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Department of Medicine
(Department of Hematology-Oncology)
Our department has been challenging to cure a variety of fatalhematological disorders with currently available methods. Themainstay to achieve our goal is, nowadays, hematopoietic stemcells (HSC) including bone marrow, peripheral blood and um-bilical cord blood cells, transplantation and cytokine therapy.
These projects have been under way at the HSCT and Hematol-ogy wards with the excellent assistance of nurses andcomedical staffs. We annually take care of more than 40 pa-tients with autologous or allogeneic HSCT. In an attempt toexpand therapeutic benefits of HSCT, we are actively participat-ing to the nation-wide project of unrelated bone marrowtransplantation as the largest HSCT center in Japan. In 1998,we furthermore have established Tokyo cord blood bank ser-vices in our hospital. Cord blood cell transplantation hasbecome strong weapon for patients sufferred from hematolog-ical malignancy without any related or unrelated stem celldonors. We have already treated more than 25 adult patientsuntil the end of March, 2001. Also since 1998, we had startedclinical gene therapy for stage IV renal cancer patients as col-laboration with other clinical departments in our hospital aswell as other hospitals including Juntendo University Hospital,Tsukuba University Hospital, and National Cancer Center.
1. Cases Reports on Clinical Studies of Immuno-
vaccinations of GM-CSF transduced autologous RCC gene Therapy Using Autologous GM-CSF
cells every two weeks for 3 patients. For our 4th pa- Transduced Tumor Vaccines(GVAX) for Stage
tients, 4 vaccinations have been done so far. The IV Renal Cell Cancer
safeties of these injections were confirmed besidesminimum adverse events including grade II local- Kenzaburo Tani, Shigetaka Asano, et al.
ized skin reactions including reddness, swelling anditching at the vaccinated sites, which spontaneously There are no effective treatment for patients with were resolved within 48 hours without special treat- Stage IV renal cell cancer (RCC). The introduction of ment. Serum GM-CSF levels were not increased after new therapy is required. Vaccination using autolo- vaccination, but the number of eosinophils was sig- gous GM-CSF transduced renal tumor cells (GVAX) nificantly increased at 24-48 hours after vaccinations.
is one of promising choices to overcome this situa- One patient experienced grade I systemic adverse ef- tion according to preclinical animal studies. Our fects of low grade fever. Immunological studies preliminary data showed that we could safely pro- showed the oligoclonal expansions of T cells in the duce lethally irradiated autologous GM-CSF peripheral blood as well as DTH sites and metastatic transduced RCC at our hands in our hospital. We re- lesions after vaccinations in three patients using port four Japanese patients suffered from Stage IV PCR-SSCP analysis of TCR Vb CDR 3 length. CTL RCC with metastasis. We have so far done total of 43 activities to autologous RCC cells were demonstrat- ed in all of the three patients but did not persist in the but all four patients with standard risk are alive 70- 1st patient sufferred originally from right RCC with 270 days after CBT. Although the number of patients multiple lung and liver metastases. His CTL activi- and periods of observation are insufficient, these re- ties decreased after 5th vaccination. Our 2nd patient, sults are comparable to that of standard bone a 71 year old man who had right RCC with the large sacral metastasis, received 3.7x108 cells over 17times. His sacral metastasis was kept stable for one 3. Cord blood transplantation for nuclear acci-
year after the final vaccination. Our 3 rd patient, a 57 dent patient
year old female who had left RCC with multiple liv-er, lung and contralateral kidney metastasis. She had Tohru Iseki, Jun Ooi, et al.
been injected with 3.2x108 cells over 15 times. Hermain liver metastasis progressed, but both of the A 39-year-old male was systemically irradiated by small lung and contralateral kidney metastasis was nuclear processing accident in October 1999. Radia- kept stable during the course of vaccinations for 7 tion dose was estimated around 8 gy equivalent.
Severe and prompt decrease of lymphocyte countwas observed and extremely hypocellular bone mar- 2. Cord blood transplantation for adult patients,
row without hematopoietic progenitor cells was including nuclear accident patient
ascertained by repeated examination from differentsites. Although the possibility of uneven radiation Tohru Iseki, Jun Ooi, et al.
exposure and subsequent autologous hematopoieticrecovery was considered possible at that point, it was Although the number of Cord Blood Transplants judged that a hematopoietic stem cell transplant was (CBT) has been rapidly increasing, most of the re- necessary. An HLA identical sibling donor was not ported cases are for children. Several reports from us available. The risk of GVHD, which could make or- and Europe described relatively high mortality rate gan damage worse over radiation damage, was of CBT patients as a result of infection. Low stem cell considered less likely to occur with CBT. Further, number resulted in delayed engraftment and re- mixed chimeric phase and late graft rejection after stricted the use of cord blood in adults. The CBT can be obtained relatively easily compared to important clinical issues such as the patient eligibili- other stem cell sources. Therefore, we selected CBT ty, optimal cell doses and the proper regimen of as stem cell source. However, because hematological conditioning and GVHD prophylaxis for adult pa- recovery after CBT is regarded to be slow in general, tients have not been defined yet. We report our we decided to cope with this risk by strict prevention experience with CBT for adult patients.
of infections and introduction of intensive support- Between August 1998 and May 2000, 15 adult pa- ive hematopoietic factors with TPO in addition to tients with hematological malignancy received CBT G-CSF. ATG, methylprednisolone and cyclosporin A from HLA mismatched unrelated donor at IMSUT.
were administered as conditioning and GVHD pro- Median age of patients was 41 years (16-51), weight phylaxis. To avoid additional damage to organs and was 53kg (40-68) and number of nucleated cells in- residual autologous hematopoietic stem cell, if there fused was 2.25X107/kg (1.2-4.1). All patients is any, we used no cytotoxic agents such as cytoxan received the standard conditioning regimen of IM- SUT according to disease status. All patients Recovery of neutrophils was observed on Day 16 received cyclosporin A and 11 received short-term after CBT followed by reticulocyte and platelet re- methotrexate (MTX) for GVHD prophylaxis. No pa- covery. No significant infection was observed.
tients received ATG. All cases received G-CSF Engraftment was ascertained by chromosome analy- administration after CBT.
sis on Day 9 after CBT. Serial FISH analysis revealed Three patients died shortly after CBT (27-39 days), that mixed chimerism was established shortly after because of RRT (2) and infection (1). The patient with CBT, following which the ratio of graft gradually de- ALL received full TBI and cytoxan and developed creased and was absolutely rejected three months autologous recovery. Among evaluable 11 patients, after CBT when autologous hematopoietic recovery median time to neutrophil and platelet engraftment was achieved. The patient died of multiple organ was 24.5 days (9-41) and 50 days (35-164, n=9), re- failure caused by radiation injury seven months after spectively which appears to be faster engraftment the accident. No clinical and pathological evidence of than previously reported, despite concomitant use of transplantation related toxicity and GVHD were not- MTX. Three patients developed Grade II and one de- ed through whole his clinical course. CBT was veloped Grade III acute GVHD. Three patients died considered to be successful in the sense to rescue of relapse 107-307 days after CBT. Overall survival bone marrow aplasia without any adverse effect.
rate at 22 months was 40 ± 19% for all patients and 53± 23% for 11 patients of first transplantation. Surviv- 4. Establishment of Stem Cell Transplantation
al rate of poor risk group (n=11) was low (33± 17%), Nursing Network in JAPAN
Yuko Ogami, Japan Stem Cell Transplant Nursing
alternative designs and only 1 was able to predict the Phase 2 dose. Based on this experience we concludethat PGDE can be alternative is challenging as imple- Nowadays, about 1,800 patients annually receive mented, while mCRM has not demonstrated.
stem cell transplantation in Japan. As the number ofpatients as well as transplantation facility has been 6. Unrelated Cord Blood Transplantation for
increasing rapidly here, it has become very impor- adult patients with MDS-related secondary
tant for nursing staff to catch up with the acute myeloid leukemia
development of the new treatment system. To an-swer various requests by nurses working at the front Jun Ooi, Tohru Iseki, et al.
in this field, we have established Stem cell Trans-plantation Nursing Network in Japan since 1997.
Between August 1998 and June 2000, 6 adult pa- Now the Network consists of more than 200 mem- tients with MDS-related secondary acute myeloid bers and the annual meeting is held at the time of leukemia (sAML) were treated with total body irra- Annual Meeting of The Japan Society of Stem Cell Transplantation. Our activities include the planning cyclophosphamide followed by unrelated cord of nursing research, lectures and symposium to edu- blood transplantation at the Institute of Medical Sci- cate members, biannual publication of periodical, ence, the University of Tokyo. Granulocyte colony- and the organization of annual meeting. Current re- stimulating factor was infused continuously at a search projects include the research protocols dose of 5 µg/kg/day, starting 12 hr before AraC concerning how to grade the severity of oral mucosi- therapy until the end of AraC therapy to increase tis and help patients overcome this difficulties anti-leukemia effect of AraC. The median recipient according to the grade.
age was 41 years (range, 27 to 50 years), weight was Transplant Nursing, highly specialized profes- 50 kg (range, 43 to 63 kg) and number of infused nu- sion, requires very matured technique and cleated cells was 2.56 x 107/kg (range, 2.09 to 4.1 x knowledge to take care of patients. Such expert nurs- 107/kg). Cyclosporine plus short-course methotrex- es should also strongly support the patient's family ate were used for GVHD prophylaxis for all patients.
members and donor bank. Such issues are supposed All patients had myeloid reconstitution and the me- to be educated in the Network. Our Network service dian time to >0.5 x 109/L ANC was 24 days (range, 19 would provide the place where every transplant to 35 days). A self-sustained platelet count greater nurse, not only members of this Network, can learn than 50 x 109/L was achieved in 5 patients at a medi- and freely communicate each other. We really hope an time of 49 days (range, 35 to 164 days). Acute our network would be helpful to improve more GVHD appeared in 4 of 6 patients (grade I; n=2, transplant nursing in Japan.
grade II; n=1, grade III; n=1) and chronic GVHD in 3of 4 evaluable patients. Four patients remain alive inCR. Two patients relapsed. Among 4 patients who 5. Comparative Review of Oncology Phase I
remain alive in CR, 3 patients had not received re- Dose Escalation Designs of New Molecular
mission induction therapy before transplantation.
These preliminary results suggest that adult sAMLpatients without suitable related or unrelated donor Fumitaka Nagamura, Steven Hirschfeld , et al.
should be considered for cord blood transplantation.
We surveyed the design and conduct of Phase I 7. Establishment of a IL-6-independent myeloma
studies for new molecular entities (NME) submitted cell line, IMS-MY1, from pleural effusion of a
to the Division of Oncology Drug Products of the patient with multiple myeloma
Center for Drug Evaluation and Research at the Foodand Drug Administration (FDA) from 1986 to 1997.
Yasushi Soda, Arinobu Tojo, Kenzaburo Tani , et al.
We identified eighty-two cytotoxic or cytostaticNME administered systemically as monotherapies.
A human myeloma cell line, IMS-MY1, was newly Eleven NME submissions that used pharmacokineti- established from pleural effusion of a patient with cally guided dose escalation (PGDE) were evaluated, advanced IgG-λ multiple myeloma. Mononuclear and 10 NMEs used modified continual reassessment cells in pleural effusion (PEMC) of the patient were method (mCRM) were evaluated. Only 7 of the 14 isolated by Ficoll-Hypaque centrifugation. These PGDE studies were able to implement the design. Of PEMC consisting of more than 99% of myeloma cells these, 6 used fewer patients than predicted using al- were cultured in RPMI1640 medium supplemented ternative designs and 5 were able to predict the with 10% FBS and 10 ng/mL rhIL-6 and a half of Phase 2 dose. Sixteen studies of the proposed 21 media was replaced biweekly. Myeloma cells began mCRM studies were able to implement the design.
growing without any support of adherent cells at Of these, 4 used more patients than predicted using day 80 after the start of culture and acquired IL-6- independence at day 160. IMS-MY1 cells were posi- cytokines, including IL-6, did not affect the cell tive for cytoplasmic IgG-λ immunoglobulin and growth. Secretion of immunoglobulin from IMS- secreted the same type of immunoglobulin into the MY1 cells, however, was increased by IL-6. PCR culture media. IMS-MY1 cells and parental PEMC analysis for HHV-8/KSHV genome was positive, but similarly showed immature phenotypic cell surface not EB virus genome in IMS-MY1 cells. Both ge- markers of CD38 (+), CD49e (-) and MPC-1 (-), but nomes were not detected in parental cells. These not CD45. All of CD10, CD56 and CD138 (syndecan- results suggested that aberrant fusion gene, not IL-6 1) were also expressed in both cells. Chromosome stimulation, is rather important for the survival and analysis of IMS-MY1 showed tetraploidy with nu- the growth of IMS-MY1 cells. And the integrated merous abberations like parental cells. Double HHV-8 genome might contribute to the progression t(11;14)(q13;32) were found in all cells and bcl-1-IgH of malignancy magnitude in this cell line. Further fusion gene was recognized by fluorescence in-situ molecular analysis is required to prove the role of the hybridization, and the rearrangement of c-myc was integrated HHV-8 genome for the establishment of also detected by Southern blot analysis. IFN-α in- IMS-MY1 cells.
duced cell death dose-dependently, but many Watari K., Tojo A., Nagamura-Inoue T., Matsuoka Machida U., Tojo A., Takahashi S., Iseki T., M., Irie S., Tani K., Yamada Y., Asano S. Hyper- Nagayama H., Shirafuji N., Mori S.,Wada Y., function of neutrophils in a BCR/ABL-negative Ogami K., Yamada Y., Sakamaki H., Maekawa T., chronic myeloid leukemia: A case report with in Tani K., Asano S. The effect of G-CSF administra- vitro studies. Cancer 89:551-560, 2000.
tion in healthy donors before bone marrow har- Nagayama H, Sato K, Kawasaki H, Enomoto M, vesting. Br J Haematol. 108(4):747-53, 2000.
Morimoto C, Tadokoro K, Juji T, Asano S, Kosugi N, Tojo A, Shinzaki H, Nagamura-Inoue T, Takahashi TA. IL-12 responsiveness and expres- Asano S. The preferential expression of CD7 and sion of IL-12 receptor in human peripheral blood CD34 in myeloid blast crisis in chronic myeloid monocyte-derived dendritic cells. J Immunol.
leukemia. Blood. 15;95(6):2188-9, 2000.
Futaki M, Yamashita T, Yagasaki H, Toda T, Yabe M, Machida U., Tojo A., Ooi J., Iseki T., Nagayama H., Kato S, Asano S, Nakahata T. The IVS4 + 4 A to T Shirafuji N., Sawada M., Nakayama K., Tani K., mutation of the Fanconi anemia gene FANCC is Asano S. Refractory facial cellulitis following cos- not associated with a severe phenotype in Japa- metic fhinoplasty after cord blood stem cell trans- nese patients. Blood. 15;95(4):1493-8, 2000.
plantation. Int J Hematol. 72(1):98-100, 2000.
Department of Surgery
We have been engaged in the surgical treatment of solid tu-mors and the renal transplantation. We have also been offeringservices, including upper and lower endoscopic examination,ultrasonic examination, and angiography, in the Department ofClinical Examination. The principal goal of our department is tocreat and conduct clinical trials (Phase I and II) for patients atResearch Hospital. We are preparing clinical phase I trial forcancer vaccine using gp100 derived peptides.
