections electron microscopy (EM) and immuno-gold EM revealed that Shigella can escape auto-phagy by secreting IcsB via the type III secretionsystem. Mutant bacteria lacking IcsB weretrapped by autophagy during multiplicationwithin the host cells. IcsB did not directly inhibitautophagy. Rather, Shigella VirG, a protein re-quired for intracellular actin-based motility, in-duced autophagy by binding to the autophagyprotein, Atg5. In non-mutant Shigella , this bind-ing is competitively inhibited by IcsB binding toVirG.

2. Targeting of enteropathogenic Escherichiacoli EspF to host mitochondria is essentialfor the bacterial pathogenesis: critical roleof the 16th leucine residue in EspF.

Takeshi Nagai, Akio Abe1 and Chihiro Sasak-awa: 1Kitasato Institute for Life Sciences, Ki-tasato University

The attachment of enteropathogenic Es-cherichia coli (EPEC) to host cells and the induc-tion of attaching and effacing (A/E) lesions are

Department of Microbiology and Immunology

Division of Bacterial Infection細菌感染分野

Professor Chihiro Sasaki, D.M.Sc.Lecturer Toshihiko Suzuki, D.M.Sc.Research Assosiate Michinaga Ogawa, D.M.Sc.Research Assosiate Hitomi Mimuro, D.M.Sc.Research Assosiate Ichiro Tatsuno, Ph.D.＊

教 授 医学博士 笹 川 千 尋講 師 医学博士 鈴 木 敏 彦助 手 医学博士 小 川 道 永助 手 医学博士 三 室 仁 美助 手 理学博士 立 野 一 郎＊

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prominent pathogenic features. EPEC infectionalso leads to host cell death and damage to theintestinal mucosa, which is partly dependentupon EspF, one of the effectors. In this study,we demonstrate that EspF is a mitochondrialimport protein with a functional mitochondrialtargeting signal (MTS), since the EspF activity toimport into mitochondria was abrogated byMTS deletion mutants. Substitution of the 16thleucine with glutamic acid (EspF(L16E)) com-pletely abolished the EspF activity. Infection ofHeLa cells with wild type but not the espF mu-tant (∆espF) decreased mitochondrial membranepotential (∆Ym), leading to cell death. The ∆Ymdecrease and cell death were restored in cells in-fected with ∆espF/pEspF but not ∆espF/pEspF(L16E), suggesting the 16th leucine in the MTSto be a critical amino acid for EspF function. Todemonstrate the impact of EspF in vivo, we ex-ploited Citrobacter rodentium by infecting C3H/HeJ mice with ∆espFCR, ∆espFCR/pEspFCR or∆espFCR/pEspF(L16E)CR. The results indicatethat EspF activity contributes to bacterial patho-genesis, as judged by murine lethality and intes-tinal histopathology, and promotion of bacterialcolonization of the intestinal mucosa.

3. A novel caspase-1/Toll-like receptor 4-independent pathway of cell death inducedby cytosolic Shigella in infected macro-phages.

Toshihiko Suzuki, Kenji Nakanishi1, HirokoTsutsui1, Hiroki Iwai, Shizuo Akira2, NaohiroInohara3, Mathias Chamaillard3,4 , GabrielNuñez3,4, and Chihiro Sasakawa: 1Departmentof Immunology and Medical Zoology, HyogoCollege of Medicine, 2Department of Host De-fense, Research Institute for Microbial Dis-eases, Osaka University, 3Department of Pa-thology and 4Comprehensive Cancer Center,The University of Michigan Medical School

Shigella-induced macrophage cell death is animportant step in the induction of acute inflam-matory responses that ultimately lead to bacil-lary dysentery. Cell death was previously re-ported to be dependent upon the activation ofcaspase-1 via interaction with IpaB secreted byintracellular Shigella , but in this study, we showthat Shigella infection of macrophages can alsoinduce cell death independent of caspase-1 or

IpaB activity. Time-lapse imaging and electronmicroscopic analyses indicated that caspase-1-dependent and -independent cell death is mor-phologically indistinguishable, and that both re-semble necrosis. Analyses of Shigella mutants orEscherichia coli using co-infection with Listeriasuggested that a component common to Gram-negative bacteria is involved in inducingcaspase-1-independent cell death. Further stud-ies revealed that translocation of bacterial lipidA into the cytosol of macrophages potentiallymediates cell death. Notably, cell death inducedby cytosolic bacteria was TLR4-independent.These results identify a novel cell death path-way induced by intracellular Gram-negativebacteria that may play a role in microbial-hostinteractions and inflammatory responses.

4. Enteropathogenic Escherichia coli acti-vates the RhoA signaling pathway via thestimulation of GEF-H1.

Takeshi Matsuzawa1, Asaomi Kuwae1, SeiYoshida, Chihiro Sasakawa and Akio Abe1:1Kitasato Institute for Life Sciences, KitasatoUniversity

Enteropathogenic Escherichia coli delivers asubset of effectors into host cells via a type IIIsecretion system, and this step is required forthe progression of disease. Here, we show thatthe type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the de-struction of the microtubule networks beneathadherent bacteria. Both effectors were shown topossess the ability to interact with tubulins, andto stimulate microtubule destabilization in vitro.A recent study showed that microtubule-boundGEF-H1, a RhoA-specific guanine nucleotide ex-change factor, was converted to its active formby microtubule destabilization, and this se-quence of events resulted in RhoA stimulation.Indeed, EspG- and Orf3-induced stress fiber for-mation was inhibited by the expression ofdominant-negative forms of GEF-H1 and RhoA,but not of Rac1 and Cdc42, and by treatmentwith a ROCK inhibitor. These results indicatethat the impact of EspG/Orf3 on microtubulenetworks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity.This report reveals for the first time that apathogen can exploit the host factor GEF-H1.

Publications

Ogawa, M., Yoshimori, T., Suzuki, T., Sagara,H., Mizushima, N. and Sasakawa, C. Escapeof Intracellular Shigella from Autophagy. Sci-

ence. 307: 727-31, 2005.Nagai, T., Abe, A. and Sasakawa, C. Targeting

of enteropathogenic Escherichia coli EspF to

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host mitochondria is essential for the bacterialpathogenesis: critical role of the 16th leucineresidue in EspF. J. Biol. Chem. 280: 2998-3011.2005

Suzuki, T., Nakanishi, K., Tsutsui, H., Iwai, H.,Akira, S., Inohara, N., Chamaillard, M.,Nuñez, G. and Sasakawa, C. A novel caspase-1/Toll-like receptor 4-independent pathway ofcell death induced by cytosolic Shigella in in-fected macrophages. J. Biol. Chem., in press.

Matsuzawa, T., Kuwae, A., Yoshida, S., Sasak-awa, C. and Abe, A. Enteropathogenic Es-cherichia coli activates the RhoA signalingpathway via the stimulation of GEF-H1.EMBO J. 23: 3570-82. 2004.

Jang, MH., Kweon, MN., Iwatani , K. ,Yamamoto, M., Terahara, K., Sasakawa, C.,Suzuki, T., Nochi, T., Yokota. Y., Rennert, PD.,Hiroi, T., Tamagawa, H., Iijima, H., Kunisawa,J., Yuki, Y. and Kiyono, H. Intestinal villousM cells: an antigen entry site in the mucosalepithelium. Proc. Natl. Acad. Sci. U.S.A. 101:6110-5. 2004.

Takahashi, T., Matsumoto, T., Nakamura, M.,Matsui, H., Kiyohara, H., Sasakawa, C. andYamada, H. A novel in vitro infection modelof Helicobacter pylori using mucin-producingmurine gastric surface mucous cells. Helico-bacter. 9: 302-12. 2004.

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Self-defense against invaded pathogenic microorganisms and foreign antigenicmolecules is strictly controlled by the immune system and inflammation. Our majorresearch interests are to elucidate cells and effector molecules in innate and ac-quired immunity and inflammation. In particular, we are focused on cellular andmolecular mechanisms of development and activation of B cells and IgH classswitch recombination under the influence of T cells, cytokines and adaptor pro-teins. We are also interested in elucidating cellular mechanisms of preferential in-duction of Th1 cells upon immunization with Mycobacterium-derived, Peptide-25and its derivatives.In 2004, Professor Dr. Fritz Melchers, Basel University, who were invited by theUniversity of Tokyo as the Eminent Scientist of JSPS, has been joining us for ourresearch projects. Dr. Melchers always encouraged us and suggested importantissues for each project. We would like to appreciate his enormous contributions.

1. Roles of IL-5 in the B cell differentiation

a. Molecular mechanisms of class switch re-combination in CD38-activated B cells.

Yumiko Tsukamoto, Atsushi Sato, KiyoshiTakatsu

Class switch recombination (CSR) is the proc-ess that changes physiological activities of anti-bodies without changing their specificities to an-tigens. Murine B cells differentiate into IgG1- orIgE- secreting cells when activated by anti-CD40and IL-4. However, IL-4 cannot induce CSR inanti-CD38-activated B cells, although IL-5 caninduce CSR in both anti-CD40- and anti-CD38-activated B cells. Anti-CD38- and IL-4-activatedB cells differentiate into IgG1-secreting cells

when stimulated with 8-mercaptugoanosine (8-SGuo). 8-SGuo is a guanosine analogue, and itinduces proliferation and differentiation of Bcells. Activation-induced cytidine deaminase(AID), which is necessary for CSR, is expressedin anti-CD38, IL-4, and 8-SGuo-activated B cells,and isn’t expressed in anti-CD38- and IL-4-activated B cells. It suggests that 8-SGuo inducesexpression of AID in B cells, and thus inducesCSR.

In anti-CD38- and 8-SGuo-activated B cells,AID is expressed; however these B cells don’tdifferentiate into IgG1-secreting B cells. Thissuggests that IL-4 also induces some factor(s)necessary for CSR in anti-CD38, IL-4, and 8-SGuo-activated B cells. Cell division, transcrip-tion of germline γ1, and looping out of γ1-µ re-ciprocal DNA are necessary for CSR. In anti-CD

Department of Microbiology and Immunology

Division of Immunology免疫調節分野

Professor Kiyoshi Takatsu, Ph.D.Associate Professor Satoshi Takaki, M.D., Ph.D.Research Associate Toshiki Tamura, Ph.D.Research Associate Ai Kariyone, Ph.D.Research Associate Taku Kouro, M.D., Ph.D.Research Associate Keisuke Horikawa, M.D., Ph.D.

教 授 医学博士 � 津 聖 志助教授 医学博士 高 木 智助 手 医学博士 田 村 敏 生助 手 医学博士 刈 米 ア イ助 手 医学博士 紅 露 拓助 手 医学博士 堀 川 啓 介＊

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38- and 8-SGuo-activated B cells and anti-CD38,IL-4, and 8-SGuo-activated B cells, cell divisionand transcription of germline γ1 were induced.However, γ1-µ reciprocal DNA didn’t exist inanti-CD38- and 8-SGuo-activated B cells. Theseresults show that anti-CD38 and 8-SGuo cannotinduce CSR because of lacking some factor(s)which are involved in CSR other than cell divi-sion, transcription of germline γ1, and AID ex-pression. This factor(s) (Factor X) may be in-duced by IL-4 in anti-CD38, IL-4, and 8-SGuo-activated B cells. We examined the expression ofUNG, Bach2, and 53BP1 genes which are neces-sary for CSR and found that they were all ex-pressed in anti-CD38 and 8-SGuo-activated Bcells. Hence Factor X is not UNG, Bach2, and 53BP1. Identification of Factor X may reveal somenew aspects of CSR.

b. IL-5 induces IgG1 isotype switch recombi-nation in CD38-stimulated murine B cells

Atsushi Sato, Yumiko Tsukamoto, KeisukeHoniara, Kiyoshi Takatsu

As we reported, IL-5 stimulation of anti-CD38-stimulated murine splenic B (B-2) cells inducesµ-γ1 class switch recombination (CSR) leading toa high level of lgG1 production. Further addi-tion of IL-4 in the system enhances IL-5-dependent µ-γ1 CSR. Stat5a- and Stat5b-deficientB-2 cells could not show µ-γ1 CSR and lgG1production, despite of the intact induction of γ1germline transcripts. Cell division cycle analysesof Stat5b deficient B-2 cells using CFSE revealedthat Stat5b deficient B cells were normally di-vided to 5 or 6 times, but they could not expresssurface IgG1. These results implied that Stat5plays pivotal roles in the µ-γ1 CSR induction. RT-PCR analysis revealed equivalent levels of AIDexpression in wild type and Stat5b deficient Bcells, while expression of the Blimp-1 gene wasimpaired in Stat5b deficient B-2 cells.

