bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor
Authors: Schulte am Esch Jan , Cruz Miguel A. , Siegel Jonathan B. , Anrather Josef , Robson Simon C. 
Published: October 2019

AbstractPlatelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.
Modulation of Idarubicin-Induced Apoptosis in Human Acute Myeloid Leukemia Blasts by All-Trans Retinoic Acid, 1,25(OH)2 Vitamin D3, and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Ketley Nicolas J. , Allen Paul D. , Kelsey Stephen M. , Newland Adrian C. 
Published: October 2019

Abstract
The relationship between differentiation of human myeloid cells and apoptosis remains unclear. Recent studies have shown that terminal differentiation need not necessarily lead to the apoptotic demise of myeloid cells, while other studies have shown that induction of differentiation is associated with increased resistance to apoptosis-inducing agents, such as chemotherapy and γ-irradiation. Such results are pertinent to the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome, where differentiating agents and hemopoietic growth factors are being combined with chemotherapy to enhance response and limit toxicity. To elucidate the factors governing apoptosis in human AML blasts, we have studied the cytotoxic effect of idarubicin on HL60, U937 and KG1 cells, after incubation with all-trans retinoic acid (ATRA), 1,25(OH)2 D3, and granulocyte-macrophage colony-stimulating factor (GM-CSF ). We show that prior incubation of human myeloid leukemic cells with ATRA or 1,25(OH)2 D3 induced resistance to idarubicin-induced apoptosis, which was modulated by coincubation with GM-CSF. The altered chemosensitivity of cells depended on the degree of G0/G1 cell-cycle arrest induced by incubation with ATRA, 1,25(OH)2 D3, and GM-CSF and was independent of differentiation status or Bcl-2 oncoprotein expression. These findings suggest that cell-cycle arrest in human leukemic cells can be induced by exogenous agents and may promote drug resistance. Determining the mechanisms by which cell-cycle arrest is induced may permit understanding of the processes by which the cells escape cytotoxic drug-mediated apoptosis.
The Human β Globin Locus Introduced by YAC Transfer Exhibits a Specific and Reproducible Pattern of Developmental Regulation in Transgenic Mice
Authors: Porcu Susanna , Kitamura Michael , Witkowska Ewa , Zhang Zemin , Mutero Annick , Lin Chin , Chang Judy , Gaensler Karin M.L. 
Published: October 2019

Abstract
The human β globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult β globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the β globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from γ to β globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human β globin mRNA levels between lines. Human β globin mRNA levels were twofold to fourfold lower than that of mouse βmaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the β globin locus.
Synchronized Cell-Cycle Induction of Engrafting Long-Term Repopulating Stem Cells
Authors: Nilsson Susan K. , Dooner Mark S. , Quesenberry Peter J. 
Published: October 2019

AbstractWe have recently defined the window for marrow stem cell homing into nonablated hosts as the first 24 hours posttransplant. Within this homing window, donor cells rapidly cleared from the peripheral blood and lungs and plateaued in the marrow. We have now assessed the cell-cycle status of the engrafting cells capable of contributing to long-term hematopoiesis using administration of hydroxyurea (HU), a chemotherapy agent with S-phase cell-cycle specificity. HU was given at very short periods following a male bone marrow transplant (0, 3, 6, 12, and 15 hours) into female nonablated hosts, and donor cell engraftment was analyzed after 6 weeks. The data show that quickly after transplant (12 hours), greater than half of the engrafting cells capable of contributing long-term to all levels of the hematopoietic hierarchy are in S-phase. Analysis after 6 weeks included whole bone marrow, peripheral blood, primitive cells with high proliferative potential, and mature lineage-restricted marrow cells. These donor cells appear to be naturally synchronized. When HU was administered at any of the other time points, there was little evidence of cell death 6 weeks postengraftment.
Inhibition of Interferon Regulatory Factor-1 Expression Results in Predominance of Cell Growth Stimulatory Effects of Interferon-γ Due to Phosphorylation of Stat1 and Stat3
Authors: Sato Tadatsugu , Selleri Carmine , Young Neal S. , Maciejewski Jaroslaw P. 
Published: October 2019