1. Summary of surgical treatments and examina-
Examinations performed in 2000 are as follows: tions performed in 2000
angiography for 85 cases, including trans-arterialembolization and trans-arterial chemotherapy, gas- Hideaki Tahara, Masazumi Eriguchi, Shinji Tomi-
troduodenal endoscopy for 506 cases, and colorectal kawa, Takuya Tsunoda, Yasutaka Takeda, Iwao
endoscopy for 166 cases.
Yoshizaki, Yoshifumi Beck, Hironobu Yanagie,
Takuya Takayama, Yasumasa Nonaka, Syogo Na-
2. Clinical phase I trial for Cancer Vaccine using
kano, Hiroyuki Mushiake
gp100 derived peptides restricted HLA-A*0201
and -A*2402 against the advanced malignant
Surgical operations have been performed in 128 cases under general anesthesia and spinal anesthe-sia. As shown in Table 1, major operations were Takuya Tsunoda, Hideaki Tahara, Yoshifumi
performed in 70 patients with malignant diseases, Beck, Yushiyuki Mushiake, Toshiyuki Baba, Shogo
and in 55 patients with benign diseases. Renal trans- plantation was performed in 3 patients.
Table 1. Major Operations Performed in 2000
Malignant Diseases Benign Diseases Cancer of the stomach Cancer of the colo-rectum Cancer of the liver Cancer of the bile duct Cancer of the pancreas Renal transplantation Cancer of the kidney Cancer of the breast Melanoma associated antigen, gp100, derived the unregulated expression of angiogenesis genes epitope peptides are used for the cancer vaccine and down regulation of cell cycle genes determined against malignant melanoma. In the restriction for by cDNA macroarray.
HLA-A*0201, the wild type gp100 derived peptide(ITDQVPFSV) and mutated gp100 peptide (IM- 4. Differential tissular expression and localiza-
DQVPFSV) are used, whereas in the restriction for tion of type IV collagen alpha1(IV), alpha2(IV),
HLA-A*2402, the newly mapped gp100 derived pep- alpha5(IV), and alpha6(IV) chains and their
tide (VYFFLPDHL) is used in this clinical trial. As the mRNA in normal breast and in benign and ma-
adjuvant, GMP grade IFA is also utilized in order to lignant breast tumors
augment for anti-tumor immunity. To analyze theimmunoresponse from the vaccinated patients, Nakano S, Iyama K4, Ogawa M4, Yoshioka H4,
HLA-Tetramer is prepared for this purpose. Our Sado Y4, Oohashi T4, Ninomiya Y4:4Department of
goal in this clinical trial is to examine its safety, ad- Surgical Pathology, Kumamoto University School
verse effect and the immunoresponse. The unique of Medicine, Japan
points of this clinical trail are that 1) wild type gp100peptide and mutate gp100 peptide are synchronous- Type IV collagen, the major component of base- ly injected and 2) the newly mapped gp100 peptide ment membrane (BM), is composed of six genetically restricted HLA-A*2402 is injected. We also investi- distinct alpha chains. We investigated the cellular gate the clinical effects by this cancer vaccine.
regulation and origin of these alpha(IV) chains innormal and neoplastic breast tissues by immunohis- 3. Determination of chemosensitivity for col-
tochemistry by using alpha(IV) chain-specific orectal cancer using macroarray
antibodies and by in situ hybridization. In normalbreast, alpha1(IV) and alpha2(IV) chains were Takuya Tsunoda1, Kazuto Nishio2, Hiroshi
stained in all BM, whereas alpha5(IV) and alpha6(IV) Tanimura3:1Department of Surgery and Bioengi-
chains were restrictively localized in a linear pattern neering (IMSUT), 2Pharmacology division, National
in the BM of the mammary gland. Similar immun- Cancer Center Research Institute and 3Second De-
ostaining profiles were observed in benign breast partment of Surgery, Wakayama Medical School
tumors and in the intraductal components of inva-sive ductal carcinoma. However, in invasive ductal It is essential for promote the clinical efficacy to carcinoma, alpha1(IV) and alpha2(1V) chains were predict the chemosensitivity for the solid tumor es- discontinuously or negatively stained in the cancer pecially the colorectal cancer. MTT assay has been cell nests, and the assembly of alpha5(IV) and performed for this purpose from the freshly isolated alpha6(IV) chains into the BM was completely inhibit- tumor. We have successfully performed the MTT as- ed. Coexpression of alpha5(IV) and alpha6(IV) chains say even the freshly isolated colorectal cancer, which was related to the localization of alpha-smooth muscle has potentially the contaminations. Furthermore, actin (alpha-SMA)-positive myoepithelial cells. By in MTT assay from the freshly isolated colorectal cancer situ hybridization, in fibroadenoma and invasive duc- was utilized to choose the anticancer agents. Espe- tal carcinoma, the signals for alpha1(IV) and cially, CPT-11 (Topoisomerase I inhibitor) was newly alpha2(IV) mRNA were abundant in stromal cells.
developed the anticancer agent that has wild spec- However, the signals for alpha5(IV) and alpha6(IV) trum including colorectal cancer. However, it is mRNA were not seen in any of these cells. In con- difficult to predict the combination with other anti- trast, in intraductal papilloma, coexpression of cancer agents. It is demonstrated that CTP-11 has alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/ synergistic effects with CDDP and MMC. It is signif- alpha6(IV) mRNA was identified in epithelial cells.
icant to analyze the chemosensitivity and gene The results indicate that the mammary gland forms a expression in order to promote the clinical efficacy second network of BM composed of alpha5(IV)/ and protect the adverse effects. Our focus is the de- alpha6(IV) chains, in addition to the classic network termination for the gene related chemosensitivity of alpha1(IV)/alpha2(IV) chains. The expression of from the freshly isolated colorectal cancer using mac- type IV collagen alpha chains seems to be differen- roarrey. Based on that, cDNA from the freshly tially regulated by the epithelial-myoepithelial isolated colorectal cancer needs to be determined.
interaction and to be associated with the invasive We successfully have demonstrated that the clinical potential of breast cancer.
samples are able to be analyzed, and have showed Tsunoda,T., Tanimura,H., Ishimoto,K., Yamaue,H., cancer patients. Biomed & Pharmacother., 54:291- Umemoto,Y., Ura,K., Tanaka, H., Matsuda,K. and Murakami,K.: Musculocutaneous rotation flap for Ishida, O., Maruyama, K., Yanagie, H., Eriguchi, M., reconstruction after curative resection of perineal Iwatsuru, M. : Targeting chemotherapy to solid tumor. Wakayama Medical Rep., in press.
tumors with long-circulating thermosensitive li- Hirao M, Onai N, Hiroishi K, Watkins SC, posomes and local hyperthermia. Japanese Jour- Matsushima K, Robbins PD, Lotze MT, Tahara H.
nal of Cancer Research, 91(1) 118-126, 2000.
CC chemokine receptor-7 on dendritic cells is in- Yamamura, Y., Sayama, K., Takeda, Y., Matsuzawa, duced after interaction with apoptotic tumor cells: A., Iguchi, T., Ohta, Y. : Metallothionein expres- critical role in migration from the tumor site to sion in transplantable mouse mammary tumors.
draining lymph nodes. Cancer Res 60: 2209- Anticancer Research, 20 : 379-384, 2000.
Tanaka F, Hashimoto W, Okamura H, Robbins PD, Lotze MT, Tahara H. Rapid generation of potentand tumor-specific cytotoxic T lymphocytes byinterleukin 18 using dendritic cells and naturalkiller cells. Cancer Res 60:4838-44, 2000.
Iwazawa, T, Chau, G-Y, Mori, T., Dookeran, KA., Rubin, JT, Watkins, Sc, Robbins, PD, Lotze, MT,Tahara, H. Potent anti-tumor effects of intra-arte-rial injection of fibroblasts genetically engineeredto express IL-12 in liver metastasis model of rat-No additional benefit of using retroviral producercell- Cancer Gene Therapy (In press) Fujii ,Y., Iwasa ,H., Hirai, J., Takahama, Y., Hasumi, K., Eriguchi ,M. : Inhibition of liver metastases andtumor cell invasion in spontaneous liver metasta-sis model (LMFS) by sodium D-Glucaro-δ-lactam(ND2001). Biomed & Pharmacother., 54:85-92,2000.
Hasebe, H., Sato, K., Nagayama, H., Takahashi, T., Eriguchi, M. : Dysfunctional regulation of the de-velopment of monocyte- derived dendritic cell in Department of Radiology
Our department consists of three major divisions: diagnosticradiology, nuclear medicine and radiation oncology. Diagnosticradiology plays a critical role in evaluating various neoplasticand infectious diseases. Clinical studies are conducted mainlyusing magnetic resonance imaging (MRI), supported by otherdepartments and other institutions. In nuclear medicine, we de-velop analytic methods to estimate in vivo physiology, as wellas studying the kinetics of radiotracers and physical character-istics of detectors. In radiation oncology, total body irradiationprior to bone marrow transplantation provides valuable advan-tage.
1. Metabolism of Tc-99m ECD in Infarcted Brain
staining. The metabolic rate of Tc-99m ECD was de- Tissue of Rats
termined in homogenates of infarcted tissue,contralateral noninfarcted tissue and tissue sampled Yusuke Inoue, Osamu Abe1, Takeshi Kawakami3,
from sham-operated rats. Infarct volume was mea- Takayuki Ozaki3, Minoru Inoue3, Ikuo Yokoyama2,
sured by direct and indirect methods to assess Kohki Yoshikawa and Kuni Ohtomo1:Departments
volume expansion due to edema, and the metabolic of 1Radiology and 2Cardiovascular Medicine, Univer-
rate in infarcted tissue was corrected for the effect of sity of Tokyo, 3Daiichi Radioisotope Laboratories,
edema. TTC staining had no effect on the metabolic rate of Tc-99m ECD. The metabolic rates in the inf-arcted tissue were 0.222±0.054%/min and 0.285± Brain SPECT with Tc-99m ethyl cysteinate dimer 0.064%/min before and after correction for edema, (ECD) visualizes subacute cerebral infarct as a hypo- respectively, and were significantly lower than those active area even in the presence of postischemic in the contralateral noninfarcted tissue (0.426± hyperperfusion. Brain retention of Tc-99m ECD de- 0.028%/min) and the tissue sampled from the sham- pends on hydrophilic conversion mediated by operated rats (0.439±0.031%/min). No substantial enzymes, and impaired enzymatic trapping is hy- difference in rates was observed between the con- pothesized to depress retention efficiency in the tralateral tissue and tissue from the sham-operated infarcted region. The aim of this study was to deter- rats. The results of this study demonstrated that inf- mine whether the metabolic rate of Tc-99m ECD is arction decreases the activity of enzymes that actually reduced in infarcted brain tissue. In 50 mM mediate the hydrophilic conversion of Tc-99m ECD phosphate buffer (pH 7.4), Tc-99m ECD was incubat- in the brain and suggest that reduced metabolic ac- ed for 30 min with homogenates of rat brain tissue tivity is related to decreased accumulation of Tc-99m with and without triphenyltetrazolium chloride ECD in hyperperfused infarcts. We are now prepar- (TTC) staining. The ratio of polar products was de- ing to examine the retention and washout of the termined by thin layer chromatography as a function tracer under physiological and ischemic conditions of incubation time, and metabolic rates were ob- using living brain slices.
tained. Permanent focal ischemia was induced byocclusion of the right middle cerebral artery (MCA) 2. Estimation of Differential Renal Function with
in rats. The brain was removed 24 hours after MCA Tc-99m MAG3
occlusion, and the infarcted area was defined by TTC Yusuke Inoue, Kohki Yoshikawa, Ikuo Yokoyama2
virtual bronchoscopy and fiberoptic bronchoscopy.
and Kuni Ohtomo1
Virtual bronchoscopy was calculated and recon-structed from the cross-sectional images on a Measurement of differential renal function is es- separate workstation. Stenoses and tumor infiltra- sential in determining therapeutic strategies for tion were classified from the fiberoptic examination.
nephrourological patients. We have studied the eval- These results were compared with the virtual bron- uation of differential renal function from renal choscopy findings. Synchonized reference images scintigraphy and developed accurate techniques ap- offered the advantage of being able to visualize rela- plicable to both children and adults. In this study, we tionship of the lesion and the bronchus. Virtual compared single-sample methods, proposed by Rus- bronchoscopy of diagnostic quality was achieved in sell et al. and Bubeck et al., and camera-based 16 of 21 patients. However, on virtual bronchoscopy methods in calculating 99mTc-MAG3 clearance, and discrete infiltration was not visible in five patients.
determined camera-based methods that provide esti- Virtual bronchoscopy using synchonized reference mates comparable to those measured by the Russell images is accurate and useful techniques in the pre- method. Twenty-one patients underwent 99mTc- operative assessment of pulmonary nodular lesions.
MAG3 renal scintigraphy, and clearance wasmeasured by the Russell method and Bubeck meth- 4. Evaluation of tissue segmentation methods
od. Various renogram parameters were determined for cerebral 1H-magnetic resonance spectro-
based on the slope of the renogram and area under scopic imaging (MRSI) using short-TE PRESS
the renogram, and correlated with the clearancemeasured by the Russell method. Camera-based Kohki Yoshikawa, Yusuke Inoue, Naoki Yoshioka,
clearance was calculated with the obtained regres- Tsuyoshi Matsuda4, Seizo Takahashi4 and Takashi
sion equations and with equations determined Ogino5:4GE Yokogawa Medical Systems, Ltd., and
previously using the Bubeck method as a standard.
5National Institute of Neuroscience
The Bubeck method provided lower measures thanthe Russell method in high renal function. Clearance The goal of our study is to evaluate the accuracy of measured by the Russell method was well correlated variable tissue segmentation methods for quantita- with renogram parameters, and clearance calculated tive cerebral proton MRSI. Tissue segmentation was with the obtained regression equation was compara- performed on the T1-W, T2-W, PD-W, T1-calculat- ble to that measured by the Russell method. When ing, and T2-calculating image data sets using camera-based clearance was predicted with the pre- own-developed software of SGI-02 and Sun SPARK vious equation, it was lower than the result obtained 20. T1- and T2-images were calculated by using the by the Russell method in high function. In conclu- images of FSE 300/7,500/7,1000/7, 6000/7 and of sion, there are systematic differences in 99mTc-MAG3 FSE 6000/7, 6000/100 respectively. Classification of clearance calculated by different methods. The cam- pixels as either CSF, gray, or white matter was per- era-based methods obtained in this study appear to formed using histogram analysis of pixel intensities.
facilitate comparison of results obtained by the Rus- The 10 or 20 slices corresponding to the thickness of sell method and camera-based method. We are now the MRSI slab were extracted from the segmented integrating our results into a semiautomated utility data sets and averaged to create images of gray mat- program and planning to conduct a multicenter vali- ter, white matter, and CSF. The metabolite dation study.
concentrations were corrected for the partial volumeeffect due to CSF. Ten healthy control subjects were 3. Virtual Bronchoscopy for Pulmonary Nodular
studied, after informed consent. The 3D-MRSI data Lesions Using Synchronized Reference Images
Naoki Yoshioka, Yusuke Inoue, Kohki Yoshikawa,
Manabu Minami1, Masaaki Akahane1 and Kuni
time=19.3minutes). A 1.5 T General Electric (GE, Milwaukee, WI) Signa Lx NV/i was used, running8.25v software. These techniques were applied to Conventional virtual bronchoscopy uses three or- study differences in metabolite concentration be- thogonal reformatted images along body axis as tween gray and white matter in normal volunteers reference views. Synchronized reference image (SRI) (n-10). The estimated ratios of metabolite concentra- mean a new technique that provides three orthogo- tion were: NAA/Cr=2.250; Cho/Cre=0.910 for gray nal reformatted images along the vector of view. In matter and NAA/Cr=1.907; Cho/Cre=1.083 for this study, we examined the usefulness of this tech- white matter. The correction of inhomogeneous B0 nique in virtual bronchoscopy for the evaluation of and B1 fields and the automatic phase correction pulmonary nodular lesions and compared it with could be done successfully by using the MRSI data corresponding fiberoptic examinations. Twenty-one without water suppression and chemical shift of the patients (sixteen men and three women) with pulmo- spectral peak of the water respectively.
nary nodular lesions were examined with both Katanoda, K., Yoshikawa, K. and Sugishita, M. Neu- Katayama, N., Inoue, Y., Harada, M., Shimoyamada, ral substrates for the recognition of newly learned K., Ikeda, M. and Tsukune, Y. Avid Ga-67 uptake faces: a functional MRI study. Neuropsychologia.
in breast metastasis from tongue cancer. Clin.