We examined the involvement of Stat5a andStat5b in anti-CD38- and 8-SGuo-activated Bcells. Cell division and transcription of germlineγ1 were induced in anti-CD38- and 8-SGuo-activated B cells, but those B cells did not un-dergo CSR similar to anti-CD38- and IL-5-stimu-lated Stat5a deficient B cells. Hence we exam-ined the phosphorylation of Stat5a and Stat5b inanti-CD38- and 8-SGuo-activated B cells. Stat5aand Stat5b were not phosphorylated. Down-stream molecules of Stat5a and Stat5b may beinvolved in the process of CSR induction by anti-CD38, IL-4, and 8-SGuo.

c. Molecular mechanisms of nuclear factor(NF)-κB and the germline γ1 transcript ex-

pression in CD38-stimulated B cells

Hiroaki Kaku and Kiyoshi Takatsu

CD38 is an ectoenzyme with both ADP ribo-syl cyclase and cADP ribosyl hydrolase activity.Ligation of CD38 on B-2 cells by anti-CD38 mAbinduces B cell proliferation, IL-5Rα expression,and the germline γ1 mRNA expression. IL-5 pro-motes µ-γ1 CSR and IgM and IgG1 productionfrom anti-CD38-stimulated B-2 cells in an IL-4independent manner. We reported that NF-κBcomplexes play critical roles in the B cell activa-tion in response to CD38 ligation, and that Btk,PKC, PI3-kinase, BASH/BLNK, PLC-γ2 and ex-tracellular Ca2＋ influx are involved in NF-κB ac-tivation induced by anti-CD38 and IgM andIgG1 production induced by anti-CD38 and IL-5co-stimulation. By analyzing the upstream mole-cules of CD38 signaling pathway, we found thatanti-CD38-mediated NF-κB can be induced viaactivation of Syk and GTP-binding protein.Moreover, GTP-binding protein but not Syk wasco-immunoprecipitated with CD38 in cell lysatesprepared using Brij-58 detergent. These datasuggest that CD38 directly associates with GTP-binding protein or that CD38 constitutively lo-calizes in GTP-binding protein- or GPCR-associated membrane micro-domain. Therefore,we speculate that GTP-binding protein may linkand modulate CD38-Syk signaling pathway orthat anti-CD38-stimulation induce colocalizationof GTP-binding protein into BCR-associatedmicro-domain.

2. Role of interleukin-5 (IL-5) and B-1 cells inmucosal immunity and elicitation of con-tact sensitivity

a. Origin and differentiation of B-1 cells

Taku Kouro, Masashi Ikutani and KiyoshiTakatsu

B-1 cells form distinctive subset of B lympho-cyte characterized by preferential distribution toperitoneal cavity and contribution to serumnatural antibodies and intestinal IgA secretion.Although several lines of evidence showed thatconstitutive B cell receptor signaling causes ex-pression of B-1 phenotype, it is still unknownhow generation and differentiation of B-1 cellsare controlled. IL-5 is indispensable for normaldevelopment of B-1 cells because IL-5R－/－ miceshow reduced number and cell size of peritonealB-1 cells. To investigate roles of IL-5 in B-1 celldevelopment, we first checked expression of IL-5R on early lymphoid progenitors in the fetalliver. IL-5Rα is not detected on lineage marker

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negative (Lin－）fetal liver cells but weakly de-tected on B220＋ proB cells. Its expression wasalso induced when Lin－ fetal liver cells wereplaced in the stroma-free, serum-free culturewith B cell differentiating condition. Then Lin－

cells were stimulated or not stimulated with IL-5 in such cultures and transferred to lymphocyte-deficient SCID mice. Progenitors from eithercondition differentiated to B-1 cells in the SCIDmice but more B-1b cells were observed in themice received IL-5-stimulated progenitors. Inter-estingly, feces IgA was only restored in the micereceived IL-5-stimulated progenitors. B-1b cellsdifferentiated from IL-5-treated progenitors arelikely responsible for intestinal IgA, becauseSCID mice transferred with B-1b cells secretedmore IgA in the feces than mice received B-1acells did. From these results, we speculate thatIL-5 induces B-1b cell differentiation in the earlystage of gestation, which eventually results inproduction of IgA in the gut.

b. Identification of IL-5 secreting cells in vivo

Masashi Ikutani, Byoung-Gon Moon, TakuKouro and Kiyoshi Takatsu

B-1 cells differ from B-2 cells in surface mark-ers, anatomical location and developmental pat-tern, and are known to be involved in naturalimmunity by producing natural antibodies. B-1cells form distinctive subset of B lymphocytescharacterized by preferential distribution to peri-toneal and pleural cavities as well as unique an-tigen reactivity. Unlike conventional B-2 cells, B-1 cells are rather maintained by self-renewalthan de novo differentiation and are activatedindependent of helper T cells though details inthe proliferation and activation of B-1 cells areunknown. We are focusing on IL-5 as a candi-date of soluble factor that controls B-1 cell de-velopment and activation. Indeed we haveshown that IL-5 is indispensable for survivaland homeostatic proliferation of peritoneal B-1cells and mucosal IgA responses. IL-5 is origi-nally found as a soluble factor secreted fromhelper T lymphocytes but recently significantamount of IL-5 is shown to be secreted fromnon-lymphoid tissues such as lung. B-1 cells alsodepend on IL-5 of non-lymphoid origin becauseneutralizing antibody against IL-5 still takes ef-fect on peritoneal B-1 cells in the host mice lack-ing lymphocytes. However, technical difficultieshave been preventing detection of IL-5 secretingcells that support B-1 cells. To overcome thisproblem, we are now generating mouse modelin which green fluorescent protein gene is in-serted in the IL-5 gene locus (IL-5/GFP knock-inmice).

c. Role of IL-5 and B-1 cells in mucosal im-munity

Atsuko Itakura, Yuji kikuchi1, Taku Kouro,Masashi Ikutani, Kiyoshi Takatsu: 1Laboratoryof Immunoregulation, Department of InfectionControl and Immunology, Kitasato Institutefor Life Sciences, Kitasato University

About a half of IgA is derived from B-1 cellsin intestinal and respiratory mucosa, whichplays an important role in primary host defensemechanism against pathogens that invadethrough mucosal tissues. B-1 cell numbers andthe size of B-1 cells are decreased in IL-5 recep-tor α chain-deficient (IL-5Rα－/－）mice, and theirserum IgM and intestinal IgA levels were lowerthan those of C57BL/6 mice. Therefore, we triedto determine if lack of IL-5/IL-5R signaling en-hances sensitivity to these pathogens.

IL-5Rα－/－ and wild type C57BL/6 mice wereorally or intranasally inoculated with Salmonellatyphimurium or influenza A PR/8 virus, respec-tively. Results revealed that IL-5Rα－/－ and wildtype mice died in a similar manner following in-oculation with S. typhimurium , and bacterialloads of Peyer’s patches, spleens and mesentericlymph nodes in IL-5Rα－/－ mice were compara-ble to those in wild type mice. We found no dif-ference in lung virus titers between IL-5Rα－/－

and wild type mice inoculated with PR/8.ELISA for IgM, IgG and IgA titers in bronchoal-veolar lavage and serum showed that PR/8-specific antibodies were induced in both IL-5Rα－/－ and wild type mice upon inoculation, andantibody titers in IL-5Rα－/－ mice were roughlyequivalent to those in wild type mice. These re-sults indicate that IL-5/IL-5R signaling does notseem to be involved in host defense against S.typhimurium and influenza virus, and antibodyresponses to these pathogens.

d. B-1 cells in elicitation of contact sensitiv-ity

Elicitation of contact sensitivity, a classic ex-ample of T cell-mediated immunity, requiresantigen-specific IgM antibodies, which are pro-duced by B-1 cells within 1 day after skin im-munization. Because IL-5 is important for main-tenance of B-1 cells, and promotes antibody pro-duction, we examined if IL-5 is involved in elici-tation of contact sensitivity. IL-5Rα－/－ and wildtype C57 BL/6 mice were immunized by paint-ing oxazolone on the chest, abdomen and feet.On day 4, mice were challenged by topical ap-plication of same antigen on the ears, and earthickness was measured at 24-hr post-challenge.

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We found that ear swelling responses were im-paired in IL-5Rα－/－ mice. Histological examina-tion of wild type mice ears showed edema ofthe connective tissue with massive accumulationof inflammatory cells including eosinophils. InIL-5Rα－/－ mice, edema and cell infiltration weremuch milder, and no eosinophils were observed.These results suggest that IL-5 is required forfull elicitation of contact sensitivity.

3. Regulatory functions of adaptor proteinsin the immune system

a. Control of hematopoietic stem/progenitorcell functions by Lnk adaptor protein,which functions as a molecular scaffoldlinking RTKs with cytoskeletal regulatorycomponents

Satoshi Takaki, Sang-Mo Kwon, Hitoshi Taki-zawa, Masanori Iseki, Kazuhiro Sudo2, HideoEma2, Jun Seita2, Mitsujiro Osawa2, HiromitsuNakauchi2 and Kiyoshi Takatsu: 2Laboratoryof Stem Cell Therapy, Center for ExperimentalMedicine, IMSUT

Lnk, a recently identified intracellular adaptorprotein, negatively regulates B-lymphopoiesisand early hematopoiesis. The lnk-deficient miceshow enhanced B cell production due to the hy-persensitivity of B precursors to stem cell factor,SCF. Competitive repopulation assays in irradi-ated host animals have demonstrated that theability of hematopoietic progenitors to generatevarious blood cells is greatly enhanced by theabsence of Lnk. We further investigated the ef-fect of lnk-deficiency on the compartment sizeand ability of hematopoietic stem cells (HSCs),which is strictly regulated in normal conditions.Measurement of competitive repopulation unit(CRU) as well as flow cytometric analysis re-vealed that there exist nearly 15-fold more func-tional HSCs in the bone marrow of adult lnk－/－

mice compared to normal mice. Clonal analysisby single cell HSC transplantation indicated thata part of lnk－/－ HSCs had highly repopulatingcapability.

Molecular mechanisms underlying Lnk-mediated regulation are not fully understood.We revealed that Lnk could control actin reor-ganization activated by receptor tyrosine kinases(RTKs), and thereby regulate cell migration. Lnkexpressing fibroblasts showed flattened, spread-ing shapes, prominent actin polymerization ac-companied by augmented Rac activation, andimpaired migration in a wound-healing assay.Lnk co-immunoprecipitated with Rac, Vav, PAKand filamin A, indicating a complex formationof cytoskeletal regulatory proteins supported by

Lnk. Adhesion and migration behavior of lnk－/－

progenitor cells are now under investigation.

b. Enhancing repopulation ability of hema-topoietic stem/progenitor cells by a tar-geted inhibition of Lnk

Hitoshi Takizawa, Chiyomi Kubo-Akashi, IkuoNobuhisa3, Sang-Mo Kwon, Masanori Iseki,Tetsuya Taga3, Kiyoshi Takatsu and SatoshiTakaki: 3Laboratory of Gene Expression andRegulation, Center for Experimental Medicine,IMSUT

Repopulating ability of HSC/Ps in irradiatedhost animals is greatly enhanced by the absenceof Lnk. In lnk-deficient mice, however, neithermalignant transformation nor functional defectof blood cells was observed. We identified func-tional domains of Lnk, generated dominant-negative (DN) Lnk mutants and tested whetherDN Lnk mutants could block functions of Lnkendogenously expressed in HSC/Ps and aug-ment repopulation ability of HSC/Ps. Lnk con-sists of an N-terminal proline-rich region, SH2-,PH-domains and a conserved tyrosine phospho-rylation site. Various Lnk mutants were gener-ated and transducted into MC9 mast cells usingretroviral vector, and their SCF-dependentgrowth was evaluated by monitoring eGFP＋

cells. While the wild-type Lnk efficiently inhib-ited growth of MC9 cells, a point mutation inthe SH2 domain completely abolished the in-hibitory effect. The SH2 mutants acted as DNmutants since they cancelled growth inhibitionof MC9 transfectants overexpressing Lnk. TheSH2 mutant with deletion of PH domain and C-terminal region was the most effective DN Lnkmutant. We, next, evaluated consequences of theDN Lnk expression in HSC/Ps by competitiverepopulation assay. HSC/Ps expressing the DNLnk was transferred into lethally irradiated hostanimals and percentage of eGFP＋ cells in pe-ripheral blood was monitored. Cells expressingDN Lnk repopulated at much higher percentagein lymphoid and myeloid lineages than cellstreated with control vectors. Inhibition of Lnkby the DN mutant could become a potent ap-proach for expansion and regulation of HSC/Ps.

