Department of Advanced Medical Science was established in September 1997.Our aim is to contribute to the performance and the development of advancedtherapeutic approach to the diseases. We have been participating in the poten-tially important clinical trials and the several projects in line with our principles. 1)First, we participated in the peptide vaccine clinical trial at IMSUT hospital. Our re-search projects were 2) analysis the role of newly identified non-coding RNA, 3)Analysis of the Gradient Expression of Genes in Human Colonic Mucosa and 4)Gene expression profiles of in acute graft-versus-host disease following cord bloodtransplantation.

Peptide vaccine clinical trial

1-1) Human leukocyte antigen (HLA)-A＊2402/A＊0201-restricted vascular endothelialgrowth factor receptor type 1 (VEGFR1)-specific peptide vaccination for ad-vanced pancreatic cancer (clinical phaseI/II trial, ClinicalTrials.gov Identifier NCT00683358 & NCT00683085)

Nagayama H. et al.

1-2) Human leukocyte antigen (HLA)-A＊0201/-A＊2402 restricted peptide vaccine ther-apy in patients with esophageal or gas-tric cancer (Clinical phase I/IIa trial, Clini-calTrials. gov Identifier NCT00681421 &NCT00681330, NCT00681421 & NCT00681330)

Ohno H. et al.

Laboratory of Molecular Medicine, Division ofAdvanced Clinical Proteomics, Human GenomeCenter, IMSUT (Prof. Nakamura’s Laboratory)has identified several new epitope-peptides thatwere up-regulated in several kinds of cancers.Based on these data, phase I/IIa clinical trial tocancer patients were developed by NakamuraLaboratory. Among these trials, we are engagedin the clinical trials on the patients with eitheresophageal, gastric or pancreatic cancer fromMay 2008 at IMSUT hospital.

Outline of the clinical trial

Purpose: Feasibility and efficacy of combinedmodality intervention using tumor-associated/tumor vessel-specific peptide vaccination withor without chemotherapeutic agents in case ofadvanced/inoperable or therapy-resistant cancerpatients. All peptides are restricted to HLA-A＊

2402 or HLA-A＊0201, both are common HLA al-leles among the Japanese human population.

Research Hospital

Department of Advanced Medical Science先端診療部

Professor Naohide Yamashita, M.D., Ph.D.Assistant Professor Takashi Nakaoka, M.D., Ph.D.Project Assistant Professor Hitomi Nagayama, M.D., Ph.D.Assistant Professor Hideki Ohno M.D., Ph.D.Assistant Professor Naoyuki Iso-O M.D., Ph.D.Assistant Professor Naoyuki Takahashi M.D., Ph.D.

教 授 医学博士 山 下 直 秀講 師 医学博士 中 岡 隆 志特任講師 医学博士 長 山 人 三助 教 医学博士 大 野 秀 樹助教（併任） 医学博士 磯 尾 直 之助教（併任） 医学博士 高 橋 直 之

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Primary endpoints: Safety of peptide vaccination(phase I) and time to progression of cancer(phase II). As these trial are phase I/IIa study toconfirm the feasibility, eligibility criteria is re-stricted to the end-stage cancer patients with noexpected alternative therapy.Secondary endpoints: Immune response (ELIS-POT, Perforin/FoxP3 FACS, in vitro CTL assayetc.) & Tumor regression (basically based on theRECIST criteria).Design: vaccination of peptides, emulsified withincomplete Freund adjuvant is planned twotimes weekly for 8 weeks with or without stan-dardized chemotherapy.Maximum Enrollment: 14. Entry period: oneyear.cancer HLA peptides Adjuvant che-

motherapyEsophagealcancer

A2402 URLC10,TTK, KOC1

none

A0201 URLC10,VEGFR1/R2

none

Gastriccancer

A2402 URLC10, KOC1,VEGFR1/R2

TS-1

A0201 URLC10,VEGFR1/R2

TS-1

Pancreaticcancer

A2402 VEGFR1 GEMA0201 VEGFR1 GEM

2. Analysis of Dnm3os, a non-coding RNAfor skeletal development

Nakaoka T. et al.

Dnm3os , a gene transcribed into a non-codingRNA (ncRNA), contains three micro RNAs;miR-199a, miR-199a＊ and miR-214, whose func-tions remain entirely unknown in mammals. Weintroduced the lacZ gene into the Dnm3os locusto recapitulate its expression pattern and disruptits function in collaboration with Department ofPhysiological Chemistry and Metabolism, Divi-sion of Biochemistry and Molecular Biology,University of Tokyo. Dnm3os＋/lacZ heterozygousembryos showed α-galactosidase activity, whichreflected the authentic expression pattern ofDnm3os RNA. Most of the Dnm3oslacZ/lacZ ho-mozygous pups died within one month of birth.After birth, Dnm3oslacZ/lacZ mice exhibited severalskeletal abnormalities, including craniofacial hy-poplasia, defects in dorsal neural arches andspinous processes of the vertebrae, and os-teopenia. Importantly, the expression of miR-199a, miR-199a＊, and miR-214 was significantlydown-regulated in Dnm3oslacZ/lacZ embryos, sup-porting the assumption that Dnm3os serves as aprecursor of these three miRNAs. Now, the pro-ject to investigate the molecular mechanisms re-sponsible for several abnormalities in Dnm3

oslacZ/lacZ mice is under way.

3. Analysis of the Gradient Expression ofGenes in Human Colonic Mucosa

Ohno H. et al.

Ulcerative colitis is characterized by continu-ous inflammation extending from rectum to oralcolonic mucosa. Epidemiological data have pro-vided incontrovertible evidence that both ge-netic and environmental factors are important inthe disease susceptibility. We speculate that thegradient expression of genes in human colonicmucosa might be related to the disease develop-ment and progression. First, we selected thegenes whose expression levels were reported toincrease toward the distal colon. Next, weevaluated the expression levels of these genesthroughout the GI tract and in other tissues bynorthern blot analysis. As a result of this analy-sis, some genes showed the expression gradientto increase toward the distal colon. We havegenerated rabbit polyclonal antibodies againstthe protein encoded by the gene. We are cur-rently examining where the protein localize inthe distal colonic mucosa.

4. Gene expression profiles of peripheralblood mononuclear cell subpopulations inacute graft-versus-host disease followingcord blood transplantation

Takahashi N.

Compared with allogeneic hematopoietic stemcell transplantation using other sources, cordblood (CB) transplantation (CBT) has clinical ad-vantages in terms of incidence and severity ofacute graft-versus-host disease (GVHD) despiteusing allogeneic stem cells with more humanleukocyte antigen mismatches. However, de-tailed pathophysiology of acute GVHD devel-oped after CBT has not yet been elucidated.

We performed microarray expression profilingof immunoregulatory genes on each of 4 sub-populations (CD4＋, CD8＋, CD14＋, and CD56＋)of peripheral blood mononuclear cells (PBMCs),which were taken from 8 patients with hema-tologic malignancies who suffered from acuteGVHD after unrelated CBT. We identified 55genes, which were differentially expressed dur-ing acute GVHD compared to recovery phase.Among them, 22 showed differential expressionconcurrently in multiple PBMC subpopulations.In particular, 5 genes (TRAIL, IL1RN, IFI27,GZMB, and CCR5) were up-regulated and 3genes (CLK1, TNFAIP3 and BTG1) were down-regulated in at least 3 out of 4 subpopulations

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during acute GVHD. These 8 genes seem to becandidates which may play key roles commonto multiple subpopulations in the pathophysiol-ogy of acute GVHD. In addition, down-regulation of anti-inflammatory factors, such asTNFAIP3, KLF2, ZFP36, and BTG1, seems to beinvolved in acceleration of immune response,thus exacerbation of acute GVHD. Further studyis required to clarify whether therapeutic aug-

mentation of these factors may ameliorate acuteGVHD. Meanwhile, differential expression ofseveral genes, such as CCL5, TNFAIP3, CD161,CD160, and COX2, was assumedly affected bythe developmental immaturity of CB-derivedcells and this might be somehow involved in therelatively low incidence and low severity ofacute GVHD following CBT.

Publications

Takahashi N, Sato N, Takahashi S, Tojo A.Gene-expression profiles of peripheral bloodmononuclear cell subpopulations in acutegraft-vs-host disease following cord bloodtransplantation. Exp. Hematol. 36: 1760-1770,2008.

Fujita S, Yamashita N, Ishii Y, Sato Y, Sato K,Eizumi K, Fukaya T, Nozawa R, Takamoto Y,Yamashita N, Taniguchi M, Sato K. Regula-tory dendritic cells protect against allergic air-way inflammation in a murine asthmaticmodel. J Allergy Clin Immunol. 121: 95-104,2008.

Watanabe T, Sato T, Amano T, Kawamura Y,Kawamura N, Kawaguchi H, Yamashita N,Kurihara H, Nakaoka T. Dnm3os , a Non-coding RNA, is Required for Normal Growthand Skeletal Development in Mice. Dev. Dyn.237: 3738-48, 2008.

Yoshikumi Y, Ohno H, Suzuki J, Isshiki M, Mor-ishita Y, Ohnishi H, Yasuda H, Omata M, Fu-jita T, Mashima H. Up-regulation of JAM-1 in

AR42J cells treated with activin A and betacel-lulin and the diabetic regenerating islets. En-docr J. 55: 757-65, 2008.

Mashima H, Suzuki J, Hirayama T, YoshikumiY, Ohno H, Ohnishi H, Yasuda H, Fujita T,Omata M. Involvement of vesicle-associatedmembrane protein 7 in human gastric epithe-lial cell vacuolation induced by Helicobacterpylori-produced VacA. Infect Immun. 76: 2296-303, 2008.

Sato K, Eizumi K, Fukaya T, Fujita S, Sato Y,Takagi H, Yamamoto M, Yamashita N, Hiji-kata A, Kitamura H, Ohara O, Yamasaki S,Saito T, and Sato K. Naturally occurring regu-latory dendritic cells regulate murine cutane-ous chronic graft-versus-host disease. Blood , inpress, 2009.

Yoshiura K, Nishishita T, Nakaoka T, YamashitaN, and Yamashita N. Inhibition of B16 mela-noma growth and metastasis in C57BL miceby vaccination with a syngenic endothelialceu line. J Exp Cancer Res in press, 2009

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We have been challenging to cure intractable hematological disorders such as leu-kemia and lymphoma mainly with the aid of hematopoietic stem cell transplanta-tion (HSCT). No less than 30 patients per year receive allogeneic HSCT in our fa-cilities. In recent years, unrelated cord blood has been our major stem cell sourcefor recipients who have no suitable family donors in HSCT. Since 1998 we haveperformed over 200 cases of cord blood transplantation (CBT) for adult patients,which appears a distinguished experience in the world. Recent advance in identifi-cation of signaling molecules activated in a tumor-specific manner or associatedwith tumor-specific genomic recombination have disclosed many candidate thera-peutic targets in tumors. In the field of hematological malignancies, we have al-ready experienced remarkable clinical efficacies of imatinib mesylate (ABL kinaseinhibitor) for CML, rituximab (chimeric anti-CD20 monoclonal antibody) for B celllymphoma as well as bortezomib (proteasome inhibitor) for multiple myeloma. Weextensively apply these molecular targeted therapies for in- and out-patients andare to be involved in clinical trial of newly developed agents including nilotinib (2nd

generation ABL inhibitor) and ICL670 (oral iron chelator).

1. Myeloablative cord blood transplantation inadults with AML.

Ooi J, Takahashi S, Tomonari A, Tsukada N,Tojo A

We analyzed the disease-specific outcomes ofadult acute myelogenous leukemia (AML) pa-tients treated with unrelated cord blood trans-

plantation (CBT) after myeloablative condition-ing. Between August 1998 and February 2008,77 adult patients with AML were treated withunrelated CBT. All patients received 4 fraction-ated 12 Gy total body irradiation (TBI) and che-motherapy as myeloablative conditioning. Themedian age was 45 years, the median weightwas 55kg, the median number of nucleated cellswas 2.44×107/kg, and the median number of

Research Hospital

Department of Medicine(Department of Hematology / Oncology)内科（血液腫瘍内科）

Professor Arinobu Tojo, M.D., D.M.Sc.Associate Professor Satoshi Takahashi, M.D., D.M.Sc.Associate Professor Kaoru Uchimaru, M.D., D.M.Sc.Assistant Professor Jun Ooi, M.D.Assistant Professor Akira Tomonari, M.D., D.M.Sc.Assistant Professor Koichiro Yuji M.D., D.M.Sc.Assistant Professor Nobuhiro Ohno M.D.Assistant Professor Nobuhiro Tsukada M.D., D.M.Sc.Assistant Professor Ai Kotani

教 授 医学博士 東 條 有 伸准教授 医学博士 高 橋 聡准教授 医学博士 内 丸 薫助 教 大 井 淳助 教 医学博士 友 成 章助 教 医学博士 湯 地 晃一郎助 教 大 野 伸 広助 教 医学博士 塚 田 信 弘助 教 医学博士 幸 谷 愛

239

CD34-positive cells was 1.00×105/kg. All pa-tients received a single and HLA mismatchedcord blood unit. The cumulative incidence ofneutrophil recovery at day 50 and platelet re-covery at day 200 was 94.8％ and 91.7％, respec-tively. A higher CD34-positive cell dose was as-sociated with faster hematopoietic recovery. Thecumulative incidence of grade III to IV acutegraft-versus-host disease ( aGVHD ) andextensive-type chronic GVHD (cGVHD) was25.1％ and 28.6％, respectively. With a medianfollow-up of 78 months, the probability of event-free survival (EFS) at 5 years was 62.8％. The 5-year cumulative incidence of treatment related-mortality (TRM) and relapse was 9.7％, 25.8％,respectively. In multivariate analyses, the riskfactor identified for event free survival (EFS)was disease status and cytogenetics. These re-sults suggest that unrelated CBT after myeloab-lative conditioning could be safely and effec-tively used for adult patients with AML.

2. Myeloablative cord blood transplantation inadults with ALL.

Ooi J, Takahashi S, Tomonari A, Tsukada N,Tojo A

We analyzed the disease-specific outcomes ofadult ALL treated with cord blood transplanta-tion (CBT) after myeloablative conditioning. Be-tween October 2000 and November 2007, 27adult patients with ALL were treated with unre-lated CBT. All patients received four fraction-ated 12 Gy TBI and chemotherapy as myeloabla-tive conditioning. The median age was 36 years,the median weight was 57kg and the mediannumber of nucleated cells was 2.47×107/kg. Allpatients received a single and HLA-mismatchedcord blood unit. The cumulative incidence ofneutrophil recovery at day 30 and platelet re-covery at day 200 was 92.6 and 92.3％, respec-tively. With a median follow-up of 47 months,the probability of EFS at 5 years was 57.2％. The5-year cumulative incidence of TRM and relapsewas 3.7 and 27.4％, respectively. These resultssuggest that unrelated CBT after myeloablativeconditioning could be safely and effectivelyused for adult patients with ALL.

3. Myeloablative unrelated cord blood trans-plantation for acute leukemia patients be-tween 50 and 55 years of age.

Konuma T, Takahashi S, Ooi J, Tomonari A,Tsukada N, Uchimaru K, Tojo A.

Increasing recipient age is a well-known riskfactor for graft-versus-host disease (GVHD) and

treatment-related mortality (TRM) and has anegative impact on allogeneic hematopoieticstem cell transplantation. Since the incidence ofsevere GVHD after cord blood transplantation(CBT) is lower than that after transplants usingbone marrow or mobilized peripheral bloodgrafts from adult cells, we should expect betteroutcomes from CBT in older patients. To evalu-ate the feasibility and efficacy of myeloablativeunrelated CBT in patients aged between 50 and55 years, we performed a retrospective compari-son of 100 patients with acute leukemia who re-ceived cord blood grafts at our institution. Nine-teen older patients (median age, 52; range, 50-55) and 81 younger patients (median, 36; range,16-49) received a myeloablative conditioningregimen including 12 Gy of total body irradia-tion and chemotherapy. GVHD prophylaxis in-cluded cyclosporine with (n＝96) or without (n＝4) methotrexate. There were no significant dif-ferences in the incidences of grades II to IVacute GVHD, extensive-type chronic GVHD,TRM, and the probability of overall and disease-free survival between these groups. These re-sults suggest that, in patients with acute leuke-mia, myeloablative CBT might be as safe and ef-fective in patients aged between 50 and 55 yearsas in younger patients.

4. Second myeloablative stem cell transplan-tation (SCT) using cord blood for leukemiarelapsed after initial allogeneic SCT.

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Uchimaru K, Tojo A.

There are many reports of second allogeneicstem cell transplantation (allo-SCT) using cordblood (CB) for graft failure after initial allo-SCT.However, the efficacy of second allo-SCT usingCB for patients with leukemia relapsed after in-itial allo-SCT is unknown. We report the resultsof second allo-SCT using CB in seven adult pa-tients with leukemia relapsed after initial allo-SCT. All patients received a myeloablative con-ditioning regimen including oral busulfan 16mg/kg, intravenously fludarabine 100mg/m2 and cy-clophosphamide 120mg/kg. All but one patienthad myeloid reconstitution and four patients re-main alive at between 4 and 40 months aftersecond SCT. We conclude that second myeloab-lative allo-SCT using CB may be feasible in se-lected patients with the relatively younger age,less organ damage and longer time interval be-tween first and second allo-SCT.

5. Pneumocystis carinii pneumonia inmyeloablative unrelated cord blood trans-plantation.

240

Tomonari A, Takahashi S, Ooi J, Tsukada N,Tojo A.