Nucl. Med. in press.
Inoue, Y., Yoshikawa, K., Suzuki, T., Katayama, N., Yoshioka, N., Minami, M., Inoue, Y., Kawauchi, N., Yokoyama, I., Kohsaka, T., Tsukune, Y. and Nakajima, J., Oka, T., Yoshikawa, K. and Ohtomo, Ohtomo, K. Attenuation correction in evaluating K. Pedunculated bronchogenic cyst mimicking renal function by a camera-based method in chil- pleural lesion. J. Comput. Assist. Tomogr. 24: 581- dren and adults. J. Nucl. Med. 41: 823-829, 2000.
Inoue, Y., Yoshikawa, K., Yokoyama, I. and Ohtomo, Okubo, T., Yoshioka, N., Hayashi, N., Abe, O., K. Estimation of 99mTc-MAG3 clearance by single- Masumoto, T., Sasaki, T. and Ohtomo, K. Con- sample methods and camera-based methods.
trast-enhanced magnetic resonance imaging of the Ann. Nucl. Med. 14: 329-332, 2000.
Inoue, Y., Yoshikawa, K., Yoshioka, N., Watanabe, schwannoma:correlation with surgical findings.
T., Saegusa, S., Kaneko, Y., Yokoyama, I. and Acta Oto-Laryngol. 542 (suppl): 13-17, 2000.
Ohtomo, K. Evaluation of renal function with99mTc-MAG3 using semiautomated regions of in-terest. J. Nucl. Med. 41: 1947-1954, 2000.
Katayama, N., Inoue, Y., Ichikawa, T., Harada, M., Itoh, T., Shimoyamada, K., Ikeda, M. and Tsukune,Y. Increased activity in benign phyllodes tumor onTc-99m MDP scintimammography. Clin. Nucl.
Med. 25: 551-552, 2000.
Yokoyama, I., Yonekura, K., Ohtake, T., Kawamura, H., Matsumoto, A., Inoue, Y., Aoyagi, T., Sugiura,S., Omata, M. and Ohtomo, K., Nagai, R. Role ofinsulin resistance in heart and skeletal muscle F-18 fluorodeoxyglusose uptake in patients withnon-insulin dependent diabetes mellitus. J. Nucl.
Cardiol. 7: 242-248, 2000.
Department of Pediatric Hematology-Oncology
Our major goal is to cure children suffering from a variety oflife-threatening hematological disorders. Attempting to achievethis, we continue the commitment to treatment and follow-upcare of such children, and to clinical and laboratory researchthat ultimately will help us devise better therapeutic approachesto these deseases. Currently efforts are directed toward he-matopoietic stem cell transplantation including ex vivoexpansion of human hematopoietic stem cells, gene therapy,immunotherapy and analysis of pathogenesis of hematopoieticdisorders.
1. Establishment of an assay for human hemato-
clinical tool for assaying human HSC.
poietic stem cells
2. Efficient expansion of human hematopoietic
Hiroshi Yoshino, Takahiro Ueda, Yasuhiro Ebihara,
stem cells by a combination of c-Kit, Flt3, c-
Atsushi Manabe, Ryuhei Tanaka, Kohichiro Tsuji,
Mpl and gp130 signals
Takahiro Ueda, Hiroshi Yoshino, Yasuhiro Ebihara,
Transplantable hematopoietic stem cells (HSC) Atsushi Manabe, Ryuhei Tanaka, Kohichiro Tsuji,
are characterized by an ability to permanently regen- erate the entire blood-forming system. An assaysystem evaluating human HSC would allow a more There has recently been great interest in the ex vivo rational approach to the development of clinical expansion of human HSC for a variety of developing treatments involving HSC transplantation, ex vivo clinical application including HSC transplantation HSC expansion and gene therapy. However, in con- and gene therapy. To obtain the optimal culture con- trast to mouse in vivo assays using syngeneic dition for human HSC expansion, many investigators recipients, until recently, no comparable system has used various combinations of cytokines which have been established in human. To address this problem, been shown to act on primitive hematopoietic cells, several strategies have been pursued to develop an since proliferation and differentiation of HSC are animal recipient for human HSC. Recently, we estab- thought to be regulated by interactions of various cy- lished a xenotransplatation system for human HSC tokine receptor signals. In particular, tyrosine kinase into NOD/Shi-scid mice, which possess lack of ma- receptor signals, such as c-Kit signal by stem cell fac- ture lymphocytes, macrophage dysfunction and tor (SCF) and Flt3 signal by Flt3 ligand (FL), play key absence of circulating complements. When treated roles in primitive hematopoiesis. Thrombopoietin with anti-asialo GM1 antibody to delete natural kill- (TPO), a ligand for c-Mpl, originally identified as a er cells, the NOD/Shi-scid mice revealed efficient primary regulator for megakaryopoiesis, has also engraftment of human cord blood (CB) CD34+ cells.
been shown to stimulate the expansion of primitive Analysis of recipient bone marrow (BM) cells 10 hematopoietic cells. In addition, we have demon- weeks after the transplantation showed the multilin- strated that gp130 signal activated by a complex of eage reconstitution, and contained a large number of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/ human CD34+ cells and colony-forming cells (CFC).
sIL-6R) synergizes with c-Kit or Flt3 signal to expand Thus, this mouse model may provide a useful pre- multipotential hematopoietic progenitor cells.
Clinically transplantable HSC should prove to re- number of myeloid progenitors in addition to eryth- tain the long-term reconstituting ability. Until roid, megakaryocytic and multipotential progenitors.
recently, however, most of human HSC expansion These results have indicated that the expression of G- studies aimed at clinical application have used the CSFR on CD34+ cells is restricted to myeloid assays for CD34+ cells, CFC and long-term culture progenitors, and erythroid, megakaryocytic and mul- initiating cells (LTC-IC) to optimize the culture con- tipotential progenitors do not express G-CSFR, ditions. These surrogate assays have been shown not suggesting that the specific activity of G-CSF on my- to reflect a stem cell activity. Recent development of elopoiesis depends on the exclusive expression of its an assay measuring the ability to reconstitute hemato- receptor on myeloid progenitors, and that the mobili- poiesis of NOD/Shi-scid mice in our department as zation of various hematopoietic progenitors is not a shown above enabled us to evaluate the stem cell ac- direct effect of G-CSF in human.
tivity of expanded hematopoietic cells. Then, usingthe xenotransplantation of human hematopoietic cells 4. Impaired Granulopoiesis in the Truncated G-
into NOD/Shi-scid mice, we established a novel ex- pansion system of human HSC with a combination ofSCF, FL, TPO and IL-6/sIL-6R. When CB CD34+ cells Tetsuo Mitsui, Sumiko Watanabe1, Kazuki Nakao2,
were cultured with the cytokine combination for 1 Motoya Katsuki2, Tatsutoshi Nakahata, Kohichiro
week, analysis of human CD45+ cells in BM of the re- Tsuji, Shigetaka Asano:1Division of Molecular
cipients transplanted with the cultured cells showed and Developmental Biology and 2Division of
significant expansion of human long-term reconsti- DNA Biology and Embryo Engineering, Center
tuting HSC in the culture. Our culture system may for Experimental Medicine, IMSUT
pave the way for the clinical application of ex vivoexpansion of human HSC.
Severe congenital neutropenia (SCN), or Kost- mann syndrome, is characterized by persistent 3. Exclusive expression of granulocyte colony-
absolute neutropenia and BM morphology that sug- stimulating factor (G-CSF) receptor on myeloid
gests maturational arrest of neutrophil precursors at progenitors in bone marrow CD34+ cells
the promyelocytic stage. In approximately 15-20% ofthe cases, mutations are found in the gene encoding Yasuhiro Ebihara, Ming-jiang Xu, Atsushi Manabe,
the G-CSFR, resulting in a cytoplasmic truncation of Ryuhei Tanaka, Kohichiro Tsuji, Shigetaka Asano
the receptor. It is supposed that these truncated re-ceptors act in a dominant negative manner to block G-CSF has been reported to act on cells of neutro- granulocyte maturation and transduces a strong philic lineage. However, the administration of growth signal. Some patients with the mutations were G-CSF to mice and human induces an increase of cir- reported to become acute myeloid leukemia after re- culating hematopoietic progenitor cells including combinant G-CSF therapy. Recently, McLemore et al.
not only myeloid but also erythroid, megakaryocytic generated mice carrying a targeted one of these trun- and multipotential progenitors. We then analyzed cations, using homologous recombination in the expression of receptors for G-CSF (G-CSFR) on embryonic stem cells. Mice heterozygous or homozy- human BM and G-CSF mobilized peripheral blood gous for the mutaion had normal levels of circulating (PB) CD34+ cells, and examined the proliferation and neutrophils and no evidence for maturational arrest.
differentiation capability of sorted CD34+G-CSFR+ In addition, Bernard et. al. described that these trunca- and CD34+G-CSFR- cells using methylcellulose tions were detected only in a minor percentage of clonal culture. Flow cytometric analysis showed that transcripts from SCN and the mutations could sponta- G-CSFR was expressed on 18.7±9.8% of BM CD34+ neously disappeared, and concluded that the gene cells, most of which were included in CD34+CD33+ abnormality has no role in etiology of SCN patients and CD34+CD38+ cell fractions, suggesting that G- and is a bystander phenomenon. On the other hand, CSFR is predominantly expressed on mature Hermans et al. reported the mice, being generated subpopulations in hematopoietic progenitors. In for the mutaion in the same way with McLemore et clonal culture, CD34+G-CSFR- cells produced eryth- al, that have reduced number of neutrophils in PB.
roid bursts, megakaryocyte and multilineage To elucidate the role of these gene abnormalities in colonies, while CD34+G-CSFR+ cells produced only SCN, we generated three types of transgenic mice myeloid colonies including granulocyte, macroph- having two types of truncated murine G-CSFR and a age, eosinophil and granulocyte-macrophage wild type receptor as a control. We made two kinds colonies. When incubated with the cytokine cocktail of truncated G-CSFR DNA fragments (Q717-stop for 5 days, CD34+G-CSFR- cells generated CD34+G- codon and Q730-stop codon), and wild type one as a CSFR+ myeloid progenitors. In G-CSF mobilized PB, control. These fragments were inserted to the expres- CD34+ cells contained 10.8±5.8 % of G-CSFR+ cells, sion vector LD2 that has murine MHC class I most of which were also myeloid progenitors, al- promoter, and transgened. The mice having the trun- though CD34+G-CSFR- cells contained a substantial cated recptors showed lower neutrophil counts in PB than those having the wild ones (225 ±225, 230±118, way existed in the proliferational process of T-ALL and 731± 301/ml in 717-truncation, 730-truncation lymphoblasts. Our current study provide a novel as- and wild type mice). BM myelogram of the mice hav- say system for the research on T-ALL lymphoblasts, ing the truncated receptors revealed reduced ratio of especially in the exploration of the the hierarchy mature myelocytes. These results suggest that the within human T-lymphoid leukemic cells, and may truncated receptors have some role in the occurrence finally contribute to a novel therapeutic modality.
of neutropenia in SCN.
6. Gene therapy for recurrent neuroblastoma
5. Growth of human T-cell acute lymphoblastic
leukemia lymphoblasts in NOD/SCID fetal thy-
Atsushi Manabe, Imiko Hirose, Naohide Yamashi-
mus organ culture
ta3, Kohichiro Tsuji, Shigetaka Asano:3Department
of Advanced Medical Science, IMSUT
Feng Ma, Atsushi Manabe, Miyuki Ito, Kohichiro
Tsuji, Shigetaka Asano
Although a notable improvement of cure rate in childhood cancer has been observed owing to devel- T-cell acute lymphoblastic leukemia (T-ALL) is a opment of multiagent chemotherapy, approximately malignant clonal desease which covers about 20 per- one half of children with cancer cannot survive with cent of all cases of ALL. Little has been understood a contemporary treatment. Even a mega-dose che- about the in vitro proliferation of T-ALL leukemic motherapy combined with stem cell support can not cells because of a lack of a appropriate culture sys- eradicate the disease completely in this subpopula- tem. We have recently established a NOD/SCID tion of patients. A novel approach is needed for these mouse fetal thymus organ culture (FTOC) that is ca- patients. Recently, cancer immunotherapy employ- pable of supporting the development of T lymphoid ing a gene therapy technique was proposed.
cells from human CD34+ hematopoietic stem/pro- Neuroblastoma (NB) is one of the most frequent genitor cells in vitro. By applying this NOD/SCID solid tumors in children and NB in children over FTOC, we found that leukemic cells from fresh (1 one-year-old is known to be very difficult to cure case) and frozen (7 cases) bone marrow (BM) sam- even with stem cell transplantation. It has been ob- ples of children with T-ALL proliferated grossly over 4 weeks in the FTOC. Re-seeding of FTOC-derived spontaneously and its mechanism has not yet been T-ALL leukemic cells into second FTOC generated elucidated. NB is derived from a neural crest which the same growth pattern. A detailed investigation of also derives malignant melanoma for which immu- of the FTOC-derived leukemic cells showed a similar notherapy has already been established after phenotype to the original one moprphologically and identification of melanoma-associated tumor specific immunophenotypically. Culturing of these FTOC- antigens such as gp100, MART-1/Melan-A, tyrosi- derived leukemic cells in suspension culture led to nase and MAGE-1. It is possible that some an expeditious death, suggesting that these FTOC- immunological mechanism may play a role in infants dependent cells were not cell lines. These with NB. Currently, we are preparing a gene therapy FTOC-derived T-ALL leukemic cells were able to for recurrent neuroblastoma. In collaboration with generate leukemia in NOD/SCID mouse and still Dr. Malcolm Brenner at Baylor College in Houston, shared clonal characteristics when probed by a PCR USA, we plan to use a tumor vaccine transduced mathod using consensus primers for TCRγ chain re- with IL-2 and lymphotactin. It was shown that the arrangement. Furthermore, a comparison of the vaccination of neuroblastoma cells transduced with original and FTOC-derived T-ALL leukemic cells re- IL-2 into patients with relapsed neuroblastoma ex- vealed that the proportion of the cells that expressed erted regression of tumors. It is possible that IL-7R increased in all 7 samples. After 4 week FTOC, gene-transduced neuroblastoma cells may become the experiment applying sorting and re-seeding of immunogenic and evoke an antitumor effect of the IL-7R+ and IL-7R- cells into second FTOC resulted in body. The addition of lymphotactin to IL-2 is expect- a predominant generation of IL-7R+ cells from both ed to attract lymphocytes to the vaccinated fractions, while IL-7R- cells proliferated more potent- neuroblastoma cells. Preclinical experiments are be- ly in the second FTOC than IL-7R+ cells did, ing performed to show the feasibility of this therapy.
suggesting that a conversion of IL-7R- to IL-7R+ path- Ueda, T., Yoshino, H., Ebihara, Y., Hisakawa, H., of human NOD/SCID-repopulating cells by stem Mitsui, T., Manabe, A., Tanaka, R., Kobayashi, K., cell factor, Flk2/Flt3 ligand, thrombopoietin, Ito, M., Nakahata, T., Tsuji, K.: Ex vivo expansion interleukin-6 and soluble interleukin-6 receptor. J.