c. Control of actin reorganization and B-1cell compartment size by adaptor moleculecontaining PH and SH2 domains, APS

Masanori Iseki, Chiyomi Kubo-Akashi, Sang-Mo Kwon, Nobuaki Yoshida3, Kiyoshi Takatsuand Satoshi Takaki

To understand functions of the Lnk family

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adaptor proteins further, we tried to identifyother members of the family, and isolatedmouse APS (adaptor molecule containing PHand SH2 domains). APS is expressed in varioustissues including spleen, bone marrow, brainand muscle, and in mature B but not in T or im-mature B cells. In cell lines, APS is tyrosinephosphorylated upon stimulation with IL-5, IL-3or anti-IgM. To investigate roles of APS in vivo,we generated APS －/－ mice. APS －/－ mice wereviable, fertile and show no anomalies or growthretardation. Lymphocyte or myeloid cell devel-opments in bone marrow, thymus, spleen andlymph nodes were not perturbed. However,APS －/－ mice had more B-1 cells in peritonealcavity, and showed enhanced humoral immuneresponses against thymus-independent type-2(TI-2) antigens, while APS －/－ B-2 cells exhibitednormal proliferative responses and tyrosinephosphorylation of intracellular proteins uponBCR crosslinking. On the other hand, in trans-genic mice overexpressing APS in lymphocytes,the numbers of peritoneal B-1 and splenic Bcells were reduced, and proliferation induced byanti-IgM stimulation was impaired. APS colocal-ized with filamentous actin (F-actin) accumu-lated during capping of BCR in APS -transgenicB cells. F-actin contents after BCR stimulationwas decreased in APS －/－ B-1 cells compared towild-type B-1 cells. Our results indicated thatAPS may have a novel regulatory role in actinreorganization and control of B-lineage cell com-partment size.

d. Roles of Lnk-family adaptor proteins, Lnk,SH2-B and APS in growth and functions ofmast cells: APS-deficiency causes aug-mented degranulation and reduced actinassembly

Chiyomi Kubo-Akashi, Masanori Iseki, Sang-Mo Kwon, Hitoshi Takizawa, Kiyoshi Takatsuand Satoshi Takaki

Lnk, SH2-B and APS form an adaptor proteinfamily conserved from drosophila. SH2-B isoriginally identified as a protein associated withimmunoreceptor tyrosine-based activation mo-tifs (ITAMs) of FcεRI γ-chain. APS is identifiedas a potential substrate of c-Kit. Since Lnk, SH2-B and APS are all expressed in bone marrow-derived mast cells (BMMCs), and may play rolesin signaling mediated through c-Kit or FcεRI,we investigated consequences of the deficiencyeither of Lnk, SH2-B or APS in mast cell func-tions. We established IL-3-dependent BMMCsfrom lnk－/－, SH2-B －/－ and APS －/－ mice. IL-3-dependent growth of those cells was compara-ble. Proliferation or adhesion mediated by c-Kit

activation as well as degranulation induced bycross-linking FcεRI were normal in the absenceof Lnk or SH2-B. In contrast, APS －/－ BMMCsshowed augmented degranulation after cross-linking FcεRI compared to wild type cells, whilec-Kit-mediated proliferation and adhesion werekept unaffected. The enhanced degranulationfrom APS －/－ BMMCs was due to augmenteddegranulation from each mast cell but not to in-creased proportion of cells that underwent de-granulation. Calcium influx and tyrosine phos-phorylation of various cellular proteins inducedby cross-linking FcεRI were normal in the ab-sence of APS, and cell survival mediated bybinding of monomeric IgE to FcεRI was alsonormal. We found, however, that APS －/－

BMMCs showed reduced F-actin assembly atsteady state. APS －/－ cells were resistant to la-trunculin, an inhibitor disrupting F-actin micro-filaments in FcεRI-mediated degranulation re-sponses. Our results suggest potential roles ofAPS in controlling actin cytoskeleton and mag-nitude of degranulation in mast cells.

4. Mechanisms of preferential induction ofTh1 response upon immunization with My-cobacteria peptide

The Ag85 of Mycobacterium (M .) tuberculosisand M. Bovis BCG is immunogenic in C57BL/6mice. Immunigation of C57BL/6 mice withAg85B expands TCRVβ11＋ CD4＋ Th1 cells inconjunction with APCs in an I-Ab-restrictedmanner. We identified the major antigenic epi-tope (Peptide-25) for Ag85B-specific Vβ11＋ Tcells as the 15-mer peptide, covering amino acidresidues 240-254 of Ag85B.

a. Role of MHC/peptide-TCR interaction inthe Peptide-25-dependent Th1 develop-ment

Haruyuki Ariga, Makiyo Nakada, TakeshiTokunaga, Yoko Shimohakamada, Ai Kari-yone, Toshiki Tamura and Kiyoshi Takatsu

Activated CD4＋ Th cells can be classified intotwo subsets, Th1 and Th2, on the basis of cy-tokine production profiles. Th1 cells play a criti-cal role in the induction of the cell-mediated im-mune responses that are important for the eradi-cation of intracellular pathogens and the devel-opment of organ-specific autoimmune diseases.In addition to the T cell antigen receptor (TCR)activation signals, other factors such as the cy-tokine environment, type of APC, genetic back-ground and co-stimulatory molecules expressedby activated APC may also be involved in thedetermination of the differentiation of naive CD

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4＋ T cells into Th1 cells. However, it is unclearwhether the TCR signaling events exert a directinfluence on Th1 differentiation. To elucidatecellular and molecular mechanisms of the induc-tion of Th1 cells by Peptide-25, transgenic mice(P25 TCR-Tg) that express the TCR-Vα5-Vβ11for recognition of Peptide-25, in conjunctionwith I-Ab molecules, were generated, and thedifferential activities of naive CD4＋ T cells fromP25 TCR-Tg were examined.

Naive CD4＋ T cells from P25 TCR-Tg prefer-entially differentiated into Th1 cells uponPeptide-25 stimulation in the presence of T andNK cell-depleted I-Ab splenic APC under neutralconditions. In contrast, a mutant of Peptide-25could induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation was observedeven in the presence of neutralizing monoclonalantibodies to IFN-γ and IL-12. Furthermore, na-ive CD4＋ T cells from STAT1 deficient P25 TCR-Tg also differentiated into Th1 cells uponPeptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovarycells (Peptide-25-I-Ab-CHO) that do not expressco-stimulatory molecules, such as CD40/80/86and ICAM-1 on their surface, and not produceany IFN-γ and IL-12 effectively, induced Th1differentiation of naive CD4＋ T cells from P25TCR-Tg. Within 3 hr after the TCR stimulationwith Peptide-25-I-Ab-CHO, IFN-γ- and IL-12-independent transient T-bet up-regulation andsuppression of GATA-3 expression were ob-served. These results imply that direct interac-tion between Peptide-25/I-Ab and TCR primarilyinfluences determination of the fate of naive CD4＋ T cells in differentiation toward the Th1 sub-sets.

b. Adjuvant activity of Peptide-25 for enhanc-ing anti-tumor immune response

Takeshi Kikuchi, Ai kariyone, Wen Xu,Toshiki Tamura and Kiyoshi Takatsu

CD8＋ cytotoxic T cells (CTL) play an impor-tant role in the protection against tumor growth.Tumor cells are thought to express an array ofantigens recognizable by CTLs that principallycontribute to tumor rejection. It still remains un-clear, however, whether CD4＋ helper T cells to-gether with CTLs mediate efficient immune re-sponses leading to tumor rejection.

As the immunization of C57BL/6 mice withPeptide-25 emulsified in incomplete Freund ad-juvant (IFA) induces Th1 response to Peptide-25,we examined adjuvant activity of Peptide-25 forCTL generation to ovalbumin (OVA) as a modeltumor antigen. Results revealed that co-immunization of C57BL/6 mice with OVA and

Peptide-25 could induce higher OVA specific-IgG2a and IFN-γ production than OVA immuni-zation. Intriguingly, a robust OVA-specific CTLgeneration was also induced when mice were co-immunized with OVA plus Peptide-25. The ad-juvant effect of Peptide-25 was not observed inCD4 deficient or IFN-γ deficient mice. Co-immunization of OVA and Peptide-25 couldprevent in vivo growth of EG.7 cell (EL4 thy-moma transfected with cDNA encoding chickenOVA) leading to the tumor rejection. Further-more, the enhancement of CTL generation byPeptide-25 was also observed when class I-binding OVA peptide (SIINFEKL) was used inplace of intact OVA. Moreover, CTL generationspecific for class I-binding B16 melanoma pep-tide (SVDFFVWL) was enhanced by co-immunization with Peptide-25. To elucidate themechanisms of this adjuvant activity of Peptide-25, we examined the dendritic cell (DC) activa-tion by Peptide-25. Results revealed that Peptide-25 stimulation alone did not enhance the ex-pression of surface markers on DC. When we co-cultured DC with CD4＋ T cells from P25 TCR-Tg mice together with Peptide-25, expressions ofMHC class I and ICAM-1 were enhanced andled to the inducation of IL-12p40 production.Such DC showed more effective OVA presenta-tion to OVA specific CD8＋ T cells and enhancedcell divisions.

These results indicate that Peptide-25 exertspotent adjuvant activity and provides efficienthelp for CTL induction against neo-tumor anti-gen when concomitantly immunized.

5. Role of mast cells, eosinophils, and IL-5 inthe development of asthma

Yoko Oe-Kikuchi and Kiyoshi Takatsu

Mast cells are though to contribute to thepathogenesis of allergic airway responsesthrough IgE dependent mechanism. Eosinophilicinflammation is clearly a hallmark of both aller-gic and non-allergic asthma. Considerable evi-dence suggests that there is association betweenpulmonary eosinophil infiltration and AHR inhuman asthma. An immunopathogenic role formast cells is suggested by the role of IL-4, IL-13,IL-5 in IgE synthesis and eosinophil differentia-tion and activation, however, the exact mecha-nism by which mast cells mediate eosinophilicinflammation and subsequent AHR are still notentirely clear.

We have studied the role of IL-5 on mast celland eosinophil activation and role of activatedmast cells in activation and survivability ofeosinophils. First, we have studied IgE-dependent production of histamine and cytokine

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(IL-5 and GM-CSF) by mast cells isolated fromIL-5Ra KO and IL-5 KO mice to observe thefunction of IL-5 on releasability. We observedthat mast cells, expressing detectable IL-5Ra, in-creased their steady state expression of IL-5mRNA after cross-linking the IgE receptor. Al-though we have observed IL-5, GM-CSF, andhistamine release from these mast cells, therewere no significant difference on each releasabil-ity, however, we have found that IL-5 was ableto reduce histamine content in mast cells in dose

dependent manner. Second, we have establishedthe system to isolate eosinophils from a long-term bone marrow culture supplemented withIL-5. Third, to understand the bioactivity of re-leased product from activated mast cells, wehave further investigated the effect of mast cellsupernatants on activation (such as degranula-tion and expression of adhesion molecule) andsurvivability of eosinophils isolated from a cul-ture of bone marrow cells.

Publications

Nagai, Y., Kobayashi, T., Motoi, Y., Ishiguro, K.,Akashi, S., Saitoh, S., Kusumoto, Y., Kaisho,T., Akira, S., Matsumoto, M., Takatsu, K. andMiyake, K. Radioprotective 105-MD-1 linkstoll-like receptors 2 (TLR2) and TLR4-MD2with antibody production to microbial mem-brane. J. Immunol. In press, 2005.

Inoue, H., Kato, R., Fukuyama, S., Nonami, A.,Taniguchi, K., Matsumoto, K., Nakano, T.,Tsuda, M., Matsumura, M., Kubo, M.,Ishikawa, F., Takatsu, K., Nakanishi, Y. andYoshimura, A. Spred-1 negatively regulatesallergen-induced airway eosinophilia and hy-perresponsiveness. J. Exp Med. 201: 73-82,2005.

