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Takao Sudo,
Takafumi Yokota, Kenji Oritani,
Yusuke Satoh,
Tatsuki Sugiyama,
Tatsuro Ishida,
Hirohiko Shibayama,
Sachiko Ezoe,
Natsuko Fujita,
Hirokazu Tanaka,
Tetsuo Maeda,
Takashi Nagasawa,
Yuzuru Kanakura
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ABSTRACT: Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.
The Journal of Immunology 05/2012; 189(1):200-10. · 5.79 Impact Factor
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Keiko Matsui,
Sachiko Ezoe, Kenji Oritani,
Masaru Shibata,
Masahiro Tokunaga,
Natsuko Fujita,
Akira Tanimura,
Takao Sudo,
Hirokazu Tanaka,
Michael W McBurney,
Itaru Matsumura,
Yuzuru Kanakura
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ABSTRACT: Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.
Biochemical and Biophysical Research Communications 02/2012; 418(4):811-7. · 2.48 Impact Factor
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01/2012; , ISBN: 978-953-307-930-1
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Masaru Shibata,
Sachiko Ezoe, Kenji Oritani,
Keiko Matsui,
Masahiro Tokunaga,
Natsuko Fujita,
Yuri Saito,
Takayuki Takahashi,
Masayuki Hino,
Itaru Matsumura,
Yuzuru Kanakura
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ABSTRACT: It would be of great value to predict the efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of individual CML patients. We propose an immunoblot system for detecting the phosphorylation of Crkl, a major target of Bcr-Abl, in blood samples after in vitro incubation with TKIs. When the remaining phosphorylated Crkl after treatment with imatinib was evaluated as the "residual index (RI)", high values were found in accordance with imatinib resistance. Moreover, RI reflected the outcome of imatinib- as well as second generation TKIs with a high sensitivity and specificity. Therefore, this system should be useful in the selection of TKIs.
Leukemia research 03/2011; 35(9):1205-11. · 2.36 Impact Factor
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ABSTRACT: With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca(2+) depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled-coil domains was massively expressed on the cell surface while mutants with the coiled-coil domains localized in ER. In addition, STIM1 mutants with the coiled-coil domains showed a longer half-life of proteins than those without them. These results are likely to indicate that the coiled-coil domains of STIM1 are essential for its ER-retention and its stability. Furthermore, we tried to comprehensively identify STIM1-associated molecules with mass spectrometry analysis of co-immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein-transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca(2+) control as well as a useful strategy to develop therapeutic drugs for intracellular Ca(2+)-related diseases including inflammation and allergy.
Journal of Cellular Biochemistry 11/2010; 112(1):147-56. · 2.87 Impact Factor
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Masahiro Tokunaga,
Sachiko Ezoe,
Hirokazu Tanaka,
Yusuke Satoh,
Kentaro Fukushima,
Keiko Matsui,
Masaru Shibata,
Akira Tanimura, Kenji Oritani,
Itaru Matsumura,
Yuzuru Kanakura
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ABSTRACT: BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage(-)Sca-1(hi)CD117(hi)) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16(INK4a)/p19(ARF)-, and p21(CIP1/WAF1)-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21(CIP1/WAF1) deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21(CIP1/WAF1), plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.
Journal of Biological Chemistry 10/2010; 285(41):31774-82. · 4.77 Impact Factor
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ABSTRACT: Progress has been slow in defining molecular requirements for human B lymphopoiesis in part because of differences from experimental animals and also because of the lack of culture conditions that efficiently support the process. We recently found that human CD10+ lymphocytes were produced when CD34+ hematopoietic stem and progenitor cells were cultured in contact with human mesenchymal stem cells (hMSC). Further investigation revealed that it occurred even when progenitors were separated from hMSC by membrane filters. Experiments with neutralizing antibodies suggested that important heat labile factors produced by hMSC are unlikely to be IL-7, TSLP, CXCL12 or hemokinin-1. Further manipulation of culture conditions revealed that optimal lymphopoiesis required careful selection of fetal calf serum lots, maintenance of high cell densities, as well as recombinant cytokines (SCF, FL and G-CSF). G-CSF was particularly important when adult bone marrow rather than umbilical cord blood derived CD34+ cells were used to initiate the cultures. These improved methods should facilitate identification of molecules that can be used to speed regeneration of the humoral immune system following chemotherapy and might suggest ways to inhibit growth of B lineage malignancies.