Clin. Invest. 105, 1013-1021, 2000.
ing intensified treatment for acute lymphoblastic Ebihara, Y., Xu, M., Manabe, A., Kikuchi A., Tanaka, leukemia of childhood. J. Clin. Oncol. 18, 1508- R., Kato, S., Nakahata, T., Tsuji, K.: Exclusive ex- pression of G-CSF receptor on myeloid progeni- Hayashi Y, Honma Y, Niitsu N, Taki T, Bessho F, Sako tors in bone marrow CD34+ cells. Br. J. Haematol.
M, Mori T, Yanagisawa M, Tsuji K, Nakahata T: SN- 109, 153-161, 2000.
1, a novel leukemic cell line with t(11;16)(q23;p13): Manabe, A., Takahashi, K., Shibata, R., Takeuchi, M., myeloid characteristics and resistance to retinoids Morita, M., Hosoya, R.: Corticosteroids, cyclo- and vitamin D3. Cancer Res. 60:1139-1145, 2000.
phosphamide, and methotrexate induced a long- Nakahata T, Xu M-J, Ueda T, Matsuoka S, Tsuji K: Ex term remission in a patient with natural killer cell vivo expansion of human hematopoietic stem cells.
leukemia. Med. Ped. Oncol. 34, 224-225, 2000.
Cell Therapy. edited by Ikeda Y, Hata J, Koyasu S, Ishii T, Manabe A, Asano S, Ebihara Y, Ueda T, Kawakami Y, Hattori Y (Springer-Verlag, Tokyo).
Yoshino H, Mitsui T, Hisakawa H, Yagasaki H, pp105-106, 2000.
Kikuchi A, Yoshimatsu T, Tanaka R, Masunaga A, Matsuoka, S., Ebihara, Y., Xu M., Yoshino, H, Ueda, K-I Sugita, Nakahata T, Asano S, Tsuji K: Improve- T, Manabe, A., Tanaka, R., Ikeda Y., Nakahata, T. , ment in bronchiolitis obliterans organizing pneu- Tsuji, K.: CD34 expression on long-term repopu- monia by combination of oral predonisolone and lating hematopoietic stem cells changes during low dose erythromycin in a child after allogegeneic developmental stages. Blood, in press.
bone marrow transplantation. Bone Marrow Trans- Yoshimasu T, Tanaka R, Suenobu S, Yagasaki H, plant. 26: 907-910, 2000.
Yoshino H, Hisakawa H, Ishii T, Mitsui T, Ebihara Yoshino H, Ueda T, Kawahata M, Kobayashi K, Y, Manabe A, Nakahata T, Maekawa T, Asano S, Ebihara Y, Manabe A, Tanaka R, Ito M, Asano S, Tsuji K: Prompt and durable hematopoietic recon- Nakahata T, Tsuji K: Natural killer cell depletion stitution by an unrelated cord blood transplanta- by anti-asialo GM1 antiserum treatment enhances tion in a child with Fanconi anemia. Bone Marrow human hematopoietic stem cell engraftment in Transplant., in press.
NOD/Shi-scid mice. Bone Marrow Transplant. 26: Manabe, A., Yoshida, Y.: Fifth international sympo- 1211-1216, 2000.
sium on myelodysplastic syndromes. Int. J.
Ueda T, Yoshino H, Kobayashi K, Kawahata M, Hematol., in press.
Ebihara Y, Ito M, Asano S, Nakahata T, Tsuji K: Manabe A: Second international symposium on Hematopoietic repopulating ability of cord blood myelodysplastic syndromes in childhood. Int J CD34+ cells in NOD/Shi-scid mice. Stem Cells 18: Hematol., in press.
Ueda T, Yoshida M, Yoshino H, Kobayashi K, Ueda, T., Manabe, A., Kikuchi, A., Yoshino, H., Kawahata M, Ebihara Y, Ito M, Asano S, Nakahata Ebihara, Y., Ishii, T., Yagasaki, H., Mitsui, T., T, Tsuji K: Hematopoietic capability of cord blood Hisakawa, H., Tsuji, K., Nakahata, T.: Massive peri- CD34+ cells: a comparison with that of adult bone cardial and pleural effusion with anasarca follow- marrow CD34+ cells. Int. J. Hamatol., in press.
ing allogeneic bone marrow transplantation. Int. J.
Tsuji K, Ueda T: Protocol for cytokine mediated ex- Hematol. 71, 394-397, 2000.
pansion of human NOD/SCID-repopulating cells.
Toyoda, Y., Manabe, A., Tsuchida, M., Hanada, R., Methods in Molecular Medicine-Cytokines and Ikuta, K., Okimoto, Y., Ohara, A., Ohkawa, Y., Colony Stimulating Factors. edited by Korholz D Mori, T., Ishimoto, K., Satoh, T., Kaneko, T., Maeda, and Kiess W (The Human Press, Totowa), in press.
M., Koike, K., Shitara, T., Hoshi, Y., Hosoya, R., Tsuji K, Ebihara Y: Expression of G-CSF receptor on Tsunematsu, Y., Bessho, F., Nakazawa, S., Saito, T.: myeloid progenitors. Leukemia and Lymphoma, Six months of maintenance chemotherapy follow- Research Hospital
Department of Infectious Diseases and
Department of Infectious Diseases and Applied Immunology(DIDAI) was founded in 1981. In 1986, clinic for patients withhuman immunodeficiency virus (HIV) infection was opened byformer professor, K. Shimada. In 2000, approximately 130 pa-tients with HIV infection visit the out-patient clinic on a monthlybasis, and 3-5 beds for HIV-infected patients in the in-patientward are usually occupied. Since the number of the staff mem-bers of DIDAI is too small to care both out-patients andin-patients, members of the Division of Infectious Diseases(DID) of the Advanced Clinical Research Center and Depart-ment of Clinical Immunology & AIDS Research Center join theclinic. Supported by clinicians of three departments, basic sci-entists of immunology and virology in DID and dedicatedmedical and paramedcal stuffs, IMSUT hospital provides themost up-to-date medical treatment to HIV-infected patients inJapan. DIDAI is also a treatment center for international infec-tious diseases such as malaria and typhoid fever.
1. Treatment of and clinical research on HIV-in-
the previous year. Although the number of admis- fection and related diseases
sion this year increased again, reasons for admissionin many cases were adverse effects caused by Tetsuya Nakamura, Takashi Takahashi1, Hitomi
HAART and the number of seriously ill patients due Nakamura1, Tomohiko Koibuchi1, Toshiyuki Miu-
to immunodeficiency-related disorders was still low.
ra1, Tokiomi Endoh1, Miou Sato1, Akihiro Hitani1,
Mieko Goto1 and Aikichi Iwamoto1:1Division of In-
ii) A Randomized, Open-Label, Phase III, International
Study of Subcutaneous Recombinant IL-2 (Proleukin)
in Patients With HIV-1 Infection and CD4+ Cell Counts
a. Treatment of HIV infection in IMSUT hospital
300/mm3 : Evaluation of Subcutaneous Proleukin in
a Randomized International Trial (ESPRIT)
i) Statistical characteristics of HIV-infected patients in
This year we joined international clinical study IMSUT hospital this year
called "ESPRIT" which is organized by National Can- On a monthly basis, approximately 130 patients cer Institute in US. IL-2 is a substance that is have visited the out-patient clinic this year. The normally produced in the body, and act to increase numbers of admission reached peak in 1996, and CD4 cells. CD4 cells assist in defense against infec- then started to decline as shown in the figure below.
tion. The concentrations of IL-2 produced in patients This is due to the success of highly active anti-retro- with HIV infection have been determined to be lower viral therapy (HAART) which was introduced to our than in normal persons. The object of this study is to clinic in 1997. This tendency is also observed in US determine whether bringing about an increase in the and Europe, and Centers for Disease Control and number of CD4 cells by administration of IL-2 leads Prevention (CDC) in US reported that the number of to a decrease in the incidence of onset of HIV-related AIDS death in 1997 decreased by half compared to diseases. In clinical studies conducted thus far, in- creases in CD4 cells have been observed in the vast genotype A carriers, 12 (92%) were men who had sex majority (but not all) patients administered IL-2. It is with men (MSM) while the other was a hemophiliac.
still unknown, however, whether these increases re- It is of note that 10 of these 13 genotype A samples ally improved the health of the patients.
were highly homologous to the strains isolated in This study will be conducted in order to investi- USA. Two of the 3 genotype B carriers reported het- gate the following matters.
erosexual intercourse as a risk factor for both HIV-1 1. Whether or not IL-2 reduces serious infections and HBV infection while the other was a hemophili- related to HIV and prolongs the survival period ac. Two patients were the carriers of genotype C. One in the case it is used concomitantly with other patient reported heterosexual intercourse as a risk factor for both HIV-1 and HBV infection. The other 2. Whether or not IL-2 can be administered safely was MSM who reported sexual intercourse as a risk over an extended period of time to HIV infection factor for HIV-1 infection but vertical transmission for HBV infection.
Approximately 4,000 subjects are scheduled to HIV subtype: The C2-V3 region of HIV-1 env gene
participate in this study from around the world. The was amplified by nested PCR in 15 out of 18 patients study is scheduled to be conducted over the course in whom HBV genotype was studied. One, 12, 1 and of 6 years and twenty patients are assigned to IMSUT 1 HIV-1 were categorized as subtype A, B, C and E, hospital. We already finished necessary paper respectively. Nine of the 12 patients with subtype B works, passed site visit evaluation and are going to HIV-1 were MSM while 2 patients were hemophiliac enroll patients next year.
and one reported heterosexual intercourse as a riskfactor. Both patients with subtype A and subtype E b. Clinical research on HIV infection
HIV-1 reported heterosexual intercourse as a riskfactor. The patient with subtype C was MSM.
i) Predominance of genotype A HBV in HBV-HIV-1 dually
In the present study we found that the genotype A positive population as compared to HIV-1-negative
HBV was predominant among HBV-HIV-1 dually- counterpart in Japan
infected Japanese MSM. Among 13 MSM, one In Annual Report 1999, we described genotypic (patient 6) with genotype C was presumably infected characterization of hepatitis B virus (HBV) in Japan.
with HBV perinatally because his mother was HBV- We extended this year the clinical research in this positive and he had been HBV-positive before he field to relationship between HIV and HBV geno- became HIV-1-positive. Consequently, all MSM pa- tients who were infected with HBV through sexual HBVgenotype: HBV DNA fragments including
intercourse had genotype A HBV. This result dis- the complete S gene were amplified from 18 Japanese closed a striking difference from the previous study patients with HIV-1-infection. These 18 sequences which showed that genotype C was the most preva- together with 16 sequences reported in the literature lent HBV in the Far East including Japan.
were compared by UPGMA method and a phyloge- Considering the decreasing rate of vertical transmis- netic tree was constructed. Thirteen out of 18 S gene sion of genotype C, the genotype map in Japan may sequences were categorized as genotype A, 3 were look different in the future.
genotype B and 2 were genotype C. Among the 13 Figure. Number of annual admission of HIV-infected patients
ii) Desensitization of fluconazole hypersensitivity
ful since the patient did not develop any adverse re- Amphotericin B and azole antifungal drugs are actions during those 7 days. Although the key agents which are used to treat cryptococcal men- desensitization protocol should be evaluated in a ingitis associated with AIDS. Fluconazole offers clinical trial involving representative number of several advantages over amphotericin B since it is HIV-infected individuals, it seems that both 7-day available in an oral formulation and has a profile of and 15-day desensitization regimens can be applied more favorable adverse effects. We reported this to HIV-infected patients with hypersensitivity to flu- year a case who experienced hypersensitivity reac- conazole under careful observation for the tion to fluconazole but was successfully desensitized development of a rash and a fever.
A 53-year-old HIV-positive Japanese man was ad- 2. Diagnosis and Treatment of Tropical Diseases
Tetsuya Nakamura, Akihiro Hitani1, Mikio Kimura2
X-ray film showed multiple nodular shadows in the and Aikichi Iwamoto1:2Infectious Disease Surveil-
right upper lobe which was diagnosed as pulmonary lance Center, National Institute of Infectious Disease
cryptococcosis. He was administered fluconazole400 mg intravenously on April 22, but developed hy- This year, we treated 16 patients with malaria among which 10 had falciparum malaria. Three cases erythematous rash on his face and body. After ob- with falciparum malaria were severely infected and taining his informed consent, we administered him successfully treated with intravenous quinine and gradually-increased doses of the drug for 7 days for hemodialysis. We also treated two cases with dengue desensitization purpose. The single oral dosages of fever, one amebiasis, one cutaneous larva migrans, fluconazole on days 1, 2, 3, 4, 5, 6 and 7 were 5, 10, 20, one ocular larva migrans, two trichuriasis, one schis- 50, 100, 200 and 400 mg, respectively. After that, he tosomiasis, one giardiasis, and two tapeworm was administered 400 mg of fluconazole orally once a day. The desensitization regimen proved success- Katano, H., Suda, T., Morishita, Y., Yamamoto, K., deficiency virus type 1 infection. Arch. Virol.
Hoshino, Y., Nakamura, K., Tachikawa, N., Sata, T., Hamaguchi, H., Iwamoto, A., and Mori, S. Hu- Taguchi, H., Yamada, T., Takahashi, T., Gotoh, M., man herpesvirus 8-assosiated solid lymphomas Nakamura, T., and Iwamoto, A. Seroprevalence of that occur in AIDS patients take anaplastic large parvovirus B19 among HIV-1-positives in Japan.
cell morphology. Modern Pathology 13:77-85, Jpn. J. Infect. Dis. 53:21, 2000.
Wu, X., Okada, N., Goto, M., Iwamoto, A., and Aoki, Y., Tosato, G., Nambu, Y., Iwamoto, A., and Okada, H. The IgM antibody level against gan- Yarchoan R. Detection of vascular endothelial glioside GM2 correlates to the disease status of growth factor in AIDS-related primary effusion HIV-1-infected patients. Microbiol. Immunol.
lymphomas. Blood 95:1109-1110, 2000.
He, L., Terunuma, H., Hanabusa, H., Iwamoto, A., Nakayama, E.E., Wasi, C., Ajisawa, A., Iwamoto, A., Oka, S., Tanabe, F., Chiba, N., Kurimoto, M., and Shioda, T. A new polymorphism in the pro- Ikeda, M., Okamura, H., Dai, J., Iwatani, Y., Ishida, moter region of the human interleukin-16 (IL-16) T., and Ito, M. Interleukin 18 and interleukin 1β gene. Genes and Immunity 1: 293-294, 2000.
production is decreased in HIV type 1-seroposi- Nakayama, E.E., Hoshino, Y., Xin, X., Liu, H., Goto, tive hemophiliacs but not in HIV type 1-seroposi- M., Watanabe, N., Taguchi, H., Hitani, A., tive nonhemophiliacs. AIDS Research and Human Kawana-Tachikawa, A., Fukushima, M., Yamada, Retroviruses 16:345-353, 2000.