The incidence of pneumonia caused by Pneu-mocystis carinii (PCP) (organism now renamedPneumocystis jiroveci) during the early periodafter cord blood transplantation (CBT) was stud-ied in 120 adults. Initially 89 patients (74％) re-ceived oral administration of 2 single-strengthtrimethoprim-sulfamethoxazole (TMP-SMZ) tab-lets twice daily from day -21. In 45 of 89 pa-tients (51％), TMP-SMZ administration for ascheduled duration was completed. In the re-maining 44 patients (49％), however, TMP-SMZadministration was discontinued prior to day -3because of toxicity. Among these patients, 42subsequently received aerosolized pentamidine(AP) on a median of day -13 (range, -20 to -6).Thirty-one patients (26％) received AP withoutTMP-SMZ administration on a median of day -14 (range, -21 to -9). None of the 120 patientswere diagnosed with PCP within 100 days or 2years after CBT; however, one patient who re-ceived AP before CBT but no prophylaxis afterCBT developed cerebral toxoplasmosis on day＋91. Pre-transplant prophylaxis against PCPdid not significantly affect transplantation-related mortality or disease-free survival at 2years after CBT. The results suggest that PCPduring the early period after CBT can be effec-tively prevented by any pre-transplant prophy-lactic method.

6. Early renal injury after myeloablative cordblood transplantation in adults.

Mae H, Ooi J, Takahashi S, Tomonari A, Tsu-kada N, Tojo A.

We report a retrospective analysis of acute re-nal failure (ARF) in a group of 54 adult patientswith hematological malignancies treated withunrelated cord blood transplantation (CBT) aftermyeloablative conditioning. All patients re-ceived four fractionated 12 Gy total body irra-diation and chemotherapy as myeloablative con-ditioning. ARF was defined as the doubling se-rum creatinine occurring within the first 100days after CBT. A statistically significant decre-ment of renal function from baseline was ob-served in days between 11 and 20. ARF oc-curred in 27.8％ of patients. Although no differ-ence was seen in maximum cyclosporine troughlevels, the maximum of vancomycin (VCM)trough levels were significantly higher in pa-tients with ARF (p＝0.01). Our result suggeststhat it is important to monitor VCM dosingmore strictly with pharmacokinetic assessment,especially in days 11-20, when the most fre-

quently observed declining renal function.

7. Cardiovascular toxicity of cryopreservedcord blood cell infusion.

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Tojo A.

Although infusion of cryopreserved bone mar-row or peripheral blood stem cell is associatedwith a variety of symptoms, there have been noreports detailing the data of infusion-related tox-icities of cryopreserved cord blood (CB) units.We prospectively evaluated the incidence andsignificance of infusion-related toxicities in 34adult patients undergoing unrelated CB trans-plantation. Cryopreserved CB units werethawed and immediately infused, unfiltered,through a central intravenous catheter withoutfurther manipulation. Heart rate, blood pres-sure, oxygen saturation and clinical symptomswere recorded during and after infusion.Twenty-four percent of patients experienced non-cardiovascular toxicities related to infusion. Theincidence of systolic and diastolic hypertensionand bradycardia was 58, 64 and 32％, respec-tively. Although three patients (9％) with severesystolic hypertension after the infusion requiredtreatment with antihypertensive agents, no pa-tients experienced life-threatening side effects orneeded discontinuation of CB unit infusion. Pa-tient or transplant characteristics had no effecton the hypertension and bradycardia related tothe infusion of CB. These data suggest that infu-sion of cryopreserved CB without further ma-nipulation after thawing is safe and well toler-ated. However, cardiovascular toxicities includ-ing hypertension and bradycardia were fre-quently observed.

8. Impact of CMV serostatus on outcome ofunrelated cord blood transplantation inadults.

Tomonari A, Takahashi S, Ooi J, Tsukada N,Tojo A.

Cytomegalovirus (CMV) disease is one of themajor infectious complications after allogeneichematopoietic stem cell transplantation (SCT).Several studies have shown that CMV-seropositive patients have a substantial survivaldisadvantage after bone marrow transplantation(BMT) or peripheral blood SCT (PBSCT). Be-tween August 1998 and February 2006, 101adult patients underwent myeloablative cordblood transplantation (CBT) from unrelated do-nors at our institution. Sixteen and 85 patientswere CMV-seronegative and CMV-seropositive,

241

respectively, prior to CBT. Outcomes of CBTwere compared between CMV-seronegative andCMV-seropositive patients. The cumulative inci-dences of neutrophil engraftment at 60 d afterCBT did not differ between CMV-seronegativeand CMV-seropositive patients (100％ and 94％,P＝0.09); however, the cumulative incidence ofplatelet engraftment at 100 d was higher inCMV-seronegative patients than CMV-seropositive patients (100％ vs. 86％, P＜0.005).The cumulative incidence of CMV antigenemiaat 100 d was lower in CMV-seronegative pa-tients than CMV-seropositive patients (0％ vs.77％, P＜0.001); however, the cumulative inci-dences of CMV disease did not differ betweenCMV-seronegative and CMV-seropositive pa-tients (0％ vs. 1％, P＝0.84). The probabilities ofdisease-free survival at 2 yr also did not differbetween CMV-seronegative and CMV-seropositive patients (92％ vs. 72％, P＝0.16).The outcomes of CBT for CMV-seropositive pa-tients as well as CMV-seronegative patients inour series were favorable. This might be due toeffective antiviral therapy for CMV infection.Large-scale studies are needed to determine theimpact of recipient CMV serostatus on the out-come of CBT for adults.

9. Factors predisposing to HTLV-1 infectionin residents of the greater Tokyo area.

Uchimaru K, Tojo A

Human T-cell leukemia virus type 1 (HTLV-1)is the etiological agent for adult T-cell leukemia.The geographic distribution of HTLV-1 carriersis quite uneven in Japan and the greatest preva-lence is in southwestern Japan. Because manypeople move from endemic areas to the greaterTokyo area, the geographic distribution mighthave changed. Therefore, we investigated thefactors predisposing to HTLV-1 infection, in-cluding birthplace, for 88 HTLV-1-infected indi-viduals in greater Tokyo who visited our outpa-tient clinic. Of these, 39.5％ were born in en-demic areas, which include Kyushu/Okinawa,south Shikoku, Kii, Tohoku, and Hokkaido,whereas 38.3％ were born in greater Tokyo andthe proportion is presumed to be increasing.Half of the HTLV-1 infected individuals ingreater Tokyo came from endemic areas,whereas around half of the remaining half waspresumed to be involved in sexual transmissionfrom a spouse from an endemic area. Overall,they constituted approximately 70％ of theHTLV-1 carriers in greater Tokyo. These migra-tion effects may increase the prevalence ofHTLV-1 in the greater Tokyo area; nationwidesurveillance is warranted.

Publications

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kato S, Sato A, Monma F, Kasa-hara S, Uchimaru K, Iseki T, Tojo A, Asano S.Second myeloablative allogeneic stem celltransplantation (SCT) using cord blood forleukemia relapsed after initial allogeneic SCT.Leuk Res. 2008 Nov 6. [Epub ahead of print]

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kato S, Sato A, Monma F, HongoE, Uchimaru K, Tojo A, Asano S. Donor cell-derived myelodysplastic syndrome after cordblood transplantation. Bone Marrow Trans-plant. 2008 Nov 3. [Epub ahead of print]

Ooi J, Takahashi S, Tomonari A, Tsukada N,Konuma T, Kato S, Kasahara S, Sato A,Monma F, Nagamura F, Iseki T, Tojo A,Asano S. Unrelated cord blood transplantationafter myeloablative conditioning in adultswith ALL. Bone Marrow Transplant. 2008 Oct27. [Epub ahead of print]

Tomonari A, Takahashi S, Ooi J, Tsukada N,Konuma T, Kato S, Kasahara S, Iseki T, TojoA, Asano S. No occurrence of Pneumocystis ji-roveci (carinii) pneumonia in 120 adults un-dergoing myeloablative unrelated cord blood

transplantation. Transpl Infect Dis. 2008 Jun18. [Epub ahead of print]

Tamai1 H, Yamaguchi H, Takahashi S, Tojo A,Hamaguchi H, Kobayashi T, Akiyama H,Sakamaki H, Okumura H, Nakao S, Arai A,Miura O, Inokuchi K, Dan K. Treatment ofadult acute myeloid leukaemia (AML) with t(6; 11)(q27; q23) by allogeneic haematopoieticstem cell transplantation (allo-HSCT) in firstcomplete remission (CR). Bone Marrow Trans-plant, 42: 553-4, 2008

Nagamura-Inoue T, Kai S, Azuma H, TakanashiM, Isoyama K, Kato K, Takahashi S,Taniguchi S, Miyamura K, Aoki1 K, Hidaka1M, Nagamura F, Tojo A, Fang1 XM, Kato Sfor Japan Cord Blood Bank Network. Unre-lated cord blood transplantation in chronicmyelogenous leukaemia: Japan Cord BloodBank Network analysis. Bone Marrow Trans-plant. 42: 241-51, 2008

Tomonari A, Takahashi S, Ooi J, Tsukada N,Konuma T, Kato S, Kasahara S, Iseki T, TojoA, Asano S. Blood eosinophilia after unrelatedcord blood transplantation for adults. BoneMarrow Transplant. 42: 63-5, 2008

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Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kato S, Kasahara S, Uchimaru K,Tojo A. Central nervous system leukemia onmagnetic resonance imaging. Intern Med. 47:477, 2008

Watanabe N, Takahashi S, Ishige M, Ishii Y, OoiJ, Tomonari A, Tsukada N, Konuma T, KatoS, Sato A, Tojo A, Nakauchi H. Recipient-derived cells after cord blood transplantation:dynamics elucidated by multicolor FACS, re-flecting graft failure and relapse. Biol BloodMarrow Transplant. 14: 693-701, 2008

Mae H, Ooi J, Takahashi S, Tomonari A, Tsu-kada N, Konuma T, Hongo E, Kato S, Kasa-hara S, Oiwa-Monna M, Kurokawa Y, Tojo A,Asano S. Early renal injury after myeloabla-tive cord blood transplantation in adults.Leuk Lymphoma. 49: 538-42, 2008

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kato S, Kasahara S, Uchimaru K,Iseki T, Tojo A, Asano S. Myeloablative unre-lated cord blood transplantation for adultacute myeloid leukemia patients with 11q23abnormalities. Eur J Haematol. 80: 545-8, 2008

Tamai H, Yamaguchi H, Hamaguchi H, Ya-gasaki F, Bessho M, Kobayashi T, Akiyama H,Sakamaki H, Takahashi S, Tojo A, Ohmine K,Ozawa K, Okumura H, Nakao S, Arai A, Mi-

ura O, Toyota S, Gomi S, Murai Y, Usui N,Miyazawa K, Ohyashiki K, Takahashi N,Sawada K, Kato A, Oshimi K, Inokuchi K,Dan K. Clinical features of adult acute leuke-mia with 11q23 abnormalities in Japan: multi-center co-operative study. Int J Hematol. 87:195-202, 2008

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kobayashi T, Sato A, Kato S, Ka-sahara S, Ebihara Y, Nagamura-Inoue T, TsujiK, Tojo A, Asano S. Cardiovascular toxicity ofcryopreserved cord blood cell infusion. BoneMarrow Transplant. 41: 861-5, 2008

Tomonari A, Takahashi S, Ooi J, Tsukada N,Konuma T, Kato S, Kasahara S, Iseki T, Yama-guchi T, Tojo A, Asano S. Impact of cy-tomegalovirus serostatus on outcome of unre-lated cord blood transplantation for adults: asingle-institute experience in Japan. Eur J Hae-matol. 80: 251-7, 2008

Tomonari A, Takahashi S, Ooi J, Tsukada N,Konuma T, Kobayashi T, Takasugi K, Iseki T,Tojo A, Asano S. Preemptive therapy withganciclovir 5mg/kg once daily for cytomega-lovirus infection after unrelated cord bloodtransplantation. Bone Marrow Transplant. 41:371-6, 2008

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Founded in 1981, Department of Infectious Diseases and Applied Immunology (DI-DAI) started HIV clinic in 1986. In 2008, 59 new patients with HIV infection havevisited or been admitted to our hospital and 399 patients in total are currently un-der our clinical management. The total number of in-patients with HIV-infectionduring 2008 was 31, and 5-7 beds in our ward have been constantly occupied bypatients with not only HIV-infection but also other infectious diseases. Since thenumber of the staff members of DIDAI is too small to care both outpatients and in-patients, members of the Division of Infectious Diseases and the Department ofInfectious Disease Control join the clinic. IMSUT hospital provides the most up-to-date medical treatment to HIV-infected patients in Japan. DIDAI is also a treat-ment center for international infectious diseases such as malaria and typhoid fe-ver.

1. Treatment of and clinical research on HIVinfection and related diseases.

a. Treatment of HIV infection in IMSUT hospi-tal: Statistical characteristics of HIV in-fected patients in IMSUT hospital this year

Takeshi, Fujii, Tokiomi Endoh, TomohikoKoibuchi1, Toshiyuki Miura2, Hitomi Nakamura1,

Michiko Koga1, Takuya Maeda2, TadashiKikuchi, Takashi Odawara1, and AikichiIwamoto1: 1Division of Infectious Diseases, TheAdvanced Clinical Research Center, 2Depart-ment of Infectious Disease Control, Interna-tional Research Center for Infectious Diseases

59 new patients with HIV-1 infection visitedour hospital this year (from January 1 to Decem-

Research Hospital

Department of Infectious Diseases andApplied Immunology感染免疫内科

Professor Aikichi Iwamoto, M.D., D.M.Sc.Lecturer Takashi Odawara, M.D., D.M.Sc.Lecturer Takeshi Fujii, M.D., D.M.Sc.Assistant Professor Tomohiko Koibuchi, M.D., D.M.Sc.Assistant Professor Tokiomi Endoh, M.D.Project Assistant Professor Takuya Maeda, M.D., ph.D.

教授（兼） 医学博士 岩 本 愛 吉＊1

講師（兼） 医学博士 小田原 隆＊1

講 師 医学博士 藤 井 毅助教（兼） 医学博士 鯉 渕 智 彦＊1

助 教 医学博士 遠 藤 宗 臣特任助教 医学博士 前 田 卓 哉＊2

【兼任】*1Division of Infectious Diseases, The Advanced Clinical Research Center（先端医療研究センター感染症分野）

*2Department of Infectious Disease Control, International Research Center for Infectious Diseases（感染症国際研究センター感染制御部門）

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ber 31, 2008), and 399 patients in total are undermedical management in our outpatient clinic.The total number of HIV-infected in-patientsduring 2008 was 31. The number of total pa-tients declined in 1997 because a part of patientsas well as medical stuffs moved to newly estab-lished AIDS Clinical Center in InternationalMedical Center of Japan. However, the numberof patients started to increase again after 1998 inaccordance with Japanese statistics of HIV-infected patients (Fig. 1). In contrast, the numberof admission has decreased since 1997 and sta-ble over the last decade (Fig. 2) because of theintroduction of highly active anti-retroviral ther-apy (HAART) which effectively suppresses thereplication of HIV. Anti-retroviral therapy hasbeen introduced to around 280 HIV-infected pa-tients in our hospital, and most of their HIV vi-ral loads have been well controlled. After oneyear of HAART, the viral loads become lessthan 400 copies/ml in more than 90% of pa-tients, and their CD4 cell counts increase by ap-proximately 200／µL in average. Consequently,the clinical management of HIV-infected pa-tients changed from how to treat opportunisticinfections into how to control patients withHAART.

2. Treatments and Clinical Research of Tropi-cal Diseases

a. Treatment of Tropical Diseases in IMSUThospital

Takeshi, Fujii, Tokiomi Endoh, TomohikoKoibuchi1, Takuya Maeda2, Toshiyuki Miura2,Tadashi Kikuchi, Takashi Odawara1, andAikichi Iwamoto1: 1Division of Infectious Dis-eases, The Advanced Clinical Research Center,2Department of Infectious Disease Control, In-ternational Research Center for Infectious Dis-eases

This year, more than hundred of overseastravelers visited our clinic. The reasons of theirvisit included prescription of malaria prophy-laxis, hepatitis A/B vaccination, other generalhealth consultation, or treatment of tropical dis-eases such as malaria, amoebic liver abscess,and post-exposure prophylaxis of rabies.

Supported by Department of Pharmacy in ourhospital, we are managing the clinical use of or-phan drugs for the treatment of tropical diseasesin Japan. We take the consultation from the doc-tors facing tropical diseases all over Japan, andprovide them with the appropriate medicinesfor the treatment of tropical diseases. In 2008,106 patients were treated with the orphan drugswe provided throughout Japan.

b. Unusual radiological findings of Fasciolahepatica infection with huge cystic andmultilocular lesions

Takuya Maeda2, Takashi Odawara1, Takeshi,Fujii, Tomohiko Koibuchi1 , and AikichiIwamoto1: 1Division of Infectious Diseases, TheAdvanced Clinical Research Center, 2Depart-ment of Infectious Disease Control, Interna-tional Research Center for Infectious Diseases

We reported a case of hepatic phase Fasciolahepatica infection presenting huge and multilo-cular lesions. The unique radiological findingsmimicked hydatid diseases and also cystic liverneoplasm. Fascioliasis should be included in thedifferential diagnosis for cystic liver diseases.