Journal of immunological methods 07/2010; 359(1-2):47-55. · 2.35 Impact Factor
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ABSTRACT: Requirements for human B lymphopoiesis are still poorly understood, and that has hampered investigation of differentiation events. For example, there are few cell surface antigens that can be used as milestones of lineage progression. The CD10 ectoenzyme is one such marker and has been used to define CLP, but we found substantial tissue specific variations in CD10 levels, and there was no information about how that corresponded to differentiation options.
The aim of the present study was to use recently developed culture methods to assess the nature and differentiation potential of progenitors sorted according to CD10 density from umbilical cord blood (CB), adult bone marrow (BM) or G-CSF mobilized peripheral blood (PB). Many CD34(+) cells in BM express high levels of CD10, while low or low/negative CD10 densities were found on CD34(+) cells in CB or G-CSF mobilized PB, respectively. The relative abundance of CD10(Lo) versus CD10(Hi) cells only accounts for some CB versus BM differences. Almost all of the CD34(+) CD10(Hi) cells expressed CD19 and lymphocyte transcription factors and corresponded to loss of myeloid potential. A high degree of immunoglobulin D(H)-J(H) gene rearrangements was characteristic only of the CD10(Hi) subset. In contrast, the CD34(+) CD10(Lo) progenitors efficiently produced plasmacytoid and conventional dendritic cells as well as myeloid cells. These findings suggest a positive correlation between CD10 density and degree of differentiation. Although freshly isolated CD34(+) CD10(Hi) cells were in cycle, those from CB or BM expanded poorly in culture, suggesting regulators of populations remain to be discovered.
Steps in human B lymphopoiesis have not been sufficiently studied, and we now show that increased CD10 expression corresponds to differentiation potential and stage. CD34(+) CD10(Hi) progenitors are obviously in the B lineage but may have progressed beyond the point where they can be expanded in culture.
PLoS ONE 01/2010; 5(9):e12954. · 4.09 Impact Factor
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ABSTRACT: CD9 belongs to the tetraspan family of proteins that facilitates the regulation of cell proliferation, motility, and adhesion. In mouse hematopoietic organs, CD9 is expressed by myeloid and stromal cells. Although the precise mechanisms are not clear, antibody ligation of CD9 on stromal cells regulates the adhesion between stromal cells and hematopoietic stem cells, the production of myeloid cells in long term bone marrow cultures and the differentiation of hematopoietic stem cells. A 100 kD protein co-precipitated with CD9 is distinct from several previously reported CD9-associated molecules with respect to size and distribution. Identification and analysis of this interesting protein may clarify the molecular mechanisms through which CD9 bearing stromal cells control the differentiation of hematopoietic stem cells and/or allow them to maintain their vital self-renewal capacity.
06/2009; 38(1-2):147-152.
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Taisei Nakamoto,
Yoko Murayama, Kenji Oritani,
Claude Boucheix,
Eric Rubinstein,
Makoto Nishida,
Fumie Katsube,
Kenji Watabe,
Shinichi Kiso,
Shusaku Tsutsui,
Shinji Tamura,
Yasuhisa Shinomura,
Norio Hayashi
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ABSTRACT: CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as motility, cell signaling, proliferation, adhesion, and metastasis. However, very little is known about the involvement of CD9 in the process of development of primary tumors. In the present study, we investigated whether anti-CD9 monoclonal antibody (ALB6) has antitumor effects in human gastric cancer cell xenografts.
Human gastric cancer cell lines (MKN-28) (5 x 10(6) cells/animal) were inoculated subcutaneously into the dorsal region of SCID mice (five mice in each group). After a tumor was visualized, animals were assigned to either the ALB6 treatment group or the control IgG treatment group (100 microg/body/time, intravenous, three times per week. Day 1, 4, and 7 of first week). Then tumor volumes were monitored every day. Proliferation of tumor was analyzed by 5-bromo-2'-deoxyuridine (BrdU) immunostaining, apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) methods, and angiogenesis was assessed by counting the number of CD34-positive endothelial cells.
Tumor volume was significantly suppressed (1,682 +/- 683 mm(3) versus 4,507 +/- 1,012 mm(3); P = 0.049), the BrdU labeling indexes were significantly decreased (10.9 +/- 1.1% versus 17.2 +/- 1.4%; P = 0.009), the apoptotic indexes were significantly increased (1.98 +/- 0.48% versus 0.72 +/- 0.09%; P = 0.034), and tumor microvessel densities were significantly suppressed (671,922 +/- 34,505 pixels/mm(2) versus 1,135,043 +/- 36,086 pixels/mm(2); P = 0.037) in the ALB6 treatment group compared with the control IgG treatment group.