K., Sugiura, W., Oka, S., Sato, H., Takebe, Ajisawa, Katano, H., Iwasaki, T., Baba, N., Terai, M., Mori, S., A., Y., Nakamura, T., Nagai, Y., Iwamoto, A., and Iwamoto, A., Kurata, T., and Sata, T. Identification Shioda, T. Polymorphism in the IL-4 promoter af- of antigenic proteins encoded by human herpes fects acquisition of HIV-1 syncytium inducing (SI) virus 8 and the seroprevalence in the general phenotype. J. Virol. 74:5452-5459, 2000.
population and among patients with and without Hosoya, N., Takahashi, T., Wada, M., Endo, T., Kaposi's sarcoma. J. Virol. 74:3478-3485, 2000.
Nakamura, T., Sakashita, H., Kimura, K., Ohnishi, Yamada, T., and Iwamoto, A. Comparison of provi- K., Nakamura, Y., Mizuochi, T., and Iwamoto, A.
ral accessory genes between long-term non- Genotyping of Pneumocystis carinii f. sp. hominis progressors and progressors of human immuno- isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of Kato, K., Miyazaki, T., Nakamura, T. and Kudo, A.
rRNA genes. Microbiol. Immunol. 44:591-596, Inducible differentiation and apoptosis of the pre- B cell receptor-positive pre-B cell line. Intern.
Yoshida, S., Yusa, N., Sato, N., Goto, M., and Immunol. 12: 325-34, 2000.
Iwamoto, A. Some problems found in HIV-1 RNA Takahashi, T., Hosoya, N., Endo, T., Nakamura, T., quantification. J. Clin. Pathol. 53:645-646, 2000.
Sakashita, H., Kimura, K., Ohnishi, K., Nakamura, Aoki, Y., Yarchoan R., Braun, J., Iwamoto, A., and Y., and Iwamoto, A. Relationship between muta- Tosato, G. Viral and cellular cytokines in AIDS-re- tions in dihydropteroate synthase of Pneumocystis lated malignant lymphomatous effusion. Blood carinii f. sp. hominis isolates in Japan and resistance 96:1599-1601, 2000.
to sulfonamide therapy. J. Clin. Microbiol. 38:3161- Miyashita, N., Niki, Y., Iwamoto, A., Yasuoka, A., Oka, S., Kawata, K., Ito, A., Tomono, K., Kohno, S., Takahashi, T., Nakamura, Y., and Iwamoto, A. De- and Matsushima, T. Seroprevalence of antibodies sensitization to fluconazole in an AIDS patient.
to Chlamydia spp. In human immunodeficiency Annals Pharmacotherapy. In press.
virus-infected subjects in Japan. Microbiol.
Koibuchi, T., Hitani, A. Nakamura, T., Nojiri, N., Immunol. 44:781-785, 2000.
Nakajima, K., Jyuji, T., and Iwamoto, A. Predomi- Hase, H., Tani, K., Nagayama, H., Watari, K., nance fo genotype A HBV in HBV-HIV-1 dually Takahashi, S., Ooi, J., Shirafuji, N., Iseki, T., positive population as compared to HIV-1-negative Nakazaki, Y., Yamashita, T., Nakamura, T., counterpart in Japan. J. Med. Virol. In press.
Masunaga, A., Maekawa, T., Tojo, A. and Asano, Shioda, T., Nakayama, E.E., Tanaka, Y., Xin, X., Liu, S. TCR-Vbeta repertoire analysis with RT-PCR H., Kawana-Tachikawa, A., Kato, A., Sakai, Y., was useful for the early detection of pulmonary Nagai, Y., and Iwamoto, A. A naturally occurring relapsed T-cell lymphoma after autologous pe- deletional mutation in the C-terminal cytoplasmic ripheral blood stem cell transplantation. Am J tail of CCR5 affects surfacee trafficking of CC5. J.
Hematol 64: 124-7, 2000.
Virol. In press.
Department of Advanced Medical Science
Department of Advanced Medical Science was established inSeptember 1997. We are investigating (1) gene therapy for GHdeficiency, (2) Identification of genes involved in outflow tractformation during embryonic heart development, (3) transfor-m a t i o n o f g r a f t e d m u s c l e b y B M P - 2 , ( 4 ) h u m a nimmunotherapy for malignancies using dendritic cells, and (5)human gene therapy for neuroblastoma. We are planning andprogressing several projects described below to develop a newtherapy for several diseases, including GH deficiency and carci-nomas.
1. Gene therapy for growth hormone (GH)-defi-
3Y1 cells together with puromycin-resistance plas- mid. Lots of colonies were picked up after selectionwith puromycin, and among them some stable GH Inazawa T. et al.
producing cell lines were obtained. These cells pro-duced 20K GH in response to glucocorticoid Growth hormone (GH) deficiency causes growth stimulation up to 6 fold of the basal level in vitro. In disturbance in children. For treatment of GH defi- vivo production of 20K GH responding to glucocorti- ciency, injection of recombinant human GH (hGH) is coid administration was also detected when the cells undertaken in general. However, the recombinant were microcapsulelated and transplanted into ab- hGH is so expensive that it is near 40% of total medi- dominal cavity of rats. Intraabdominal application of cal cost for chronic diseases of children in Japan. In dexamethasone increased plasma hGH concentra- addition, it is troublesome for children to inject hGH tion, indicating that hGH expression is also every day. Therefore more convenient and economic regulated in vivo. We are incorporating hGH gene therapy for GH deficiency is expected. We intended into an AAV vector, which is expected to express to treat GH deficiency using ex vivo gene therapy.
hGH more efficiently.
Human 20K GH gene was incorporated into VSV-Gpseudotype retrovirus vector, and introduced into 2. Identification of genes involved in outflow
human skin fibroblasts, primate skin fibroblasts, or tract formation during embryonic heart devel-
bone marrow stromal cells. Not only production of 20KGH by those cells has been detected in vitro, butin vivo expression of 20K GH for more than a month Nakaoka T. et al.
was also confirmed in primates which was intrave-nously injected with transduced bone marrow Malformations of the cardiovascular system ac- stromal cells.
count for most of the premature deaths caused by We have another project to make a circadian congenital abnormalities. Of these, the majorities are rhythm of 20KGH. Naturally GH is secreted during congenital heart defects that arise from the abnormal sleep. We intend to create GH secretion rhythm using remodeling of the single heart tube into four separate endogenous circadian rhythm of glucocorticoids.
and properly aligned chambers. The importance of The 20K GH gene was inserted into downstream of understanding the origin and fate of the outflow glucocorticoid-sensitive LTR of pMSG plasmid. The tract segment and associated cushions is that this site obtained 20K GH-pMSG plasmid was lipofected into is most likely to be related to birth defects of the heart of the histological sections and roentgenographical in humans. In fact, this is a very common site of mal- radio-opacity of the region. It was also found that the formations that result from a wide variety of BMP-2 transgene over-expressed in grafted muscles experimental perturbations in vertebrate models of inhibited muscle regeneration, which should other- congenital heart disease. The heart defect (hdf) wise follow the muscle degeneration. It is mouse is a recessive lethal that arises from a trans- noteworthy that a thick cortex with vast marrow si- gene insertional mutation on chromosome 13.
nus was formed and bone remained even one year Embryos homozygous for the transgene die in utero after the gene transfer. In conclusion, over-expres- by some 11.5 d.p.c. The future right ventricle and sion of BMP-2 gene induced massive heterotopic outflow tract fail to form and endocardial cushions ossification in skeletal muscles under graft-induced are absent in this homozygote. As a result of pursuit ischemic degeneration, which possibly up-regulates of responsible gene, it was found Cspg2 (versican) osteoprogenitor cells in situ. This modality requires a gene is disrupted in hdf mouse. This information is modification in terms of clinical application, because helpful to understand the importance of extra-cellu- muscle regeneration differs between human and ro- lar matrix in the process of heart formation; however, dents. We are now under way.
it is still enigmatic how lack of expression of Cspg2gene leads to defect of embryonic heart develop- 4. Immunotherapy for malignancies using den-
ment. In order to address this question, we collected dritic cells
total RNA from the embryonic heart of homozygote,heterozygote and normal littermate at 9.5 d.p.c. Af- Yamashita N. & Morishita M. et al.
ter subtractive hybridization, several candidateclones for differentially expressed gene in hdf mouse Malignant melanoma is an intractable disease and at 9.5 d.p.c. were obtained. We are now under pro- its prognosis is poor when the disease progresses to cess of elucidation of truly up-regulated or down- stage IV. We are finishing phase I study of immuno- regulated genes among them and involvement of therapy using dendritic cells (DCs) to stage IV these genes for the embryonic heart development.
melanoma patients. The procedure to make matureDCs is as follows: periferal mononuclear cells are col- 3. Transformation of grafted muscle by BMP-2
lected using apheresis. Adherent cells to culturedishes are collected and GM-CSF and IL-4 are added Nakaoka T. et al.
to culture medium to make immature DCs. Tumorlysate is applied to immature DCs and further cul- Bone transformation of a grafted skeletal muscle tured in the presence of TNF-α. Thus obtained would be very useful for treating impaired regional mature DCs are intracutaneously injected to the pa- bone formation, including delayed or non-bone frac- tients once a week. In conjunction with application of ture union, congenital pseudoarthrosis occurring DCs rIL-2 is subcutaneoulsy injected. During 10 either alone or in association with von Recklinghaus- weeks the administration of DCs is continued. After en's disease, and segmental bone defects after therapy clinical evaluations were done. From 1999 to trauma, osteomyelitis, or tumor resection. First to 2000 ten patients entered this study protocol. Tumor test whether high level of expression of BMP-2 in- progression was stopped in one patient. In two pa- duces bone formation in vivo, adenoviruses carrying tients obvious regressions of metastatic tumors were BMP-2 gene (AxCABMP2) were directly injected into observed. Safety of this therapy was proved and the the soleus muscle of adult rat. The BMP-2 gene was activation of tumor immunity has been suggested.
successfully over-expressed in the target muscle by Thyroglobulin (Tg) plays a central role in thyroid adenovirus-mediated transfer, whereas bone forma- pathophysiology. Most differentiated thyroid carci- tion in and around the muscle failed to occur in this nomas and some anaplastic thyroid carcinomas case. It was hypothesized that this failure was due to express thyroglobulin, and the tissue-specific origin a lack of putative osteoprogenitor cells in the normal of Tg has led to its use as a marker for thyroid cancer muscle. Therefore, in order to recruit putative os- especially in patients without residual normal thy- teoprogenitor cells, we then induced ischemic roid tissue. In general, the majority of patients with degeneration of the target muscle by orthotopically differentiated thyroid carcinoma have a good prog- grafting it simultaneously with the gene transfer.
nosis, but some high risk cases with large metastases The combination of BMP-2 gene transfer and ortho- cause death despite surgical resection, radioiodine, topic muscle grafting resulted in successful or external beam irradiation. Therefore we intended ossification of entire region of the grafted muscle, to treat metastatic thyroid cancer by immunotherapy whereas neither muscle grafting alone nor the com- using Tg pulsed dendritic cells (DCs). Some experi- bination of muscle grafting and adenovirus- mental autoimmune thyroiditis caused by Tg have mediated transfer of reporter gene, LacZ, induced been reported in animals. Now we are trying to de- any bone formation in the muscle. The ossification tect human T cell clones stimulated by Tg pulsed process was evident by positive von Kossa staining DCs in vitro and to project clinical study.
long-term survival. This failure has lead to a resur-gence of interest in alternative methods of disease 5. Human gene therapy for neuroblastoma
eradication, immune modulation in particular. Wehave finished the preparation of clinical gene thera- Yamashita N. et al.
py protocol for neuroblastoma in collaborations withProfessor Brenner in Baylor University of Texas. The Neuroblastoma is the most common extracranial aims of this study are as follows; (1) to determine the solid tumor of childhood. When the tumor occurs in safety up to four subcutaneous (SC) injections of au- infants (< 1 year age), it is frequently localized and tologous neuroblastoma cells, which have been responds well to therapy. However in older children genetically modified by adenoviral vectors to secrete (> 1year age) the prognosis is far worse. Although lymphotactin and interleukin-2, (2) to determine the patients with localized disease may still be cured by safety of up to eight (total) injections in patients who conventional therapy, 80% or more of those with dis- have received the first four injections without unac- seminated tumor can be expected to relapse within 3 ceptable toxicity and have evidence of stable disease years, and virtually none of this subgroup will be- or better after receiving these injections, (3) to deter- come long-term survivors. Over the past decade, mine whether MHC restricted or unrestricted attempts to improve the outcome of advanced neuro- antitumor immune responses are induced by SC in- blastoma have focused on greater intensification of jection of modified autologous neuroblastomas and the induction and consolidation phases of chemo-ra- the cell doses required to produce these effects, (4) to diotherapy, with or without stem cell rescue.
obtain preliminary data on the antitumor effects of Although remission rates have been increased, there this treatment regimen. After permission by ethical is no evidence at all of significant improvement in committees this clinical study is going to start.
Gonda K, Nakaoka T, Yoshimura K, Otawara- Maekawa T, Tojo A, & Asano S. TCR-Vbeta reper- Hamamoto Y, & Harii K. Heterotopic Ossification toire analysis with RT-PCR was useful for the of Degenerating Rat Skeletal Muscle Induced by early detection of pulmonary relapsed T-cell lym- Adenovirus-Mediated Transfer of Bone Morpho- phoma after autologous peripheral blood stem cell genetic Protein-2 Gene. J Bone Miner Res 15:1056- transplantation. Am J Hematol 64:124-7, 2000 Machida U, Tojo A, Takahashi S, Iseki T, Ooi J, Yamashita N, Hashimoto Y, Honjo M. & Yamashita Nagayama H, Shirafuji N, Mori S, Wada Y, Ogami N. The effect of growth hormone on the prolifera- K, Yamada Y, Sakamaki H, Maekawa T, Tani K, & tion of human Th cell clones. Life Sciences, Asano S. The effect of granulocyte colony-stimu- 66:1929-1935, 2000 lating factor administration in healthy donors be- Machida U, Tojo A, Ooi J, Iseki T, Nagayama H, fore bone marrow harvesting. Br J Haematol Shirafuji N, Sawada M, Nakayama K, Tani K, & Asano S. Refractory facial cellulitis following cos- Mori, N., Morishita, M., Tsukazaki, T., Giam, C-Z., metic rhinoplasty after cord-blood stem cell trans- Kumatori, A., Tanaka, Y., & Yamamoto, N. The plantation. Int J Hematol 72:98-100, 2000 human T-cell leukemia virus type-1 oncoprotein Hase H, Tani K, Nagayama H, Watari K, Takahashi Tax represses Smad-dependent transforming S, Ooi J, Shirafuji N, Iseki T, Nakazaki Y, growth factor beta signaling via interaction with Yamashita T, Nakamura T, Masunaga A, CREB binding protein/p300. Blood, in press.
Department of Laboratory Medicine
Our research interest is divided into two major projects. Theone is to understand the molecular mechanisms underlying dif-ferentiation of normal granulocytes from multipotent stemcells. And the other is to find single-nucleotide polymorphisms(SNPs) in genes relevant to the progression and drug-sensitiv-ities of chronic myelogenous leukemia.