3. Development of an early detection methodfor pathogens causing acute respiratoryinfection

Takuya Maeda2, Tadashi Kikuchi, TokiomiEndoh, Tomohiko Koibuchi1, Takashi Odawara1,Aikichi Iwamoto1, and Takeshi Fujii: 1Divisionof Infectious Diseases, The Advanced ClinicalResearch Center, 2Department of Infectious

Figure 1. Number of HIV-infected outpatients inIMSUT Hospital

Figure 2. Number of HIV-infected inpatients inIMSUT Hospital

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Disease Control, International Research Centerfor Infectious Diseases

To develop a rapid detection method forpathogens causing acute respiratory infection,we have employed Loop-mediated isothermalamplification (LAMP), which is capable of am-plifying nucleic acids under isothermal condi-tions with high specificity and efficiency. Previ-ously we developed appropriate primers for thedetection of each pathogen in vitro. The targetpathogens included RNA viruses causing respi-ratory infection such as influenzavirus, respira-tory syncytial virus (RSV) and human coronavi-rus. Last year we tested the feasibility of thismethod for clinical samples, i.e., 236 nasal swabsamples (remnants of immunochromatography(IC) test for influenza at clinics), and could de-tect influenzavirus A or B from 137 samples(58.1%), which was 1.5 times more sensitivethan IC detection. These results showed the po-tential clinical feasibility of genetic diagnostictools with high specificity and efficiency, butRNA extraction step requires cost and time. Thisyear we tested an RT-LAMP method withoutRNA extraction and purification (we called thismethod ‘Direct-LAMP’) from clinical nasopharyngeal-swab specimens for the detection of influenzavirus. Nasopharyngeal swab specimens werecollected from 104 Japanese outpatients (agerange 0-13 years) presenting influenza orinfluenza-like illnesses. The samples were testedimmediately with commercial IC kits to detectthe antigens of influenza A/B virus at clinics.The remaining samples stored at －4°C weresent to our laboratory and used for genetic diag-nosis. Although the conventional RT-LAMPmethod demonstrated the highest sensitivityand specificity in comparison with the real-timePCR and IC test, “Direct LAMP” could detectinfluenza genomes in 51 (94.4%) of 54 positivesamples of conventional RT-LAMP. The specific-ity was also as high as conventional RT-LAMPmethod. “Direct LAMP” method developed inthis study would be useful for clinical diagnosisof respiratory RNA viral diseases, especially inthose countries where only minimal laboratoryfacilities are available and inexpensive tech-

niques are desired.

4. Comprehensive preoperative evaluation ofpatients with hemophiliac arthropathy

Tomohiko Koibuchi1, Tokiomi Endoh, TakuyaMaeda2, Takeshi, Fujii, Takashi Odawara1, andAikichi Iwamoto1: 1 Division of Infectious Dis-eases, The Advanced Clinical Research Center,2 Department of Infectious Disease Control, In-ternational Research Center for Infectious Dis-eases

In collaboration with the Department of JointSurgery we evaluate medical status of patientswith hemophiliac arthropathy who are going tobe given surgery. The majority of hemophiliacpatients who had received factor concentrates inthe early 1980s were infected with hepatitis Cvirus (HCV). A considerable percentage of themwere also infected with Human immunodefi-ciency virus (HIV). Among the hemophiliac pa-tients who had orthopedic surgery in 2008, 85%were HCV-Ab-positive and 30% were HIV-positive. Appropriate preoperative evaluation ofliver function and immunological status is es-sential to reduce the morbidity associated withthe surgery and improve the clinical outcomesof these patients.

We have developed a comprehensive preop-erative assessment system with a flow chart toevaluate the liver function and immunologicalstatus of hemophiliac patients. The flow chartemploys several indices for evaluation, such asCD4 cell count, ICG retention test, prothrombintime, fibrosis markers (type IV collagen,hyaluronic acid), the finding of abdominal ultra-sound and Child-Pugh score. Enhanced abdomi-nal CT and/or upper gastrointestinal endoscopyare also required when we suspect the existenceof hepatocellular carcinoma or esophageal varix.Based on the result of these indices, our multi-disciplinary team assesses the risks and benefitsof the surgery. The check-up system by themulti-disciplinary team using the flow chart hasimproved the care of the hemophiliac patientsundergoing surgical operations.

Publications

1. Miyazaki E, Kawana-Tachikawa A, TomizawaM, Nunoya J, Odawara T, Fujii T, Shi Y, GaoFG, Iwamoto A. Highly restricted TCR reper-toire in the CD8＋ T-cell response against anHIV-1 epitope with a stereotypic amino acidsubstitution. AIDS. In press.

2. Hou W, Aoki C, Yu L, Wen X, Xue Y, Gao B,

Liu W, Gao GF, Iwamoto A, Kitamura Y. Arecombinant replication-competent hepatitis Cvirus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visu-alization of infected cells. Biochem BiophysRes Commun. 377: 7-11, 2008.

3. Zong L, Chen Y, Peng H, Gao F, Iwamoto A,

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Gao GF. Rhesus macaque: A tight ho-modimeric CD8 alphaalpha. Proteins. 75: 241-244, 2008.

4. Nagai H, Iwasaki N, Odawara T, Okada S.Actual status of AIDS-related lymphoma man-agement in Japan. Int J Hematol. 87: 442-443,2008.

5. Saito T, Nishi M, Lim MI, Wu B, Maeda T,Hashimoto H, Takeuchi T, Roos DS, Asai T. Anovel GDP-dependent pyruvate kinaseisozyme from Toxoplasma gondii localizes toboth the apicoplast and the mitochondrion. JBiol Chem. 283: 14041-52, 2008.

6. Maeda T, Yamada H, Akao N, Iga M, Endo T,Koibuchi T, Nakamura T, Odawara T,Iwamoto A, Fujii T. Unusual radiological find-ings of Fasciola hepatica infection with hugecystic and multilocular lesions. Intern Med.47: 449-52, 2008.

7. Hosoya N, Miura T, Kawana-Tachikawa A,Koibuchi T, Shioda T, Odawara T, NakamuraT, Kitamura Y, Kano M, Kato A, HasegawaM, Nagai Y, Iwamoto A. Comparison betweenSendai virus and adenovirus vectors to trans-duce HIV-1 genes into human dendritic cells.J Med Virol. 80: 373-82, 2008.

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Our major goal is to cure children suffering from a variety of life-threatening hema-tological disorders. Attempting to achieve it, we continue the commitment to treat-ment and follow-up care of such children, and to clinical and laboratory researchthat ultimately will help us devise better therapeutic approaches to the diseases.Currently efforts are directed toward establishment of novel therapies including he-matopoietic stem cell transplantation (HSCT) and regenerative medicine using hu-man embryonic stem cells (hESC), induced pluripotent stem cells (hiPSC) andbome marrow (BM)-derived mesenchymal stem cells (MSC), and analysis ofpathogenesis of hematopoietic disorders, especially pediatric myelodysplastic syn-drome (MDS).

1. Hematopoietic stem cell transplantation forchildren with high-risk leukemia

Yasuhiro Ebihara, Kohichiro Tsuji

Although a standard regimen in HSCT hasbeen available for children with acute lym-phoblastic leukemia (ALL) and acute myeloidleukemia (AML), it has not been standardizedfor those with rare diseases including congenitalbone marrow failure syndrome (CBMFS) andnatural killer (NK) cell leukemia. A multi-institutional trial using regimens with a ration-ale should be proposed in a prospective manner.For CBMFS, we conducted in vitro and in vivoassays to assess the sensitivity of granulocytecolony-stimulating factor (G-CSF), and trans-planted the patients whose leukemic cells had ahigh sensitivity to G-CSF using a regime includ-ing G-CSF. Thus, we could avoid intensive che-motherapy before HSCT for patients with a vul-nerable normal bone marrow reserve. For pa-tients with Fanconi anemia, in particular, weemployed a regimen containing fludarabine toreduce the dose of alkylating agents and irradia-

tion to avoid the toxicity, which was otherwiselikely to occur in those patients. For patientswith NK cell disease, we used a regimen com-bining alkylating agents (cyclophosphamide andthiotepa) and total body irradiation based on theresults that NK leukemic cells strongly ex-pressed multidrug-registant genes. Now weplan to extend our experience in nationwide col-laborative studies.

2. Cooperative clinical trial for pediatric myel-odysplastic syndrome

Kohichiro Tsuji, Yasuhiro Ebihara, AtsushiManabe1, Yuji Zaike2: 1St. Luke’s InternationalHospital, 2Department of Laboratory Medicine,Research Hospital

Pediatric MDS is a rare disease, and only 50-100 children under the age of 16 suffer from thedisease annually. The diagnosis and treatmenthave not been standardized and it should be de-termined in a nationwide manner. On behalf ofthe MDS committee of the Japanese Society ofPediatric Hematology, we began the pathologic

Research Hospital

Department of Pediatric Hematology-Oncology小児細胞移植科

Associate Professor Kohichiro Tsuji, M.D., D.M.Sc.Assistant Professor Yasuhiro Ebihara, M.D., D.M.Sc.

准教授 医学博士 辻 浩一郎助 教 医学博士 海老原 康 博

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central review in 1999 and reviewed all samplesof patients suspected of having MDS. At pre-sent, over 300 patients have been enrolled, andstandard diagnostic criteria have been proposedfor juvenile myelomonocytic leukemia (JMML),a subset of MDS. We also tested in vitro cellgrowth for patients with JMML using diagnosticsamples. The results showed that spontaneousgrowth and hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF)were observed in most children with JMML. Weproposed a cooperative trial to establish thetreatment for MDS (MDS99) and have enrolledover 50 patients from the whole country.

3. Novel approach to therapy in juvenilemyelomonocytic leukemia

Yasuhiro Ebihara, Yoshitoshi Ohtsuka3, At-sushi Manabe1, Yuji Zaike2, Kohichiro Tsuji:3Department of Pediatrics, Hyogo College ofMedicine

JMML is a clonal myeloproliferative/myelo-dysplastic disorder of early childhood with poorprognosis. JMML cells are characterized by hy-persensitivity to GM-CSF caused by continu-ously activated GM-CSF receptor-RAS signaltransduction pathway through various molecu-lar mechanisms, resulting in spontaneous colonyformation in vitro. Bisphosphonate zoledronicacid (ZOL), a RAS-blocking compound, sup-pressed colony formation from BM cells ofJMML patients and normal volunteers withoutand with GM-CSF, respectively, in a dose-dependent manner in clonal culture. At 10 µMof ZOL, however, spontaneous colony formationdecreased, but formation of granulocyte (G)colonies containing only granulocytes, but nomacrophages was enhanced in culture of JMMLBM cells, while granulocyte-macrophage (GM)colonies containing both granulocytes andmacrophages retained and G colony formationwas not affected in culture of normal BM cellswith GM-CSF. In suspension culture, 10 µM ofZOL also inhibited spontaneous proliferationand differentiation along monocyte/macrophagelineage of JMML BM cells, but not developmentof normal BM cells by GM-CSF assessed in cyto-chemical and flow cytometric analyses. The in-hibitory effect of ZOL on JMML cells was con-firmed at a single-clone level, and observedeven at 3 µM. The current result offers a novelapproach to therapy in JMML.

4. Novel method for efficient production ofmultipotential hematopoetic progenitorsfrom hESC

Feng Ma5, Yasuhiro Ebihara, Sachiyo Hanada,Yuji Zaike2, Hiromitsu Nakauchi5, KohichiroTsuji: 5Division of Stem Cell Therapy, Centerfor Stem Cells and Regenerative Medicine

ESC are pluripotent cells derived from the in-ner cell mass of preimplantation embryos. SinceESC have the ability to be maintained in cultureindefinitely as undifferentiated cells, yet theyare capable of forming more differentiated celltypes, hESC recently established are expected asa novel source of human transplantable cells.We then planed to produce HSC for HSCT andfunctional blood cells for transfusion medicinefrom human ES cells. This study was started onDecember 20, 2003 with the permission by theethical committee of the Japanese Government.On beginning this study, we thought that in vi-tro reconstitution of the circumstance surround-ing embryonic hematopoietic cells is importantto induce the differentiation of hESC into HSCor functional blood cells. To achieve this, we de-termined to use stromal cells from murine em-bryonic hematopoietic tissues to coculture hESCwith them, since some mouse-derived stromalcells have been reported to be able to act on hu-man hematopoietic cells.

We then proposed a novel method for the ef-ficient production of hematopoietic progenitorsfrom hESC by co-culture with stromal cells de-rived from murine FL (mFLSC) at 14 to 15 dayspost coitus (dpc), in which embryonic hema-topoiesis dramatically expands at midgestation.In the co-culture, various hematopoietic progeni-tors were generated, and this hematopoietic ac-tivity was concentrated in cobblestone-like (CS)cells within differentiated hESC colonies. The CScells expressed CD34 and retained a potentialfor endothelial cells. They also contained hema-topoietic colony-forming cells, especially eryth-roid and multilineage colony-forming cells athigh frequency. The multipotential hematopoie-tic progenitors abundant among the CS cellsproduced all types of mature blood cells, includ-ing adult type β globin-expressing erythrocytesand tryptase and chymase-double positive mastcells (MC). They showed neither immatureproperties of ESC nor potentials to differentiateinto endoderm and ectoderm at a clonal level.The developed co-culture system of hESC canprovide a novel source for hematopoietic andblood cells applicable to cellular therapies anddrug screenings.

5. Generation of functional erythrocytes fromhESC-derived definitive hematopoiesis

Feng Ma5, Yasuhiro Ebihara, Sachiyo Hanada,Yuji Zaike2, Hiromitsu Nakauchi5, Kohichiro

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Tsuji

A critical issue for utilization of hESC in pos-sible clinical use is whether they can derive ter-minally mature progenies with the normal func-tion. To solve this, we examined hESC-derivederythroid cells in coculture with mFLSC. By thecoculture, large quantity of hESC-derived eryth-roid progenitors allowed us to analyze the de-velopment of erythropoiesis at a clone level andto investigate their function as oxygen carrier.The results showed that the globin expression inthe erythroid cells in individual clones changedin a time-dependent manner. In particular, em-bryonic ε globin positive erythrocytes decreased,while adult-type β globin positive cells in-creased to almost 100％ in all single clones weexamined, indicating they had already beenfated to definitive hematopoiesis. Enucleatederythrocytes also appeared in the clonal eryth-roid progenies. A comparison analysis showedthat hESC-derived erythroid cells took a similarpathway in differentiation to human cord bloodCD34＋ progenitor-derived erythrocytes whentraced by glycophorin A, CD71 and CD81. Fur-thermore, these hESC-derived erythroid cellscould function as oxygen carrier, and had a suf-ficient glucose-6-phosphate dehydrogenase ac-tivity. The present study provided an experi-mental model to explore early development ofhuman erythropoiesis, hemoglobin switching,erythroid pathogenesis, and to discover drugsfor hereditary diseases in erythrocyte develop-ment. The quantitative production and theirfunctional maturation indicate that hESC-derived erythrocytes can be a novel potentialsource for therapeutic transfusion.

6. Differential production of connectivetissue-type and mucosal mast cells fromhESC for anti-allergy drug screening

Feng Ma5, Yasuhiro Ebihara, Sachiyo Hanada,Hiromitsu Nakauchi5, Kohichiro Tsuji

MC function as effector cells in allergy andatopic disease. Therefore, anti-allergy drugshave been established to diminish MC function.However, since the acquisition of an abundanceof human MC (hMC) is difficult because of noculture method producing massive hMC, mostanti-allergy drugs targeted animal MC. Thus, ef-ficient discovery of effective anti-allergy drugsneeds to establish the culture system of massivehMC. Then, hESC are considered as a potentialcell source for hMC.

In human, two types of MC have been charac-terized; connective tissue-type and mucosal MC(CTMC and MMC, respectively). CTMC contain

tryptase, chymase, MC carboxypeptidase andcathepsin G in their secretory granules, are pre-dominantly located in normal skin and in intes-tinal submucosa, and involve in atopic dermati-tis. MMC contain tryptase in their secretorygranules, but lack the other proteases, are themain type of MC in normal alveolar wall and insmall intestinal mucosa, and involve in allergicrhinitis or bronchial asthma. Although MC canbe generated from human adult CD34＋ hema-topoietic progenitor cells in vitro, these MC aremainly MMC. So far, there lacks an evidence forthe direct derivation of CTMC from adult hema-topoietic progenitors.

We achieved successful production of hESC-derived CD34＋ hematopoietic progenitors, usingco-culture with mFLSC for 1-2 weeks. In suspen-sion culture favoring MC differentiation within3weeks, hESC-derived progenitors generatedmature MC that shared a chymase/tryptasedouble positive phenotype and strongly ex-pressed c-Kit, similar to human skin derivedCTMC. On the other hand, hESC-derived mul-tipotential hematopoietic progenitors obtainedin clonal culture developed into MC for a longertime (over 5 weeks) and only expressed tryp-tase, with no or few chymase, similar to humanCD34＋ cell-derived MMC. Since the current cul-ture system of hESC can produce differentially alarge number of CTMC and MMC, our studymay highlight a new understanding for MC de-velopment and finally benefit the screening foranto-allergy drugs.

7. Derivation of multipotential hematopoieticprogenitors and mature blood cells fromhuman induced pluripotent stem cells

Feng Ma5, Yasuhiro Ebihara, Sachiyo Hanada,Daisuke Tomizawa, Hiromitsu Nakauchi5 andKohichiro Tsuji

The establishments of hESC and hiPSC haveconstructed a firm base for regenerative medi-cine. As mentioned above, we developed a cul-ture system for efficient production of hESC-derived multipotential hematopoietic progeni-tors and functionally mature erythrocytes, thusproviding an experimental model to exploreearly events in human erythropoiesis. We thenapplied a similar culture system to inducehiPSC to differentiate into hematopoietic proge-nies.

hiPSC (235G1, 235G4, 201B6 and 201B7,kindly provided from Dr. Yamanaka, KyotoUniversity) were used in our experiments. Toinduce differentiation to hematopoietic cells, un-differentiated hiPSC were cocultured with amouse feeder stromal cell line derived from

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AGM region (AGMS-3). On different times,cocultured cells were harvested by 0.05％ tryp-sin/EDTA solution and re-culture in a semisolidculture to produce hematopoietic colonies. Inthe consequential experiments, erythrocytes de-veloped in colonies were examined with Hb ex-pression, while other myeloid cells, mast cellsand magakaryocytes were examined with theirmaturity.

Although all hiPSC lines were capable of gen-erating hematopoietic cells in clonal culture, 235G1 revealed most efficiently, which producedapproximately a hundred colonies (Mix colonies:5％) from 30 undifferentiated hiPSC colonies.Nine tenths of clonal erythroid cells expresseddefinitive β-globin, which is comparable tohESC-derived ones. Furthermore, hiPSC-derivedblood cells (granulocytes, mast cells and mega-karyocytes) expressed various mature markers,indicating their fully functional maturation.