Tamura, T., Ariga, H., Kinashi, T., Uehara, S.,Kikuchi, T., Nakada, M., Tokunaga, T., Xu,W., Kariyone, A., Saito, T., Kitamura, T., Max-well, G., Takaki, S. and Takatsu, K. The roleof antigenic peptide in CD4＋ T helper pheno-type development in a T cell receptor trans-genic model. Int. Immunol. 15: 1691-1699,2004.

Wen, X., Zhang, D., Abe, M., Jiang, Y., Mat-suoka, S., Nakamura, K., Hamano, Y.,Kikuchi, Y., Takatsu, K., Shirai, T. and Hirose,S. Transgene-mediated over-expression ofinterleukin-5 suppresses autoimmune disease,but increases the risk of B cell chronic lym-phocyte leukemia. Eur. J. Immunol, 34: 2740-2749, 2004.

Moon, B.G., Takaki, S., Nishizumi, H.,Yamamoto, T. and Takatsu, K. Abrogation ofautoimmune disease in Lyn-deficient mice bythe deletion of IL-5 receptor alpha chain gene.Cell. Immunol. 228: 110-118, 2004.

Tanaka, H., Komai, M., Nagao, K., Ishizaki, M.,

Kajiwara, D., Takatsu, K., Delespesse, G. andNagai, H. Role of IL-5 and eosinophils inallergen-induced airway remodeling in mice.Am. J. Resp. Cell. Mol. Biol. 31: 62-68, 2004.

Hirano, M., Kikuchi, Y., Nisitani, S., Yamaguchi,A., Satoh, A., Ito, T., Iba, H. and Takatsu K.Bruton’s tyrosine kinase (Btk) enhances tran-scriptional co-activation activity of BAM11, aBtk-associated molecule of a subunit of SWI/SNF complexes. Int Immunol. 16: 747-757,2004.

Moon, B.G., Takaki, S., Miyake, K. and Takatsu,K. The role of IL-5 for mature B-1 cells in ho-meostatic proliferation, cell survival, and Igproduction. J. Immunol. 172: 6020-6029, 2004.

Kubo-Akashi, C., Iseki, M., Kweon, S.M. Taki-zawa, H., Takatsu, K. and Takaki S. Roles oconserved family of adaptor proteins, Lnk, SH2-B, and APS for mast cell developmentgrowth, and functions: APS-deficiency causesimpaired degranulation and reduced actin as-sembly. Biochem Biophys Res Commun. 315:356-362, 2004.

Iseki, M., Kubo, C., Kwon, S.M., Yamaguchi, A.,Kataoka, Y., Yoshida, N., Takatsu, K. andTakaki, S. Negative regulatory role of APS,adaptor molecule containing PH and SH2 do-mains in B-1 cells and B cell receptor-mediated proliferation. Mol. Cell. Biol. 24:2243-2250, 2004.

Nakamura, K., Kouro, T., Kincade, P.W., Malyk-hin, A., Maeda, K., and Coggeshall, K.M. SrcHomology 2-containing 5-inositol phosphatase(SHIP) suppresses an early stage of lymphoidcell development through elevated interleukin-6 production by myeloid cells in bone mar-row. J Exp Med 199: 243-254, 2004.

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Cellular mechanisms for the surveillance and transcriptional suppression of in-trogenomic parasites such as retoroviruses and transposons are now being recog-nized as an important host cell defense system through epigenetical regulation ofchromosome. In Drosophila and plants, transcripts of retrotransposon are post-transcriptionally suppressed by a mechanism designated as RNA silencing (orRNA interference), but it is not clear whether a similar mechanism is operating inhuman retroviral gene silencing. Our goal is to elucidate the entire human cellulardefence system and counter-defence viral strategy. These studies would give usnew ideas for latent infection observed in many human viruses including HIV orHTLV and also for the design of unique retroviral vectors that would achieve long-term transgene expression providing strong tools for human gene therapy and re-generation medicine.

1. Epigenetical regulation and SWI/SNF chro-matin remodeling complex

Since retroviruses that are once integrated intohost chromosomes cannot be excised, host cellsinevitably uses epigenetical regulation systemsto shut-off the virus gene expression. Thereforeelucidation of epigenetical regulation mecha-nisms is now essential to understand both hostand viral strategies in this post-genome era. Incellular nuclei, DNA methylation, histone ace-tylation and chromatin remodeling play majorroles in epigenetical regulations. On the otherhand, some RNA transcripts of exogenous aswell as endogenous genes are regulated at post-transcriptional level by a mechanism designatedas RNA silencing (RNA interference). There is agrowing body of evidence indicating that chro-mosomal regulation and RNA silencing are in-terconnected.

For the understanding of epigenetics, we have

been concentrated on the major chromatin re-modeling factor in human, SWI/SNF complex,which is composed of 10 protein subunits. Thecatalytic subunits, BRG1 and Brm, have ATPaseactivity with helicase motifs. Each SWI/SNFcomplex contains a single molecule of eitherBRG1 or Brm, but not both. We previouslyshowed mechanistic links between chromatin re-modeling factor SWI/SNF complex and tran-scriptional factor AP-1, which is composed ofheterodimers between Fos family proteins andJun family proteins. AP-1 is known to play im-portant roles in wide variety of biological func-tion, such as host and viral immediate early re-sponses, cellular growth, differentiation and tu-mor formation. Our results showed that a spe-cific subset of Fos/Jun dimers specifically bindto the BAF60a subunit of SWI/SNF complexand recruits the entire complex to the AP-1DNA binding sites located in a relatively inac-tive context of chromatin. The recruited SWI/

Department of Microbiology and Immunology

Division of Host-Parasite Interaction宿主寄生体学

Professor Hideo Iba, Ph. D.Research Associate Taiji Ito, Ph. D.Research Associate Shigeru Minoguchi, Ph. D.Research Associate Taketoshi Mizutani, Ph. D.

教 授 理学博士 伊 庭 英 夫助 手 理学博士 伊 藤 太 二助 手 医学博士 箕 口 滋助 手 医学博士 水 谷 壮 利

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SNF complex remodels flanking nucleosomes toinitiate the transcription. From these observa-tions, we have concluded that BAF60a is themajor determinant of AP-1 transactivating activ-ity.

In 2002, we showed that MuLV-based retrovi-rus vector transgene expression is rapidly si-lenced in human tumor cell lines lacking expres-sion of Brm even though these vectors can suc-cessfully enter, integrate, and initiate transcrip-tion. We detected this gene silencing as a reduc-tion in the ratio of cells expressing the exoge-nous gene rather than a reduction in the aver-age expression level, indicating that down-regulation occurs in an all-or-none manner.Retroviral gene expression was protected fromsilencing and maintained in Brm-deficient hostcells by exogenous expression of Brm but notBRG1, an alternative ATPase subunit in theSWI/SNF complex. Introduction of exogenousBrm to these cells suppressed recruitment ofprotein complexes containing YY1 and histonedeacetylase (HDAC) -1 and -2 to the 5’-LTR re-gion of the integrated provirus, leading to theenhancement of acetylation of specific lysineresidues in histone H4 located in this region.These results suggest that the Brm-containingSWI/SNF complex subfamily (trithorax-G) and acomplex including YY1, EZH2, EED andHDACs (Polycomb-G) counteract each other tomaintain transcription of exogenously intro-duced genes.

Since the host protein, Brm plays pivotal rolesin epigenetical regulation of retrovirus expres-sion, we were next interested in the biogenesisof Brm and started initial studies using a humancell line, SW13, which lacks expression of bothBrm and BRG-1. This year, we extended thisearly study by analysing seven human cell linesdeficient in Brm (designated as Brm-deficientcell lines thereafter) (a). We showed that in eachcell line examined, the functional Brm gene ispresent and actively transcribed but is not ex-pressed as mRNA, indicating that an uniquemolecular mechanism of post-transcriptionalgene silencing (PTGS) is operating in these celllines.

In the process of these studies, we have no-ticed that SWI/SNF complex does not necessar-ily function as a positive regulator of transcrip-tion; this complex strongly suppresses severalneuron-specific genes in some non-neuronalcells. Therefore we have also concentrated onthe molecular mechanisms underlaying this phe-nomenon this year and found that SWI/SNFcomplex can supress of transcription in a spe-cific context and plays rather broad biologicalfunction depending upon each promoter even inthe same cell (b).

(a) The Brm gene suppressed at the post-transcriptional level in various human celllines is inducible by transient HDAC in-hibitor treatment, which exhibits anti-oncogenic potential.

Nobutake Yamamichi, Mitsue Yamamichi-Nishina, Taketoshi Mizutani, Hirotaka Watan-abe, Shigeru Minoguchi, Nao Kobayashi, Sa-toko Kimura, Taiji Ito, Naohisa Yahagi1,Masao Ichinose2, Masao Omata1, and HideoIba: 1Department of Gastroenterology, Facultyof Medicine, University of Tokyo, Tokyo, 2Sec-ond Department of Internal Medicine ,Wakayama Medical College, Wakayama, Ja-pan

To understand the biological function and bio-genesis of Brm protein, we examined seven celllines derived from various human tumors thatdo not produce Brm protein. We show thatthese Brm-deficient cell lines transcribe the Brmgenes efficiently as detected by nuclear run-ontranscription assay, whereas Brm mRNA wasundetectable by reverse transcription-polyme-rase chain reaction analysis. These results indi-cate that expression of Brm is strongly sup-pressed at the post-transcriptional level throughprocessing and transport of the primary tran-script or through stability of mature BrmmRNA. This suppression was strongly attenu-ated by transient treatment of these cell lineswith HDAC inhibitors through indirect mecha-nism. Importantly, all of the treated cellsshowed prolonged induction of Brm expressionafter the removal of HDAC inhibitors, and ac-quired the ability to maintain retroviral gene ex-pression. Since we previously reported that lossof Brm but not of BRG1 causes transcriptionalgene silencing of murine leukemia virus-basedretrovirus vectors, these results indicate thatthese Brm-deficient human tumor cell linescarry a functional Brm gene. Treatment withHDAC inhibitors or introduction of exogenousBrm into Brm-deficient cell lines significantly re-duced the oncogenic potential as assessed bycolony-forming activity in soft agar or invasioninto collagen gel, indicating that, like BRG1 , Brmis involved in tumor suppression.

(b) SWI/SNF complex is a key negative regu-lator of neuronal gene expression in hu-man non-small cell lung carcinoma celllines.

Hirotaka Watanabe, Taketoshi Mizutani, Take-shi Haraguchi, Nobutake Yamamichi, ShigeruMinoguchi, Mitsue Yamamichi-Nishina, No-zomu Mori3, and Hideo Iba: 3Department of

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Molecular Anatomy and Neurobiology, Na-gasaki University School of Medicine, Japan.

In this year, we found that specific neuronallineage marker genes such as synaptophysin andSCG10 are expressed in several human non-small cell lung carcinoma cell lines deficient inboth Brm and BRG1 expression. Exogenous ex-pression of either Brm or BRG1 in these celllines induced expression of IL-6 but decreasedexpression of these neuron-specific genes, indi-cating that the SWI/SNF complex can functionas either a positive or negative regulator in thesame cell. In addition, retrovirus vectors ex-pressing siRNAs designed to suppress expres-sion of Brm , BRG1 , or Ini1 , which encodes an-other integral component of the SWI/SNF com-plex, induced expression of the neuronal geneseven in SWI/SNF-competent lung carcinomacell lines. These results reveal that the SWI/SNFcomplex is necessary to suppress expression ofthese neuron-specific genes in lung epithelialcells. We present evidence that this suppressionrequires association of the SWI/SNF complexwith a complex that includes neuron-restrictivesilencer factor (NRSF), CoREST, mSin3A, andHDAC1/2. This larger complex induced efficientand specific deacetylation of histone H4 in thesynaptophysin gene, when SWI/SNF complexwas recruited to the NRSF binding site by mSin3A and CoREST. Patients with Brm/BRG1-deficient lung carcinoma are reported to havepoor prognosis; epigenetic disturbance of theseneuron-specific genes could enhance tumori-genicity and possibly provide selective markersfor targeting these tumors.