These results suggest that administration of anti-CD9 antibody to mice bearing human gastric cancer cells successfully inhibits tumor progression via antiproliferative, proapoptotic, and antiangiogenetic effects.
Journal of Gastroenterology 06/2009; 44(9):889-96. · 4.16 Impact Factor
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ABSTRACT: Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity, more information about their characteristics will improve our understanding of their development and stage-related functions. Here, using microarray technology, we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1(-) c-kit(Hi) Sca1(+) HSC-enriched fraction, but sharply down-regulated with activation of the Rag1 locus, a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors, ESAM expression showed intimate correlation with HSC activity. The ESAM(Hi) population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity, and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2(+) c-kit(+) lymphohematopoietic cells in the E9.5-10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore, ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly, ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life.
Blood 01/2009; 113(13):2914-23. · 9.90 Impact Factor
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ABSTRACT: It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally simulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker(-) c-kit(high) ScaI(+), hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen-inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-alpha was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized beta-catenin and blocked early lymphoid progression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differentiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels.
The Journal of Immunology 12/2008; 181(9):6061-72. · 5.79 Impact Factor
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Yoko Murayama,
Yasuhisa Shinomura, Kenji Oritani,
Jun-Ichiro Miyagawa,
Hitoshi Yoshida,
Makoto Nishida,
Fumie Katsube,
Masamichi Shiraga,
Tamana Miyazaki,
Taisei Nakamoto,
Shusaku Tsutsui,
Shinji Tamura,
Shigeki Higashiyama,
Iichirou Shimomura,
Norio Hayashi
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ABSTRACT: CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, and adhesion. In addition, it has been known that CD9 can associate with other proteins. Here we demonstrated the physical and functional association of CD9 with epidermal growth factor receptor (EGFR) on MKN-28 cells. Double-immunofluorescent staining and immunoprecipitation demonstrated the complex formation of CD9-EGFR and CD9-beta(1) integrin, and that both complexes are colocalized on the cell surface especially at the cell-cell contact site. Anti-CD9 monoclonal antibody ALB6 induced a dotted or patch-like aggregation pattern of both CD9-EGFR and CD9-beta(1) integrin. The internalization of EGFR after EGF-stimulation was significantly enhanced by the treatment with ALB6. CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO-HER/CD9), which were transfected with pTJ/human EGFR/CD9. Furthermore expression of CD9 specifically attenuated EGFR signaling in CHO-HER/CD9 cells through the down regulation of surface expression of EGFR. These results suggest that CD9 might have an important role that attenuates EGFR signaling. Therefore, CD9 not only associates EGFR but also a new regulator, which may affect EGF-induced signaling in cancer cells.
Journal of Cellular Physiology 08/2008; 216(1):135-43. · 3.87 Impact Factor
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Michiko Ichii, Kenji Oritani,
Takafumi Yokota,
Makoto Nishida,
Isao Takahashi,
Takahiro Shirogane,
Sachiko Ezoe,
Norimitsu Saitoh,
Rie Tanigawa,
Paul W Kincade,
Yuzuru Kanakura
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ABSTRACT: To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology.
We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development.
Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells.
hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.
Experimental Hematology 05/2008; 36(5):587-97. · 2.90 Impact Factor
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Hiroaki Masaie, Kenji Oritani,
Takafumi Yokota,
Isao Takahashi,
Takahiro Shirogane,
Hidetoshi Ujiie,
Michiko Ichii,
Norimitsu Saitoh,
Tetsuo Maeda,
Rie Tanigawa,
Kazumasa Oka,
Yoshihiko Hoshida,
Yoshiaki Tomiyama,
Yuzuru Kanakura
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ABSTRACT: Adiponectin, a fat cell-derived protein, has been attracting considerable attention because of its antidiabetic and antiatherogenic activities. The aim of the present study is to identify molecules physiologically associating with adiponectin and to understand how the protein displays diverse biological activities.
We used an expression cloning method combined with enzyme-linked immunosorbent assay to clone adiponectin-binding proteins from the MS-5 complementary DNA library.
We successfully isolated two chemokines, stromal cell-derived factor-1 (SDF-1) and CCF18, and verified that adiponectin bound to them via its globular head. Adiponectin bound with various chemokines in vitro, such as macrophage-inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemoattractant protein-1 (MCP-1), suggesting that the protein had a feature commonly to bind to the chemokine family. The middle part of chemokines, dispensable for interacting with their receptors, was found to be important for the adiponectin binding. Although the interaction of adiponectin to SDF-1 affected neither the SDF-1-CXCR4 binding nor the SDF-1 signaling in Jurkat cells, adiponectin and heparin mutually interfered in their association to SDF-1 and MCP-1 in vitro, implying that their association might influence the distribution of adiponectin and SDF-1 in inflammatory sites. Indeed, both adiponectin and SDF-1 was positively immunostained in vascular walls in guts from acute graft-vs-host disease patients. In addition, peripheral blood of adiponectin-deficient mice contained more hematopoietic progenitors than that of wild-type mice.
