bluuebunny/crossref_metadata_2025_split huggingface dataset:
Necessity of tyrosine 719 and phosphatidylinositol 3′-kinase–mediated signal pathway in constitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the Asp814Val mutation
Authors: Hashimoto Koji , Matsumura Itaru , Tsujimura Tohru , Kim Dae-Ki , Ogihara Hideki , Ikeda Hirokazu , Ueda Shuji , Mizuki Masao , Sugahara Hiroyuki , Shibayama Hirohiko , Kitamura Yukihiko , Kanakura Yuzuru 
Published: January 2003

AbstractSubstitution of valine (Val) for aspartic acid (Asp) at codon 814 constitutively activates murine c-kit receptor tyrosine kinase (KIT), and Asp816Val mutation, corresponding to murine Asp814Val mutation, is found in patients with mastocytosis and acute myelocytic leukemia. However, the signal transduction pathways responsible for oncogenesis by the Asp814Val mutant (KITVal814) are not fully understood. To examine the oncogenic signal transduction of KITVal814, we converted 20 tyrosine (Tyr) residues to phenylalanine (Phe) in the cytoplasmic domain of KITVal814 or deleted the C-terminal region containing 2 other tyrosine residues (Del). Among various KITVal814- derived mutants, KITVal814-Tyr719Phe and KITVal814-Delseverely impaired receptor tyrosine phosphorylation and association with the p85 subunit of phosphatidylinositol 3′-kinase (p85PI3-K). Moreover, KITVal814-Tyr719Pheand KITVal814-Del failed to induce ligand-independent growth in Ba/F3 cells, indicating that Tyr719, the binding site for p85PI3-K, and the C-terminal region are indispensable for factor-independent growth by KITVal814. Although the C-terminal region was also required for ligand-dependent growth by wild-type KIT (KITWT), the Tyr719Phe substitution had negligible effects on ligand-dependent growth by KITWT. Furthermore, dominant-negative PI3-K significantly inhibited ligand-independent growth by KITVal814. These results demonstrate that Tyr719 is crucial for constitutive activation of KITVal814, but not for the ligand-induced activation of KITWT, and that the downstream signaling of PI3-K plays an important role in ligand-independent growth and tumorigenicity by KITVal814, thereby suggesting that KITVal814 is a unique activating mutation that leads to a distinguishable function from the effects of KITWT.
Aberrant iron accumulation and oxidized status of erythroid-specific δ-aminolevulinate synthase (ALAS2)–deficient definitive erythroblasts
Authors: Harigae Hideo , Nakajima Osamu , Suwabe Naruyoshi , Yokoyama Hisayuki , Furuyama Kazumichi , Sasaki Takeshi , Kaku Mitsuo , Yamamoto Masayuki , Sassa Shigeru 
Published: January 2003

AbstractAlas2 encodes the erythroid-specific δ-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with theAlas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking theAlas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype ofAlas2-null definitive erythroblasts was examined.Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes inAlas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate thatAlas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron,Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.
The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development
Authors: Schotte Remko , Rissoan Marie-Clotilde , Bendriss-Vermare Nathalie , Bridon Jean-Michel , Duhen Thomas , Weijer Kees , Brière Francine , Spits Hergen 
Published: January 2003

AbstractHuman plasmacytoid dendritic cells (pDCs), also called type 2 dendritic cell precursors or natural interferon (IFN)–producing cells, represent a cell type with distinctive phenotypic and functional features. They are present in the thymus and probably share a common precursor with T and natural killer (NK) cells. In an effort to identify genes that control pDC development we searched for genes of which the expression is restricted to human pDC using a cDNA subtraction technique with activated monocyte-derived DCs (Mo-DCs) as competitor. We identified the transcription factor Spi-B to be expressed in pDCs but not in Mo-DCs. Spi-B expression in pDCs was maintained on in vitro maturation of pDCs. Spi-B was expressed in early CD34+CD38− hematopoietic progenitors and in CD34+CD1a− thymic precursors. Spi-B expression is down-regulated when uncommitted CD34+CD1a− thymic precursors differentiate into committed CD34+CD1a+ pre-T cells. Overexpression of Spi-B in hematopoietic progenitor cells resulted in inhibition of development of T cells both in vitro and in vivo. In addition, development of progenitor cells into B and NK cells in vitro was also inhibited by Spi-B overexpression. Our results indicate that Spi-B is involved in the control of pDC development by limiting the capacity of progenitor cells to develop into other lymphoid lineages.
In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas
Authors: Manches Olivier , Lui Gabrielle , Chaperot Laurence , Gressin Rémy , Molens Jean-Paul , Jacob Marie-Christine , Sotto Jean-Jacques , Leroux Dominique , Bensa Jean-Claude , Plumas Joël 
Published: January 2003