2. Design and establishment of new retrovi-rus vectors that express shRNA efficiently.

Taketoshi Mizutani, Takeshi Haraguchi, Nobu-take Yamamichi, Takashi Kameda4, ToshihiroSugiyama4, and Hideo Iba: 4Department of Bio-chemistry, Akita University, School of Medi-cine, Akita Japan.

In many organisms such as plant, nematoda,Drosophila, RNA silencing systems, which in-clude such molecules as short interfering (si)RNA and micro (mi) RNA, are now known toregulate expression of specific endogenousgenes, exogenously introduced viral genomesand intragenomic parasites such as endogenousretrovirus and retrotransposons. Whereas bio-logical function of RNA silencing (or RNA inter-ference) in human remain largely elusive, RNAsilencing has recently emerged as a specific and

efficient method to silence gene expression inhuman cells either by introducing siRNA di-rectly or short hairpin (sh) RNA that functionsas an artificial precursor of siRNA.

This year, we have designed several retrovi-rus vectors carrying expression units for shRNA.We have prepared all of them as VSV-G pseu-dotypes and selected the vectors that efficientlysuppress the expression of a target gene, (weused the exogenous GFP gene). We finally estab-lished a MuLV-based retrovirus vector that canreduce the expression level of exogenous GFPup to 1％. To achieve such an efficient “kcock-down”, following vector structures and trans-duction procedure were needed.1. The MuLV based vector DNA was deleted in

a large region of 3’-LTR U3, and to the de-leted region, shRNA expression unit was in-serted. Therefore after vector preparation byDNA transfection, the provirus lost func-tional Pol II promoter activity in both 5’-and3’-LTR, and have a structure so called a “self-inactivating vector”. The shRNA expressionunit is composed of U6 promoter (drived byPol III), the hairpin region which contains aloop sequence originated from mir-23a andsubsequently oligoT (T6) sequence (termina-tion signal of Pol III). This unit is present atboth LTRs of the provirus.

2. The vector should be prepared as VSV-Gpseudotyped vector to ensure introduction ofmulti-copy proviruses into the human cell bya single transduction.

3. This shRNA expression vector shows dosage-dependent suppressing activity when intro-duced up to 3～7 copies per cell and can re-duse the expression levels of the target geneto 10％ of that of uninfected cell or emptyvector transduced cell. But higher vector dos-age of the same vector shows no additionalsuppression. When these cells were trans-duced with another vector that expresses adifferent shRNA of the same target gene, theexpression levels of the target gene was re-duced up to 1％ of the original cell.

This procedure was applicable to most of thecell lines originated from human tumors, but wefound that knock-down effects was significantlyreduced in mouse embryonic stem cells or non-dividing human cells. Therefore, we are now in-troducing the same shRNA expression unit intolentivirus vectors for broader application. Weare also analyzing the molecular mechanisms ofRNA silencing using the cell lines establishedabove, in which the target GFP gene is effi-ciently suppressed.

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Publications

Yamamichi, N., Yamamichi-Nishina, M., Mizu-tani, T., Watanabe, H., Minoguchi, S., Kobay-ashi, N., Kimura, S., Ito, T., Yahagi, N., Ichi-nose M., Omata, M., and Iba, H. The Brmgene suppressed at the post-transcriptionallevel in various human cell lines is inducibleby transient HDAC inhibitor treatment, whichexhibits anti-oncogenic potential. Oncogene inpress (2005)

Joo, A., Aburatani, H., Morii, E., Iba, H. andYoshimura, A. STAT3 and MITF cooperativelyinduce cellular transformation throughupregulation of c-fos expression. Oncogene. 23:726-734 (2004)

Hirano, M., kikuchi, Y., Nisitani, S., Yamaguchi,A., Satoh, A., Ito, T., Iba, H. and Takatsu, K.Bruton’s tyrosine kinase (Btk) enhances tran-scriptional co-activation activity of BAM11, aBtk-associated molecule of a subunit of SWI/SNF complexes. International Immunology(2004)伊庭英夫，山道―仁科光恵，水谷壮利：レトロウイルスのジーンサイレンシング―ウイルス学はエピジェネティクスの分子機構の解明を担うウイルス ５３：１３３―１３９，２００３．水谷壮利，原口健，伊庭英夫「shRNA発現ユニットを搭載したウイルスベクター」遺伝子医学MOOK「RNAと創薬」印刷中

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Viruses can cause devastating diseases. The long-term goal of our research is tounderstand the molecular pathogenesis of viral diseases, using influenza andEbola virus infections as models. Interactions between viral and host gene prod-ucts during viral replication cycles determine the consequences of infection (i.e.,the characteristics of disease manifestation, whether limited or widespread);hence, our research has centered on such interactions in these viral infections.

1. Production of novel Ebola virus-like parti-cles from cDNAs: an alternative to Ebolavirus generation by reverse genetics.

Watanabe S, Watanabe T, Noda T, Takada A,Feldmann H, Jasenosky LD, Kawaoka Y.

We established a plasmid-based system forgenerating infectious Ebola virus-like particles(VLPs), which contain an Ebola virus-like mini-genome consisting of a negative-sense copy ofthe green fluorescent protein gene. This systemproduced nearly 103 infectious particles per mlof supernatant, equivalent to the titer of Ebolavirus generated by a reverse genetics system. In-terestingly, infectious Ebola VLPs were gener-ated, even without expression of VP24. Trans-mission and scanning electron microscopicanalyses showed that the morphology of theEbola VLPs was indistinguishable from that ofauthentic Ebola virus. Thus, this system allowsus to study Ebola virus entry, replication, andassembly without biosafety level 4 containment.Furthermore, it may be useful in vaccine pro-

duction against this highly pathogenic agent.

2. A Human Macrophage C-Type Lectin Spe-cific for Galactose and N -Acetylgalacto-samine Promotes Filovirus Entry.

Takada A, Fujioka K, Tsuiji M, Morikawa A,Higashi N, Ebihara H, Kobasa D, Feldmann H,Irimura T, Kawaoka Y.

Filoviruses cause lethal hemorrhagic diseasein humans and nonhuman primates. An initialtarget of filovirus infection is the mononuclearphagocytic cell. Calcium-dependent (C-type)lectins such as dendritic cell- or liver／lymphnode-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as thehepatic asialoglycoprotein receptor, bind toEbola or Marburg virus glycoprotein (GP) andenhance the infectivity of these viruses in vitro.Here, we demonstrate that a recently identifiedhuman macrophage galactose- and N -acetylga-

Department of Microbiology and Immunology

Division of Virologyウイルス感染分野

Professor Yoshihiro Kawaoka, DVM, Ph.D.Associate Professor Taisuke Horimoto, DVM, Ph.D.Research Associate Hidio Goto, DVM, Ph.D.Research Associate Ayato Takada, DVM, Ph.D.Research Associate Yuko Sakai-Tagawa, Ph.D.Faculty Member Kiyoko Iwatsuki-Horimoto, DVM, Ph.D.

教 授 獣医学博士 河 岡 義 裕助教授 獣医学博士 堀 本 泰 介助 手 獣医学博士 五 藤 秀 男助 手 獣医学博士 高 田 礼 人助 手 医学博士 坂井（田川）優子特任教員 獣医学博士 岩附（堀本）研子

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lactosamine-specific C-type lectin (hMGL),whose ligand specificity differs from DC-SIGNand L-SIGN, also enhances the infectivity offiloviruses. This enhancement was substantiallyweaker for the Reston and Marburg viruses thanfor the highly pathogenic Zaire virus. We alsoshow that the heavily glycosylated, mucin-likedomain on the filovirus GP is required for effi-cient interaction with this lectin. Furthermore,hMGL, like DC-SIGN and L-SIGN, is present oncells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggestinga role for these C-type lectins in viral replicationin vivo. We propose that filoviruses use differ-ent C-type lectins to gain cellular entry, depend-ing on the cell type, and promote efficient viralreplication.

3. A protective immune response in mice toviral components other than hemaggluti-nin in a live influenza A virus vaccinemodel.

Horimoto T, Takada A, Iwatsuki-Horimoto K,Kawaoka Y.

Previously, we generated influenza A virusesthat possess chimeric type (A／B) hemagglutinins(HA), in which immunogenic regions of type AHA were replaced with those of type B HA, andshowed that these viruses were attenuated inmice. Here we intranasally immunized micewith these viruses and then challenged themwith a wild-type A virus to assess a protectiveimmune response to viral components otherthan HA in the form of a live virus. All immu-nized mice survived challenge with a lethal doseof wild-type virus; none or a limited amount ofvirus, if any, was recovered from nasal tur-binates or lungs of the mice 3 days post-challenge. These results provide direct evidencethat immune responses to viral componentsother than HA confer protection against influ-enza A virus infection in a mouse model, sug-gesting the usefulness of live vaccines for vi-ruses that have undergone antigenic drift withrespect to HA, or for viruses with heterosub-typic HAs.

4. Influenza B Virus Requires BM2 Protein forReplication.

Hatta M, Goto H, Kawaoka Y.

The BM2 protein of influenza B virus func-tions as an ion channel, which is suggested tobe important for virus uncoating in endosomesof virus-infected cells. Because direct support forthis function is lacking, whether BM2 plays an

essential role in the viral life cycle remains un-known. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutantviruses possessing M segments with the mu-tated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still ex-pressed BM2. The introduction of multiple stopcodons and a one-nucleotide deletion down-stream of the stop-start pentanucleotide, in addi-tion to disablement of the BM2 initiation codon,failed to generate viable mutant viruses, but themutant M segments still expressed proteins thatreacted with the BM2 peptide antiserum. Tocompletely abolish BM2 expression, we gener-ated a mutant M gene whose BM2 open readingframe was deleted. Although this mutant wasnot able to replicate in normal MDCK cells, itdid replicate in a cell line that we establishedwhich constitutively expresses BM2. Further-more, a virus possessing the mutant M genelacking the BM2 open reading frame and a mu-tant NA gene containing the BM2 open readingframe instead of the NA open reading frame un-derwent multiple cycles of replication in MDCKcells, with exogenous sialidase used to supple-ment the deleted viral sialidase activity. Thesefindings demonstrate that the BM2 protein is es-sential for influenza B virus replication.

5. Characterization of a neuraminidase-de-ficient influenza A virus as a potentialgene delivery vector and a live vaccine.

Shinya K, Fujii Y, Ito H, Ito T, Kawaoka Y.

We recently identified a packaging signal inthe neuraminidase (NA) viral RNA (vRNA) seg-ment of an influenza A virus, allowing us toproduce a mutant virus [GFP(NA)-Flu] thatlacks most of the NA open reading frame butcontains instead the gene encoding green fluo-rescent protein (GFP). To exploit the expandingknowledge of vRNA packaging signals to estab-lish influenza virus vectors for the expression offoreign genes, we studied the replicative proper-ties of this virus in cell culture and mice. Com-pared to wild-type virus, GFP(NA)-Flu washighly attenuated in normal cultured cells butwas able to grow to a titer of >106 PFU／ml in amutant cell line expressing reduced levels ofsialic acid on the cell surface. GFP expressionfrom this virus was stable even after five pas-sages in the latter cells. In intranasally infectedmice, GFP was detected in the epithelial cells ofnasal mucosa, bronchioles, and alveoli for up to4 days postinfection. We attribute the attenuatedgrowth of GFP(NA)-Flu to virion aggregation atthe surface of bronchiolar epithelia. In studies totest the potential of this mutant as a live attenu-

29

ated influenza vaccine, all mice vaccinated with>_105 PFU of GFP(NA)-Flu survived when chal-lenged with lethal doses of the parent virus.These results suggest that influenza virus couldbe a useful vector for expressing foreign genesand that a sialidase-deficient virus may offer analternative to the live influenza vaccines recentlyapproved for human use.

6. Generation of influenza A virus NS2(NEP)mutants with an altered nuclear export sig-nal sequence.

Iwatsuki-Horimoto K, Horimoto T, Fujii Y,Kawaoka Y.