AbstractInterferon-γ (IFN-γ) is a potent inhibitor of hematopoiesis in vitro and has been implicated in the pathophysiology of human bone marrow failure syndromes. IFN-γ both inhibits cell cycling and induces expression of the Fas-receptor, resulting in subsequent apoptosis of hematopoietic progenitor cells. IFN regulatory factor-1 (IRF-1) mediates some of these suppressive effects by activation of downstream inducible genes, such as double-stranded RNA-activatable protein kinase and inducible nitric oxide synthase. However, under certain experimental conditions, IFN-γ appears to stimulate proliferation of hematopoietic cells. Based on the hypothesis that IFN-γ–receptor triggering may activate diverse signaling cascades, we designed experiments to determine which intracellular mechanisms (in addition to the IRF-1 transduction pathway) influence the biologic effects of IFN-γ. Using antisense technique, we inhibited the IRF-1–mediated pathway in KG1a cells stimulated with IFN-γ. In contrast to the suppressive effects of IFN-γ observed in control cells, untreated and IFN-γ–treated KG-1a cells that were transduced with retroviral vectors expressing IRF-1 antisense mRNA showed enhanced proliferation. The increased growth rate was associated with decreased levels of IRF-1 mRNA and protein but unchanged levels of IRF-2. We inferred that IFN-γ could also activate a stimulatory transduction pathway that, under specific conditions, may control the cellular response to this cytokine. The family of Stat proteins is involved in signal transduction of hematopoietic growth factors. We showed that, in KG-1a cells, IFN-γ also induced phosphorylation of Stat1 and Stat3, whereas p42 MAP kinase was phosphorylated regardless of the presence of IFN-γ. Using electrophoresis mobility shift assays, IFN-γ enhanced Stat1-Stat1 homodimer and Stat1-Stat3 heterodimer formation, suggesting that, in addition to inhibitory signals mediated by IRF-1, IFN-γ may activate proliferative signals by phosphorylation of Stat1 and Stat3 proteins. The observations made in experiments with KG-1a cells were confirmed in primary hematopoietic cells. After inhibition of the IRF-1 pathway by transduction of an antisense IRF-1 retrovirus into human CD34+ cells, IFN-γ produced an aberrant stimulatory effect on hematopoietic colony formation. Conversely, in control vector-transduced CD34+ cells, the typical inhibitory response to IFN-γ was seen. Our results indicate that inhibitory cytokines such as IFN-γ may exhibit diverse biologic effects depending on the intracellular balance of transcriptional regulators, in turn influenced by the activation and differentiation status of the target cells.
Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor
Authors: Okuda Keiko , Smith Lorie , Griffin James D. , Foster Rosemary 
Published: October 2019

AbstractThe granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.
Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Dissemination and Induces Remission of Juvenile Myelomonocytic Leukemia in Engrafted Immunodeficient Mice
Authors: Iversen Per O. , Lewis Ian D. , Turczynowicz Suzanne , Hasle Henrik , Niemeyer Charlotte , Schmiegelow Kjeld , Bastiras Stan , Biondi Andrea , Hughes Timothy P. , Lopez Angel F. 
Published: October 2019

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor α (TNFα) have been implicated in the pathogenesis of the fatal childhood disease termed juvenile myelomonocytic leukemia (JMML). We used a severe combined immunodeficient/nonobese diabetic (SCID/NOD) mouse model of JMML and examined the effect of inhibiting these cytokines in vivo with the human GM-CSF antagonist and apoptotic agent E21R and the anti-TNFα monoclonal antibody (MoAb) cA2 on JMML cell growth and dissemination in vivo. We show here that JMML cells repopulated to high levels in the absence of exogeneous growth factors. Administration of E21R at the time of transplantation or 4 weeks after profoundly reduced JMML cell load in the mouse bone marrow. In contrast, MoAb cA2 had no effect on its own, but synergized with E21R in virtually eliminating JMML cells from the mouse bone marrow. In the spleen and peripheral blood, E21R eliminated JMML cells, while MoAb cA2 had no effect. Importantly, studies of mice engrafted simultaneously with cells from both normal donors and from JMML patients showed that E21R preferentially eliminated leukemic cells. This is the first time a specific GM-CSF inhibitor has been used in vivo, and the results suggest that GM-CSF plays a major role in the pathogenesis of JMML. E21R might offer a novel and specific approach for the treatment of this aggressive leukemia in man.
Molecular Follow-Up of Disease Progression and Interferon Therapy in Chronic Myelocytic Leukemia
Authors: Ben-Yehuda Dina , Krichevsky Svetlana , Rachmilewitz Eliezer A. , Avraham Ayelet , Palumbo Giuseppe A. , Frassoni Francesco , Sahar Dvora , Rosenbaum Hanna , Paltiel Ora , Zion Michal , Ben-Neriah Yinon 
Published: October 2019