AbstractTo assess the sensitivity of primary non-Hodgkin lymphoma cells to rituximab-mediated cytotoxicity, we compared the potency of several rituximab-mediated killing mechanisms on fresh lymphoma cells. All lymphoma cells tested were equally sensitive to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis of tumor cells, and rituximab-induced apoptosis. However, they were differentially lysed by complement-dependent cytotoxicity (CDC). We found that taking into account both CD20 and complement regulatory protein expression on tumor cells could predict CDC sensitivity in vitro. Importantly, the sensitivity of lymphoma cells to CDC was consistent with the reported different clinical response rates of lymphomas: rituximab induced high CDC killing of follicular lymphoma cells, whereas mantle cell lymphoma and diffuse large cell lymphoma cells were moderately sensible to CDC, and small lymphocytic lymphoma cells were almost all resistant. We propose that CDC is a determinant mechanism of rituximab-induced killing in vivo. Poor sensitivity to CDC in vitro might predict a poor clinical response, whereas high sensitivity to CDC would only indicate a likelihood of response to rituximab treatment.
Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity
Authors: Amrolia Persis J. , Reid Steven D. , Gao Liquan , Schultheis Beate , Dotti Gianpietro , Brenner Malcolm K. , Melo Junia V. , Goldman John M. , Stauss Hans J. 
Published: January 2003

AbstractRecent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2− donors by sequential stimulation with peptide-pulsed HLA-A2+ stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2+ patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2+ patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2+ fibroblasts or HLA-A2− normal PBMCs. Coculture of CD34+ PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit–granulocyte macrophage (CFU-GM) formation in HLA-A2+healthy controls and CML patients but resulted in no significant inhibition in HLA-A2− healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2–mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2+ progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.
A macrophage colony-stimulating factor receptor–green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse
Authors: Sasmono R. Tedjo , Oceandy Delvac , Pollard Jeffrey W. , Tong Wei , Pavli Paul , Wainwright Brandon J. , Ostrowski Michael C. , Himes S. Roy , Hume David A. 
Published: January 2003

AbstractThe c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fmstranscripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5′ flanking sequence and the downstream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms–positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (theFms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function.
Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)–1promoter/enhancer during mouse embryogenesis
Authors: Hirai Hideyo , Ogawa Minetaro , Suzuki Norio , Yamamoto Masayuki , Breier Georg , Mazda Osam , Imanishi Jiro , Nishikawa Shin-Ichi 
Published: January 2003

AbstractAccumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFP gene conjugated to this unit. The expression ofGFP differed from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+ lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)–cadherin+ ECs. Emergence of GFP− ECs preceded that of GFP+ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP− ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP+ and GFP− ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.
The zinc finger transcription factor ZBP-89 is a repressor of the human β2-integrin CD11b gene
Authors: Park Heiyoung , Shelley C. Simon , Arnaout M. Amin 
Published: January 2003

AbstractIntegrin CD11b is a differentiation marker of the myelomonocytic lineage and an important mediator of inflammation. Expression of theCD11b gene is transcriptionally induced as myeloid precursors differentiate into mature cells, then drops as monocytes further differentiate into macrophages. Previous studies have identified elements and factors involved in the transcriptional activation of the CD11b gene during myeloid differentiation, but no data exist regarding potential down-regulatory factors, especially in the later stages of differentiation. Using 2 copies of a GC-rich element (−141 to −110) in the CD11bpromoter, we probed a cDNA expression library for interacting proteins. Three clones were identified among 9.1 million screened, all encoding the DNA-binding domain of the zinc finger factor ZBP-89. Overexpression of ZBP-89 in the monocyte precursor cell line U937 reducedCD11b promoter-driven luciferase activity when U937 cells were induced to differentiate into monocytelike cells using phorbol esters. To identify the differentiation stage at which ZBP-89 repression of the CD11b gene is exerted, the protein level of ZBP-89 was correlated with that of CD11b mRNA in differentiating U937 as well as in normal human monocytes undergoing in vitro differentiation into macrophages. A clear inverse relationship was observed in the latter but not the former state, suggesting that ZBP-89 represses CD11b gene expression during the further differentiation of monocytes into macrophages.
Identification of a novel class of human adherent CD34− stem cells that give rise to SCID-repopulating cells
Authors: Kuçi Selim , Wessels Johannes T. , Bühring Hans-Jörg , Schilbach Karin , Schumm Michael , Seitz Gabriele , Löffler Jürgen , Bader Peter , Schlegel Paul G. , Niethammer Dietrich , Handgretinger Rupert 
Published: January 2003