Thus, we have established a culture system toinduce hiPSCs to multipotential hematopoieticprogenitors and mature blood cells. Our studymay be used as an experimental model and fi-nally help in clinical cures for hereditary blooddiseases.

8. hESC-derived MSC capable of efficientlymaintaining hESC and hiPSC under animalserum-free conditions

Yasuhiro Ebihara, Feng Ma5, Sachiyo Hanada,Daisuke Tomizawa, Hiromitsu Nakauchi5,Haruo Onoda2, Naoki Oyaizu2, Kohichiro Tsuji

hESC and hiPSC have the ability to differenti-ate into all cell types in the body and hold greatpromise for regenerative medicine. However,the culture maintaining undifferentiated hESCand hiPSC depends on animal feeder cells and/or serum in most cases. Such a culture system isa huge obstacle for clinical applications of thesestem cells because of xenogeneic pathogen con-tamination. To solve this issue, we developed anovel culture method for maintaining undiffer-entiated hESC and hiPSC using MSC as feedercells and platlet lysate (PL) instead of animal se-rum.

When undifferentiated hESC (line H1) cul-tured on murine embryonic fibroblast (MEF)feeder cells were recultured on gelatin-coatedculture dishes with PL-containing media in theabsence of MEF feeder cells. Cells were pas-saged several times with PL containing media,and then stromal cells were induced after 6 to 8weeks. The stromal cells were spindle-likeshaped, revealed a phenotype of CD45-, CD34-,CD14-, CD105＋, CD166＋, CD31-, and SEA-4-,and had the ability to differentiate into mesen-

chymal tissues such as bone, cartilage and fat invitro, indicating these cells were MSC. MEFfeeder cells and undifferentiated hESC were un-detectable in the hESC-derived MSC by reversetranscription polymerase chain reaction analysis.

In the coculture with the hESC-derived MSCin the presence of PL-containing media, undif-ferentiated hESC (line H1) were maintained atleast for four weeks. The cocultured hESC ex-pressed specific markers for undifferentiatedESC, such as Oct-3/4, Sox-2, and Nanog, andformed teratoma containing ectodermal, endo-dermal, and mesodermal tissues in the trans-plantation into non-obese diabetic/severe com-bined immunodeficient mice. These hESC-derived MSC also have the ability to keep an-other hESC (line khES-1, kindly provided by Dr.Nakatsuji, Kyoto University) and hiPSC (line235/G1, kindly provided by Dr. Yamanaka,Kyoto University) undifferentiated state. In ad-dition, khES-1 cells generated MSC with theability same to H1 cell-derived MSC. Interest-ingly, hESC or hiPSC isolated by passingthrough 100 micro-meter filter were capable offorming undifferentiated stem cell colonies inthe coculture with hESC-derived MSC.

These results indicate that hESC-derived MSCare able to be substituted for MEF feeder cells inthe absence of animal serum but in the presenceof PL in the maintenance of hESC and hiPSC,and efficiently support the proliferation of un-differentiated stem cells, even from single hESCor hiPSC. The current culture system can be use-ful for the clinical application of hESC andhiPSC.

9. Establishment of human BM-derived MSCfor the treatment of hemophilic arthropa-thy

Yasuhiro Ebihara, Hideyuki Takedani6, TokikoNagamura-Inoue7, Shigeyuki Wakitani8, Ari-nobu Tojo9, Hiromitsu Nakauchi5, KohichiroTsuji: 6Department of Joint Surgery, ResearchHospital, 7Department of Cell Processing andTransfusion, 8Department of Orthopaedic Sur-gery, Osaka City University Graduate Schoolof Medicine, 9Division of Molecular Therapy,Advanced Clinical Research Center

Hemophilia is the congenital disease with alack of coagulation factors. One to two thirds ofthe patients had arthropathy because of recur-rent intra-articular bleeding. Most of surgicaltreatment for the arthropathy, such as synovec-tomy or total joint arthroplasty, in Japan is per-formed by Department of Joint Surgery in ourhospital. So far, however, the efficacy of thetreatment has been insufficient. We then planed

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the transplantation of autologous culture-expanded BM-derived MSC into the articularcartilage defect in the hemophilic arthropathy

patients. For the project, we are establishing theculture system of MSC from the patient BM us-ing autologous serum.

Publications

Ma F, Ebihara Y, Umeda K, Sakai H, Hanada S,Zhang H, Zaike Y, Tsuchida E, Nakahata T,Nakauchi H, Tsuji K. Generation of functionalerythrocytes from human embryonic stemcell-derived definitive hematopoiesis. ProcNatl Acad Sci USA 105: 13087-13092, 2008.

Ma F, Kambe N, Wang D, Shinoda G, Fujino H,Umeda K, Fujisawa A, Ma L, Suemori H,Nakatsuji N, Miyachi Y, Torii R, Tsuji K,Heike T, Nakahata T. Direct development offunctionally mature tryptase/chymase doublepositive tissue-type mast cells from primateES cells. Stem Cells 26: 705-714, 2008.

Ebihara Y. Identification and clinical applicationof fibroblasts/myofibroblasts derived from he-matopoietic stem cells. Japan J Pediatr Hema-tol 36: 1-6, 2008.

Mizutani T, Tsuji K, Ebihara Y, Ohba Y,Taniguchi T , and Kenya Honda K. Homeo-static erythropoiesis by the transcription factorIRF2 through attenuation of type I interferonsignaling. Exp Hematol 36: 255-264, 2008.

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kobayashi T, Sato A, Kato S, Ka-

sahara S, Ebihara Y, Nagamura-Inoue T, TsujiK, Tojo A, Asano S: Cardiovascular toxicity ofcryopreserved cord blood cell infusion. BoneMarrrow Transplant 41: 861-865, 2008.

Tauchi H, Tomizawa D, Eguchi M, Eguchi-Ishimae M, Koh K, Hirayama M, MiyamuraN, Kinukawa N, Hayashi Y, Horibe K, Ishii E.Clinical features and outcome of MLL generearranged acute lymphoblastic leukemia ininfants with additional chromosomal abnor-malities other than 11q23 translocation. LeukRes 32: 1523-1529, 2008.

Kulkeaw K, Mizuochi C, Horio Y, Osumi N,Tsuji K, Sugiyama D. Application of wholemouse embryo culture system on stem cell re-search. Stem Cell Rev & Rep, in press.

Tomizawa D, Koh K, Hirayama M, Miyamura T,Hatanaka M, Saikawa Y, Ishii E. Outcome ofrecurrent or refractory acute lymphoblasticleukemia in infants with MLL gene rearrange-ments: A report from the Japan Infant Leuke-mia Study Group. Pediatr Blood Cancer, inpress.

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We participate in cutting edge science of autoimmune, rheumatic and allergic dis-ease and novel treatments for patients with these disorders. In addition to conven-tional drug studies aimed to improve the efficacy and safety of current therapies,we are going to carry out experimental protocols of particular interest for patientsnot responding to conventional therapy and to perform the translational research.

I. Study on CD26 molecule in normal immuneresponse and in patients with immune-mediated diseases

Osamu Hosono, Kei Ohnuma, NoritadaYoshikawa, Hiroshi Kawasaki , HirotoshiTanaka, Chikao Morimoto. (Department ofRheumatology and Allergy), Emi kumagai,Akiko Souta-Kuribara, (Division of ClinicalImmunology)

CD26 is a T cell costimulatory molecule aswell as an activation antigen with dipeptidylpeptidase IV (DPPIV) enzyme activity in its ex-tracellular region that is preferentially expressedon memory T cells. The soluble form of CD26(sCD26) is present in serum and recombinantsoluble CD26 can enhance peripheral blood Tcell proliferation induced by the recall antigen.We demonstrated that CD26 binds Caveolin-1on antigen presenting cells, and that followingCD26-caveolin-1 interaction on recall antigen-loaded monocytes, caveolin-1 is phosphorylated,with linkage to NF-κB activation, followed by

upregulation of CD86. In addition, reducedcaveolin-1 expression on monocytes inhibits CD26-mediated CD86 upregulation and abrogatesCD26 effect on recall antigen-induced T cell pro-liferation, and immunohistochemical studies re-vealed an infiltration of CD26＋ T cells in thesublining region of rheumatoid synovium andhigh expression of caveolin-1 in the increasedvasculature and synoviocytes of the rheumatoidsynovium. Taken together, these results stronglysuggest that CD26-caveolin-1 interaction plays arole in the upregulation of CD86 on recallantigen-loaded monocytes and subsequent en-gagement with CD28 on T cells, leading toantigen-specific T cell activation such as the T-cell-mediated antigen-specific response in rheu-matoid arthritis (RA).

Currently we are focusing on the translationalresearch of utilization of anti-CD26 mAb as wellas recombinant soluble CD26 for treatment ofmalignant tumors, immune-mediated disordersand immune deficiency diseases. Hopefully wewill perform phase I/II clinical trial utilizing hu-manized CD26 antibody for the treatment of the

Research Hospital

Department of Rheumatology and Allergyアレルギー免疫科

Professor Chikao Morimoto, M.D., D.M. Sc.Associate Professor Hirotoshi Tanaka, M.D., D.M. Sc.Assistant Professor Osamu Hosono, M.D., D.M. Sc.Assistant Professor Hiroshi Kawasaki, M.D., D.M. Sc.Assistant Professor Noritada Yoshikawa, M.D., D.M. Sc.Assistant Professor Kei Ohnuma, M.D., D.M. Sc.

教 授 医学博士 森 本 幾 夫准教授 医学博士 田 中 廣 壽病院講師 医学博士 細 野 治助 教 医学博士 河 崎 寛助 教 医学博士 吉 川 賢 忠助 教 医学博士 大 沼 圭

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above diseases, such as malignant mesotheliomaand other CD26 positive malignant tumors soon.

a. Clinical significance of soluble CD26/DPPIV in various disease conditions

(i) Soluble CD26/DPPIV in autoimmune andother immune-mediated disorders

Our previous studies demonstrated that CD26-caveolin-1 interaction plays a role in theupregulation of CD86 on recall antigen-loadedmonocytes and subsequent engagement withCD28 on T cells, leading to antigen-specific Tcell. Possible substrates of CD26/DPPIV includeseveral critical cytokines and chemokines. CD26could modulate function of several cytokinesand chemokines such as RANTES (CCL5), SDF-1α (CXCL12) and glucagons-like peptide 1 (GLIP-1) through its DPPIV enzyme activity. We haveshown that the DPPIV enzyme activity ofplasma sCD26 was low in HIV-1-infected indi-viduals, and was inversely correlated with HIV-1 RNA, and that the in vitro addition of recom-binant sCD26 could enhance purified proteinderivative-induced lymphocyte proliferation.These DPPIV enzyme activity of sCD26 in HIV-1-infected individuals contributes to the im-munopathogenesis of HIV infection. Further-more, we have shown that serum levels of sCD26 and its specific DPPIV activity were signifi-cantly decreased in SLE and were inversely cor-related with SLE disease activity index score,but not with clinical variables or clinical subsetsof SLE. Serum levels of sCD26 may be involvedin the pathophysiology of SLE, and appear to beuseful as a new disease activity measure forSLE.

We examined sCD26 and its specific DPPIVactivity in serum of patients with inflammatorybowel diseases (IBD), such as Crohn’s disease orulcerative colitis in collaboration with Gastroin-testinal Unit, School of Medicine, Keio Univer-sity. The DPPIV activity was reduced in patientswith IBD and was significantly lower in patientswith Crohn’s disease compared to with ulcera-tive colitis (P＜0.05). We have also measuredsCD26/DPPIV levels in sera and synovial fluidfrom patients with RA and found significant de-crease of serum sCD26 and its specific DPPIVactivity. These findings indicate that CD26 maybe potentially important for the pathophysiol-ogy of IBD and RA. Furthermore, we have in-vestigated autoantibodies against CD26 in se-rum using ELISA and Western blotting meth-ods. We have not found anti-CD26 autoantibodywhich could reduce DPPIV activity so far. Weplan to examine the effect of TNF-α blockingtherapy (infliximab, etanercept, adalimumab) onserum levels of sCD26/DPPIV in patients with

RA and its clinical significance.(ii) Soluble CD26/DPPIV in malignancies associ-

ated with asbestos exposureCD26/DPPIV is able to cleave selected bio-

logical factors to alter their functions and regu-lates topoisomerase II α level in hematologicmalignancies, affecting sensitivity to doxorubicinand etoposide. Expressed on various tissues, CD26 is involved in the development of certain hu-man cancers. We have shown CD26 is highlyexpressed on the cell surface of malignant meso-thelioma and that a newly developed human-ized anti-CD26 monoclonal antibody has an in-hibitory effect on malignant mesothelioma cellsin both in vitro and in vivo experiments.

We examined sCD26 and its specific DPPIVactivity in serum of patients with asbestosis incollaboration with Okayama Rosai Hospital. Se-rum levels of sCD26 and its specific DPPIV ac-tivity were significantly increased in patientswith pleural plaque compared to healthy indi-viduals (P＜0.05). However, serum levels of sCD26 and its specific DPPIV activity was signifi-cantly reduced in patients with both malignantmesothelioma and primary lung cancer associ-ated with asbestos exposure compared to pa-tients with pleural plaque. We also examinedsCD26 and its specific DPPIV activity in pleuraleffusion of patients with mesothelioma, primarylung cancer and tuberculosis. In mesotheliomathere seems to be a relationship between pleuralCD26/DPPIV and prognosis. We are doing se-rial studies and measuring sCD26/DPPIV in se-rum and pleural fluid to confirm their clinicalsignificance.

b. CD26-based molecular target therapy forgraft-versus-host disease in hematopoieticstem cell transplantation

Graft-versus-host disease (GVHD) remains amajor cause of morbidity and mortality in allo-geneic hematopoietic stem cell transplantation(alloHSCT). In GVHD, mature donor T cells thataccompany the stem cell graft attack recipienttissues, especially the skin, liver, gastrointestinaltract, and lung. Therefore, all patients undergo-ing alloHSCT receive GVHD prophylaxis to im-pair T cell function; however, treatment to pre-vent GVHD can be deleterious since mature do-nor T cells play a critical role in mediating re-constitution of the adaptive immune system. Re-cipients of alloHSCT are thus at great risk forinfections, particularly when prolonged im-munosuppression is required for treatment ofGVHD. Although the role of CD26/DPPIV inGVHD needs to be studied in more detail, treat-ment with a murine antibody against humanCD26 was reported to have an effect in patients

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with steroid-resistant acute GVHD following al-loSCT (Bacigalupo A., et al., Acta Haematol1985: 73: 185, de Meester, et al., Immunobiology1993: 188: 145). To examine the efficacy of CD26-targeting therapy in GVHD more profoundly,we established mouse GVHD model using hu-man peripheral blood lymphocytes (huPBL)(xenograft GVHD mouse model; x-GVHD). Af-ter NOD/LtSz-scid or NOD/Cg-Prkdcscidil2rgtm1Sug/Jic mice were injected with appropri-ate numbers of huPBL, mice show symptoms ofGVHD such as loss of weight, loss of hair, dete-rioration of activity, and thinning of ear pads.Histopathological examination revealed that CD3＋CD26＋ human lymphocytes were infiltratedin the skin, intestinal mucosa, salivary gland,lung and liver of the x-GVHD mice. In thismouse model, humanized anti-CD26 monoclo-nal antibody (mAb) was injected two weekslater of onset of x-GVHD, and the symptoms ofGVHD were improved after ten injections of hu-manized anti-CD26 mAb. Moreover, x-GVHDwas observed to be suppressed when human-ized anti-CD26 mAb was prophylactically ad-ministered. Taken together, it may be possiblethat the full therapeutic potential of alloSCT willbe realized by approaches that aim to minimizeGVHD by targeting CD26-mediated T cell regu-lation.

II. Therapeutically targeting transcription fac-tors

Hirotoshi Tanaka, Noritada Yoshikawa, Nori-aki Shimizu, Chikao Morimoto.

We are interested in the mechanism of eu-karyotic gene expression and development ofnovel therapy and/or drugs which target tran-scriptional machineries. For this purpose, our re-cent work is mainly focused on conditionalregulation of transcription factors including theglucocorticoid receptor and hypoxia-induciblefactor-1α.

a. Glucocorticoid receptor (GR) project

Glucocorticoid hormones are effective in con-trolling inflammation and immunity, but under-lying mechanisms are largely unknown. It hasbeen shown that both positive and negativeregulations of gene expression are necessary forthis process. The genes whose activity is nega-tively modulated in the anti-inflammatory proc-ess code for several cytokines, adhesion mole-cules. Most of them do not carry a classicalbinding site for regulation by the GR, but haveinstead regulatory sequences for transcriptionfactors such as AP-1 or NF-κB. Considering

various severe side effects of glucocorticoids, itmay be pharmacologically important to dissoci-ate these negative regulatory function of the GRfrom induction of genes for metabolic enzymes,expression of which have been shown to bepositively regulated by the GR. We propose thata certain class of compounds (surprisingly, someof them are non-steridal chemicals) may dissoci-ate transactivation and transrepression functionof the GR and offer opportunities for the designof such compounds that could function more ef-fectively as antiinflammatory drugs. In this line,we are developing novel therapeutic strategy.(i) Redox Regulation of the GR

Redox regulation is currently considered as amode of signal transduction for coordinatedregulation of a variety of cellular processes.Transcriptional regulation of gene expression isalso influenced by cellular redox state, mostpossibly through the oxido-reductive modifica-tion of transcription factors. The glucocorticoidreceptor belongs to a nuclear receptor super-family and acts as a ligand-dependent transcrip-tion factor. We demonstrate that the glucocorti-coid receptor function is regulated via redox-dependent mechanisms at multiple levels. More-over, it is suggested that redox regulation of thereceptor function is one of dynamic cellular re-sponses to environmental stimuli and plays animportant role in orchestrated crosstalk betweencentral and peripheral stress responses.(ii) Development of Dissociating Ligand for the

GRThe GR function could be differencially regu-

lated by ligands. We have recently shown thatnot only synthetic glucocorticoids but also cer-tain bile acids could differentially modulate GRfunction. Moreover, the effects of those com-pounds are indicated to be ascrived to theligand binding domain of the receptor. In thisline, we are going to isolate the dissociatingligand that preferencially promotes transrepres-sion function of the GR. Recently we have dem-onstrated that certain ligands can modulate in-terdomain communication of the GR, which willeventually contribute to isolation of novel cate-gory of ligands. On the other hand, receptorspecificity is another important aspect of novelGR regulator. In this line, we have shown thatcortivazol is extremely specific for GR and doesnot bind to MR. We are studying the molecularbasis for this receptor specificity of the ligandusing cortivazol as a model. Our recent microar-ray study demonstrated that GR and MR havedifferential role in homeostatic regulation innon-classical corticosteroid target tissues includ-ing the heart. Notably, collaboration with Pro-fessor Miyano’s laboratory greatly contributedto development of this program.