The NS2 (NEP) protein of influenza A viruscontains a highly conserved nuclear export sig-nal (NES) motif in its amino-terminal region (12

ILMRMSKMQL21, A／WSN／33), which is thoughtto be required for nuclear export of viral ribonu-cleoprotein complexes (vRNPs) mediated by acellular export factor, CRM1. However, simulta-neous replacement of three hydrophobic resi-dues in the NES with alanine does not affect NS2 (NEP) binding to CRM1, although the viruswith these mutations is not viable. To determinethe extent of sequence conservation required bythe NS2 (NEP) NES for its export function dur-ing viral replication, we randomly introducedmutations by degenerative mutagenesis into theregion of NS cDNA encoding the NS2 (NEP)NES and then attempted to generate mutant vi-ruses containing these alterations by reverse ge-netics. Sequence analysis of the recovered vi-ruses showed that although some of the mutantspossessed amino acids other than those con-served in the NES, hydrophobicity within thismotif was maintained. Nuclear export of vRNPsrepresenting all of the mutant viruses was com-pletely inhibited in the presence of a CRM1 in-hibitor, leptomycin B, as was the transport ofwild-type virus, indicating that the CRM1-mediated pathway is responsible for the nuclearexport of both wild-type and mutant vRNPs.The vRNPs of some of the mutant viruses wereexported in a delayed manner, resulting in lim-ited viral growth in cell culture and in mice.These results suggest that the NES motif may bean attractive target for the introduction of at-tenuating mutations in the production of livevaccine viruses.

7. Resistant influenza A viruses in childrentreated with oseltamivir: descriptive study.

Kiso M, Mitamura K, Sakai-Tagawa Y,Shiraishi K, Kawakami C, Kimura K, HaydenFG, Sugaya N, Kawaoka Y.

Oseltamivir is an effective inhibitor of influ-enza virus neuraminidase. Although viruses re-sistant to oseltamivir emerge less frequentlythan those resistant to amantadine or rimanta-dine, information on oseltamivir-resistant vi-ruses arising during clinical use of the drug inchildren is limited. Our aim was to investigateoseltamivir resistance in a group of childrentreated for influenza. We analysed influenza Aviruses (H3N2) collected from 50 children beforeand during treatment with oseltamivir. We se-quenced the genes for neuraminidase and hae-magglutinin and studied the mutant neuramini-dases for their sensitivity to oseltamivir carbox-ylate. We found neuraminidase mutations in vi-ruses from nine patients (18％), six of whomhad mutations at position 292 (Arg292Lys) andtwo at position 119 (Glu119Val), which areknown to confer resistance to neuraminidase in-hibitors. We also identified another mutation(Asn294Ser) in one patient. Sensitivity testing tooseltamivir carboxylate revealed that the neu-raminidases of viruses that have an Arg292Lys,Glu119Val, or Asn294Ser mutation were about10(4)-10(5)-fold, 500-fold, or 300-fold more resis-tant than their pretreatment neuraminidases, re-spectively. Oseltamivir-resistant viruses werefirst detected at day 4 of treatment and on eachsuccessive day of the study. More than 10(3) in-fectious units per mL of virus were detected insome of the patients who did not shed drug-resistant viruses, even after 5 days of treatment.Oseltamivir-resistant mutants in children beingtreated for influenza with oseltamivir arise morefrequently than previously reported. Further-more, children can be a source of viral transmis-sion, even after 5 days of treatment withoseltamivir.

8. Enhanced virulence of influenza A viruseswith the haemagglutinin of the 1918 pan-demic virus.

Kobasa D, Takada A, Shinya K, Hatta M,Halfmann P, Theriault S, Suzuki H, NishimuraH, Mitamura K, Sugaya N, Usui T, Murata T,Maeda Y, Watanabe S, Suresh M, Suzuki T,Suzuki Y, Feldmann H, Kawaoka Y.

The‘Spanish’influenza pandemic of 1918-19was the most devastating outbreak of infectiousdisease in recorded history. At least 20 millionpeople died from their illness, which was char-acterized by an unusually severe and rapidclinical course. The complete sequencing of sev-eral genes of the 1918 influenza virus has madeit possible to study the functions of the proteinsencoded by these genes in viruses generated byreverse genetics, a technique that permits the

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generation of infectious viruses entirely fromcloned complementary DNA. Thus, to identifyproperties of the 1918 pandemic influenza Astrain that might be related to its extraordinaryvirulence, viruses were produced containing theviral haemagglutinin (HA) and neuraminidase(NA) genes of the 1918 strain. The HA of thisstrain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demon-strate that the HA of the 1918 virus confers en-

hanced pathogenicity in mice to recent humanviruses that are otherwise non-pathogenic in thishost. Moreover, these highly virulent recombi-nant viruses expressing the 1918 viral HA couldinfect the entire lung and induce high levels ofmacrophage-derived chemokines and cytokines,which resulted in infiltration of inflammatorycells and severe haemorrhage, hallmarks of theillness produced during the original pandemic.

Publications

Original papersKondo T, McGregor M, Chu O, Chen D, Hori-

moto T, Kawaoka Y. Protective effect of epi-dermal powder immunization in a mousemodel of equine herpesvirus-1 infection. Vi-rology 318: 414-419, 2004.

Watanabe S, Watanabe T, Noda T, Takada A,Feldmann H, Jasenosky LD, Kawaoka Y. Pro-duction of novel Ebola virus-like particlesfrom cDNAs: an alternative to Ebola virusgeneration by reverse genetics. J Virol 78: 999-1005, 2004.

Maeda Y, Goto H, Horimito T, Takada A,Kawaoka Y. Biological Significance of the UResidue at the -3 Position of the mRNA Se-quences of Influenza A Viral Segments PB1and NA. Virus Res 100: 153-157, 2004.

Takada A, Fujioka K, Tsuiji M, Morikawa A, Hi-gashi N, Ebihara H, Kobasa D, Feldmann H,Irimura T, Kawaoka Y. A Human Macro-phage C-Type Lectin Specific for Galactoseand N -Acetylgalactosamine Promotes Filovi-rus Entry. J Virol 78: 2943-2947, 2004.

Garulli B, Kawaoka Y, Castrucci MR. Mucosaland systemic immune responses to a humanimmunodeficiency virus type 1 epitope in-duced upon vaginal infection with a recombi-nant influenza A virus. J Virol 78: 1020-1025,2004.

Horimoto T, Takada A, Iwatsuki-Horimoto K,Kawaoka Y. A protective immune response inmice to viral components other than hemag-glutinin in a live influenza A virus vaccinemodel. Vaccine 22: 2244-2247, 2004.

Horimoto H, Fukuda N, Iwatsuki-Horimoto K,Guan Y, Lim W5, Peiris M, Sugii S, Odagiri T,Tashiro M, Kawaoka Y. Antigenic Differencesbetween H5N1 Human Influenza Viruses Iso-lated in 1997 and 2003. J Vet Sci 66: 303-305,2004.

Iwatsuki-Horimoto K, Kanazawa R, Sugii S,Kawaoka Y, Horimoto T. The index influenzaA virus subtype H5N1 isolated from a humanin 1997 differs in its receptor binding proper-ties from a virulent avian influenza virus. J

Gen Virol 85: 1001-1005, 2004.Shinya K, Hamm S, Hatta M, Ito H, Ito T,

Kawaoka Y. PB2 amino acid at position 627affects replicative efficiency, but not cell tro-pism, of Hong Kong H5N1 influenza A vi-ruses in mice. Virology 320: 258-266, 2004.

Shinya K, Fujii Y, Ito H, Ito T, Kawaoka Y.Characterization of a neuraminidase-deficientinfluenza A virus as a potential gene deliveryvector and a live vaccine. J Virol 78: 3083-3088, 2004.

Hatta M, Goto H, Kawaoka Y. Influenza B VirusRequires BM2 Protein for Replication. J Virol78: 5576-5583, 2004.

Barman S, Adhikary L, Chakrabarti AK, BernasC, Kawaoka Y, Nayak DP. Role of Transmem-brane Domain and Cytoplasmic Tail AminoAcid Sequences of Influenza A Virus Neu-raminidase in Raft Association and VirusBudding. J Virol 78: 5258-5269, 2004.

Iwatsuki-Horimoto K, Horimoto T, Fujii Y,Kawaoka Y. Generation of influenza A virusNS2(NEP) mutants with an altered nuclear ex-port signal sequence. J Virol 78: 10149-10155,2004.

Horimoto T, Iwatsuki-Horimoto K, Hatta M,Kawaoka Y. Influenza A viruses possessingtype B hemagglutinin and neuraminidase: po-tential as vaccine components. Microbes andInfection 6: 579-583, 2004.

Theriault S, Groseth A, Neumann G, KawaokaY, Feldmann H. Rescue of Ebola virus fromcDNA using heterologous support proteins.Virus Res 106: 43-50, 2004

Kiso M, Mitamura K, Sakai-Tagawa Y, ShiraishiK, Kawakami C, Kimura K, Hayden FG,Sugaya N, Kawaoka Y. Resistant influenza Aviruses in children treated with oseltamivir:descriptive study. Lancet 364: 759-765, 2004.

Kobasa D, Takada A, Shinya K, Hatta M, Half-mann P, Theriault S, Suzuki H, Nishimura H,Mitamura K, Sugaya N, Usui T, Murata T,Maeda Y, Watanabe S, Suresh M, Suzuki T,Suzuki Y, Feldmann H, Kawaoka Y. Enhancedvirulence of influenza A viruses with the hae-

31

magglutinin of the 1918 pandemic virus. Na-ture 431: 703-707, 2004.

ReviewsNeumann G, Noda T, Takada A, Jasenosky LD,

Kawaoka Y. Roles of filovirus matrix- andglycoproteins in the viral life cycle. In: Ebolaand Marburg Viruses: Molecular and CellularBiology, Feldmann and Klenk, Eds., HorizonBioscience, Wymondham, U.K. pp 137-179,2004.

Neumann G, Kawaoka, Y. Reverse genetics sys-tems for the generation of segmented negative-sense RNA viruses entirely from cloned

cDNA, In: Biology of Negative Strand RNAViruses: The Power of Reverse Genetics,Kawaoka ed., Springer, London, UK. CurrTop Microbiol Immunol 283: 43-60, 2004.

Neumann G, Brownlee G, Fodor E, Kawaoka Y.,Orthomyxovirus replication, transcription, andpolyadenylation In: Biology of NegativeStrand RNA Viruses: The Power of ReverseGenetics, Kawaoka ed., Springer, London, UK.Curr Top Microbiol Immunol 283: 121-143,2004.

Jasenosky LD, Kawaoka Y. Filovirus budding.Virus Res 106: 181-188, 2004.

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Our research main focuses on molecular mechanism underlying innate pathogenrecognition by Toll-like receptors (TLRs). TLRs have been implicated in microbialrecognition in the innate immune system but their microbial recognition mechanismremains enigmatic. We have proposed a concept that TLR requires a coreceptor,by discovering MD-2 that is associated with TLR4 and indispensable for endotoxinrecognition by TLR4. Molecular cloning of MD-2 led us to a search for coreceptorsof other TLRs. Such molecules would reveal molecular mechanisms underlying in-nate microbial recognition.

1. Anti-Toll like receptor 4 /MD-2 AntibodyProtects Mice from Acute Lethal Hepatitisinduced by Tumor Necrosis Factor-α

Sachiko Akashi-Takamura1, Natsuko Tani-mura1, Shin-ichiroh Saitoh1, Kohichiroh Taka-hashi1, and Kensuke Miyake1,2: 1Div. of Infec-tious Genetics, Dept of Microbiol. and Immu-nol., The Inst. of Med. Sch., The Univ. of To-kyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, JAPAN, 2CREST, JST, Tokyo, JAPAN

MD-2 is associated with the extracellular do-main of TLR4, and TLR4/MD-2 complex recog-nizes LPS. However, the function of TLR4/MD-2 for recognition of LPS has not been fully eluci-dated yet. To investigate the function of TLR4/MD-2, we established two-type of mAbs tomouse TLR4/MD-2, Sa15-21 and MTS510.

Sa15-21, which is able to co-precipitate theLPS directly bound to TLR4/MD-2, preventsmice from endotoxin shock induced by LPS andD-galactosamine (D-GalN) completely. On theother hand, MTS510, which is not able to recog-nize the LPS/TLR4/MD-2 complex but it inhib-

its LPS signaling, does not prevent mice fromLPS/(D-GalN) induced endotoxin shock.