1. Studies on the nuclear factors participating in
tablished stable NAP-expressing cell line. The result- the transcriptional regulation of alkaline phos-
ing transformants showed increased capacity to phatase gene in neutrophils
phagocytose zymosan, suggesting the role of alka-line phosphatase in active phagocytosis. In addition Nozomi Yusa, Satoru Yoshida, Kunihito Watanabe1,
they showed increased ability to adhere to type I col- Shigetaka Asano2, Kenzaburo Tani2 and Noriharu
lagen, suggesting its role in the case of neutrophil Sato:1Institute of Bio-medical Research, Teijin Ltd.
migration in the extravascular tissue.
and 2Department of Hematology-Oncology
3. Molecular diagnosis of chronic myelogenous
Using transient transfection assay, we found that leukemia and acute promyelocytic leukemia
about 150 bp DNA fragment in the promoter region by nested RT-PCR
that contained TATA box was essential for its basalpromoter activity. Since the fragment also contained Mayumi Karikomi, Nozomi Yusa, Satoru Yoshida
3 GC-boxes, we examined Sp1 family of transcrip- and Noriharu Sato
tional factors if they bound to the DNA. Using gelshift assay and Southwestern method, we found Sp3 Using nested RT-PCR, we are now able to detect could bind to the fragment. When the expression minimal residual leukemic cells in patients with vector with minimal promoter sequence was co- CML or APL who were treated by allogeneic stem transfected with Sp3 expression vector into cell transplantation. We are carefully monitoring Schneider cells, Sp3 was shown to activate basal pro- these patients after hematopoietic stem cell trans- moter of alkaline phosphatase gene.
2. Functional characterization of alkaline phos-
4. Quantitative HIV-1 RNA testing
phatase in granulocytes
Satoru Yoshida, Nozomi Yusa and Noriharu Sato
Satoru Yoshida, Nozomi Yusa, Mayumi Karikomi
and Noriharu Sato
We have began to quantitate HIV-1 RNA levels since January of 1998 and the number of assayed Alkaline phosphatase is expressed only in cells samples is going to amount to 2,200 as of the end of belonging to neutrophilic lineage in the hematopoie- this year. This test provides reliable data for disease tic tissue and called neutrophil alkaline phosphatase prognosis and important informations for treatment (NAP). Although NAP is regulated by G-CSF in vivo, of the disease, since serial monitoring of HIV levels its function in neutrophls is largely unknown. In or- let us know when to start the treatment or to change der to explore the function of NAP, we transfected the drugs. In addition, since HIV-1 RNA subtype A alkaline phosphatase cDNA into U937 cells and es- and E have some problems of amplification with cur- rent assay kits, we have studied the application of 6. Functional role of Fas and Fas-ligand in glio-
improved primers for these subtypes. In order to ma cell lines
monitor HIV-1 RNA levels under the current detec-tion limit (<400 copies/ml), we are also using Hisaaki Shinihara3, Hideo Yagita4, Yoji Ikawa3 and
hype-sentitive assay methods of HIV-1 RNA.
Naoki Oyaizu:3Tokyo Medical and Dental Univer-
sit and 4Juntendo University School of Medicine
5. Assay for phagocytic activity of neutrophils
and other hematopoietic cells.
We found that Fas transduce signals leading to ap- optosis and, to our surprise, cell growth as well in Satoru Yoshida, Mayumi Karikomi, Nozomi Yusa
gliomas. All the glioma cell lines examined were and Noriharu Sato
found to express functional FasL and blocking en-dogenous Fas/FasL interaction resulted in reduced We developed E.coli expressing green fluorescent cell growth. Further, we show evidence that Fas-me- protein (GFP). Using these cells as the targets of ph- diated growth signal was closely linked to agocytic cells, we can measure the percentage of cells extracellular signal regulated kinase (ERK) activa- that phagocytized these GFP positive-cells. In addi- tion. Regarding the role of Fas/FasL system in tion this system may pave the way for multi-colour tumorigenesis, our present study provide an impor- analysis with various kinds of antibodies to define tant implication that some tumors may make use of the characteristics of individual phagocytozing cells.
this system for further progression not only by evad-ing immune surveillance but also by promoting itsown growth.
Satoru Yoshida, Nozomi Yusa, Noriharu Sato, Mieko relation of loss of CD4 T cells with plasma levels of Goto, Aikichi Iwamoto. Some problems found in both soluble form Fas (CD95) and Fas ligand HIV-RNA quantification. J. Clin. Pathol. 53:645- (FasL) in HIV-infected patients. Clin. Immunol.
Nozomi Yusa, Kunihito Watanabe, Satoru Yoshida, Tateyama, M., Oyaizu, N. McCloskey TW, Than S, Naoki Shirafuji, Satoshi Shimomura, Kenzaburo and Pahwa S. Priming for the induction of FasL- Tani, Shigetaka Asano, Noriharu Sato. Transcrip- expressing CD4+ cytotoxic T lymphocytes by CD4 tion factor Sp3 activates the liver/bone/kidney- crosslinking: Possible contribution to bystander type alkaline phosphatase promoter in hemato- CD8+ T cell death in HIV infection. Blood (in poietic cells. J. Leukoc. Biol 68: 772-777, 2000 Shinohara, H., Yagita, H., Ikawa, Y., and Oyaizu, N. : Fas drives cell cycle progression in glioma cells viaextracellular signal regulated kinase (ERK1/2) ac-tivation. Cancer Res. 60:1766-1772, 2000 Hosaka, N., Oyaizu, N., Than, S., and Pahwa, S. Cor- Research Hospital
Department of Clinical AIDS Research
Department of Clinical Immunology & AIDS Research wasfounded in 1995 to provide medical care for patients with hu-man immunodeficiency virus (HIV) infection and HIV-relatedopportunistic disorders. In 2000, approximately 130 patientswith HIV infection visit the out-patient clinic on a monthly ba-sis, and 3-5 beds for HIV-infected patients in the in-patientward are usually occupied. In collaboration with Department ofInfectious Diseases & Applied Immunology and Division of In-fectious Diseases (DID) of the Advanced Clinical ResearchCenter, our department contributes medical management ofHIV-infected patients in IMSUT hospital.
1. Treatment of and clinical research on HIV-in-
ii) A Randomized, Open-Label, Phase III, International
fection and related diseases
Study of Subcutaneous Recombinant IL-2 (Proleukin)
in Patients With HIV-1 Infection and CD4+ Cell Counts
Tetsuya Nakamura, Takashi Takahashi1, Hitomi
300/mm3 : Evaluation of Subcutaneous Proleukin in
Nakamura1, Tomohiko Koibuchi1, Toshiyuki Miu-
a Randomized International Trial (ESPRIT)
ra1, Tokiomi Endoh1, Miou Sato1, Akihiro Hitani1,
This year we joined international clinical study Mieko Goto1 and Aikichi Iwamoto1:1Division of In-
called "ESPRIT" which is organized by National Can- cer Institute in US. IL-2 is a substance that isnormally produced in the body, and act to increase a. Treatment of HIV infection in IMSUT hospital
CD4 cells. CD4 cells assist in defense against infec-tion. The concentrations of IL-2 produced in patients i) Statistical characteristics of HIV-infected patients in
with HIV infection have been determined to be lower IMSUT hospital this year
than in normal persons. The object of this study is to On a monthly basis, approximately 130 patients determine whether bringing about an increase in the have visited the out-patient clinic this year. The number of CD4 cells by administration of IL-2 leads numbers of admission reached peak in 1996, and to a decrease in the incidence of onset of HIV-related then started to decline as shown in the figure below.
diseases. In clinical studies conducted thus far, in- This is due to the success of highly active anti-retro- creases in CD4 cells have been observed in the vast viral therapy (HAART) which was introduced to our majority (but not all) patients administered IL-2. It is clinic in 1997. This tendency is also observed in US still unknown, however, whether these increases re- and Europe, and Centers for Disease Control and ally improved the health of the patients.
Prevention (CDC) in US reported that the number of This study will be conducted in order to investi- AIDS death in 1997 decreased by half compared to gate the following matters.
the previous year. Although the number of admis- 1. Whether or not IL-2 reduces serious infections re- sion this year increased again, reasons for admission lated to HIV and prolongs the survival period in in many cases were adverse effects caused by the case it is used concomitantly with other HIV HAART and the number of seriously ill patients due to immunodeficiency-related disorders was still low.
2. Whether or not IL-2 can be administered safely over an extended period of time to HIV infectionpatients.
Approximately 4,000 subjects are scheduled to closed a striking difference from the previous study participate in this study from around the world. The which showed that genotype C was the most preva- study is scheduled to be conducted over the course lent HBV in the Far East including Japan.
of 6 years and twenty patients are assigned to IMSUT Considering the decreasing rate of vertical transmis- hospital. We already finished necessary paper sion of genotype C, the genotype map in Japan may works, passed site visit evaluation and are going to look different in the future.
enroll patients next year.
ii) Desensitization of fluconazole hypersensitivity
b. Clinical research on HIV infection
Amphotericin B and azole antifungal drugs are i) Predominance of genotype A HBV in HBV-HIV-1 dually
key agents which are used to treat cryptococcal men- positive population as compared to HIV-1-negative
ingitis associated with AIDS. Fluconazole offers counterpart in Japan
several advantages over amphotericin B since it is In Annual Report 1999, we described genotypic available in an oral formulation and has a profile of characterization of hepatitis B virus (HBV) in Japan.
more favorable adverse effects. We reported this We extended this year the clinical research in this year a case who experienced hypersensitivity reac- field to relationship between HIV and HBV geno- tion to fluconazole but was successfully desensitized HBVgenotype: HBV DNA fragments including A 53-year-old HIV-positive Japanese man was ad- the complete S gene were amplified from 18 Japanese patients with HIV-1-infection. These 18 sequences cytomegalovirus retinitis on March 29, 2000. Chest X- together with 16 sequences reported in the literature ray film showed multiple nodular shadows in the were compared by UPGMA method and a phyloge- right upper lobe which was diagnosed as pulmonary netic tree was constructed. Thirteen out of 18 S gene cryptococcosis. He was administered fluconazole 400 sequences were categorized as genotype A, 3 were mg intravenously on April 22, but developed hyper- genotype B and 2 were genotype C. Among the 13 sensitivity reaction such as fever and erythematous genotype A carriers, 12 (92%) were men who had sex rash on his face and body. After obtaining his in- with men (MSM) while the other was a hemophiliac.
formed consent, we administered him gradually- It is of note that 10 of these 13 genotype A samples increased doses of the drug for 7 days for desensitiza- were highly homologous to the strains isolated in tion purpose. The single oral dosages of fluconazole USA. Two of the 3 genotype B carriers reported het- on days 1, 2, 3, 4, 5, 6 and 7 were 5, 10, 20, 50, 100, 200 erosexual intercourse as a risk factor for both HIV-1 and 400 mg, respectively. After that, he was adminis- and HBV infection while the other was a hemophili- tered 400 mg of fluconazole orally once a day. The ac. Two patients were the carriers of genotype C. One desensitization regimen proved successful since the patient reported heterosexual intercourse as a risk patient did not develop any adverse reactions during factor for both HIV-1 and HBV infection. The other those 7 days. Although the desensitization protocol was MSM who reported sexual intercourse as a risk should be evaluated in a clinical trial involving repre- factor for HIV-1 infection but vertical transmission sentative number of HIV-infected individuals, it for HBV infection.
seems that both 7-day and 15-day desensitization reg- HIV subtype: The C2-V3 region of HIV-1 env gene
imens can be applied to HIV-infected patients with was amplified by nested PCR in 15 out of 18 patients hypersensitivity to fluconazole under careful observa- in whom HBV genotype was studied. One, 12, 1 and tion for the development of a rash and a fever.
1 HIV-1 were categorized as subtype A, B, C and E,respectively. Nine of the 12 patients with subtype B 2. Diagnosis and Treatment of Tropical Diseases
HIV-1 were MSM while 2 patients were hemophiliacand one reported heterosexual intercourse as a risk Tetsuya Nakamura, Akihiro Hitani1, Mikio Kimura2
factor. Both patients with subtype A and subtype E and Aikichi Iwamoto1:2Infectious Disease Surveil-
HIV-1 reported heterosexual intercourse as a risk lance Center, National Institute of Infectious Disease
factor. The patient with subtype C was MSM.
In the present study we found that the genotype A This year, we treated 16 patients with malaria HBV was predominant among HBV-HIV-1 dually- among which 10 had falciparum malaria. Three cases infected Japanese MSM. Among 13 MSM, one with falciparum malaria were severely infected and (patient 6) with genotype C was presumably infected successfully treated with intravenous quinine and with HBV perinatally because his mother was HBV- hemodialysis. We also treated two cases with dengue positive and he had been HBV-positive before he fever, one amebiasis, one cutaneous larva migrans, became HIV-1-positive. Consequently, all MSM pa- one ocular larva migrans, two trichuriasis, one schis- tients who were infected with HBV through sexual tosomiasis, one giardiasis, and two tapeworm intercourse had genotype A HBV. This result dis- Katano, H., Suda, T., Morishita, Y., Yamamoto, K., K., Nakamura, Y., Mizuochi, T., and Iwamoto, A.
Hoshino, Y., Nakamura, K., Tachikawa, N., Sata, Genotyping of Pneumocystis carinii f. sp. hominis T., Hamaguchi, H., Iwamoto, A., and Mori, S.
isolates in Japan based on nucleotide sequence Human herpesvirus 8-assosiated solid lympho- variations in internal transcribed spacer regions of mas that occur in AIDS patients take anaplastic rRNA genes. Microbiol. Immunol. 44:591-596, large cell morphology. Modern Pathology 13:77- Yoshida, S., Yusa, N., Sato, N., Goto, M., and Aoki, Y., Tosato, G., Nambu, Y., Iwamoto, A., and Iwamoto, A. Some problems found in HIV-1 RNA Yarchoan R. Detection of vascular endothelial quantification. J. Clin. Pathol. 53:645-646, 2000.
growth factor in AIDS-related primary effusion Aoki, Y., Yarchoan R., Braun, J., Iwamoto, A., and lymphomas. Blood 95:1109-1110, 2000.
Tosato, G. Viral and cellular cytokines in AIDS-re- He, L., Terunuma, H., Hanabusa, H., Iwamoto, A., lated malignant lymphomatous effusion. Blood Oka, S., Tanabe, F., Chiba, N., Kurimoto, M., 96:1599-1601, 2000.
Ikeda, M., Okamura, H., Dai, J., Iwatani, Y., Ishida, Miyashita, N., Niki, Y., Iwamoto, A., Yasuoka, A., T., and Ito, M. Interleukin 18 and interleukin 1β Oka, S., Kawata, K., Ito, A., Tomono, K., Kohno, S., production is decreased in HIV type 1-seroposi- and Matsushima, T. Seroprevalence of antibodies tive hemophiliacs but not in HIV type 1-seroposi- to Chlamydia spp. In human immunodeficiency tive nonhemophiliacs. AIDS Research and Human virus-infected subjects in Japan. Microbiol.
Retroviruses 16:345-353, 2000.
Immunol. 44:781-785, 2000.
Katano, H., Iwasaki, T., Baba, N., Terai, M., Mori, S., Hase, H., Tani, K., Nagayama, H., Watari, K., Iwamoto, A., Kurata, T., and Sata, T. Identification Takahashi, S., Ooi, J., Shirafuji, N., Iseki, T., of antigenic proteins encoded by human herpes Nakazaki, Y., Yamashita, T., Nakamura, T., virus 8 and the seroprevalence in the general Masunaga, A., Maekawa, T., Tojo, A. and Asano, S.
population and among patients with and without TCR-Vbeta repertoire analysis with RT-PCR was Kaposi's sarcoma. J. Virol. 74:3478-3485, 2000.
useful for the early detection of pulmonary re- Yamada, T., and Iwamoto, A. Comparison of provi- lapsed T-cell lymphoma after autologous periph- ral accessory genes between long-term non- eral blood stem cell transplantation. Am J Hematol progressors and progressors of human immuno- 64: 124-7, 2000.
deficiency virus type 1 infection. Arch. Virol.