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(iii) Molecular biology of small nuclear RNAbinding protein HEXIM1

Expression of HEXIM1 is induced by treat-ment of vascular smooth muscle cells with a dif-ferentiation inducer hexamethylane bisaceta-mide. It is shown that HEXIM1 binds 7SKsnRNA and inhibits P-TEFb-mediated transcrip-tional elongation process. On the other hand, wehave found that HEXIM1 directly associateswith the GR in the absence of 7SK and repressesGR-mediated transcription. We are currentlyworking on regulation of HEXIM1 expression,physiological role of HEXIM1 in GR action. In-deed, HEXIM1 has differential roles in generegulation in a context and gene specific fashion.We have recently characterized that HEXIM1may play an important role in tissue-specificregulation of glucocorticoid-mediated gene ex-pression. Physiological significance of HEXIM1is being studied using newly generated trans-genic mice.

b. Hypoxia-inducible Factor (HIF)-1α project

HIF-1α is essential for not only angiogenesisbut also development of certain organs. In thisline, molecular biology of HIF-1α will provideus possible advantage to characterize and man-upilate such processes. Peripheral T cells en-counter rapid decrease in oxygen tension asthey are activated by antigen recognition andmigrate into inflammatory sites or tumors. Acti-vated T cells, therefore, are thought to havesuch machineries that enable them to adapt tohypoxic conditions and execute immune regula-tion in situ. We have recently shown that sur-vival of CD3-engaged human peripheral bloodT cells is prolonged under hypoxic conditionsand HIF-1 and its target gene product adreno-medullin play a critical role for the process. It isalso shown that hypoxia alone is not sufficientbut TCR-mediated signal is required for accu-mulation of HIF-1α in human peripheral T cells.In the present study, we showed that TCR-engagement does not influence hypoxia-depen-dent stabilization but stimulates protein synthe-sis of HIF-1α, most possibly via PI3K/mTORsystem, and that expression of HIF-1α and itstarget gene is blocked by treatment with rapa-mycin. Since some of those gene products, e.g.,glucose transporters and phosphoglycerate kina-se-1, are considered to be essential for glycolysisand energy production under hypoxic condi-tions and adequate immune reaction in T cells,this TCR-mediated synthesis of HIF-1α mayplay a pivotal role in peripheral immune re-sponse. Taken together, our results may high-light a novel aspect of downstream signal fromantigen recognition by TCR with giving insight

of a unique pharmacological role of rapamycin.We are currently working with the mechanismof translational regulation of HIF-1α.

III. Case Reports

Osamu Hosono, Kei Ohnuma, NoritadaYoshikawa , Tomoki Katayose , HiroshiKawasaki, Hirotoshi Tanaka, Chikao Mori-moto (Department of Rheumatology and Al-lergy), Naoki Oyaizu (Department of Labora-tory Medicine)

a. An adult case of Henoch-Schönlein pur-pura complicating common peroneal nervemononeuropathy.

We present an adult patient with Henoch-Schönlein Purpura who developed mononeuro-pathy in the common peroneal nerve. Upon ad-mission, the patient had palpable purpura in thearms and legs, polyarthralgia, abdominal pain,and leukocytoclastic vasculitis in the skin bi-opsy. These symptoms disappeared with 30 mgdaily of oral prednisolone. One month later, af-ter induction therapy, fever, livedo reticularisand peripheral mononeuropathy developed withhypocomplementemia and the patient was trea-ted successfully with glucocorticoid pulse ther-apy.

b. Three cases of MPO-ANCA associatedvasculitis: Clinical features and therapeu-tic options

We report here 3 cases of MPO-ANCA associ-ated vasculitis. [Case 1] A 50-year-old womanwith proteinuria and MPO-ANCA for 2 years,developed alveolar hemorrhage. She was im-proved with PSL 20mg/d, but 1 year laterstarted on hemodialysis. [Case 2] A 64-year-oldwoman developed myalgia and hematuria withMPO-ANCA in 2006, and then alveolar hemor-rhage and renal dysfunction. She was improvedby both PSL and cyclophosphamide (CY). [Case3] A 76-year-old woman had hemoptysis withabnormal CT findings, hematuria and MPO-ANCA, and treated with PSL and IVCY. 7 yearsago she was treated with PSL 15mg/d due toSjS and renal impairment. [Discussion] Even ifcases of MPO-ANCA associated vasculitis werenot diagnosed as MPA, they should be treatedearly with CY and PSL to prevent renal failure.

IV. Medical Genome Science Program

Satoshi Iwata (Division of Clinical Immunol-ogy), Osamu Hosono, Chikao Morimoto.

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We have made contribution to MedicalGenome Science Program, the Institute of Medi-cal Science, the University of Tokyo. Our mainlyinvolving courses are, “Introduction of Medicineand Medical Ethics” and “Experience and Prac-tice of Medicine”, arranged for non-M.D. stu-dents of the program. The former is a series oflectures drawing outline of Medicine (past, pre-sent & future), and the latter is a weekly prac-tice aiming to make attendants to get experi-enced in practical Medicine while rounding atthe Research Hospital of the Institute of Medical

Science. The attendants are supposed to visit avariety of divisions in the hospital, such as Ra-diology, Laboratory Medicine, Blood Transfu-sion, Surgical Center, Nursing Quarters, as wellas the patient round by Department of Hematol-ogy/Oncology, and the joint patient round byDepartment of Advanced Medical Science/Infec-tious Disease and Applied Immunology/Rheu-matology and Allergy. We especially thank allthe people in the Research Hospital of the Insti-tute of Medical Science, who participate in “Ex-perience and Practice of Medicine”.

Publications

Nishida, H., Yamada, T., Iwata, S., Dang, NH.,Inukai, T., Sugita, K., Ikeda, Y. and Morimoto,C. CD9 correlates with cancer stem cell poten-tials in human B-acute lymphoblasticleukemiacells. Biochem. Biophys. Res. Commun. Inpress (2009).

Harima, A., Nakaseko, C., Yokota, A., Kitagawa,M., Morimoto, C., Harigaya, K. and Saito, Y.Fibronectin promotes cell proliferation of hu-man pre-B cell line via its interactions withVLA-4 and VLA-5. Hematology. 13: 236-43,2008.

Iwata, S., Nori, M., Hashizume, Y., You, K. andMorimoto, C. Effect of H-1-receptor antago-nists on proliferative response, cytokine pro-duction, and cellular migration of human Tcells and macrophages. Clinical and Experi-mental Allergy Reviews 8: 21-29, 2008.

Ohnuma, K., Hosono, O., Kawasaki, H.,Yoshikawa, N., Katayose, T., Oyaizu, N.,Tanaka, H. and Morimoto, C. An adult case ofHenoch-Schönlein purpura complicating com-mon peroneal nerve mononeuropathy. ModRheumatol. 19: 73-9, 2009.

Fujimaki, W., Takahashi, N., Ohnuma, K., Na-gatsu, M., Kurosawa, H., Yoshida, S., Dang,NH., Uchiyama, T. and Morimoto, C. Com-parative study of regulatory T cell function ofhuman CD25＋CD4＋ T cells from thymo-cytes, cord blood, and adult peripheral blood.Clin. Dev. Immunol. 2008: 305859, 2008.

Ohnuma, K., Dang, NH. and Morimoto, C. Re-visiting an old acquaintance: CD26 and itsmolecular mechanisms in T cell function.Trends Immunol. 29: 295-301, 2008.

Yamochi, T., Ohnuma, K., Hosno, O., Tanaka,H., Kanai, Y. and Morimoto, C. SSA/Ro52autoantigen interacts with Dcp2 to enhance itsdecapping activity. Biochem. Biophys. Res.Commun. 370: 195-199, 2008.

Yamashita, T., Ohneda, O., Nagano, M., Iemitsu,

M., Makino, Y., Tanaka, H., Miyauchi, T.,Goto, K., Ohneda, K., Fujii-Kuriyama, Y.,Poellinger, L. and Yamamoto, M. Abnormalheart development and lung remodeling inmice lacking the hypoxia-inducible factor-related basic helix-loop-helix PAS proteinNEPAS. Mol Cell Biol. 28: 1285-1297, 2008.

Kagawa, T., Watanabe, N., Mochizuki, K., Nu-mari, A., Ikeno, Y., Itoh, J., Tanaka, H., Arias,IM. and Mine, T. Phenotypic differences inPFIC2 and BRIC2 correlate with protein stabil-ity of mutant Bsep and impaired taurocholatesecretion in MDCK II cells. Am. J Physiol.Gastrointest. Liver Physiol. 294: G58-67, 2008.

Yoshikawa, N., Shimizu, N., Sano, M., Ohnuma,K., Iwata, S., Hosono, O., Fukuda, K., Mori-moto, C. and Tanaka, H. Role of the hinge re-gion of glucocorticoid receptor for HEXIM1-mediated transcriptional repression. Biochem.Biophys. Res. Commun. 371: 44-49, 2008.

Shimizu, N., Yoshikawa, N., Wada, T., Handa,H., Sano, M., Fukuda, K., Suematsu, M., Sa-wai, T., Morimoto, C. and Tanaka, H. Tissue-and context-dependent modulation of hormo-nal sensitivity of glucocorticoid-responsive ge-nes by hexamethylene bisacetamide-inducibleprotein 1. Mol Endocrinol. 22: 2609-2623, 2008.

Shibata, S., Nagase, M., Yoshida, S., Kawarazaki,W., Kurihara, H., Tanaka, H., Miyoshi, J.,Takai, Y. and Fujita, T. Modification of miner-alocorticoid receptor function by Rac 1GTPase: implication in proteinuric kidney dis-ease. Nat Med. 14: 1370-1376, 2008.

Hozawa, S., Nakamura, T., Nakano, M., Adachi,M., Tanaka, H., Takahashi, Y., Tetsuya, M.,Miyata, N., Soma, H. and Hibi, T. Inductionof matrix metalloproteinase-1 gene transcrip-tion by tumour necrosis factor alpha via the p50/p50 homodimer of nuclear factor-kappa Binactivated human hepatic stellate cells. LiverInt. 28: 1418-1425, 2008.

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Our department was established to support the translational researches of ourhospital. Currently, we are studying a correlation between expression profiles ofperipheral blood mononuclear cells in patients with hematologic malignancy andseverity of graft-versus-host disease developed after hematopoietic stem cell trans-plantation. We have been helping prospective studies such as prediction of thesensitivity of Gefenitib to adenocarcinoma of the lung, and that of Imatinib tochronic myeloid leukemia (CML). In addition to the involvement in these projects,we are in charge of outpatient clinic for genetic counseling in Research Hospital,IMSUT.

1. Analysis of gene expression profiles of in-flammatory cells in acute graft-versus-hostdisease following umbilical cord bloodtransplantation

Naoyuki Takahashi, Noriharu Sato, SatoshiTakahashi1, and Arinobu Tojo1: 1Department ofHematology-Oncology

Compared with allogeneic hematopoietic stemcell transplantation using other sources, cordblood (CB) transplantation (CBT) has clinical ad-vantages in terms of incidence and severity ofacute graft-versus-host disease (GVHD) despiteusing allogeneic stem cells with more humanleukocyte antigen mismatches. However, de-tailed pathophysiology of acute GVHD devel-oped after CBT has not yet been elucidated.

We performed microarray expression profilingof immunoregulatory genes on each of 4 sub-populations (CD4＋, CD8＋, CD14＋, and CD56＋)of peripheral blood mononuclear cells (PBMCs),which were taken from 8 patients with hema-tologic malignancies who suffered from acute

GVHD after unrelated CBT. We identified 55genes, which were differentially expressed dur-ing acute GVHD compared to recovery phase.Among them, 22 showed differential expressionconcurrently in multiple PBMC subpopulations.In particular, 5 genes (TRAIL, IL1RN, IFI27,GZMB, and CCR5) were up-regulated and 3genes (CLK1, TNFAIP3 and BTG1) were down-regulated in at least 3 out of 4 subpopulationsduring acute GVHD. These 8 genes seem to becandidates which may play key roles commonto multiple subpopulations in the pathophy-siology of acute GVHD. In addition, down-regulation of anti-inflammatory factors, such asTNFAIP3, KLF2, ZFP36, and BTG1, seems to beinvolved in acceleration of immune response,thus exacerbation of acute GVHD. Further studyis required to clarify whether therapeutic aug-mentation of these factors may ameliorate acuteGVHD. Meanwhile, differential expression ofseveral genes, such as CCL5, TNFAIP3, CD161,CD160, and COX2, was assumedly affected bythe developmental immaturity of CB-derivedcells and this might be somehow involved in the

Research Hospital

Department of Applied Genomicsゲノム診療部

Professor Yoichi Furukawa, M.D., Ph.D.Professor Yusuke Nakamura, M.D., Ph.D.Professor Yoshinori Murakami, M.D., Ph.D.Assistant Professor Naoyuki Takahashi, M.D., D.M.Sc.

教 授 医学博士 古 川 洋 一（併任）教 授 医学博士 中 村 祐 輔（併任）教 授 医学博士 村 上 善 則（併任）助 教 医学博士 高 橋 直 之（併任）

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relatively low incidence and low severity ofacute GVHD following CBT.

2. Genetic diagnosis of HNPCC

Yoichi Furukawa, Yusuke Nakamura1: 1Labo-ratory of Molecular Medicine, Human GenomeCenter, IMSUT

Hereditary non-polyposis colorectal cancer(HNPCC) is an autosomal dominant hereditarydisease accompanied by tumors arising mainlyin the colon and other associated organs, suchas stomach, renal pelvis, and endometrium. Thefrequency of HNPCC in Caucasian patients withcolorectal cancer is estimated between two andfive percent. However the frequency in Japanesepatients with colorectal cancer remains undeter-mined. Therefore, Japanese Study Group forColorectal Cancer started a collaborative projectof registration of Japanese HNPCC patients andgenetic analysis of mutations in MSH2 , MLH1 ,and MSH6 , the responsible genes for HNPCC.All patients with colorectal cancer and thosewho are diagnosed as HNPCC by Amsterdam’sII criteria in the collaborative hospitals havebeen registered, and the frequency of HNPCC inregistered patients with colon cancer has beendetermined. Collaborating to this project, wehave analyzed genetic alteration in a total of 131patients using PCR-direct sequencing and Multi-plex Ligation-dependent Probe Amplification.Among the 131 cases, 69 cases harbored patho-genic mutation in one of the three responsiblegenes. We have clarified that gastric cancer isfrequently observed in HNPCC pedigrees, and

that it should be considered as an HNPCC-related tumor in Japanese. In addition, we gen-erated an algorithm to predict patients with apathogenic mutation. These data will providevaluable information for the understanding ofthe frequency, penetrance and phenotypes ofJapanese HNPCC. The results will be helpful forthe identification and diagnosis of JapaneseHNPCC patients.

3. Genetic counseling and related activities.

Naoyuki Takahashi, Yoshinori Murakami, Yoi-chi Furukawa, Reiko Sada1, Momoyo Ohki2,Kohichiro Tsuji3, Koichiro Yuji4, Rie Tada5,Kisako Sato5, Masae Ono6, Shiro Ikegawa7,Toshihiro Tanaka7, Mayumi Tamari7, TsuyoshiSakamoto8, and Yusuke Nakamura9: 1Divisionof Bioengineering, 2Bunky University, 3Depart-ment of Pediatric Hematology-Oncology, 4De-partment of Hematology-Oncology, 5Depart-ment of Nursing, 6Department of Pediatrics,Tokyo Teishin Hospital, 7SNP Research Centerof RIKEN, 8Department of Neurology, JikeiMedical University, 9Laboratory of MolecularMedicine, Human Genome Center

In the genetic counseling clinic, we providedgenetic counseling for clients who suffered fromor had family members of hereditary disease.Genetic diseases and related problems seen atthe clinic in 2008 include spinocerebellar ataxia,hereditary breast cancer, and bipolar disease.We also took psychological care of the clients incollaboration with clinical psychologists.

Publications

1. Obama K, Satoh S, Hamamoto R, Sakai Y,Nakamura Y, Furukawa Y. Enhanced expres-sion of RAD51AP1 is involved in the growthof intrahepatic choloangiocarcinoma cells.Clin. Cancer Res 14: 1333-1339, 2008.

2. Silva FP, Hamamoto R, Kunizaki M, TsugeM, Nakamura Y, Furukawa Y. Enhancedmethyltransferase activity of SMYD3 by the

cleavage of its N-terminal region in humancancer cells. Oncogene 27, 2686-2692, 2008.

3. Takahashi N, Sato N, Takahashi S, Tojo A.Gene-expression profiles of peripheral bloodmononuclear cell subpopulations in acutegraft-vs-host disease following cord bloodtransplantation. Exp. Hematol. 36: 1760-1770,2008.