Abstract
Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched, mobilized CD133+ peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells, endothelial cells, mesenchymal cells, dendritic cells, and stromal fibroblasts. However, all adherent cells expressed the adhesion molecules VE-cadherin, CD54, and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-α) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable, nonadherent CD133+CD34−stem cell subset. These cells do not generate in vitro hematopoietic colonies. However, their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34+ cells, suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage, nonadherent CD34− cells were able to give rise to human cells with B-, T-, and natural killer–cell phenotype. Hence, these cells possess a distinct in vivo differentiation potential compared with that of CD34+ stem cells and may therefore provide an alternative to CD34+ progenitor cells for transplantation.
Constitutive overexpression of IL-5 induces extramedullary hematopoiesis in the spleen
Authors: Khaldoyanidi Sophia , Sikora Lyudmila , Broide David H. , Rothenberg Marc E. , Sriramarao P. 
Published: January 2003

AbstractThe differentiation of eosinophils from hematopoietic precursors and their subsequent maturation, chemotaxis, and activation is primarily regulated by interleukin-5 (IL-5). To examine the effect of chronic IL-5 exposure on hematopoiesis, IL-5 transgenic (IL-5trg) mice and wild-type BALB/c (WT) mice were examined. In comparison to WT mice, a significant alteration in bone marrow hematopoiesis was observed in IL-5trg mice. Although the total number of myeloid progenitors in the bone marrow of IL-5trg mice was not significantly altered, the number of long-term culture-initiating cells (LTC-ICs) was 1.5-fold lower than that observed in WT mice. Furthermore, IL-5trg mice failed to demonstrate hematopoietic activity in long-term bone marrow cultures, which correlated with a significant decrease in the number of bone marrow mesenchymal/stromal progenitor (MSP) cells in these mice. In comparison to WT mice, a 10-fold decrease was observed in the number of fibroblast colony-forming units (CFU-Fs) in IL-5trg bone marrow. Hematopoietic activity of IL-5trg bone marrow cells was rescued by cultivation on preestablished layers of bone marrow-derived stromal cells. However, in contrast to bone marrow, increased hematopoietic activity was observed in the spleen and peripheral blood of IL-5trg mice. Likewise, the numbers of LTC-ICs and granulocyte-macrophage, macrophage, eosinophil, B-lymphocyte progenitors in the peripheral blood and spleen of IL-5trg mice were approximately 20-fold higher than in WT mice. A significant increase in CFU-F numbers was also observed in the spleens of IL-5trg mice compared with WT mice. Overall, our results suggest that constitutive overexpression of IL-5 can potentially induce colonization of spleen with MSP cells, which provides the necessary microenvironment for establishment of hematopoiesis in extramedullary sites.
Involvement of p38 mitogen–activated protein kinase in different stages of thymocyte development
Authors: Hsu Shu-Ching , Wu Chia-Cheng , Han Jiahuai , Lai Ming-Zong 
Published: January 2003

AbstractPositive selection of thymocytes during T-cell development is mediated by T-cell receptor (TCR)–activated signals. For different mitogen-activated protein kinases (MAPKs) activated by TCR complex, a selective involvement of extracellular signal–regulated kinase, but not p38 MAPK, in positive selection has been suggested. Using transgenic mice with dominant-negative mutation of both MAP kinase kinase 3 (MMK3) and MKK6, we obtained mice with different extents of inhibition of p38 MAPK activation. Partial inhibition of p38 MAPK impaired CD4−CD8− thymocyte development and T-cell proliferation, but not positive selection. Interference with thymocyte positive selection was observed in mice with effective suppression of p38 MAPK. Our results suggest that, in addition to early thymocyte development, p38 is involved in positive selection.
Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia
Authors: Rezvany Mohammad-Reza , Jeddi-Tehrani Mahmood , Wigzell Hans , Österborg Anders , Mellstedt Håkan 
Published: January 2003

AbstractT-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.