Adiponectin may be involved in regulation of inflammation via binding to specific chemokines. Additionally, the interaction possibly enables adiponectin to gather and play its role in inflammatory sites.
Experimental Hematology 07/2007; 35(6):947-56. · 2.90 Impact Factor
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ABSTRACT: Allogeneic immune responses during hematopoietic reconstitution play central roles in beneficial and adverse effects after allogeneic bone marrow transplantation (allo-BMT). Appropriate regulation of the immune responses might improve the outcome of allo-BMT. However, a useful marker for monitoring allogeneic immune responses remains to be established. We enrolled 22 consecutive patients who underwent myeloablative allo-BMT between March 2002 and March 2006 and examined the relationship between CD27 expression on peripheral blood T-lymphocytes, a possible marker for naive/effector phenotypes, and clinical events, especially acute graft-versus-host disease (aGVHD). In 8 patients with aGVHD of grades II to IV, the CD27+/CD27- ratios of CD4+ (but not CD8+) T-lymphocytes were significantly higher after allo-BMT, even at day 21, than the ratios in patients with aGVHD of grade 0 or I and remained high after day 21. In contrast, the ratios were low after day 21 following allo-BMT in 14 patients with aGVHD of grade 0 or I. Moreover, the clinical analysis suggested a relationship between the ratio and aGVHD grade. Thus, we showed that the CD27+/CD27- ratio in CD4+ T-lymphocytes may have value in predicting the development of severe aGVHD and may correlate with clinical symptoms of aGVHD.
International Journal of Hematology 12/2006; 84(4):367-76. · 1.27 Impact Factor
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Hidetoshi Ujiie, Kenji Oritani,
Hisashi Kato,
Takafumi Yokota,
Isao Takahashi,
Tetsuo Maeda,
Hiroaki Masaie,
Michiko Ichii,
Yoshihiro Kamada,
Shinji Tamura,
Shinji Kihara,
Tohru Funahashi,
Yoshiaki Tomiyama,
Yuzuru Kanakura
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ABSTRACT: Adiponectin is an abundant adipose-specific protein, which acts as an anti-diabetic, anti-atherogenic, and anti-inflammatory adipokine. Although recent advances in the field of adiponectin have been made by the identification of adiponectin receptors and by the understanding about relationship between its multimerization and functions, detailed molecular background remains unclear. Our established anti-human adiponectin antibodies, ANOC 9103 and ANOC 9104, blocked some adiponectin functions such as the growth inhibition of B-lymphocytes on stromal cells and the inhibition of acetylated LDL uptake in macrophages, suggesting that they may recognize important functional regions of adiponectin. As a result of epitope mapping based on the ability to bind to the deleted adiponectin mutants, we identified that these antibodies recognize amino-terminal region of adiponectin before the beginning of the collagen-like domain. Notably, a peptide fragment (DQETTTQGPGVLLPLPKGACTGWMA) corresponding to amino acid residues 17-41 of human adiponectin could bind to restricted types of cells and block adiponectin-induced cyclooxygenase-2 gene expression and prostaglandin E2 production in MS-5 stromal cells. Moreover, the deletion of its amino-terminal region reduced the abilities to inhibit not only collagen-induced platelet aggregation but also diet-induced hepatic steatosis. These data indicate that amino-terminal region of adiponectin is a physiologically functional domain and that a novel receptor, which recognizes amino-terminal region of adiponectin, may exist on some types of cells. Further investigations will contribute to the understanding of molecular mechanisms about adiponectin functions as well as to the designing of novel strategies for the treatment of patients with insulin-resistance, vascular dysfunction, and chronic inflammation.
Journal of Cellular Biochemistry 06/2006; 98(1):194-207. · 2.87 Impact Factor
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ABSTRACT: Although a novel IFN-zeta/limitin uses IFN-alpha/beta receptor, it lacks some common activities of type I IFNs. We compared effects on megakaryocyte proliferation and differentiation as well as signals for their biological activities.
Recombinant IFN-zeta/limitin and IFN-alpha titrated with a cytopathic effect dye binding assay, were used in this study. Colony assays and serum-free suspension cultures for megakaryocytes were performed to compare their growth inhibitory effects. To analyze signals, megakaryocytes cultured in serum-free suspension cultures were stimulated and Western blotted with the indicated antibody.