In this article, we show that serum TNF-α in-duced by LPS/D-GalN is upregulated by the Sa15-21 pretreatment. We also show that the Sa15-21 protects mice from lethal liver failure in-duced by TNF-α/D-GalN. Lastly, we show thatSa15-21 activates NF-κB activity as judged by areporter cell line, and induces NF-κB-dependentanti-apoptotic genes in the liver, and these effectof Sa15-21 is dependent on TLR4-signaling. Weconclude that Sa15-21 mAb inhibits mice hepa-tocyte apoptosis and liver damage caused by theagonistic signal through TLR4. These resultssuggest therapeutic application of an agonisticanti-TLR4/MD-2 mAb. (Japanese Ministry ofEducation and Science Research; the NaitoFoundation)

2. Protective roles of tumor necrosis factorderived from mast cells in murine malaria

Takahisa Furuta: Div. of Infectious Genetics,Dept of Microbiol. and Immunol., The Inst. ofMed. Sch., The Univ. of Tokyo 4-6-1 Shiro-

Department of Microbiology and Immunology

Division of Infectious Genetics感染遺伝学分野

Professor Kensuke Miyake, M.D., Ph.D.Research Associate Takahisa Furuta, D.V.M., Ph.D.Research Associate Sachiko (Takamura) Akashi, M.D., Ph.D.Research Associate Shin-Ichiroh Saitoh, Ph.D.

教 授 医学博士 三 宅 健 介助 手 農学博士 古 田 隆 久助 手 医学博士 赤司（高村）祥子助 手 医学博士 斉 藤 伸一郎

33

kanedai, Minato-ku, Tokyo 108-8639, JAPAN

In this study, we first demonstrated the im-portance of mast cells for host immunity in ma-laria. The key molecule responsible for protec-tion in this malaria model is TNF from mastcells. Association between mast cell activationand elevation of serum TNF levels resulted inenhanced expression of protection. Critical evi-dences on the essential role of mast cell-derivedTNF for protection against P. berghei ANKA in-fection were obtained by experiments using re-constitution of mast cell deficient mice withBMMCs of ＋/＋ or TNF deficient mice. It hasbeen well known that TNF is a major cytokineresponsible for protection and pathogenesis inmalaria. TNF controls both protective immunityand severity of malaria. TNF levels are used asan indicator of clinical severity in human ma-laria. The importance of TNF in protection wasalso confirmed in our studies with anti-TNFtreatment. The sources of TNF have been con-sidered to be macrophages and/or T cells withinteraction of these cells. We proposed that mastcells are a critical source of TNF in addition tomacrophages and T cells. Distinctive features ofmast cells include their ability to storage and re-lease large amounts of TNF, and their close as-sociation with blood vessels. It is noteworthythat presynthesized TNF in mast cells is a candi-date for the immediate initiation of the host re-sponse to infection. Since the secretion of TNFby other cell types is greatly delayed by thetime required to complete de novo synthesis ofthis cytokine, involvement of mast cell activa-tion through innate immunity or direct stimula-tion is likely to function in the present model.Indeed, higher parasitemia was found in W /W v

mice in an early stage of infection when ac-quired immune responses might not operate.After the stage of innate immunity, mast cellsare activated with IgE antibody andcomplement-fixing antibody, through FcεR andanaphylatoxin respectively, as an acquired im-munity. These features play special importanceduring malaria.

3. Lipid A Antagonist Revealed a Role for MD-2 in a Link between LPS interaction andsubsequent Toll-like receptor 4-oligomeri-zation

Shin-ichiroh Saitoh1, Sachiko Akashi1, TakenaoYamada2, Natsuko Tanimura2, Makiko Kobay-ashi1, Kazunori Konno1, Fumi Matsumoto2,Koichi Fukase3, Shoichi Kusumoto3, YoshinoriNagai1, Yutaka Kusumoto1, Atsushi Kosugi2,4,and Kensuke Miyake1,4: 1Division of InfectiousGenetics, The Institute of Medical Science, The

University of Tokyo, Tokyo, Japan, 2MedicalTechnology and Science, Graduate School ofMedicine, Osaka University, 3Department ofChemistry, Graduate School of Science, OsakaUniversity, 4CREST, Japan Science and Tech-nology Corporation, Tokyo, Japan

Lipopolysaccharide (LPS) is a membrane com-ponent of Gram negative bacteria with potentimmuno-stimulating activity. Toll-like receptor 4(TLR4) delivers a transmembrane activation sig-nal upon LPS stimulation. MD-2, an extracellu-lar molecule that is associated with the extracel-lular domain of TLR4, is indispensable for cellsurface expression of and LPS recognition byTLR4. Despite identification of TLR4-MD-2 asthe LPS recognition molecules, little is knownabout a molecular mechanism for LPS recogni-tion. Recently we demonstrated the direct inter-action between LPS and TLR4-MD-2 on the cellsurface. We here show a subsequent event, LPS-dependent TLR4-MD-2 oligomerization. Epitope-tagged TLR4 was coprecipitated with TLR4tagged with another epitope during 5-150 minafter LPS stimulation. TLR4 oligomerization re-quired membrane CD14, but not the cytoplasmicsignaling portion of TLR4. TLR4 oligomerizationwas triggered by lipid A but not by a lipid Aantagonist lipid IVa, although both bind simi-larly to TLR4-MD-2 (although lipid IVa binds toTLR4-MD2 even better than lipid A). To addressa role for MD-2 in TLR4-oligomerization, wetook an advantage that lipid IVa is agonistic onmouse TLR4-MD-2 but antagonistic whenmouse MD-2 is replaced with human MD-2. Inkeeping with this, Lipid IVa-dependent mouseTLR4 oligomerization was observed with mouseMD-2 but not with human MD-2. Taken to-gether, MD-2 plays an important role in LPSrecognition by linking LPS interaction with TLR4-MD-2 to TLR4 oligomerization. We also dem-onstrate the antagonistic mechanism of lipid IVaby the competitive inhibition of TLR 4-oligomerization. More effective drug to treatseptic shock will be designed from these resultsin near future.

4. The search of the association moleculeswith toll-like receptors to recognize micro-bial components

Yutaka Kusumoto1,2, Kazunori Konno1, Shin-Ichiroh Saitoh1, Koutarou Ishiguro1, TakashiIshii1, Toshihiko Kobayashi1, Yuji Motoi1, Sa-chiko Takamura Akashi1 and Kensuke Mi-yake1,2: 1Div. of Infectious Genetics, Dept of Mi-crobiol. and Immunol., The Inst. of Med. Sch.,The Univ. of Tokyo 4-6-1 Shirokanedai,Minato-ku, Tokyo 108-8639, JAPAN, 2CREST,

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JST, Tokyo, JAPAN

We research the mechanisms of recognition ofbacterial components by toll-like receptors(TLRs). TLR4 and MD-2 make a complex andrecognize lipopolysaccharide (LPS). It is consid-ered that some cell surface proteins or serumproteins work in cooperated with TLR4/MD-2complex or other TLRs to recognize the bacterialcomponents. CD14 and LBP play the roles of theeffective transfer of LPS to TLR4/MD-2 com-plex. Several findings indicate that other mole-cules associate with the binding of LPS to TLR4/MD-2. Immunoprecipitation of TLR4/MD-2 inpresence of LPS indicated that plasma gelsolin

was one candidate as an associated molecule torecognize LPS by TLR4/MD-2. In regard to TLR2, it was reported that this TLR make complexwith other TLRs, TLR1 or TLR6, and recognizedbacterial lipopeptide. However, splenocyte ob-tained from TLR1 or TLR6 deficient mice showus the existence of other molecules to recognizelipopeptide. We also search the associated mole-cules with TLR2 when it recognizes lipopeptideutilizing the artificially mutated cells in the re-sponses to TLR2 ligands. We have continuouslyanalyzed the function of gelsolin or other associ-ating molecules to TLR4/MD-2 and alsosearched the associating molecules to TLR2.

Publications

Saitoh S, Akashi S, Yamada T, Tanimura N,Matsumoto F, Fukase K, Kusumoto S, KosugiA, Miyake K. Ligand-dependent Toll-like re-ceptor 4 (TLR4)-oligomerization is directlylinked with TLR4-signaling. J Endotoxin Res.10(4): 257-60, 2004.

Saitoh S, Akashi S, Yamada T, Tanimura N, Ko-bayashi M, Konno K, Matsumoto F, Fukase K,Kusumoto S, Nagai Y, Kusumoto Y, KosugiA, Miyake K. Lipid A antagonist, lipid IVa, isdistinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization. Int. Immunol.2004 Jul; 16(7): 961-9. Epub 2004.

Iwahashi M, Yamamura M, Aita T, Okamoto A,Ueno A, Ogawa N, Akashi S, Miyake K, Go-dowski PJ, Makino H. Expression of Toll-likereceptor 2 on CD16＋ blood monocytes andsynovial tissue macrophages in rheumatoidarthritis. Arthritis Rheum. 50(5): 1457-67, 2004.

Okamoto M, Oh-E G, Oshikawa T, Furuichi S,Tano T, Ahmed SU, Akashi S, Miyake K,Takeuchi O, Akira S, Himeno K, Sato M, Oh-kubo S. Toll-like receptor 4 mediates the anti-tumor host response induced by a 55-kilodalton protein isolated from Aeginetia in-dica L., a parasitic plant. Clin Diagn Lab Im-munol. 11(3): 483-95, 2004.赤司祥子．CD１４，TLR４，MD-２とLPSの相互作用．臨床免疫，科学評論社，vol４２，p１１５―

１２１，２００４．高村（赤司）祥子．エンドトキシンショックとTLR―TLRによるエンドトキシンの認識機構― 炎症と免疫，先端医学社，vol１３，p３７―４４，２００５．

Furuta, T,. Kikuchi, T,.1, Akira, S. andYoshikawa, Y. Toll-like receptor 4-mediatedinnate resistance to murine toxoplasmosis isinduced by peroral but not intraperitoneal in-oculation of parasites. Int. Immunol. (inpress).

Y. Kusumoto, H. Hirano, K. Saitoh, S. Yamada,M. Takedachi, T. Nozaki, Y. Ozawa, Y. Naka-hira, T. Saho, H. Ogo, Y. Shimabukuro, H.Okada, S. Murakami Human gingival epithe-lial cells produce chemotactic factors inter-leukin 8 and monocyte chemoattractantprotein-1 after stimulation with Porphyromonasgingivalis via toll-like receptor 2. J. Periodontol(3) 370-379, 2004.斉藤伸一郎，三宅健介．自然免疫とToll-like re-

ceptor（TLR）のシグナル伝達，シグナル伝達イラストマップ，羊土社，１７０―１７９，２００４．斉藤伸一郎，三宅健介．エンドトキシン（LPS）の認識機構とTLR４の活性化機構，免疫研究のフロンティア，実験医学，Vol.２２ №５，２１―２７，２００４．斉藤伸一郎，三宅健介．LPSの認識とTLR４/MD-２の多量体形成，免疫２００５，中山書店，２４５―２５３，２００４.

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The mucosal surface provides a first line of defense for the host. The goal of ourresearch is to understand the molecular and cellular aspects of the mucosal im-mune system and their contribution for the host defense against infectious dis-eases, inflammation and immunological disorders. Further, it is important to applyour fundamental findings of the mucosal immune system for the development ofmucosal vaccine and mucosal immunotherapy with all haste.

1. Mucosal Vaccine:

I. Development of M-cell Target MucosalVaccine

Kazutaka Terahara, Tomonori Nochi, OsamuIgarashi, Yoshikazu Yuki, Shiho Kurokawaand Hiroshi Kiyono: Division of Mucosal Im-munology, Department of Microbiology andImmunology, Institute for Medical Science, TheUniversity of Tokyo

Peyer’s patch (PP) and nasopharynx-associ-ated lymphoid tissue (NALT) possess a uniquesubset of antigen-sampling cell called M-cell.Mucosally delivered antigens are generallytaken up by M-cells and delivered to profes-sional antigen presenting cells, including macro-phages and dendritic cells located in the pocketof M-cell. In addition to PP associated M cells,we have identified the development of villousM cells, as a new and novel gateway for theoutside environment. Elucidation of the molecu-lar and cellular characteristics of PP associatedand villous M-cells would not only greatly fa-cilitate the fundamental aspect of M cell biology,

but also lead to the design of effective mucosalvaccine and the discovery of novel targets fordeveloping new mucosal antigen and drug de-livery vehicle to combat pathogens and aller-gens. To this end, our efforts have been focusingon the identification of M-cell-specific gene(s)and corresponding proteins by using DNA mi-cro array and proteomics approaches.