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The Department of Radiology works in general diagnostic radiology, neuroradiol-ogy, clinical nuclear medicine, radiation therapy, and molecular imaging. For clini-cal imaging, we have a multi-detector row CT scanner, high-field MRI unit, andgamma camera. We perform all examinations of CT, MRI, angiography, and nu-clear medicine, and official reports on all the examinations are made by board-certified radiologists and a nuclear medicine physician. Molecular imaging tech-niques permit repeated assessment of individual animals to evaluate disease pro-gression, therapeutic effect, and pharmacokinetics. They contribute to preclinicalstudies and have potentials of application to clinical assessments.

Bioluminescent evaluation of the therapeuticeffects of total body irradiation in a murinehematological malignancy model

Yusuke Inoue and Arinobu Tojo1: 1Division ofMolecular Therapy, Advanced Clinical Re-search Center

We investigated the utility of in vivo biolumi-nescence imaging (BLI) in assessing the thera-peutic effects of total body irradiation (TBI) in amurine hematological malignancy model. Thesuspension of Ba/F3 cells transduced with fire-fly luciferase and p190 BCR-ABL genes was ex-posed to ionizing radiation, and viable cell num-bers and bioluminescent signals were measuredserially. Mice intravenously inoculated with thecells underwent TBI at various doses. In vivoBLI was performed repeatedly until spontane-ous death, and whole-body bioluminescence sig-nals were determined as an indicator of whole-body tumor burden. In the cell culture study,bioluminescence signals generally reflected vi-able cell numbers, despite some overestimationimmediately after irradiation. Sublethal TBI in

mice transiently depressed the increase inwhole-body signals and prolonged survival.Spontaneous death occurred at similar signallevels regardless of radiation dose. A significantnegative correlation was found between survivaland whole-body signal early after TBI. Signifi-cant dose dependence was demonstrated forboth survival and signal increase early after TBIand was more evident for signal increase. Le-thally irradiated mice without bone marrowtransplantation died while showing weak sig-nals. In mice receiving lethal TBI and syngeneicbone marrow transplantation, signal reductionand prolongation of survival were prominent,and whole-body signals at death were similar tothose in unirradiated or sublethally irradiatedmice. In vivo BLI allows longitudinal, quantita-tive evaluation of the response to TBI in mice ofa hematological malignancy model. Antitumoreffects can be assessed early and reliably usingin vivo BLI.

Comparison of subcutaneous and intraperito-neal injection of D-luciferin for in vivo biolu-minescence imaging

Research Hospital

Department of Radiology放射線科

Associate Professor Yusuke Inoue, M.D., D.M.Sc.Lecturer Shigeru Kiryu, M.D., D.M.Sc.Assistant Professor Makoto Watanabe, M.D., D.M.Sc

准教授 医学博士 井 上 優 介講 師 医学博士 桐 生 茂助 教 医学博士 渡 辺 慎

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Yusuke Inoue, Kiyoko Izawa1, and ArinobuTojo1

We compared subcutaneous (SC) injection andintraperitoneal (IP) injection of D-luciferin for invivo bioluminescence imaging (BLI) to deter-mine the utility of SC injection. Mice bearing SCtumors stably expressing firefly luciferase un-derwent in vivo BLI using SC and IP injection ofD-luciferin. BLI studies were repeated, with aninterval of 3 h, using a given injection route toassess repeatability and using different injectionroutes to assess correlation. In mice bearing bothSC and IP tumors, BLI was performed succes-sively using intravenous (IV), SC, and IP injec-tion of D-luciferin. Hematological malignancymodel mice underwent BLI using SC and IP in-jection. In SC tumors, the peak time was slightlyshorter and the peak signal was greater usingSC injection compared with IP injection. The re-peatability of determining peak signals wascomparable between the two injection routes,and a good correlation was observed betweenthem. In mice bearing both SC and IP tumors,signals from IP tumors relative to those from SCtumors were much greater using IP injectionthan using IV or SC injection. In the hematologi-cal malignancy model, signals from the spleenrelative to those from the bone marrow weregreater using IP injection than using SC injec-tion. In addition to rare injection failure, the IPinjection of D-luciferin led to the overestimationof signals from IP tissues. For BLI, SC injectionwas shown to be a convenient alternative to IPinjection.

Diet and gastrointestinal signal on T1-weighted magnetic resonance imaging ofmice

Shigeru Kiryu, Yusuke Inoue, and KohkiYoshikawa2: 2Department of RadiotechnicalSciences, Faculty of Radiological Health Sci-ences, Komazawa University.

Magnetic resonance (MR) imaging providesthree-dimensional information about both anat-omy and function without radiation exposure,and is accepted as a powerful modality in smallanimal experiments. In MR imaging of smallanimals, the gastrointestinal contents may giverise to intense signals on T1-weighted imagesand it can cause significant problems in MR im-aging of the abdomen of a small animal, e.g., byincreasing image degradation due to motion ar-tifacts related to peristalsis and respiratory mo-tion. We investigated to determine the optimaldietary preparation to reduce gastrointestinalsignals in mice and to evaluate the usefulness of

this approach. Images of the mouse trunk wereobtained using a T1-weighted, three-dimen-sional fast low-angle shot sequence under vari-ous dietary conditions and were compared withrespect to the gastrointestinal signals and imagequality. The dietary preparation studied in-cluded giving alternative diets for 24 hours, in-testinal cleansing, and 6-hour fasting. Mice withand without dietary preparation underwent MRlymphography using gadofluorine 8, and thevisualization of abdominal lymph nodes wascompared. In the absence of dietary preparation,hyperintense areas were conspicuous in the gas-trointestinal system, whereas on the imagestaken from mice fed potato or sweet potato for24 hours before imaging, gastrointestinal hyper-intensity was less prominent. This preparationalso reduced artifactual signals and resulted inhigher-quality images of the kidneys. Intestinalcleansing, which consisted of 24-hour fastingand laxative intake, did not reduce the gastroin-testinal signals and caused signal changes thatwere indicative of fatty liver development. Someof the abdominal lymph nodes of the mice thatdid not receive dietary preparation were visual-ized on MR lymphography source images butnot on maximum intensity projection (MIP) im-ages. In contrast, on the MIP images of mice fedpotato, all the lymph nodes delineated on thesource images were successfully visualized. Inconclusion, feeding mice potato or sweet potatofor 24 hours before MR imaging reduces thegastrointestinal signals and image degradationdue to artifacts. Appropriate dietary prepara-tions facilitate the display of target structures onMIP images and are expected to enhance the ca-pabilities of small animal MR imaging.

Free-breathing diffusion-weighted imaging forthe assessment of inflammatory activity inCrohn’s disease

Shigeru Kiryu, Masakazu Takazoe3, and Rikis-aburo Sahara4: 3Inflammatory Bowel DiseaseCenter and 4Department of Proctology, SocialInsurance Central General Hospital.

Crohn’s disease is an inflammatory disorderof unknown cause that affects mainly youngpeople and the repeated evaluation to monitordisease activity with minimal burden to the pa-tient is desirable for the assessment of Crohn’sdisease. Diffusion-weighted MR imaging (DWI)reflects molecular diffusion without the admini-stration of contrast material, and has the capa-bility to detect inflammatory foci. We investi-gated the application of free-breathing DWI tothe assessment of disease activity in Crohn’s dis-ease. Thirty-one patients with Crohn’s disease

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were investigated using free-breathing DWIwithout special patient preparation or neither IVnor intraluminal contrast agent. The bowel wasdivided into seven segments, and disease activ-ity was assessed visually on DWI. For quantita-tive analysis, the apparent diffusion coefficient(ADC) was measured in each segment. The find-ings of a conventional barium study or surgerywere regarded as the gold standard for evaluat-ing the diagnostic ability of DWI to assess dis-ease activity. Upon visual assessment, the sensi-tivity, specificity, and accuracy for the detectionof disease-active segments were 86.0, 81.4, and82.4％, respectively. In the quantitative assess-ment, the ADC value in the disease active areawas lower than that in disease inactive area insmall and large bowels. Free-breathing DWI isuseful in the assessment of Crohn’s disease. Theaccuracy of DWI is high in evaluating diseaseactivity, especially in the small bowel, and theADC may facilitate quantitative analysis of dis-ease activity.

Development of integrated techniques usingbioluminescence imaging and magnetic reso-nance imaging

Yusuke Inoue, Shigeru Kiryu, YoshitakaMasutani5, and Arinobu Tojo1: 5Department ofRadiology, Graduate School of Medicine, Uni-versity of Tokyo.

In vivo bioluminescence imaging (BLI) detectsluciferase expression in living mice easily andsensitively. However, because of the projectionalnature, lack of anatomical information, and lowspatial resolution, the localization of biolumines-cent sources is often difficult and unreliable.Magnetic resonance imaging (MRI) is anotherimaging technique applicable to small animalimaging and allows the assessment of detailedmorphology. We have demonstrated the feasibil-ity and usefulness of combined BLI and MRIevaluations in evaluating a mouse model of ahematological malignancy. For further sophisti-cation of the multi-modality imaging approach,we are now studying to develop BLI/MRI fu-sion imaging. MR images suffer from geometricdistortion due to gradient nonlinearity and in-homogeneity in static magnetic field. We devel-oped a method to correct such distortion andvalidated its application to the distortion correc-tion of mouse MR images. Distortion of CCDcamera images was also evaluated, and a tech-nique for minimizing the distortion was estab-lished. With the aid of point markers that is de-tectable by fluorescence imaging and MRI, accu-rate registration was achieved.

Evaluation of disseminated intraperitonealdisease using in vivo bioluminescence imag-ing

Makoto Watanabe, Yusuke Inoue, and Ari-nobu Tojo1

Quantitative assessment of tumor burden isdifficult in animal models of disseminated in-traperitoneal disease. In vivo bioluminescenceimaging (BLI) allows to assess the tumor burdenquantitatively and noninvasively, and has beenused for the studies regarding disseminated in-traperitoneal disease. We are investigating thetechniques for the quantitative evaluation of tu-mor burden in disseminated intraperitoneal dis-ease. For in vivo BLI, mice commonly receive in-traperitoneal injection of D-luciferin, are fixed inthe imaging chamber, and are imaged at a fixedtime point. We determine the relationship ofquantitative indicators with the injection route,mouse posture, and imaging timing. Our aim isto improve the reliability of the experimental re-sults, which should enable to decrease the num-ber of mice to be examined.

Lymph node mapping in the mouse using im-aging techniques.

Yusuke Inoue and Shigeru Kiryu

Lymph nodes are important as sites of metas-tatic involvement, and animal models of lymphnode metastasis are being developed. However,lymph nodes of mice are small and difficult todetect. Even normal lymphatic pathway in themouse has not been well established. We are de-veloping the image-based methods for the as-sessment of the lymphatic system in living miceand applying them to the investigation of themurine lymphatic system. MRI visualized in-flammatory lymph nodes after subcutaneous in-jection of gadofluorine 8. Fluorescence imagingdemonstrated normal lymph nodes after subcu-taneous injection of quantum dots. Althoughfluorescence imaging is generally unsuitable tothe delineation of deep structures because ofstrong scattering and attenuation of light pho-tons, we achieved clear demonstration of deeplymph nodes using a novel, simple technique.Strong signals are originated from gastrointesti-nal contents in both MRI and fluorescence imag-ing, and our techniques for reduction in gastro-intestinal signals aid the detection of abdominallymph nodes.

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Publications

Inoue, Y., Izawa, K., Kiryu, S., Tojo, A., and Oh-tomo, K. Diet and abdominal autofluorescencedetected by in vivo fluorescence imaging ofliving mice. Mol. Imaging. 7: 21-7, 2008.

Inoue, Y., Izawa, K., Kiryu, S., Kobayashi, S.,Tojo, A., and Ohtomo, K. Bioluminescentevaluation of the therapeutic effects of totalbody irradiation in a murine hematologicalmalignancy model. Exp. Hematol. 36: 1634-41,2008.

Yokoyama, I., Inoue, Y., Kinoshita, T., Itoh, H.,Kanno, I., and Iida, H. Heart and brain circu-lation and CO2 in healthy men. Acta. Physiol.193:303-8, 2008.

Kiryu, S., Sundaram, T., Kubo, S., Ohtomo, K.,Asakura, T., Gee, JC., Hatabu, H., and Taka-hashi, M. MRI assessment of lung parenchy-mal motion in normal mice and transgenicmice with sickle cell disease.J. Magn. Reson.

Imaging. 27: 49-56, 2008.Sakai, M., Aoki, S., Inoue, Y., Ashida, R.,

Yamada, Y., Kiryu, S., Inano, S., Mori, H.,Masutani, Y., Ohtomo, K., and Nakamura, H.Silent white matter lesion in linearscleroderma en coup de sabre. J. Comput. As-sist. Tomogr. 32:822-4, 2008.

Inoue, Y., Kiryu, S., Izawa, K., Watanabe, M.,Tojo, A., and Ohtomo, K. Comparison of sub-cutaneous and intraperitoneal injection of D-luciferin for in vivo bioluminescence imaging.Eur. J. Nucl. Med. Mol. Imaging. In press井上優介，東條有伸．血液学研究における疾患動物モデルの作製と応用：生体イメージング技術を用いた疾患モデルマウスの病態解析．臨床血液 ４９�：４３７―４４４，２００８．桐生茂．９後腹膜・大血管．画像診断臨時増刊号．大友邦，吉岡直己編著．秀潤社 ２８�：２０６―２２５，２００８．

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We have been engaged in the surgical treatment of solid tumors and the immuno-therapy of various malignancies. We have also been offering diagnostic services,including upper and lower endoscopic examination. The principal goal of our de-partment is to provide surgical service of malignancy and inflammatory bowel dis-ease and to develop and conduct clinical trials in the early stages (Phase I and II)at the Research Hospital.

1. Summary of surgical treatment in 2008

Masaru Shinozaki, Akihito Itoh, GiichiroTsurita, Akira Kanamoto, Kimiyasu Yone-yama, Keisuke Hata

We performed surgical operations on 128 pa-tients. Each procedure was classified and listedin Table 1. Malignancy is the leading indicationfor operation, followed by benign diseases, suchas inflammatory bowel disease (IBD) and hernia.In 2008, three colorectal specialists (M.S., G.T.,and K.H.) came to the Department, and we ex-perienced more IBD patients. Two cases withIBD had neoplasm: high grade dysplasia in ul-cerative colitis and invasive cancer in Crohn’sdisease. Laparoscopic surgery for colorectal dis-ease was introduced: ileocecal resection and to-tal proctocolectomy were performed. We up-dated operative procedures to avoid surgical siteinfection. Furthermore, routine endoscopic ob-servation after colorectal anastomosis was initi-ated. Intractable diseases, including malignancy

and IBD, are our main target in surgical treat-ment and we are seeking for less invasive proce-dures and new strategies for patients’ cure/quality of life.

2. Summary of endoscopic examination in2008

Giichiro Tsurita, Keisuke Hata, Masaru Shino-zaki, Akira Kanamoto, Masahisa Jinushi

Under the cooperation with Department ofAdvanced Medical Science, we performed 485upper gastrointestinal endoscopies and 340colonoscopies without major complications. InApril, 2008, Dr. Tsurita became the chief of Divi-sion of Endoscopy. Since then, the number ofendoscopy cases has increased gradually. Forthe patients’ satisfaction, we aggressively per-form endoscopic treatment and avoid operationas much as possible.

Research Hospital

Department of Surgery外科

Associate Professor Masaru Shinozaki, M.D., D.M.Sc.Professor Hideaki Tahara, M.D., D.M.Sc.Lecturer Akihiko Itoh, M.D.Assistant Professor Giichiro Tsurita, M.D., D.M.Sc.Assistant Professor Akira Kanamoto, M.D., D.M.Sc.Assistant Professor Keisuke Hata, M.D., D.M.Sc.Assistant Professor Masahisa Jinushi, M.D., D.M.Sc.

准教授 医学博士 篠 崎 大教 授 医学博士 田 原 秀 晃講 師 伊 藤 精 彦助 教 医学博士 釣 田 義一郎助 教 医学博士 金 本 彰助 教 医学博士 畑 啓 介助 教 医学博士 地 主 将 久

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3. Clinical trials completed in/before 2008

Akira Kanamoto, Kenji Nakano, KimiyasuYoneyama, Akihiko Itoh, Hideaki Tahara

� Phase I clinical trial of tumor specific vac-

cine using epitope peptides derived from anovel tumor associated antigen RNF43 againstadvanced colorectal cancer.� Phase I clinical trial of epitope peptides

based vaccine targeting tumor vascular endo-thelial cells against advanced cancer patients

Table 1. Surgical procedures performed in 2008

Disease Procedure

Esophageal cancer Subtotal esophagectomy 1Gastric cancer Total gastrectomy 3

Distal gastrectomy 4Colorectal cancer/tumor Laparoscopic ileocecal resection 1

Ileoceal resection 2Right hemicolectomy 4Left hemicolectomy 2Anterior resection 4Abdomino-perineal resection 2Partial resection 3Hartmann’s operation 1Transanal resection 1

Inflammatory bowel disease Laparoscopic total proctocolectomy 1Abdomino-perineal resection 2Ileectomy 1Total colectomy 1Subtotal colectomy 1Ileal pouch anal canal anastomosis 1Ileostomy closure 1

The other intestinal diseases Hemorrhoidectomy 3Transverse colostomy 2Ileectomy 1Subtotal colectomy 2Partial colectomy＋jejunectomy 1

Hepatoma Partial hepatectomy 2Metastatic liver tumor Partial hepatectomy 1Gallbladder stone Laparoscopic cholecystectomy 3

Cholecystectomy 3Pancreas cancer Distal pancreatectomy 1Breast cancer Mastectomy 1

Partial mastectomy 7Breast tumor Tumor excision 4

Fistulectomy 2Hernia 12Miscellaneous 48

Total 129

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such as gastric cancer, colorectal cancer, breastcancer and GIST.� Phase I clinical trial: Adjuvant use of epi-

tope peptides based vaccine targeting tumorvascular endothelial cells in patients with sur-gically resected pancreatic cancer.� Phase I clinical trial of epitope peptides

based vaccines both targeting RNF43 andVEGFR2 in patients with advanced colorectalcancer.