Both IFN-zeta/limitin and IFN-alpha suppressed the proliferation of megakaryocyte progenitors without influencing their differentiation. However, much higher concentrations of IFN-zeta/limitin were required for the growth inhibition than IFN-alpha. The growth inhibition by IFN-zeta/limitin and IFN-alpha was significantly reduced when either Tyk2 or STAT1 was disrupted. In addition, the antisense oligonucleotides against Crk and Daxx, downstream molecules of Tyk2, greatly rescued the IFN-zeta/limitin- and IFN-alpha-induced reduction of megakaryocyte colony numbers. In cultured megakaryocytes, IFN-zeta/limitin induced the expression of SOCS-1 as strongly as IFN-alpha. However, IFN-zeta/limitin induced weaker phosphorylation of Crk and lower induction of Daxx than IFN-alpha.
Weaker signals for Crk and Daxx may participate in less megakaryocyte suppressive activity of IFN-zeta/limitin and may distinguish IFN-zeta/limitin from IFN-alpha in megakaryocytes. Our results extend the understanding about thrombocytopenia in patients with IFN-alpha treatment as well as the possibility for the clinical application of human homologue of IFN-zeta/limitin or an engineered cytokine with useful features of the IFN-zeta/limitin structure.
Experimental Hematology 05/2005; 33(4):495-503. · 2.90 Impact Factor
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ABSTRACT: Interferon zeta (IFN-zeta)/limitin has been regarded as a novel type I IFN by the Nomenclature Committee of the International Society for Interferon and Cytokine Research. IFN-zeta/limitin, which has some sequence homology with IFN-alpha and IFN-beta, has a globular structure with 5 alpha helices and 4 loops and recognizes IFN-alpha/beta receptor. Although it displays antiviral, immunomodulatory, and antitumor effects, IFN-zeta/limitin has much less lymphomyelosuppressive activity than IFN-alpha. Unique interactions between IFN-zeta/limitin and the receptor probably led to the narrow range of signals and biological activities. A human homologue of IFN-zeta/limitin may be clinically more effective than IFN-alpha and IFN-beta because it has fewer adverse effects. Moreover, further analysis of the structure-function relationship may establish an engineered cytokine with the useful features of IFN-zeta/limitin.
International Journal of Hematology 12/2004; 80(4):325-31. · 1.27 Impact Factor
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Shin-ichiro Kawamoto, Kenji Oritani,
Eiji Asakura,
Jun Ishikawa,
Mamoru Koyama,
Kenmi Miyano,
Minori Iwamoto,
Shin-ichiro Yasuda,
Hirosi Nakakubo,
Fumihiro Hirayama,
Naoko Ishida,
Hidetoshi Ujiie,
Hiroaki Masaie,
Yoshiaki Tomiyama
[show abstract]
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ABSTRACT: Limitin is a new member of type I interferon (IFN) identified with an expression cloning based on the growth suppression of a myelomonocytic leukemia cell line WEHI3. Although limitin uses the IFN-alpha/beta receptor, its signal transduction pathways to express the antiviral effects are different from those of IFN-alpha. To clarify the characteristics of limitin, we compared the biological activities of limitin, such as the antiviral, immunomodulatory, antitumor, and myelosuppressive effects, with IFN-alpha.
Limitin and IFN-alpha were titered with a cytopathic effect dye binding assay. Induction of MHC class I on a keratinocyte cell line PAM212 was estimated with flow cytometry. Induction of OVA-restricted cytotoxic T lymphocyte (CTL) activity was analyzed with 51Cr release assay. Antiproliferative effects were evaluated with 3H-thymidine incorporation assay using WEHI3 and a lymphoblast cell line L1210. Myelosuppresive effects were evaluated with colony assay. In vivo side effects were estimated after the injection of limitin or IFN-alpha.
Limitin had relatively higher antiviral activity than IFN-alpha. Limitin induced the surface expression of MHC class I, the enhancement of CTL activity, and the growth inhibition of lymphohematopoietic cell lines as strong as IFN-alpha. Nevertheless, the treatment of mice with limitin showed neither myelosuppression nor fever that are common adverse effects of IFN-alpha.
Strong immunomodulatory, antitumor, and antiviral effects with weak myelosuppressive and weak acute toxic effects of limitin indicate that it may be useful as a new therapeutic drug for virus-hepatitis and cancers.
Experimental Hematology 10/2004; 32(9):797-805. · 2.90 Impact Factor