Kato, K., Miyazaki, T., Nakamura, T. and Kudo, A.
Inducible differentiation and apoptosis of the pre- Taguchi, H., Yamada, T., Takahashi, T., Gotoh, M., B cell receptor-positive pre-B cell line. Intern.
Nakamura, T., and Iwamoto, A. Seroprevalence of Immunol. 12: 325-34, 2000.
parvovirus B19 among HIV-1-positives in Japan.
Takahashi, T., Hosoya, N., Endo, T., Nakamura, T., Jpn. J. Infect. Dis. 53:21, 2000.
Sakashita, H., Kimura, K., Ohnishi, K., Nakamura, Wu, X., Okada, N., Goto, M., Iwamoto, A., and Y., and Iwamoto, A. Relationship between muta- Okada, H. The IgM antibody level against gan- tions in dihydropteroate synthase of Pneumocystis glioside GM2 correlates to the disease status of carinii f. sp. hominis isolates in Japan and resistance HIV-1-infected patients. Microbiol. Immunol.
to sulfonamide therapy. J. Clin. Microbiol. 38:3161- 44:405-410, 2000.
8. Nakayama, E.E., Wasi, C., Ajisawa, A., Iwamoto, Takahashi, T., Nakamura, Y., and Iwamoto, A. De- A., and Shioda, T. A new polymorphism in the sensitization to fluconazole in an AIDS patient.
promoter region of the human interleukin-16 (IL- Annals Pharmacotherapy. In press.
16) gene. Genes and Immunity 1: 293-294, 2000.
Koibuchi, T., Hitani, A. Nakamura, T., Nojiri, N., Nakayama, E. E., Hoshino, Y., Xin, X., Liu, H., Goto, Nakajima, K., Jyuji, T., and Iwamoto, A. Predomi- M., Watanabe, N., Taguchi, H., Hitani, A., nance fo genotype A HBV in HBV-HIV-1 dually Kawana-Tachikawa, A., Fukushima, M., Yamada, positive population as compared to HIV-1-nega- K., Sugiura, W., Oka, S., Sato, H., Takebe, Ajisawa, tive counterpart in Japan. J. Med. Virol. In press.
A., Y., Nakamura, T., Nagai, Y., Iwamoto, A., and Shioda, T., Nakayama, E. E., Tanaka, Y., Xin, X., Liu, Shioda, T. Polymorphism in the IL-4 promoter af- H., Kawana-Tachikawa, A., Kato, A., Sakai, Y., fects acquisition of HIV-1 syncytium inducing (SI) Nagai, Y., and Iwamoto, A. A naturally occurring phenotype. J. Virol. 74:5452-5459, 2000.
deletional mutation in the C-terminal cytoplasmic Hosoya, N., Takahashi, T., Wada, M., Endo, T., tail of CCR5 affects surfacee trafficking of CC5. J.
Nakamura, T., Sakashita, H., Kimura, K., Ohnishi, Virol. In press.
Department of Transfusion Medicine
Improving the clinical outcome of hematopoietic stem celltransplantation, we have been engaged in researches to clarifythe cytokine network acting on normal hematopoiesis, and toestablish the ex vivo expansion system of hematopoietic stemcells. For the successful engraftment of cord blood cells, we areengaging in the basic researches to establish novel culture sys-tems for progenitor B cells, and to clarify the mechanisms howtransplanted stem cells are homing to the bone marrow envi-ronment. We are also engaging in the development for the newtherapeutic strategies using antisense oligodeoxynucleotides.
As the clinically-based department, we supply purified hemato-poietic stem cells to clinical trials for allogeneic bone marrowand peripheral blood transplantation. The depletion of T lym-phocytes from the donor cells is undertaken to reduce the rateof graft-versus-host disease, and the purification of CD34+ cellsis performed in order to purge grafts of tumor cells. These clin-ical-oriented researches are focused not only on cytokinetherapy and immunotherapy, but also on gene therapy, anti-sense therapy, and cell therapy that are being generated in theInstitute.
1. Purification of human hematopoietic progeni-
have been developed such as the panning and the tor and stem cells for bone marrow, peripheral
culumn filtration methods with immunobeads. The blood transplantations in the clinical setting
purity and recovery efficiencies after separation us-ing immunobeads in our department are more than Taira Maekawa, Kazuo Ogami, Yuka Wada, Shino-
98% and 50-60%, respectively. The administration of bu Hosoda, Tetsu Yoshimasu, Hitomi Nagayama1,
more than 5 x 105/kg CD34+ cells purified from bone Tsuneo A. Takahashi1, Tohru Iseki2, Jun Ooi2,
marrow and 2 x 106/kg CD34+ cells purified from PB Ryuhei Tanaka3, Atsushi Manabe3, Kouichiroh
after mobilization by granulocyte colony-stimulat- Tsuji3, and Shigetaka Asano2: 1Division of Cell Pro-
ing factor (G-CSF) are capable of inducing a rapid cessing, 2Department of Hematology-Oncology,
and permanent recovery of the hematopoiesis after and 3Pediatric Hematology-Oncology, The Insti-
transplantation. Allogeneic peripheral blood stem tute of Medical Science, The University of Tokyo
cells have now been used as an alternative methodfor clinical transplantation (allo-PBSCT). In order to Cell surface antigen CD34+ cells contain the major- obtain less graft versus host disease (GVHD), bone ity of human hematopoietic progenitors and stem marrow and peripheral blood transplantations using cells, that can produce a variety of hemopoietic colo- allograft of purified CD34+ cells are now used in the nies and reconstitute the hematopoiesis after clinical settings.
myeloablative chemotherapy. Several methods topurify a large number of CD34+ cells from the bone 2. Establishment of enzyme linked immunosor-
marrow (BM) and peripheral blood (PB) samples bent assay to detect soluble HLA class I
antigens in serum from patients received allo-
revealed to to be a coreceptor for T-tropic HIV-1 en- geneic stem cell transplantations
try. The physiological roles of PBSF/SDF-1 andCXCR4 were studied using mutant mice with a tar- Taira Maekawa, Shinobu Hosoda, Yuka Wada Y,
geted these genes, showing that they are responsible Kazuo Ogami, Tetsu Yoshimasu, Tohru Iseki, Hito-
for B-lymphopoiesis and marrow myelopoiesis. Re- mi Nagayama, Jun Ooi, Arinobu Tojo, Kenzaburo
cently, it has been reported that the expression of Tani, Ryuhei Tanaka, Atsushi Manabe, Kohichiro
CXCR4 is critical for the engraftment of CB cells into Tsuji, and Shigetaka Asano2
Besides being expressed on the membrane of most progenitors, but give to lymphoid progenitors, nucleated cells, HLA class I antigens are present in wheres CD34+CXCR4– BM cells can generate both of serum. We established the emzyme linked immun- these. To address these questions, we have studied osorbent assay to detect soluble HLA class I antigens the proliferative and diffentiative potentials of CB in serum. An increase in the serum HLA class I anti- and PB (G-CSF mobilized) CD34+ cells along the my- gen level has been seen in acute rejection episodes eloid and lymphoid pathway, and compare these following heart, liver, and kidney transplants. We results to those with BM cells. The myeloid colony found that soluble HLA class I level significantly in- frming potentials were as follows; creases in patients suffering from acute graft versushost disease (GVHD) episodes following allogeneic Unlike BM and PB cells, both CD34+CXCR4+ and bone marrow transplantation (allo-BMT) whereas it CD34+CXCR4- CB cells similarily had myeloid colony does not change in patients without GVHD. We are forming potentials. Concerning lymphoid progeni- now investigating whether the increase of this soluble tors, CD34+CXCR4– BM cells could generate pre-B HLA Class I antigen levels in serum from patients re- cells more abundantly than CD34+CXCR4+ BM cells.
In contrast, both CD34+CXCR4+ and CD34+CXCR4- transplantation (CBT) can modulate the immunoregu- CB cells have the similar potentials in producing pre-B latory systems leading to less onsets of GVHD, cells. these results suggest that the requirement of the comparing with other cytokine levels including inter- functional expression of CXCR4 for CB cells may dif- leukins, interferons, and tumor necrosis factor.
fer from that for adult BM and PB cells. Furtherstudies were needed to understand the significance ofexpression in the homing, engraftment, and repopula- 3. Functional expression of chemokine receptor
tion of hematopoietic stem cells in the different organ.
CXCR4 for CB cells differ from adult BM and
PB cells in culture
4. Erythroid progenitos differentiate and mature
in response to endogenous erythropoietin
Takefumi Ishii, Masamichi Nishihara, Feng Ma3,
Yasuhiro Ebihara3, Kohichiro Tsuji, Shigetaka
Sato T3, Maekawa T, Watanabe S6, Tsuji K and
Asano, Tatsutoshi Nakahata5, and Taira Maekawa:
Nakahata T5:6Division of Molecular and Develop-
5Department of Pediatrics, Kyoto University
mental Biology, The Institute of Medical Science,
The University of Tokyo
Pre-B-cell growth-stimulating factor/stromal cell derived factor 1 (PBSF/SDF-1) is a member of CXC It has been reported that stimulation of glycopro- chemokines, and its receptor CXCR4 has also been tein 130 (gp130) by a combination of human soluble Number of Colonies / 300 cells
IL-6R (sIL-6R) and IL-6 could support proliferation, 6. Megakaryopoiesis of cord blood cells are ef-
differentiation and terminal maturation of erythroid fectively enhanced by stromal cells derived
cells in the absence of erythropoietin (EPO) from hu- from bone marrow mesenchymal stem cells
man CD34+ cells in culture with SCF. Thisobservation suggested that differentiation of hemato- Taira Maekawa, Kazuo Ogami, and Shigetaka
poietic stem/progenitor cells to erythroid cells was progressed along an intrinsic program, and that EPORcould be replaced by other cytokine receptors and was Mesenchymal stem cells (MSCs) give rise to mar- dispensable for erythropoiesis. We examined the role row stromal cells that produce the spongy stromal of EPOR in erythropoiesis stimulated by SCF, sIL-6R, matrix comprising the bone marrow microenviron- and IL-6. Surprisingly, elimination of EPOR expres- ment. These marrow stromal cells contribute directly sion by antisense oligodeoxynucleotide (AS ODN) to blood cell formation by producing the extracellular suppressed erythropoiesis in the absence of EPO. EPO matrix where blood cell development takes place and mRNA was detected in the erythroid cells but not by providing cytokines and other molecules that di- myeloid cells cultured in the presence of SCF, sIL-6R, rect or stimulate the production of mature blood cells.
and IL-6. Furthermore, high concentrations of anti- MSCs are rarely contained in cord blood, that may EPO neutralizing antibody abrogated erythropoiesis cause the delayed engraftment of megakaryopoiesis in cultures without extrinsic EPO. Based on these re- in a clinical setting of cord blood transplantation. Pre- sults, we suggest that erythroid progenitors per se clinical studies in animals have demonstrated that secrete EPO and have the potential to differentiate culture-expanded mesenchymal stem cells can be and maturate in an autocrine manner by endogenous used to repair bone defects, full-thickness articular cartilage defects, bone marrow stroma, and tendon.
We are now seeking to develop human therapeutic 5. Development of antisense therapeutics for he-
products based on the role of hMSCs in megakaryo- cytopoiesis. We found that stromal cells derivedfrom MSCs effectively suuport the megakaryocy- Taira Maekawa, and Shigetaka Asano2
topiesis of cord blood cells in vitro. Cloning and sequencing of pathogenic genes have 7. Establishment of Room for Clinical Cellular
provided useful informations for preventive medi- cine and conventional therapies through moleculardiagnosis of various diseases including hereditary Taira Maekawa, Tsuneo A. Takahashi1, Kenzoburo
disease, cancer, and AIDS. They have also made pos- Tani2, Naohide Yamashita8, Tatsutoshi Nakahata5,
sible new therapeutic approaches through gene and Shigetaka Asano2:8Department of Advanced
manipulation. Oligodeoxynucleotides (ODNs) show Medical Science, The Institute of Medical Science,
great promise as therapeutic agents because of their The University of Tokyo
potential to inhibit gene expression by sequence-spe-cific mechanisms. The elegant specificity of Cell therapy including stem cell transplantation Watson-Crick base pairing between the antisense and gene therapy being ultimate therapeutic ap- (AS) ODNs and the target mRNA or gene could form proaches for incurable diseases, their establishments the basis for a highly specific and effective drug.
are urgently needed. It is also mandatory to separate Clinical trials are now in progress to the United and manipulate cells under quality-controlled steril- States, Italy and the United Kingdom. However, be- ized circumstances that can meet with GMP cause chemically modified AS ODNs, especially PS approvals, and provide powerfully enegineered cells ODNs, have been reported to cause a number of non- to clinical settings. For this purpose, the center hav- specific effects as described above, we are now ing clean rooms (Room for Clinical Cellular examining the efficacy of new ODN analogues with Technology:RCCT) with clinical P2 and P3 facilities mixed backbone structure and N3'→P5' phosphroa- is now operating in the Institute. The banking of cord midates targetting oncogenes such as BCR-ABL, blood cells for cord blood transplantation, the inser- c-myc, and Bcl-2, and investigating the feasibility to tion of GM-CSF gene to renal tumor cells using establish antisense therapeutics for leukemias and retrovirus vector for gene therapy, and the genera- lymphomas. We have recently establish the novel tion of antigen-pulsed denderitic cells against drug delivery system named transmembrane carrier malignant melanoma cells are on going in RCCT.
system (TCS) to increase the efficacy of cellular up-take of AS ODNs.
Maekawa, T., Ishii, T.: Chemokine/receptor dynam- production by hematopoietic cells. Int J Hematol ics in the regulation of hematopoiesis. Internal 72:455-462, 2000.
Med, 39: 90-100, 2000.
Miyamoto, S., Tsuji, K., Maekawa, T., Asano, S., Machida, U., Tojo, A., Takahashi, S., Iseki, T., Ooi, J., Nakahata, T.: Inhibitory effect of interleukin-3 on Nagayama, H., Shirafuji, N., Mori, S., Wada, Y., early development of human B-lymphopoiesis.
Ogami, K., Yamada, H., Sakamaki, H., Maekawa, Brit J Haematol (in press) T., Tani, K., Asano, S.: The effect of granulocyte Yoshimasu, T., Tanaka, R., Suenobu, S., Yagasaki, H., colony-stimulating factor administration in Yoshino, H., Ueda, T., Hisakawa, H., Ishii, T., healthy donors before bone marrow harvesting.
Mitsui, T., Ebihara, Y., Manabe, A., Iseki, T., Brit J Haematol, 108: 747-753, 2000.