4. Phase I/II colorectal cancer and breast can-cer trial

Masaru Shinozaki, Giichiro Tsurita, KimiyasuYoneyama, Keisuke Hata

Genome-wide exploration using cDNA Mi-croarray Profiling enabled a new tumor-associated antigen that can induce potent cyto-toxic T-cells (CTLs) specific to tumor cells.Among them, we selected RNF43 (ring fingerprotein 43), TOMM34 (translocase of outer mito-chondrial membrane 34), and TTK proteinkinase. We also picked up angiogenesis as acritical mechanism of tumor development.VEGFR1 (vascular endothelial growth factor re-ceptor 1) and VEGFR2 were selected as candi-dates for vaccine therapy. Specific CTLs for eachpeptide were induced in cancer patients. We setthe combination of peptide for colorectal cancerpatients and breast cancer patients with HLA-A*0201 and HLA-A*2402.

Three colorectal cancer patients (3 HLA-A*0201 and 1 HLA-A*2402) and four breast cancerpatients (3 HLA-A*2402 and 1 HLA-A*0201)were completed without major complications.

5. Phase I/IIa clinical trial of melanoma vac-cine using gp100 derived peptides re-stricted to HLA-A*2402.

Akira Kanamoto, Kenji Nakano, KimiyasuYoneyama, Akihiko Itoh, Hideaki Tahara

From the results of phase I clinical trial ofmelanoma vaccine using gp100 derived pep-tides, phase I/IIa clinical trial of melanoma vac-cine using gp100 derived peptides were per-formed. HLA-A*2402-restricted gp100 derivedpeptide (gp100-int4: VYFFLPDHL) was usedwith IFA and interleukin (IL-2) in order to aug-

ment for anti-tumor immunity. Our goals in thisclinical trial are to examine these clinical effi-cacy, furthermore, safety and immune responsesassociated with the peptide vaccination. Wehave enrolled 25 melanoma patients until 2008.So far, the protocols were well tolerated, and nocardiac, hematological, hepatic, or renal toxicitywas noted.

6. Phase I clinical trial of melanoma vaccineusing gp100 derived peptides restricted toHLA-A*2402 with fully matured dendriticcells to induce Th1 type immune re-sponses.

Akira Kanamoto, Kenji Nakano, KimiyasuYoneyama, Akihiko Itoh, Hideaki Tahara

Dendritic cells (DC) administration appears tobe very promising approach for immunotherapyagainst cancer. To further magnify the immuneresponses and obtain the clinical benefits, wehave focused on the gp100-int4peptide loadedDC vaccination. However, what we found fromthe results of phase I clinical trial using DC inour institute was the dysfunction of immatureDC derived from cancer patients. Thus, we de-veloped a new culture method to obtain thefully matured DC that is capable of T helpertype 1 (Th1) polarization. From these back-grounds, we are going to utilize this fully ma-tured DC to the phase I clinical trial of peptidevaccinations.

The goals in this clinical trial using our propa-gated DC are to examine the safety and immuneresponses, furthermore, the clinical efficacy asso-ciated with the peptide loaded fully maturedDC vaccinations. We have enrolled 7 melanomapatients until 2008. So far, the protocols werewell tolerated, and no cardiac, hematological,hepatic, or renal toxicity was noted.

7. Clinical trials under development: Develop-ment of gene therapy using dendritic cells

Akira Kanamoto, Kimiyasu Yoneyama, Aki-hiko Itoh, Hideaki Tahara

In close collaboration with Core Facility forTherapeutic Vectors, we are developing clinicalapplication of IL-12 gene transducted dendriticcells for cancer patients.

Publications

Maki T, Carville A, Stillman IE, Sato K, KodakaT, Minamimura K, Ogawa N, Kanamoto A,Gottschalk R, Monaco AP, Marr-Belvin A,

Westmoreland SV, Sehgal P. SV40 infectionassociated with rituximab treatment after kid-ney transplantation in nonhuman primates.

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Transplantation 85: 893-902, 2008.Suzuki T, Ogawa S, Tanabe K, Tahara H, Abe R,

Kishimoto H. Induction of antitumor immuneresponse by homeostatic proliferation and CD28 signaling. J Immunol 80: 4596-605, 2008.

Sunami E, Shinozaki M, Sim MS, Nguyen SL,Vu AT, Giuliano AE, Hoon DS. Estrogen re-ceptor and HER2/neu status affect epigeneticdifferences of tumor-related genes in primarybreast tumors. Breast Cancer Res 10: R46,2008.

Takahashi H, Ishizaki H, Tahara H, Tamaki Y,Yanagi Y. Suppression of choroidal neovascu-larization by vaccination with epitope peptidederived from human VEGF receptor 2 in an

animal model. Invest Ophthalmol Vis Sci 49:2143-7, 2008.

Saito T, Takayama T, Osaki T, Nagai S, SuzukiT, Sato M, Kuwano H, Tahara H. Combinedmobilization and stimulation of tumor-infiltrating dendritic cells and natural killercells with Flt3 ligand and IL-18 in vivo in-duces systemic antitumor immunity. CancerSci 99: 2028-36, 2008.

Suzuki H, Tsurita G, Ishihara S, Akahane M, Ki-tayama J, Nagawa H. Resovist-Enhanced MRIfor Preoperative Assessment of Colorectal He-patic Metastases. Case Rep Gastroenterol 2:509-16, 2008.

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Department of Joint Surgery was established in 2006. Our mission is evaluationand treatment of hemophilic arthropathy. In Japan, some hospitals are able tocontrol bleeding for hemophilia using concentrates, however there are no hospitalsfocus on surgical treatments except us. Many hemophilia patients come to our de-partment from all over Japan. We evaluate their joint function and condition roent-genographically and physiotherapically and explain joint status and indication ofsurgical treatments. After that, we make surgical plans for some of them and per-formed surgical treatments mainly joint arthroplasty and arthroscopic synovectomyto improve their quality of life.

Surgical treatment for haemophilia

Hideyuki Takedani

In 2008, there are 20 surgical treatments forhemophilia (15 for hemophilia A, five for hemo-philia B) included three inhibitor cases (one he-

mophilia A and two hemophilia B). We per-formed 15 joint arthroplasties (eight primary to-tal knee arthroplasties, one revision total kneearthroplasties, seven total hip arthroplasties, onebipolar hip arthroplasties and one total elbowarthroplasties) and three arthroscopic synovecto-mies and two others.

Publications

１）松下正，天野景裕，瀧正志，岡敏明，酒井道夫，白幡聡，高田昇，高松純樹，竹谷英之，花房秀次，日笠聡，福武勝幸，藤井輝久，田中一郎，三間屋純一，吉岡章，嶋緑倫，インヒビターのない血友病患者の急性出血，処置・手術における凝固因子補充療法のガイドライン，日本血栓止血学会誌，５１０―５１９，２００８

２）田中一郎，天野景裕，瀧正志，岡敏明，酒井道夫，白幡聡，高田昇，高松純樹，竹谷英之，花房秀次，日笠聡，福武勝幸，藤井輝久，松下正，三間屋純一，吉岡章，嶋緑倫，インヒビター保有先天性血友病患者に対する止血治療ガイドライン，日本血栓止血学会誌，５２０―５３９，２００８

Research Hospital

Department of Joint Surgery関節外科

Lecturer Hideyuki Takedani, M.D. D.M.Sc. 講 師 医学博士 竹 谷 英 之

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Our clinical practice and clinical studies have been focused on (1) anestheticmanagement in patients undergoing major surgery, (2) management of intraopera-tive and postoperative pain, and (3) management of chronic intractable pain, and(4) assessment of the impact of anesthesia and surgery on autonomic nervousactivity. We have published several works on these subjects.

1. Safety in anesthetic management, espe-cially focusing on cerebral circulation dur-ing anesthesia and surgery.

The Bispectral Index (BIS) is a recently devel-oped derivative of processed electroencephalo-gram that has been proven to closely correlatewith the level of consciousness during generalanesthesia. It has been widely used in the areaof anesthesia to evaluate sedative/hypnotic statein patients undergoing surgery under generalanesthesia.

We have also found that BIS is useful to de-tect cerebral ischemia during pediatric and adultcardiac surgery sepecially when used in combi-nation with the near-infrared spectroscopy(NIRS) to measure oxygen saturation of thebrain. Simultaneous monitoring with BIS andNIRS revealed that in children, especially in in-fants, cerebral ischemia occurred frequently dur-ing cardiac surgery presumably due to immatur-ity of the cerebral vascular autoregulation. Wealso reported successful anesthetic managementof patients with compromised circulation.

2. Management of intraoperative and postop-erative pain.

We have published several works on manage-ment of intraoperative and postoperative pain.We have developed a rabbit model of surgicalanesthesia/analgesia, which allows for repeatedand quantitative evaluation of depth of surgicalanesthesia/analgesia provided by a variety ofanesthetics/analgsics. We also published severalreview articles on how to manage postoperativepain, and original articles comparing variousmodalities of postoperative pain management.

3. Management of chronic intractable pain

We published several works on new treat-ment modalities for chronic intractable pain syn-drome with various drugs including ketamineand ATP, after application of drug tests to dif-ferentiate the mechanism underlying the pain.We also reviewed usefulness of epiduloscopy inpain management in patients with chronic in-tractable low back pain.

4. Assessment of the impact of anesthesiaand surgery on autonomic nervous activ-ity.

It is generally accepted that the parameters

Research Hospital

Surgical Center手術部

Associate Professor Mieko Chinzei, M.D., M.D.Sc.Assistant Professor Sachiko Imamura, M.D.Clinical Engineer Osamu Ichimura

准教授 医学博士 鎮 西 美栄子助 教 医学博士 今 村 佐知子

臨床工学技士 市 村 理

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derived from power spectral analysis (PSA) ofheart rate variability (HRV) can provide a noninvasive measure of autonomic nervous activity.We published several works on assessment of

the impact of anesthetics on autonomic nervousactivity during perioperative period using realtime monitor for PSA of HRV.

Publications

1) Oshima, N,.Chinzei, M. Application of heartrate variability analysis for evaluation of theeffects of psychiatric occupationa therapy. JJpn Soc Balneol Climatol Phys Med, 71 (2),97-100, 2008,

2) 長友香苗，富岡俊也，金信秀，小川真，鎮西美栄子，山田芳嗣．トリーチャー・コリンズ症候群とピエール・ロバン症候群における気管挿管難易度の加齢による推移について．麻酔５８巻；１６５―１６９，２００９

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Division of Clinical Trial Safety Management (DCTSM) owes two major missions.One is the risk management of the Research Hospital (RH) and the other is thesupport for the conduct of clinical trials, especially for Translational Research (TR).Our roles on TR varies from the assistance for planning study design and writingprotocol to the data confirmation by Case Report Form which is managed byTranslational Research Coordinator (TRC) and the quality assurance of TRs bymonitoring/audit. To protect the right of participants into TR and to conduct TRscientifically and ethically appropriately, we have organized TRC, which consistsnurse, pharmacist, clinical laboratory technologist, dietitian, and clinical psycho-therapist.

1. Risk management of Research Hospital

Fumitaka Nagamura, Seiichiro Kobayashi,Hatsue Narita

We engage in the risk management of RH.We have promoted the report system on medi-cal incidents and accidents, and quick corre-sponding scheme such as “Medical Accident-Response Meeting” and “Council of Risk Man-agement in the RH”. We take place at least twoseminars for staffs of RH on medical safetyevery year. Almost all of the workers of RH par-ticipated into these seminars. We have revisedmanuals on the risk management and StandardOperating Procedures (SOP) on operations ofRH every year. As the result of these actions, nosevere medical accident has been observed in2008.

2. Assistance and oversight of Clinical trials/TRs

Kazufumi Matsumoto , Kumiko Sumino ,Noriko Fujiwara, Ikue Nakajima, Minako

Kohno, Makiko Tajima, Fumitaka Nagamura

The assistance by TRC is indispensable for theconduct of clinical trials, especially for TR. In2008, we participated in 31 protocols, and thir-teen of them were newly started in this period.Details of protocols are summarized in Table 1.Table 2 shows the number of patients enrolledinto clinical trials at RH in 2008. In addition tothese patients, around two hundred patients vis-ited RH for the purpose of the screening. Fur-thermore, around 500 patients called to ask onparticipation into clinical trials. TRCs managedthe visit for screening and telephone consulta-tion.

3. The development of the scholastic pro-gram for the graduate students of nursesin the area of Translational Research.

Kazufumi Matsumoto, Fumitaka Nagamura.

High-educated nurses are required to performTRs in terms of the protection of study patientsand the conducts of scientifically appropriate

Research Hospital

Department of Clinical Trial Safety Management附属病院・医療安全管理部

Associate Professor Fumitaka Nagamura, M.D., D.M.Sc.Project Assistant Professor Seiichiro Kobayashi, M.D., D.M.Sc.

准教授 医学博士 長 村 文 孝特任助教（兼） 医学博士 小 林 誠一郎

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studies. We developed the scholastic programfor the graduate students of nurses in the areaof TR. We planed and implemented the two-weeks program aimed to acquire the expertiseof research nurse for the graduate students ofnurses. It consists of lectures on the featurepoints of TR (e.g. ethical considerations of TRs,and the role of research nurse), role-plays of In-stitutional Review Board and obtaining In-formed Consent, case conference, and the expe-rience of the actual operations. We evaluatedthe reports and questionnaires from students toexplore the degree of their understandings andsatisfactions for this program. These reports andquestionnaires were analyzed in accordancewith the qualitative method.Six students partici-pated in the program this year, and we receivedthe reports and questionnaires. Students couldunderstand the role of research nurse includingthe supports of investigators, cares and the pro-tection of participants, the coordination betweenparticipants and medical staffs, and the neces-sary ability to conduct appropriately. They weresatisfied with the content and the quality of lec-tures and role-plays, however, the experiencesof the actual operations did not meet their de-mands due to the less acquisition of the practi-cal expertise. Generally, our program meets thedemands of students, however, the improve-ment of the content on the experience of the ac-tual operations is the next issue.

4. Preliminary research for patients partici-pating in translational research on the in-fluence of conflict of interest factors ontheir decision-making.

Momoyo Ohki1, Fumitaka Nagamura. 1Depart-ment of Psychology, Faculty of Human Science,Bunkyo University.

We analyzed results in the preliminary re-search stage for participating in TranslationalResearch (TR) on the influence of Conflict of In-terest (COI) factors on their decision-making.Analysis revealed the following tendencies: (1)patients participating in TR paid more heed tointrinsic COIs, which are ubiquitous in researchand include an investigator’s perceived need toengage in and publish research to achieve careeradvancement, to receive accolades from peersand professional societies, and to be competitivefor grand funding, rather than financial COIs;(2) Information on COI has little effect ondecision-making as part of TR participation; (3)Desired methods of specifically describing COIsdiffer among participants; (4) Opinions on therelationship to the company also differed amongparticipants; (5) efforts of the Institute of Medi-cal Science, the University of Tokyo, along withthe activities of Translational Research Coordi-nators, in dealing with COI problems, have beenrecognized to an extent from the perspective ofcompliance with ethics.

Table 1.

Number of protocol Started in 2008 Continuation before 2008 Total

TR 12 11 23

Clinical trials frompharmaceutical companies

0 4 4

Multi-center studies 1 3 4

13 18 31

Table 2.

Number of patients Enrolled in 2008 Continuation before 2008 Total

TR 52 7 59

Clinical trials frompharmaceutical companies

4 2 6

Multi-center studies 3 6 9

59 15 74

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Publications

Nagamura-Inoue T, Kai S, Azuma H, TakanashiM, Isoyama K, Kato K, Takahashi S,Taniguchi S, Miyamura K, Aoki K, Hidaka M,Nagamura F, Tojo A, Fang X, Kato S; JapanCord Blood Bank Network. Unrelated cordblood transplantation in CML: Japan CordBlood Bank Network analysis. Bone MarrowTransplant. 42: 241-51, 2008

Ooi J, Takahashi S, Tomonari A, Tsukada N,Konuma T, Kato S, Kasahara S, Sato A,Monma F, Nagamura F, Iseki T, Tojo A,Asano S. Unrelated cord blood transplantationafter myeloablative conditioning in adultswith ALL. Bone Marrow Transplant. (in press)

Ooi J, Takahashi S, Tomonari A, Tsukada N,Konuma T, Kato S, Kasahara S, Sato A,Monma F, Nagamura F, Iseki T, Tojo A,Asano S. Unrelated cord blood transplantationafter myeloablative conditioning in adults

with acute myelogenous leukemia. Biol BloodMarrow Transplant. 14: 1341-7, 2008.

Futami M, Hatano T, Soda Y, Kobayashi S, Miy-agishi M, Tojo A. RNAi-mediated silencing ofp190Bcr-Abl inactivates Stat5 and cooperateswith imatinib mesylate and 17-allylamino-17-demetoxygeldanamycin in selective killing ofp190Bcr-Abl-expressing leukemia cells. Leuke-mia. 22: 1131-8, 2008.

Inoue Y, Izawa K, Kiryu S, Kobayashi S, Tojo A,Ohtomo K. Bioluminescent evaluation of thetherapeutic effects of total body irradiation ina murine hematological malignancy model.Exp Hematol. 36: 1634-41, 2008.大木桃代，長村文孝．探索型臨床研究において利益相反問題が参加者の意思決定と人権に及ぼす影響の予備的検討 人間科学研究 ３０：１１５‐１２０，２００８．

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Our department is established in 1990, in order to manage the transfusion medi-cine and the cell processing for hematopoietic stem cell transplantation. We aretransferring the processing facility of Tokyo Cord Blood Bank (Tokyo CBB) forclinical use to Yotsugi Facility and instead we have newly established the re-search cord blood stem cell bank corporate with Tokyo CBB.Also we have been engaged to study for the development of various cell thera-pies together with other departments, as follows.