Abstract
We previously reported that the abl promoter (Pa) undergoes de novo DNA methylation in the course of chronic myelocytic leukemia (CML). The clinical implications of this finding are the subject of the present study in which samples of CML patients, including a group treated with interferon α (IFNα) were surveyed. The methylation status of the abl promoter was monitored by polymerase chain reaction (PCR) amplification of the Pa region after digestion with several site-methylation sensitive restriction enzymes. Some 74% of the DNA samples from blood and marrow drawn in the chronic phase were nonmethylated, similar to control samples from non-CML patients. The remaining 26% were partially methylated in the abl Pa region. The latter samples were derived from patients who were indistinguishable from the others on the basis of clinical presentation. Methylated samples were mostly derived from patients known to have a disease of longer duration (26 months v 7.5 months, P = .01). Samples of 30 IFNα-treated patients were sequentially analyzed in the course of treatment. Fifteen patients with no evidence of Pa methylation before treatment remained methylation-free. The remainder, who displayed Pa methylation before treatment, reverted to the methylation-free status. The outcome is attributed to IFNα therapy, as the Pa methylation status was not reversed in any of the patients treated with hydroxyurea. Methylation of the abl promoter indicates a disease of long-standing, most likely associated with a higher probability of imminent blastic transformation. It appears to predict the outcome of IFNα therapy far better than the cytogenetic response.
Incidence of TEL/AML1 Fusion Gene Analyzed Consecutively in Children With Acute Lymphoblastic Leukemia in Relapse
Authors: Harbott J. , Viehmann S. , Borkhardt A. , Henze G. , Lampert F. 
Published: October 2019

Abstract
The translocation t(12; 21)(p13; q22) is difficult to detect by classic cytogenetics. However, using fluorescence in situ hybridization (FISH) and by screening for the TEL/AML1 rearrangement by the polymerase chain reaction (PCR), it has been demonstrated to be the most frequent known structural chromosomal abnormality in childhood acute lymphoblastic leukemia (ALL). It is closely correlated with a B-cell precursor (BCP) phenotype and is considered a favorable prognostic factor. However, little is known about the incidence of the translocation in relapsed patients and the duration of complete remission (CR) in children expressing the TEL/AML1 fusion gene. We therefore examined 49 bone marrow samples from children with ALL at first or second relapse that were consecutively mailed to our laboratory to test for the presence of t(12; 21) using reverse transcriptase (RT)-PCR. The TEL/AML1 rearrangement could be identified in nine of 44 (20%) of the patients, a result similar to the reported incidence at diagnosis. Most of the TEL/AML1–positive children showed no adverse clinical features at diagnosis (eg, white blood cell [WBC] count <100 × 109/L or age <10 years), and regarding these data, there were no differences versus children who were negative for the fusion gene. However, the period of remission was about 1 year longer in children expressing TEL/AML1 (P = .046), and the majority of relapses in this group appeared late (<2 years after diagnosis). Our findings therefore reinforce the urgent need for further prospective studies with a long follow-up period to determine the true prognostic significance of t(12; 21) and to avoid premature changes of treatment strategies.
Abnormal Expression of the B-Cell Homing Chemokine Receptor BLR1 During the Progression of Acquired Immunodeficiency Syndrome
Authors: Förster Reinhold , Schweigard Georgina , Johann Sabine , Emrich Thomas , Kremmer Elisabeth , Nerl Christoph , Lipp Martin 
Published: October 2019