Maekawa, T., Nakahata, T., Asano, S., Tsuji, K.: Hase, H., Tani, K., Nagayama, H., Watari, K., Prompt and durable hematopoietic reconstitution Takahashi, S., Ooi, J., Shirafuji, N., Iseki, T., by unrelated cord blood transplantation in a child Nakazaki, Y., Yamashita, T., Nakamura, T., with Fanconi anemia. Bone Marrow Transplant (in Masunaga, A., Maekawa, T., Tojo, A., Asano, S.: TCR-Vβ repertoire analysis with RT-PCR was use- Ma, F.A., Wada, M., Yoshino, H., Ebihara, Y., Ishii, ful for the early detection of pulmonary relapsed T., Manabe, A., Tanaka, R., Maekawa, T., Itoh, M., T-cell lymphoma after autologous peripheral Mugishima, H., Asano, S., Nakahata, T., Asano, S.: blood stem cell transplantation. Am J Hematol, Development of human lymphohematopoietic stem/progenitor cells defined by CD34 and CD81 Sato, T., Maekawa, T., Watanabe, S., Tsuji, K., expressions. Blood (in press ) Nakahata, T.: Erythroid progenitors differentiateand maturate in response to endogenous erythro-poietin. J. Clin. Invest. 106: 263-270, 2000.
Sasaki, S., Ito, E., Toki, T., Maekawa, T., Kanezaki, R., Umenai, T., Muto, A., Nagai, H., Kinoshita, T.,Yamamoto, M., Inazawa, J., Taketo, M.M.,Nakahata, T., Igarashi, K., Yokoyama, M.: Growthinhibition of a Burkitt cell line by B-cell specifictranscription factor BACH2. Oncogene 19(33):3739-3749, 2000.
Tani, K., Nakazaki, Y., Hase, H., Takahashi, K., Azuma, M., Ohata, J., Kitamura, R., Komine, F.,Oiwa, M., Masunaga, A., Maekawa, T., Satoh, N.,Adachi, D., Soda, Y., Machida, U., Endo, M.,Yamazaki, T., Watari, K., Tojo, A., Yamashita, N.,Tomikawa, S., Eriguchi, M., Hamada, H.,Wakumoto, Y., Hanazawa, K., Okumura, K.:Progress reports on immune gene therapy for stageIV renal cell cancer using lethally irradiated granu-locyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells. CancerChemother Pharmacol 46 Suppl: S73-6, 2000.
Matsui, T., Sato, T., Maekawa, T., Asano, S., Nakahata, T., Tsuji, K.: Glycoprotein 130 and c-kitsignals synergistically induce thrombopoietin Department of Surgical Center
Basically our purpose is to improve quality of life of the pa-tients in surgery or in intensive care. In that real direction ofclinically based advanced medicine, especially we have in-vestigated recently 1) mechanisms of pain and better analgesicregimen, and 2) coagulation, fibrinolysis, and hemolysis in var-ious conditions. In addition, we support some investigationand clinical reports in other institutions.
1. Tomoki Nishiyama. Interaction between in-
The NMDA (N-methyl-D-aspartate) receptor an- trathecal morphine and glutamate receptor
tagonists and the NMDA glycine site antagonists antagonists in formalin test
given alone have minimal effects on acute nocicep-tion. In contrast, the AMPA (alpha-amino-3-hydroxy- The analgesic interaction between intrathecally 5-methylisoxazole-4-propionic acid) receptor antago- administered morphine and the NMDA receptor an- nists have a great role in acute nociception. We investigated the interactions among these three antag- AP-5), the NMDA receptor glycine site antagonist onists in acute nociception. Sprague-Dawley rats ,(5-nitro-6,7-dichloro-2,3-quinoxaline dion; ACEA (250-300 g) were implanted with chronic lumbar in- 1021), or the AMPA (α-amino-3-hydroxy-5-methyl- trathecal catheters and were tested for their thermal isoxazole-4-propionic acid) receptor antagonist withdrawal response using the hot plate test after in- (ACEA 2752) in the formalin test was investigated trathecal administration of AP-5 (NMDA receptor with a rat model of chronic lumbar intrathecal cathe- antagonist), ACEA 1021 (NMDA glycine site antago- terization. After obtaining dose-response curves for nist), or ACEA 2085 (AMPA receptor antagonist).
each agent, combinations of morphine and AP-5, The combinations of these three agents were also ACEA 1021 or ACEA 2752 were tested for their effect tested. Intrathecal administration of ACEA 2085 had on the number of flinches in the formalin test and for a dose dependent analgesic effect while intrathecal associated side-effects, such as motor disturbances, AP-5 or ACEA 1021 could not induce dose depen- flaccidity, and agitation/allodynia. Using isobolo- dent effect. Coadministration of AP-5 10 µg and graphic analyses, a potent analgesic synergy was ACEA 2085 intrathecally showed no changes in the observed with decreased side-effects between mor- thermal response latency compared to ACEA 2085 phine and ACEA 2752 or AP-5. ACEA 1021 increased alone. ACEA 1021, 12 µg, and AP-5 showed the left the analgesic effect of low-dose morphine. Spinal µ- ward shift of the dose effect curve only with small opioid receptor activation and NMDA or AMPA doses of AP-5 (1 µg and 3 µg). Only the smallest dose receptor antagonism showed a synergistic antinoci- of ACEA 2085 (0.1 ng) with ACEA 1021 12 µg in- ception against tonic pain. These results suggest an duced antinociception compared with that of ACEA important direction in the management of inflamma- 2085 alone. The combination of the NMDA glycine site antagonist and low doses of the NMDA receptorantagonist or the AMPA receptor antagonist in- 2. Tomoki Nishiyama. Interaction among NMDA
creased the analgesic effect on acute thermal receptor-, NMDA glycine site-, andAMPA re-
nociception with increased side effects, while the ceptor antagonists in spinally mediated
NMDA receptor antagonist and the AMPA receptor antagonist had no such interaction.
3. Tomoki Nishiyama, Rodney JY Ho, Danny D
potentiated the analgesic effect of epidural morphine Shen, Tony L Yaksh. The effects of intrathecal
at both weak and strong nociceptive stimuli similar- morphine encapsulated in L- and D-dipalmi-
ly, while increasing the dose of epidural morphine toylphosphatidyl choline liposomes on acute
was not as effective for strong nociceptive stimula- nociception in rats
tion. Therefore, adding bupivacaine might be moreeffective than increasing the dose of epidural mor- Liposomes can serve as a sustained-release carrier phine for strong nociception.
system permitting the spinal delivery of large opioiddoses, while restricting the dose for acute systemicuptake. The purpose of this study was to evaluate the 5. Tomoki Nishiyama, Kazuo Hanaoka. Free he-
antinociceptive effects of morphine encapsulated in moglobin concentrations in patients receiving
liposomes of two isomeric phospholipids, L-DPPC massive blood transfusion during emergency
(L-dipalmitoylphosphatidyl choline) and D-DPPC, surgery for trauma.
in comparison with morphine in saline. Sprague-Dawley rats with chronic lumbar intrathecal To investigate free hemoglobin concentration in catheters were tested for their acute nociceptive re- patients who received massive blood transfusion sponse using a hind paw thermal escape test. Their during emergency surgery for trauma with consider- general behavior, motor function, pinna reflex, and ation of the storage of the transfused blood. Fifteen corneal reflex were also examined. The duration of patients undergoing emergency surgery for multiple antinociception was longer in both liposomal mor- trauma and who received blood transfusion of more phine groups than in the free morphine group. The than 5,000 mL were studied. Transfusion of the peak antinociceptive effects were observed within 30 stored whole blood in citrate-phosphate glucose so- minutes after intrathecal morphine, L-DPPC or D- lution using a micropore filter was started before DPPC morphine injection. The rank order of the area surgery. Serum concentrations of hemoglobin under the effect-time curve for antinociception was (total:THb and free:fHb) and total haptoglobin L-DPPC morphine > D-DPPC morphine > morphine.
(THp) were measured until 5,000 mL of blood had The ED was: 2.7 µg (morphine), 4.6 µg (L-DPPC been transfused. Serum free haptoglobin (fHp) con- morphine) and 6.4 µg (D-DPPC morphine). D-DPPC centration was calculated. The correlation between morphine had less side effects for a given antinocicep- the changes in hemoglobin or haptoglobin concen- tive AUC than morphine. In conclusion, L- and trations and total storage days of the transfused D-DPPC liposome encapsulation of morphine pro- blood was analyzed by a simple regression analysis.
longed antinociceptive effect on acute thermal Free hemoglobin was detected after 2,000 mL trans- stimulation and could decrease side effects compared fusion. THp and fHp decreased after 1,000 mL of to morphine alone.
transfusion. Total storage time (days) of transfusedblood had correlated with the changes of THp (P < 4. Tomoki Nishiyama, Kazuo Hanaoka. The ef-
0.0001) and fHp (P = 0.0027) but not with the changes fects of epidural bupivacaine, morphine, and
of THb (P = 0.984) and fHb (P = 0.834). After blood their combination on thermal nociception with
transfusion during surgery for trauma, serum hapto- different stimulus intensity in rats
globin concentration decreased with transfusion of 1,000 mL of whole blood with mean storage time The analgesic effect of the drugs depends on the of 12.2 days. Free hemoglobin was detected after stimulus intensity as well as the potency of the 2,000 mL transfusion when THp decreased to 1,000 drugs. We investigated the effects of stimulus inten- mg/L. Serum haptoglobin concentrations correlated sity on antinociceptive potencies of epidural negatively with storage time (days) of transfused bupivacaine + morphine. Sprague-Dawley rats im- planted with chronic lumbar epidural catheters weretested for paw withdrawal response to thermal stim- 6. Tomoki Nishiyama, Takashi Matsukawa, Ka-
ulation after epidural injection of bupivacaine, zuo Hanaoka. Is protease inhibitor a choice for
morphine, or bupivacaine + morphine Two stimula- the treatment of pre- or mild dissected intra-
tion currents were employed; 5.1 and 4.6 A to vascular coagulation ?
provide baseline response latency of approximately5.0 sec (high intensity) and 10.0 sec (low intensity), To investigate the effect of a protease inhibitor, respectively. Increasing the dose of epidural mor- gabexate mesilate (FOY) on pre- disseminated intra- phine in a dose range, which had a maximum effect vascular coagulation (DIC) or mild DIC in on low intensity stimulation, was not effective for comparison with the control without any anticoagu- high intensity stimulation. Bupivacaine, which alone lation therapy, 40 adult patients with DIC score had no effect, potentiated the antinociceptive effect between 6 and 8 (pre- or mild DIC) were divided into of epidural morphine at both high and low intensity two groups. In 20 patients FOY 2 mg/kg/h was in- stimuli similarly. We concluded that bupivacaine fused with 2 ml/h (FOY group) and in other 20 patients saline 2 ml/h (Control group) was infused during the study (seven days). The following param-eters were determined at the time of admission to the To investigate the changes of renin-angiotensin- intensive care unit (before treatment), and 1, 3, 5, and aldosterone system by nicardipine administration 7 days thereafter: platelet count, AT III activity, se- during isoflurane or sevoflurane anesthesia, 20 pa- rum or plasma levels of fibrinogen, FDP, D-dimer, tients aged 40 to 70 for elective neurosurgery were fibrin monomer (FM), thrombin-antithrombin III studied. Anesthesia was induced with thiopental, complex (TAT), and plasmin-plasmin inhibitor com- midazolam and fentanyl and was maintained with plex (PIC), prothrombin time (PT) ratio, and DIC nitrous oxide in oxygen and isoflurane or sevoflu- score. Two patients in the FOY group and four in the rane. When blood pressure was constant, 0.017 mg/ Control group were excluded from the study be- kg nicardipine was administered as a bolus. Blood cause they expired during the study, therefore 34 pressure, heart rate, and plasma concentrations of patients were analyzed. The measured variables of nicardipine, angiotensin I and II, aldosterone and re- coagulation and fibrinolysis were not significantly nin activity were measured for 30 min. after different between the two groups except for the D- nicardipine administration. Blood pressure decreased dimer on the day 3 (FOY group showed higher level).
significantly for 30 min. after nicardipine administra- D-dimer level and DIC score went down more quick- tion in both groups with lower values in sevoflurane ly in the Control group than the FOY group, but not anesthesia. Heart rate increased with significance only significantly. Mortality in one month was 40% (8/20) in isoflurane group. Plasma nicardipine concentra- in the FOY group and 35% (7/20) in the Control tion did not differ between isoflurane and sevoflurane group without any differences between the two groups. Plasma renin activity and concentrations of groups. In a limited number of patients (N=34), FOY angiotensin II and aldosterone did not change in both 2mg/kg/h could not inhibit coagulation or fibrinol- groups and had no differences between the groups.
ysis and FOY could not improve DIC score or Plasma concentration of angiotensin I increased at 20 mortality in pre-DIC or mild DIC.
and 30 min. after nicardipine administration in isoflu-rane group but did not increase in sevoflurane group.
7. Tomoki Nishiyama, Kazuo Hanaoka. Nicar-
The activity of renin-angiotensin-aldosterone system dipine did not activate renin-angiotensin-
did not increase by a single shot administration of aldosterone system during isoflurane or
nicardipine in isoflurane or sevoflurane anesthesia.
Matsukawa T, Ozaki M, Nishiyama T, Imamura M, trations in patients receiving massive blood trans- Kumazawa T. Comparison of infrared thermom- fusion during emergency surgery for trauma. Can eter with thermocouple for monitoring skin tem- J Anesth 49:881-885,2000 perature. Crit Care Med 28:532-536,2000 Nishiyama T, Hanaoka K. A traumatic asphyxia in a Nishiyama T, Matsukawa T, Hanaoka K. Is protease child. Can J Anesth 49:1099-1102,2000 inhibitor a choice for the treatment of pre- or mild Nishiyama T, Yokoyama T, Matsukawa T, Hanaoka dissected intravascular coagulation? Crit Care K. Continuous nicardipine infusion to control Med 28:1419-1422,2000 blood pressure after evacuation of acute cerebral Nishiyama T. Interaction between intrathecal mor- hemorrhage. Can J Anesth 49:1196-1201,2000 phine and glutamate receptor antagonists in for- Nishiyama T, Hanaoka K. Nicardipine did not acti- malin test. Eur J Pharmacol 395:203-210,2000 vate renin-angiotensin-aldosterone system during Nishiyama T. Interaction among NMDA receptor-, isoflurane or sevoflurane anesthesia. Can J Anesth NMDA glycine site-, andAMPA receptor antago- nists in spinally mediated analgesia. Can J Anesth47:693-698,2000 Nishiyama T, Ho RJY, Shen DD, Yaksh TL. The ef- fects of intrathecal morphine encapsulated in L-and D-dipalmitoylphosphatidyl choline lipo-somes on acute nociception in rats. Anesth Analg91:423-428,2000 Nishiyama T, Hanaoka K. The effects of epidural bupivacaine, morphine, and their combination onthermal nociception with different stimulus inten-sity in rats. Anesth Analg 91:652-656,2000 Nishiyama T, Hanaoka K. Free hemoglobin concen-
LE COMMERCE EN FRANCE GROUPE LOUIS DELHAIZE- PROVERA FRANCE LES AUTRES ENSEIGNES DU GROUPE LE COMMERCE EN FRANCE Cible des " gros", Cora s'en est toujours sortie : " attaquée" successivement par Carrefour puis par Casino, tous deux entrés au capital à hauteur de 42%, respectivement entre 1996 et 2001 et entre 2001 et 2006, Cora les a fait plier, pour conserver, aujourd'hui, 100% de son indépendance
A. Les McDonald, Founding CNAC Executive Director (Editor's Note: The following article appeared in the Globe & Mail "Lives Lived" section on April 3, 2008. It waswritten by Les' good friend, Ron Jette. I am pleased to share it with you. It's hard for me to believe it has already beensix months. Ron really captured Les' spirit and made me hear my good friend's laugh one more time.Thanks Ron. – Cheryl)