1. Expansion and regulatory T cells for im-munosuppressive therapy and immu-nological analysis of the patients with he-matological disease:

Tokiko Nagamura-Inoue , Kazuo Ogami ,Kazuaki Yokoyama1, Seiko Izawa, SeiichiroKobayashi and Arinobu Tojo: 1Department ofHematology/Oncology, The Institute of MedicalScience, The University of Tokyo

Regulatory T cells harbored the immunosup-pressive effects and were related to the onset ofgraft-versus-host disease (GVHD), rejection oforgan transplantation and autoimmune disease.We developed the system of ex vivo expansionof CD25＋FOXP3＋regulatory T cells from thesmall amount of peripheral blood, to apply thecell therapy for severe GVHD, autoimmune dis-eases (Collaboration with Division of MolecularTherapy). In addition, we have been monitoringthe immunological change of the patients withhematological disease, especially CML.

2. Research Cord Blood Stem Cell Bank:

T. Nagamura-Inoue, I. Ishige, Miki Yuzawa, K.Ogami and A. Tojo

“Research Cord Blood Stem Cell Bank” (for-mer named ‘Research Stem Cell Resource Bank’)was established with the support of MEXT(Ministry of Education, Culture, Sports, Scienceand Technology) for the achievement of the de-velopment of the medicine including Regenera-tive Medicine and drug discovery in Japan since2004. The research banks perform cord bloodprocessing for non-conforming units for clinicaluse incorporated public clinical cord blood bankand cryopreserve and provide to domestic re-searchers for research use via RIKEN Biore-source Center. Visit our home page http://scb.ims.u-tokyo.ac.jp/

3. Exploring mesenchymal stem cells derivedfrom Umbilical Cord:

Ikuo Ishige1, Tokiko Nagamura-Inoue, ArinobuTojo: 1Department of Stem cell processing,Lab. of Stem cell Therapy, The Institute ofMedical Science, The University of Tokyo

In addition to contribute the research use ofcord blood banking as the regenerative leadingproject, we have been explored the new source,mesenchymal stem cells derived from umbilicalcord (Warton Jelly) (Collaboration with Division

Research Hospital

Department of Cell Processing and Transfusionセルプロセッシング・輸血部

Professor Arinobu Tojo, M.D., D.M.Sc.Lecturer Tokiko Nagamura-Inoue, M.D., D.M.Sc.

教 授 医学博士 東 條 有 伸講 師 医学博士 長村（井上）登紀子

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of Molecular Therapy and Department of Stemcell processing).

4. Room for Clinical Cellular Technology(RCCT):

Tokiko Nagamura-Inoue , Kazuo Ogami ,Masako Hirai, Arinobu Tojo and RCCT Exe-cution Committee

To promote the cell therapy related to transla-tional research, RCCT has been established in1997. Until now, the following projects were im-plemented; 1) Cord blood cell processing forbanking (for Tokyo Cord Blood Bank and Re-search cord blood stem cell bank), 2) Dendriticcell therapy, 3) Regenerative therapy of alveolarbone derived from bone marrow mesenchymalcells, 4) Gene therapy for renal cancer.

Publications

Ikeda K, Nagamura-Inoue T, Kai S, Fujii Y,Ozaki S, Sagawa K, Takamatsu J, TakahashiK, Ohto H. Questionnaire surveys on cellprocessing in Japan by the Japanese Society ofTransfusion Medicine and Cell Therapy/Japa-nese Association of Medical Technologists(2007) and by Working Group for AdverseEvents of the Transfusion Conference of theUniversity Hospitals (2006)., Jap. J. Transfu-sion and cell Therapy, in press

Atsuta Y, Suzuki R, Nagamura-Inoue T,Taniguchi S, Takahashi S, Kai S, Sakamaki H,Kouzai Y, Kasai M, Fukuda T, Azuma H,Takanashi M, Okamoto S, Tsuchida M, KawaK, Morishima Y, Kodera Y, and Kato S, forthe Japan Marrow Donor Program and the Ja-pan Cord Blood Bank Network; Disease-specific analyses of unrelated cord bloodtransplant compared with unrelated bonemarrow transplant in adult patients withacute leukemia. Blood, 113, 1631-8. 2009.

Yoshimi A, Kojima S, Taniguchi S, Hara J, Mat-sui T, Takahashi Y, Azuma H, Kato K,Nagamura-Inoue T, Kai S, Kato S; Japan CordBlood Bank Network., Unrelated cord bloodtransplantation for severe aplastic anemia.Biol Blood Marrow Transplant. 9, 1057-63.2008

Nagamura-Inoue T, Kai S, Azuma H, TakanashiM, Isoyama K, Kato K, Takahashi S,Taniguchi S, Miyamura K, Aoki K, Hidaka M,Nagamura F, Tojo A, Fang X, Kato S; JapanCord Blood Bank Network. Unrelated cordblood transplantation in CML: Japan CordBlood Bank Network analysis. Bone MarrowTransplant. 42, 241-51, 2008

Konuma T, Ooi J, Takahashi S, Tomonari A,Tsukada N, Kobayashi T, Sato A, Kato S, Ka-sahara S, Ebihara Y, Nagamura-Inoue T, TsujiK, Tojo A, Asano S. Cardiovascular toxicity ofcryopreserved cord blood cell infusion. BoneMarrow Transplant. 41, 861-5. 2008

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The primary function of the Core Facility for Therapeutic Vectors (CFTV) is to sup-port clinical trials that require the genetic modification and/or ex vivo manipulationof patients’ tissue or cells under current Good Manufacturing Practice (cGMP)conditions defined by FDA of USA. The CFTV is the first facility established inJapanese academia to produce genetic or cellular vectors of clinical grade. Usingthis facility, clinical lots of the adenoviral vector and herpes vector are scheduledto be produced in CFTV in 2008-2009.

1. Preparation of Standard Operating Proce-dures (SOPs)

The cGMP compliance is maintained usingwritten SOPs prepared by ourselves. The SOPscodify all aspects of laboratory activities includ-ing facility design and operations of the person-nel. The SOPs enables the staff not only to pro-duce the reagents with high quality in the stablemanners but also to help identify areas for im-provement.

2. Adoption of ISO

In order to continuously improve our activi-ties, quality management system of the CFTVhas been assessed and found to be in accor-dance with the requirements of the quality stan-dards detailed ISO9001:2000; in the scope of de-velopment and manufacture of cell and genetherapy products.

3. Validation of CFTV

The CFTV consists of two distinct units; 1)Vector Unit, the primary viral vector productionsuite which may also function as ex vivo trans-

duction suite; 2) Cell Unit, cell processing suitecapable of generating dendritic cells for immu-notherapy and gene therapy. There are two self-contained vector production suites in the VectorUnit and two self-contained tissue culture suitesin the Cell Unit. These suites are kept Class10,000. There are many features incorporatedinto the design of this CFTV to minimize therisk of cross-contamination between products; i.e., unidirectional traffic flow, individual airlocksto each production suite, single-pass HEPA fil-tered supply air, 100 percent exhaust from thebiological safety cabinets through dedicateducts, among others. Periodical validation hasbeen performed on the facility and the equip-ments in CFTV to ensure cGMP compliance.

4. Projects in CFTV

Four projects are now in progress in theCFTV.

I. Cancer gene therapy using dendritic cellstransfected with IL-12 genes

Collaboration with Division of Bioengineeringof Advanced Clinical Research Center (ACRC)

Research Hospital

Core Facility for Therapeutic Vectors治療ベクター開発室

Professor Hideaki Tahara, M.D., D.M.Sc.Guest Researcher Takafumi Nakamura, Ph.D.Project Assistant Professor Hisako Katano, D.D.S., Ph.D.

教授（室長） 医学博士 田 原 秀 晃（併）客員研究員 生命科学博士 中 村 貴 史特任助教 歯学博士 片 野 尚 子

276

and Department of Surgery of the research hos-pital.・Preparation of the Clinical Lot

We have been preparing the replication-defective recombinant adenoviral vector encod-ing human interleukin-12, which is an immune-stimulatory cytokine. The backbone of this vec-tor is based on the E1- and E3-deleted serotype5 adenovirus with a modified fiber, harboringan integrin-binding CDCRGDCDC-motif withinthe HI-loop of its knob protein. The IL-12 genesare driven by a CA promoter (CMV-IE enhancerwith the chicken β-actin promoter). The mastervirus seed stock (MVSS) and purified final mate-rial have been prepared following the optimiza-tion of purified method for the production ofhigh-titer vector. The purified material is now inthe process of quality examination for use ofearly phase trials.

II. Vaccine therapy with peptide-loaded den-dritic cells for advanced melanoma

Collaboration with Division of Bioengineeringof ACRC and Department of Surgery of the re-search hospital.・Preparation of Peptide-Loaded Dendritic Cells

(DCs)We have been supporting phase I clinical tri-

als against melanoma. Based on the results ofthe basic research performed in Division of Bio-engineering, the SOPs of the DC preparationhave been written and used. The cellular re-agents have been successfully prepared in theCell Unit and offered for clinical trials withoutserious problems.

III. Oncolytic viral therapy using geneticallyengineered herpes simplex viruses formalignant brain tumors.

Department of Neurosurgery , GraduateSchool of Medicine, The University of Tokyo,・Manufacture of the viral vector

In collaboration, we have been preparing on-colytic herpes simplex virus. This preparationwill be used for phase I clinical trial for braincancer patients. We have supported the estab-lishment of the master and working cell banksof Vero cells to produce genetically engineeredherpes simplex viruses. The cGMP compliantMVSS, which contains a replication-competentherpes simplex virus type 1 vector defective forthe α47 gene, was successfully produced. Thepurified final products have been successfullyprepared and are now in the process of regula-tory approvals for use of early phase trials.

IV. Development of robotized cell culturesystem

In collaboration with Dr. Wakitani of OsakaCity University and Kawasaki Heavy Industries,Inc., we are developing robotized cell culturesystem which could be applied to a variety ofprocedures including virus production as afunded project by NEDO.

5. Financial Support

This CFTV has been supported in large by 21st Century COE Program (P.I. Dr. Yusuke Naka-mura) from Japan Society for the Promotion ofScience (2003-2007). From 2007, it has been alsosupported by Coordination, Support and Train-ing Program for Translational Research fromMinistry of Education, Culture, Sports, Scienceand Technology (2007-2011).

Publications

Hummel, HD., Kuntz, G., Russell, SJ., Naka-mura, T., Greiner, A., Einsele, H. and Topp,MS. Genetically engineered attenuated mea-sles virus specifically infects and kills primarymultiple myeloma cells. J. Gen. Virol. 90: 693-701, 2009.

Jing, Y., Tong, C., Zhang, J., Nakamura, T.,Iankov, I., Russell, SJ. and Merchan, JR. Tu-mor and vascular targeting of a novel onco-lytic virus retargeted against the urokinase re-ceptor. Cancer Res. 69: 1459-1468, 2009.

Pham, L., Nakamura, T., Gabriela Rosales, A.,Carlson, SK., Bailey, KR., Peng, KW. and Rus-sell, SJ. Concordant activity of transgene ex-pression cassettes inserted into E1, E3 and E4

cloning sites in the adenovirus genome. J.Gene Med. 11: 197-206, 2009.

Takeda, M., Ohno, S., Tahara, M., Takeuchi, H.,Shirogane, Y., Ohmura, H., Nakamura, T. andYanagi, Y. Measles viruses possessing the po-lymerase protein genes of the Edmonston vac-cine strain exhibit attenuated gene expressionand growth in cultured cells and SLAM-knockin mice. J. Virol. 82: 11979-11984, 2008.

Inoue, H., Iga, M,., Xin, M., Asahi, S., Naka-mura, T., Kurita, R., Nakayama, M., Nakazaki,Y., Takayama, K., Nakanishi, Y. and Tani, K.TARC and RANTES enhance antitumor im-munity induced by the GM-CSF-transducedtumor vaccine in a mouse tumor model. Can-

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cer immunology immunotherapy. 57: 1399-1411, 2008.

Nakamura, T. and Russell, SJ. Chapter 18 of en-gineering oncolytic measles viruses for tar-

geted cancer therapy. molecular targeting inoncology. Edited by Kaufman, HL., Wadler, S.and Antman, K. (Humana Press). pp 431-445,2008.

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The Department of Laboratory Medicine consists of eight divisions-clinical physiol-ogy, hematology, biochemistry, serology, bacteriology, molecular diagnosis and pa-thology, and a division of flow cytometrical analysis which has been added re-cently. This Department engages in the laboratory analysis and gives diagnosis ofclinical materials in the hospital. While facilitating the ongoing translational re-search (TR) projects in the research hospital, the Department also functions as anintegrated diagnosis & monitoring laboratory that evaluates the safety and effec-tiveness of experimental therapeutic approaches. These functions are now offciallyoperating under the name of “laboratory of TR vererification”.

Overview

Our basic research strategies include the fol-lowing approaches: characterizing molecularmechanisms underlying the pathology, develop-ing a novel method to measure the disease-defining mechanism in the clinical materials andevaluating the effectiveness of molecular-targeted therapies thereby contributing to thetranslational research conducted in the institute.Integrating molecular-/biochemical-based labo-ratory assays on the solid background of patho-logical examinations enables us to evaluate theeffectiveness of experimental clinical trials andleads to correct experimental therapies that fur-ther promote translational research. Our depart-ment also functions as an integrated diagnosis &safety-monitoring laboratory as well as the divi-sion of quality control by examining/evaluatingthe safety of investigational new drugs underGMP conditions.

1. Evaluation of “proof of concept” of the TRclinical trials and validate safety of the bio-medical reagents under GMP standard

Evaluation of “proof of concept” of the TRclinical trial is one of our important missionswhich are described as follows. In addition tothis, the missions include validate the safety ofthe biomedical reagents such as vector con-structs are cellular therapeutic materials whichwill be used for patients as gene therapy andcell therapy, respectively. These are critical toconduct TR clinical trials in a safe manner. Wethus established a new division, which specifi-cally aimed to fulfill this purpose under thequality of GMP regulation. We are now ready tovalidate the safety by conducting aseptic test,endotoxin-free test and micoplasma-free test un-der strictly regulated GMP facility.

2. Pathological evaluation of cancer immuno-therapy

We have initiated the analysis of surgicalspecimen obtained from the patients under can-cer immuno-therapy conducted in the researchhospital. By applying sophisticated immunohis-tochemical techniques, we now are intensivelyanalyzing materials from cases including GM-CSF-based gene therapy for renal cell carcinoma

Research Hospital

Department of Laboratory Medicine附属病院 検査部

Associate Professor Naoki Oyaizu, M.D., Ph.D.Assistant Professor Naoyuki Isoo, M.D., Ph.D.

准教授，部長 医学博士 小柳津 直 樹助 教 医学博士 磯 尾 直 之

279

and dendritic cell-based or peptide-pulsed anti-melanoma immuno-therapy. One of our goals isto evaluate the effectiveness of the therapies andto elucidate the mechanisms of anti-tumor im-mune response elicited by the therapy in situ .

3. Elucidation of immunopathological mecha-nisms of autoimmune-based hematologicaldisorders

We found the presence of characteristic patho-logical findings in bone marrow specimen fromsome patients with MDS-RA, aplastic anemia, orpure red cell aplasia, which implicates that com-mon immunopathological mechanism, may beoperative in these hematological abnormalities;that is destruction of erythroid precursors byimmune-based mechanisms in the bone marrow.In collaboration with the Department of Hema-tology, the Department of Laboratory Medicinewill elucidate molecular mechanisms based onthe pathological consideration to establish newdisease entities and develop new therapeutic in-terventions.

4. Analysis of the chimeric gene expressionof hematological disorder

We have initiated the analysis of bcr-abl geneexpression in specimen from patients with CMLand Ph1＋ve ALL by real-time PCR and nestedRT-PCR techniques. In addition, we sequencedthe amplified products to provide informationfor the molecular resistance to STI571 treatment.We are expanding the target molecules to non-

hematological disorder, which includes c-kit,PDGF-R genes that is associated with gastro-intestinal stromal cell tumor (GIST).

5. Developing quick & inclusive diagnosissystem for infectious disease

Since the introduction of new therapeutic ma-neuver, host-pathogen interactions have drasti-cally altered drawing attention. This has re-sulted in altered recognition and molecular in-teraction of infected cells with immune cells,leading to atypical pathological as well as clini-cal manifestations. While distinguishing infec-tious disease and immunological disorder callsfor urgent attention, it may be difficult toachieve these tasks in some cases due to modi-fied manifestations. To avoid such cases, it isimperative to establish a comprehensive diagno-sis system of infectious disease to the earliestpossible opportunity.

6. Immunopathogical analysis of hematopoie-tic cell transplantation

The number of allogeneic hematopoietic stemcell transplantation (HSCT), mainly cord bloodtransplantation, has been performed for thetreatment of hematological malignancies. Graft-versus-host disease (GVHD), a life-threateningcomplication, occurs as a complication of alloge-neic HSCT. Our prime goal is to develop a newway to detect GVHD and make an accurateevaluation of GVHD at our laboratory.

Publications

1. Ohnuma K, Hosono O, Kawasaki H,Yoshikawa N, Katayose T, Oyaizu N, TanakaH, Morimoto C. An adult case of Henoch-Schönlein purpura complicating common per-oneal nerve mononeuropathy. Mod Rheuma-tol. 2009; 19: 73-9.

2. Togo M, Hashimoto Y, Iso-O N, Kurano M,Hara M, Kadowaki T, Koike K, Tsukamoto K.Identification of a novel mutation for phytos-terolemia. Genetic analyses of 2 cases. ClinChim Acta. 2009 401 (1-2): 165-9.

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