II. Development of M Cell Targeted EdibleVaccine

Yoshikazu Yuki, Seiji Yokoyama, MasakoMori, Tomohiro Akashima, Tomoko Yok-oyama and Hiroshi Kiyono: Division of Mu-cosal Immunology, Department of Microbiol-ogy and Immunology, Institute for Medical Sci-ence, The University of Tokyo

The production of vaccine antigens in plantshas many potential advantages for new genera-tion of vaccine which will contribute further ad-vancement of clinical medicine for public health.For example, the plant system more economi-cally produces vaccine antigen than the indus-trial fermentation system. Second, the purifica-

Department of Microbiology and Immunology

Division of Mucosal Immunology炎症免疫学分野

Professor Hiroshi Kiyono, D.D.S. Ph.D.Research Associate Yoshikazu Yuki, M.B.A. Ph.D.Research Associate Takachika Hiroi, D.D.S. Ph.D.Research Associate Jun Kunisawa, Ph.D.

教 授 医学博士 清 野 宏助 手 医学博士 幸 義 和助 手 歯学博士 廣 井 隆 親助 手 薬学博士 國 澤 純

36

tion requirement can be eliminated when planttissue containing vaccine is used as food orgiven via the physiological route of ingestion.Third, the plant system can direct accumulationof vaccine antigen into the intracellular compart-ment where they are more stable. Finally therisk arising from contamination with potentialhuman pathogens can be minimized. Taking to-gether, plant-based subunit vaccine is consid-ered to be not only effective but also practicalmucosal vaccine. To this end, our efforts havebeen focusing on the development of new gen-eration of mucosal vaccine, M cell targeted ed-ible vaccine for combating infectious diseasesand bioterrorism.

III. NALT Target Mucosal Vaccine: Mecha-nism of NALT Organogenesis

Satoshi Fukuyama, Takahiro Nagatake, andHiroshi Kiyono: Division of Mucosal Immunol-ogy, Department of Microbiology and Immu-nology, Institute for Medical Science, The Uni-versity of Tokyo

NALT is thought to be a key targeted lym-phoid tissue for the nasal vaccination. Themechanism of NALT organogenesis is quite dif-ferent from that of Peyer’s patch (PP) in spite ofthe similarity of immunological function of thesetwo inductive tissues. Lymphotoxin (LT) β re-ceptor signals have been shown to play a piv-otal role for the organogenesis program of sec-ondary lymphoid tissue development. However,our findings demonstrated that NALT organo-genesis is independent of LTβ receptor signalingpathway. CD3－CD4＋CD45＋ cells which are dif-ferentiated from fetal liver progenitors inducethe organogenesis of secondary lymphoid tis-sues (e.g., NALT, PP, and lymph node). CXCL13and its ligand CXCR5 are essential for the in-duction of PP organogenesis by CD3－CD4＋CD45＋ cells. Thus, our current efforts are aimed atthe elucidation of CXCL13／CXCR5 interactionfor the genesis of NALT.

2. Mucosal allergy: DNA Vaccine for TheControl of Allergic Diarrhea

Ayako Hino1, Satoshi Fukuyama1, NaokoTakayama, Kosuke Kataoka3, Mi-Na Kweon1,2

Kotaro Fujihashi3, Jerry R. McGree3 and Hiro-shi Kiyono1: 1Division of Mucosal Immunology,Department of Microbiology and Immunology,Institute for Medical Science, The University ofTokyo, 2Mucosal Immunology Section, Interna-tional Vaccine Institute, 3The ImmunobiologyVaccine Center, The University of Alabama atBirmingham

To develop therapic vaccine against food al-lergy, we demonstrated that nasally adminis-trated murine IL-12 naked DNA expressionplasmids, containing both the p35 and p40subunits (IL-12p70) resulted in the synthesis ofco-responding cytokine in large intestine leadingto the inhibition of antigen-specific Th2 type re-sponse. The development of the allergic diarrheawas prevented in mice nasally treated with IL-12p70 DNA with the inhibition of clinical symp-tom including OVA-specific IgE Ab synthesis.To clarify the distribution of co-responding pro-tein treated by naked DNA nasally, the expres-sion of nasally administrated naked GFP＋DNAwas chronologically demonstrated by the usingin vivo image analysis and confocal microscopy.Our further analysis revealed that nasally ad-ministrated DNA resulted in the preferentialprotein expression of IL12p70 by CD11c positivecells in NALT, spleen and intestine. In addition,IL-12p70 producing DCs were located nearlymph vessel of large intestine. The nasal IL-12p70 DNA treatment was also effective even afterthe establishment of allergic diarrhea. Our find-ings provide new possibility for the use of theclinical investigation of mucosal DNA vaccineand mucosal gene therapy for the control of mu-cosal immune disease.

3. Uniqueness of mucosal antigen presentingcells

I. Classical and non-classical MHC-mediatedantigen presentation for the regulation ofmucosal immune system

Jun Kunisawa1, Yosuke Kurashima1, MikiShimizu1, NamJu Kim1, Nilabh Shastri2, andHiroshi Kiyono1: 1Division of Mucosal Immu-nology, Department of Microbiology and Im-munology, The Institute of Medical Science,The University of Tokyo, 2Division of Immu-nology, Department of Molecular and Cell Bi-ology, University of California, Berkeley

Understanding of antigen processing andpresentation pathway at mucosal sites will pro-vide us important information for the develop-ment of efficient mucosal vaccine. However,very minimum information is currently avail-able about the molecular aspect of antigen proc-essing and presentation at mucosal compart-ment. To address this, we developed a novel en-zymatic method (KOVAK system) to detect anti-genic intermediates generated in living cells,which revealed that intracellular proteolysispathway for the generation of antigenic interme-diates and the function of molecular chaperonesin the antigen-processing pathway. One of our

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current efforts is aimed to clarify the molecularpathway for the antigen processing and presen-tation at the mucosal site using the KOVAK sys-tem, and to apply the novel information to thedevelopment of mucosal vaccine. Additionally,recent study has proposed that non-classicalMHC molecules expressed on the mucosal epi-thelial cells also play an important role in themaintenance of mucosal homeostasis mediatedby intraepithelial lymphocytes (IELs). Thus, anadditional purpose of our study is to identifythe resemblance and discrepancy between classi-cal MHC- and non-classical MHC-mediatedantigen-presentation pathway and to revealtheir function for the regulation of mucosal im-mune system.

II. Discovery and characterization of intesti-nal eosinophilic dendritic cells for theregulation of oral tolerance

Takachika Hiroi, Masaki Yoda and HiroshiKiyono: Division of Mucosal Immunology, De-partment of Microbiology and Immunology,The Institute of Medical Science, The Univer-

sity of Tokyo

Recently, we found the novel type of F4／80＋

Gr-1＋CD11b＋CD11c＋ cells as segmented cells inintestinal lamina propria (i-LP). Since these cellspossessed morphological characteristics of neu-trophiles like fraction of dendritic cells, it namedeosinophilic dendritic cells (EDCs). Interestingly,newly identified EDCs were essential for the in-duction of oral tolerance (OT) independent onthe T regulatory by adoptive transfer of in vivoOVA-primed EDCs into anti-CD25 treated Tregulatory deficient mice. Transferred EDCswith i-LP origin migrated to spleen and ex-pressed high levels of MHC class-II. Ag-specificCD4＋ T cell proliferation was also inhibited byCpG／Flt3-L stimulated EDCs with dendriticmorphology. Taken together, Intestinal EDCsare a prime candidate for the immediate re-sponder cells that can negatively regulate im-mune responses against orally administered an-tigens. Thus, our study has provided new ave-nue that EDCs may be key target cells as atherapeutic treatment for the control of allergyand autoimmune diseases.

Publications

Jang, M.H., Kweon, M.N., Iwatani, K.,Yamamoto, M., Terahara, K., Sasakawa, C.,Suzuki, T., Nochi, T., Yokota, Y., Rennert, P.D., Hiroi, T., Tamagawa, H., Iijima, H., Kuni-sawa, J., Yuki, Y. and Kiyono, H. Intestinalvillous M cells: an antigen entry site in themucosal epithelium. Proc Natl Acad Sci USA.101: 6110-6115, 2004.

Kiyono, H. and Fukuyama, S. NALT- versusPeyer’s-patch-mediated mucosal immunity.Nat Rev Immunol. 4: 699-710, 2004.

Hino, A., Kweon, M.N., Fujihashi, K., McGhee,J.R. and Kiyono, H. Pathological role of largeintestinal IL-12p40 for the induction of Th2-type allergic diarrhea. Am J Pathol. 164: 1327-1335, 2004.

Nochi, T., Yuki, Y., Terahara, K., Hino, A., Kuni-sawa, J., Kweon, M.N., Yamaguchi, T. and Ki-yono, H. Biological role of Ep-CAM in thephysical interaction between epithelial cellsand lymphocytes in intestinal epithelium. ClinImmunol. 113: 326-339, 2004.

Shikina, T., Hiroi, T., Iwatani, K., Jang, M.H.,Fukuyama, S., Tamura, M., Kubo, T.,Ishikawa, H. and Kiyono, H. IgA class switchoccurs in the organized nasopharynx- and gut-associated lymphoid tissue, but not in thediffuse lamina propria of airways and gut. JImmunol. 172: 6259-6264, 2004.

Yamamoto, M., Kweon, M.N., Rennert, P.D., Hi-

roi, T., Fujihashi, K., McGhee, J.R. and Ki-yono, H. Role of gut-associated lymphoreticu-lar tissues in antigen-specific intestinal IgAimmunity. J Immunol. 173: 762-769, 2004.

Ueta, M., Nochi, T., Jang, M.H., Park, E.J., Iga-rashi, O., Hino, A., Kawasaki, S., Shikina, T.,Hiroi, T., Kinoshita, S. and Kiyono, H. Intra-cellularly expressed TLR2s and TLR4s contri-bution to an immunosilent environment at theocular mucosal epithelium. J Immunol. 173:3337-3347, 2004.

Ohmura-Hoshino, M., Yamamoto, M., Yuki, Y.,Takeda, Y. and Kiyono, H. Non-toxic Stx de-rivatives from Escherichia coli possess adju-vant activity for mucosal immunity. Vaccine.22: 3751-2361, 2004.

Mizushima, T., Ito, T., Kishi, D., Kai, Y., Tama-gawa, H., Nezu, R., Kiyono, H. and Matsuda,H. Therapeutic effects of a new lymphocytehoming reagent FTY720 in interleukin-10 gene-deficient mice with colitis. Inflamm BowelDis. 10: 182-192, 2004.

Yoshino, N., Lu, F.X., Fujihashi, K., Hagiwara,Y., Kataoka, K., Lu, D., Hirst, L., Honda, M.,van Ginkel, F.W., Takeda, Y., Miller, C.J., Ki-yono, H. and McGhee, J.R. A novel adjuvantfor mucosal immunity to HIV-1 gp120 in non-human primates. J Immunol. 173: 6850-6857,2004.

Kai, Y., Takahashi, I., Ishikawa, H., Hiroi, T.,

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Mizushima, T., Kishi, D., Hamada, H., Tama-gawa, H., Ito, T., Yoshizaki, K., Kishimato, T.,Matsuda, H. and Kiyono, H. Absence of com-mon cytokine receptor g chain results in thedevelopment of colitis by IL-6-producing CD4＋ T cells. Gastroenterology (in press).

Kobayashi, R., Kohda, T., Kataoka, K., Ihara, H.,Kozaki, S., Pascual, D.W., Staats, H.F., Kiyono,H., McGhee, J.R. and Fujihashi, K. A nevelbotulinum neurotoxoid vaccine prevents mu-cosal botulism. J. Immunol. (in press).

Kweon, MN., Yamamoto, M., Rennert, P.D.,Park, EJ., Lee, AY., Chang, SN., Hiroi, T.,Nanno, M. and Kiyono, H. Prenatal blockage

of LTbR and TNFRp55 signaling cascade re-sulted in the acceleration of tissue genesis forisolated lymphoid follicles in the large intes-tine. J. Immunol. (in press).

Kunisawa, J., and Kiyono, H. A marvel of mu-cosal T cells and secretory antibodies for thecreation of first lines of defense. Cell. Mol.Life. Sci. (in press).

Kunisawa, J., Fukuyama, S., and Kiyono, H. Re-semblance and discrepancy of mucosa-associated lymphoid tissues in aerodigestivetract for the orchestration of mucosal immunesystem. Curr. Mol. Med. (in press).

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