bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hematopoietic Potential and Retroviral Transduction of CD34+Thy-1+ Peripheral Blood Stem Cells From Asymptomatic Human Immunodeficiency Virus Type-1–Infected Individuals Mobilized With Granulocyte Colony-Stimulating Factor
Authors: Junker Uwe , Moon James J. , Kalfoglou Creton S. , Sniecinski Irena , Forman Stephen J. , Zaia John A. , Kaneshima Hideto , Böhnlein Ernst 
Published: October 2019

AbstractThe potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)–infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1–infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 μg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+Thy-1+ cell population of high purity (76% to 92% CD34+Thy-1+ cells). This cell population showed no evidence of HIV-1–containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1–infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.
In Vitro Maintenance of Highly Purified, Transplantable Hematopoietic Stem Cells
Authors: Moore Kateri A. , Ema Hideo , Lemischka Ihor R. 
Published: October 2019

AbstractThe cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity. In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells. This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.
Role of Glycoprotein V in the Formation of the Platelet High-Affinity Thrombin-Binding Site
Authors: Dong Jing-Fei , Sae-Tung Garunee , López José A. 
Published: October 2019

AbstractThe glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ibα, GP Ibβ, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ibα, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ibα was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.
The Missense Mutation Arg593 → Cys Is Related to Antibody Formation in a Patient With Mild Hemophilia A
Authors: Fijnvandraat Karin , Turenhout Ellen A.M. , van den Brink Edward N. , ten Cate Jan W. , van Mourik Jan A. , Peters Marjolein , Voorberg Jan 
Published: October 2019

AbstractThe development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII < 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593 by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593 → Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593 → Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593 → Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.
Reassessment of Protein Tyrosine Phosphorylation in Thrombasthenic Platelets: Evidence That Phosphorylation of Cortactin and a 64-kD Protein Is Dependent on Thrombin Activation and Integrin αIIbβ3
Authors: Rosa Jean-Philippe , Artçanuthurry Valérie , Grelac Françoise , Maclouf Jacques , Caen Jacques P. , Lévy-Toledano Sylviane 
Published: October 2019

AbstractTyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin αIIbβ3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack αIIbβ3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% αIIbβ3, functional), and variant type (50% αIIbβ3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional αIIbβ3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by αIIbβ3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of αIIbβ3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.
The Conversion of Fibrinogen to Fibrin: Recombinant Fibrinogen Typifies Plasma Fibrinogen
Authors: Gorkun Oleg V. , Veklich Yuri I. , Weisel John W. , Lord Susan T. 
Published: October 2019

AbstractPlasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.
Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties
Authors: Hornick Jason L. , Khawli Leslie A. , Hu Peisheng , Lynch Maureen , Anderson Peter M. , Epstein Alan L. 
Published: October 2019

AbstractAlthough monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.
GABP Cooperates With c-Myb and C/EBP to Activate the Neutrophil Elastase Promoter
Authors: Nuchprayoon Issarang , Simkevich Carl P. , Luo Menglin , Friedman Alan D. , Rosmarin Alan G. 
Published: October 2019

AbstractNeutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells — GABP — binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABPα, an ets factor, and GABPβ, a Notch-related protein. GABPα bound to the mNE ets site and, in turn, recruited GABPβ to form a transcriptionally active complex. GABPα and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBPα to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.
Pertussis Toxin Shows Distinct Early Signalling Events in Platelet-Activating Factor–, Leukotriene B4–, and C5a-Induced Eosinophil Homotypic Aggregation In Vitro and Recruitment In Vivo
Authors: Teixeira Mauro M. , Giembycz Mark A. , Lindsay Mark A. , Hellewell Paul G. 
Published: October 2019

AbstractThe present study was performed to investigate the early signalling events responsible for eosinophil activation in response to platelet-activating factor (PAF ), C5a, and leukotriene B4 (LTB4 ). We evaluated the effect of pertussis toxin (PTX) on eosinophil aggregation in vitro and cutaneous eosinophil recruitment in vivo. Further studies using the protein kinase inhibitors Ro 31-8220 and staurosporine were performed in vitro to assess in more detail the early signalling events induced by these three mediators. Our results show that C5a and LTB4 signal predominantly or exclusively through a PTX-sensitive G protein that is negatively modulated by protein kinase C, possibly at the level of phospholipase C-β. In contrast, PAF activates eosinophils independent of Gi by a mechanism that is abolished by Ro 31-8220, a selective protein kinase C inhibitor. In addition, these results show for the first time that a receptor-operated event on the eosinophil is essential for chemoattractant-induced eosinophil recruitment in vivo.
FLT3 Ligand Preserves the Ability of Human CD34+ Progenitors to Sustain Long-Term Hematopoiesis in Immune-Deficient Mice After Ex Vivo Retroviral-Mediated Transduction
Authors: Dao Mo A. , Hannum Charles H. , Kohn Donald B. , Nolta Jan A. 
Published: October 2019