AbstractThe putative chemokine receptor BLR1 has been identified as the first G-protein–coupled receptor involved in B-cell migration and in microenvironmental homing to B-cell follicles and to germinal centers. In healthy individuals, expression of BLR1 is restricted to all mature recirculating B cells and to a subpopulation of T-helper memory cells. In the present study, we analyzed the distribution of BLR1 on defined lymphocyte subsets during the progression of acquired immunodeficiency syndrome. It is shown that the proportion of T-helper memory cells coexpressing BLR1 continuously decreases during the infection, whereas a high proportion of γ/δ T cells expressing BLR1 can be found in peripheral blood. The latter subpopulation is restricted to lymphoid tissues in healthy individuals. Most interestingly, in 75% of all human immunodeficiency virus (HIV)+ individuals, peripheral blood B cells were identified as not expressing BLR1 and phenotypically resembling germinal center cells of lymphoid tissue. Using BLR1 as a marker molecule, this study identifies peripheral blood lymphocytes in HIV+ individuals that are usually restricted to lymphoid tissue in healthy individuals. Because HIV infection is active in lymphoid tissue even at the clinically latent stage, aberrant expression of the B-cell homing chemokine receptor BLR1 might be an early indicator for the onset of destruction of lymphoid tissue.
Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells
Authors: Sui Xingwei , Krantz Sanford B. , Zhao Zhizhuang 
Published: October 2019

AbstractPolycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.
Signaling Pathways Regulating CD44-Dependent Cytolysis in Natural Killer Cells
Authors: Sconocchia Giuseppe , Titus Julie A. , Segal David M. 
Published: October 2019

AbstractCD44 is a cytotoxic triggering molecule on activated, but not fresh natural killer (NK) cells. In the current study, metabolic pathways used in CD44-directed lysis (CD44DL) were examined using activated human NK cells as effectors. We found that CD44 expressed by activated NK cells was indistinguishable in isoform and molecular weight from CD44 on unactivated cells. However, de novo protein expression was required for the induction of CD44DL, suggesting that activated NK cells contain proteins not present in fresh NK cells that couple CD44 to the lytic machinery. Concanimycin A, a selective inhibitor of perforin-based cytolysis, totally blocked CD44DL, natural cytototoxicity, and antibody-dependent cell-mediated cytolysis (ADCC). Moreover, studies in which kinase inhibitors were added during the effector phase of lysis indicated that protein-tyrosine and ser/thr kinases were required for all three cytolytic activities and that protein kinase C played a nonessential role in lysis. By contrast, wortmannin totally inhibited CD44DL, but failed to block natural cytotoxicity and only partially blocked ADCC, suggesting that phosphatidylinositol 3-kinase (PI 3-kinase) is required at an early, receptor-specific stage of CD44DL. Finally, cytochalasin B enhanced CD44DL, but not ADCC, indicating that CD44DL is modulated by actin polymerization. Taken together, our data suggest that CD44 in NK cells interacts with proteins induced during interleukin-2 activation in a triggering pathway that induces perforin release, requires PI 3-kinase, and is modulated by the cytoskeleton.
Ig DH Gene Segment Transcription and Rearrangement Before Surface Expression of the Pan-B–Cell Marker CD19 in Normal Human Bone Marrow
Authors: Bertrand F.E. , Billips L.G. , Burrows P.D. , Gartland G.L. , Kubagawa H. , Schroeder H.W. 
Published: October 2019

AbstractThe onset of IgH transcription and rearrangement is a defining characteristic of the progenitor population in which B-lineage commitment occurs. These features were used to better define the earliest stage of B-cell commitment in humans and to determine if these stages differ as a function of human ontogeny. Fetal and adult bone marrow mononuclear cells were sorted into B-lineage subpopulations on the basis of surface expression of the stem cell marker CD34, the pan-B–cell marker CD19, and IgM and analyzed for transcription and rearrangement of the IgH locus. The locus was found to be transcriptionally active before surface expression of CD19, as indicated by the presence of germline Iμ, Cμ, and DHQ52 transcripts in the CD34+ CD19− subpopulation. Transcripts from IgH alleles that had undergone DJCμ rearrangements were also detected in the CD34+ CD19− subpopulation. Within this subpopulation, low levels of DXP-containing DJCμ transcripts were detected in both fetal and adult cells. Although DHQ52 DJCμ transcripts were abundant in fetal CD34+ CD19− cells, they were not detected in cells of the same phenotype derived from adult bone marrow. In both fetus and adult, VH3-and VH6-containing VDJCμ transcripts were detected only in the CD19+ subpopulations. These data indicate that transcription of DHQ52-JH and DXP-JH rearrangements differs during fetal and adult B lymphopoiesis. Moreover, in both fetus and adult, transcription of unrearranged components of the IgH locus and DJ rearrangements can proceed before the surface expression of CD19.