Abstract
Stromal support is required during retroviral-mediated transduction of human bone marrow-derived CD34+ cells to maintain the clonogenicity of the primitive progenitors. We hypothesized that the cytokine FLT3 ligand (FL) might be able to replace the maintenance role provided by the stroma. CD34+ progenitors from human bone marrow were transduced by the retroviral vector LN with the cytokines interleukin-3 (IL-3), IL-6, and stem cell factor (SCF ) present in all cultures. Transductions were performed with or without stromal support and with or without the inclusion of 100 U/mL FL. No significant increase in gene transfer into colony-forming cells was obtained by the addition of FL to the cultures. Transduction and survival of more primitive human hematopoietic cells was determined by growth in immune-deficient mice for 7 to 8 months. Human myeloid cells, T lymphocytes, and colony-forming progenitors were recovered from the marrow of mice that had received human cells transduced on stroma or in suspension culture with IL-3, IL-6, SCF, and FL, but not with IL-3, IL-6, and SCF alone. LN provirus was detected by polymerase chain reaction in the marrow recovered from 9 of 10 mice transplanted with human CD34+ cells transduced with stromal support, 5 of 11 mice that received human cells transduced in suspension culture with FL, but none of the 10 mice that received human cells transduced in suspension culture without FL. We conclude that FLT3 ligand, in conjunction with IL-3, IL-6, and SCF, preserves the generative capacity of primitive human hematopoietic cells during in vitro transductions in suspension culture.
Homodimerization of Erythropoietin Receptor by a Bivalent Monoclonal Antibody Triggers Cell Proliferation and Differentiation of Erythroid Precursors
Authors: Schneider Helmut , Chaovapong Warak , Matthews David J. , Karkaria Cyrus , Cass Robert T. , Zhan Hangjun , Boyle Mark , Lorenzini Tony , Elliott Steve G. , Giebel Lutz B. 
Published: October 2019

AbstractErythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Several lines of evidence indicate that the most likely mechanism of EPO receptor (EPO-R) activation by EPO is homodimerization of the receptor on the surface of erythrocyte precursors. Therefore, we argued that it should be possible to raise EPO-R monoclonal antibodies (MoAbs) that would activate the receptor by dimerization and thus mimic EPO action. We have identified such an agonist MoAb (MoAb34) directed against the extracellular EPO binding domain of the EPO-R. This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. In contrast, the monovalent Fab fragment, which cannot dimerize the receptor, is completely inactive. The mechanism of receptor activation by homodimerization implies that at high ligand concentrations the formation of 1:1 receptor/ligand complexes is favored over 2:1 complexes, thereby turning the ligand agonist into an antagonist. Thus, EPO and MoAb34 should self-antagonize at high concentrations in both cell proliferation and differentiation assays. Our data indeed demonstrate that EPO and MoAb34 antagonize ligand-dependent cell proliferation with IC50 values of approximately 20 and 2 μmol/L, respectively. Erythroid colony formation (BFUe) is inhibited at MoAb34 concentrations above 1 μmol/L. Furthermore, we analyzed the MoAb34:EPO-R interaction using a mathematic model describing antibody-mediated receptor dimerization. The data for proliferation and differentiation activity were consistent with the receptor dimer formation on the cell surface predicted by the model.
Induction of Hypoxia-Inducible Factor-1, Erythropoietin, Vascular Endothelial Growth Factor, and Glucose Transporter-1 by Hypoxia: Evidence Against a Regulatory Role for Src Kinase
Authors: Gleadle Jonathan M. , Ratcliffe Peter J. 
Published: October 2019

AbstractThe induction by hypoxia of genes such as erythropoietin, vascular endothelial growth factor (VEGF ), and glucose transporter-1 (Glut-1) is mediated in part by a transcriptional complex termed hypoxia-inducible factor-1 (HIF-1). Several lines of evidence have implicated protein phosphorylation in the mechanism of activation of HIF-1 by hypoxia. Recent reports have described the activation of the tyrosine kinase src by severe hypoxia, and a role in the induction of VEGF by severe hypoxia has been proposed. This led us to examine whether src and related kinases operated more widely in the hypoxic induction of HIF-1 and HIF-1–dependent genes regulated by hypoxia. Measurements of src kinase activity in cells exposed to varying severities of hypoxia showed activation by severe hypoxia (0.1% oxygen or catalyst induced anoxia), but not 1% oxygen. This contrasted with the marked induction of HIF-1 by exposure to 1% oxygen. Manipulations of src activity were produced by transient and stable transfection of Hep3B cells. Despite substantial changes in src activity, no alteration was seen in the normoxic or hypoxic expression of erythropoietin, VEGF, or Glut-1, or in the regulation of HIF-1–dependent reporter genes inducible by hypoxia. Similarly, we found that the expression of these genes in src- or c-src kinase-deficient cells did not differ from wild-type cells at either 1% oxygen or more severe hypoxia. These results indicate that src is not critical for the hypoxic induction of HIF-1, erythropoietin, VEGF, or Glut-1.
Exocytotic Competence and Intergranular Fusion in Cord Blood-Derived Eosinophils During Differentiation
Authors: Scepek Susanne , Lindau Manfred 
Published: October 2019

AbstractWe studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5′-O-(3-thiotriphosphate) (GTP) γS after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from ≈700 μm2 to ≈400 μm2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.