bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Influences Determining Progenitor Cell Mobilization and Leukocytosis Induced by Granulocyte Colony-Stimulating Factor
Authors: Roberts Andrew W. , Foote Simon , Alexander Warren S. , Scott Clare , Robb Lorraine , Metcalf Donald 
Published: October 2019

Abstract
The mechanisms involved in the mobilization of progenitor cells into the blood by granulocyte colony-stimulating factor (G-CSF ) and other cytokines are poorly understood. To identify important influences on this complex process, in vivo murine models were used. Granulocyte-macrophage colony-stimulating factor (GM-CSF ) transgenic, Max41 transgenic, W/WV, Mpl-null, GM-CSF receptor (β chain)-null mice, wild-type littermate controls, and six inbred strains of mice were injected with 200 μg/kg/d G-CSF for 5 days. Three parameters of response were monitored: white blood cell count (WCC), peripheral blood progenitor cell (PBPC) numbers, and spleen weight. In all genotypes studied, G-CSF induced increases in these three parameters. However, PBPC mobilization in W/WV and Mpl-null mice was only 30% and 9%, respectively, of that observed in wild-type mice. In contrast, perturbations of GM-CSF signalling had no demonstrable effect on in vivo responses to G-CSF. Broad variability was evident between inbred strains for each parameter of the response to G-CSF. A 10-fold range in response was observed for circulating progenitor cell numbers, similar to that observed for normal human subjects receiving G-CSF. The interstrain differences were in the distribution of mature and progenitor cells between peripheral blood, bone marrow, and spleen rather than in the total numbers of these cells in the body. Results of an F2 intercross of low-responding C57BL/6 and intermediate-responding SJL mice indicated that regulation of progenitor cell mobilization is a complex genetic trait, that there is a correlation between this trait and WCC response (r2 = .5), and that this approach may serve as a useful model for the identification of genes involved in the mobilization process.
Purification and Molecular Cloning of SH2- and SH3-Containing Inositol Polyphosphate-5-Phosphatase, Which Is Involved in the Signaling Pathway of Granulocyte-Macrophage Colony-Stimulating Factor, Erythropoietin, and Bcr-Abl
Authors: Odai Hideharu , Sasaki Ko , Iwamatsu Akihiro , Nakamoto Tetsuya , Ueno Hiroo , Yamagata Tetsuya , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru 
Published: October 2019

AbstractGrb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF ). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.
Functional Studies on Platelet Adhesion With Recombinant von Willebrand Factor Type 2B Mutants R543Q and R543W Under Conditions of Flow
Authors: Lankhof Hanneke , Damas Conchi , Schiphorst Marion E. , IJsseldijk Martin J.W. , Bracke Madelon , Sixma Jan J. , Vink Tom , de Groot Philip G. 
Published: October 2019

AbstractType 2B von Willebrand disease (vWD) is characterized by the absence of the very high molecular weight von Willebrand factor (vWF ) multimers from plasma, which is caused by spontaneous binding to platelet receptor glycoprotein Ib (GPIb). We studied two mutations in the A1 domain at position 543 in which arginine (R) was replaced by glutamine (Q) or tryptophan (W), respectively. Both mutations were previously identified in vWD type 2B patients. The mutations R543Q and R543W were cloned into a eukaryotic expression vector and subsequently transfected in baby hamster kidney cells overexpressing furin (fur-BHK). Stable cell lines were established by which the mutants were secreted in the cell culture supernatant. The subunit composition and multimeric structure of R543Q and R543W were similar to wild-type (WT) vWF. The mutants showed a spontaneous binding to GPIb. R543Q and R543W showed normal binding to collagen type III or heparin. Both mutants supported platelet adhesion under conditions of flow, usually when preincubated on a collagen type III surface. A low dose (2.5% of the concentration present in normal pooled plasma) of recombinant R543Q or R543W added to normal whole blood inhibited platelet adhesion to collagen type III. No inhibition was found when vWF was used as an adhesive surface. These results indicate that point mutations identified in vWD type 2B cause bleeding symptoms by two mechanisms: (1) the mutants cause platelet aggregation, which in vivo is followed by removal of the aggregates leading to the loss of high molecular weight multimers and thrombocytopenia, (2) on binding to circulating platelets the mutants block platelet adhesion. Relatively few molecules are required for the latter effect.
Human Platelets as a Model for the Binding and Degradation of Thrombopoietin
Authors: Fielder Paul J. , Hass Philip , Nagel Mark , Stefanich Eric , Widmer Ramon , Bennett Gregory L. , Keller Gilbert-André , de Sauvage Frederic J. , Eaton Dan 
Published: October 2019

Abstract
Recent studies have shown that plasma thrombopoietin (TPO) levels appear to be directly regulated by platelet mass and that removal of plasma TPO by platelets via binding to the c-Mpl receptor is involved in the clearance of TPO in rodents. To help elucidate the role of platelets in the clearance of TPO in humans, we studied the in vitro specific binding of recombinant human TPO (rhTPO) to human platelet-rich plasma (PRP), washed platelets (WP), and cloned c-Mpl. Using a four-parameter fit and/or Scatchard analysis, the approximate affinity of rhTPO for its receptor, which was calculated from multiple experiments using different PRP preparations, was between 128 and 846 pmol/L, with ∼25 to 224 receptors per platelet. WP preparations gave an affinity of 260 to 540 pmol/L, with ∼25 to 35 receptors per platelet, and erythropoietin failed to compete with 125I-rhTPO for binding to WP. Binding and dissociation studies conducted with a BiaCore apparatus yielded an affinity of 350 pmol/L for rhTPO binding to cloned c-Mpl receptors. The ability of PRP to bind and degrade 125I-rhTPO was both time- and temperature-dependent and was blocked by the addition of excess cold rhTPO. Analysis of platelet pellets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 125I-rhTPO was degraded into a major fragment of ∼45 to 50 kD. When 125I-rhTPO was incubated with a platelet homogenate at pH = 7.4, a degradation pattern similar to intact platelets was observed. Together, these data show that human platelets specifically bind rhTPO with high affinity, internalize, and then degrade the rhTPO.
CD6 Ligation Modulates the Bcl-2/Bax Ratio and Protects Chronic Lymphocytic Leukemia B Cells From Apoptosis Induced by Anti-IgM
Authors: Osorio Lyda M. , De Santiago Angelina , Aguilar-SantelisesHå Miguel , Mellstedt kan , Jondal Mikael 
Published: October 2019

AbstractCD6 and CD5 belong to a scavenger-receptor cysteine-rich (SRCR) super family of membrane glycoproteins that are expressed on chronic lymphocytic leukemia B (B-CLL) cells, normal T cells, and a small subset of normal B cells. CD6 configures in the membrane in relation to the cellular activation level and can act as a coreceptor for T-cell activation. We have examined a group of progressive and nonprogressive B-CLL cells. Most B-CLL cells were positive for CD6 and the expression of CD6 was increased after activation with Staphylococcus aureus Cowan I plus interleukin-2 or 12-O-tetradecanoylphorbol 13-acetate, although anti-CD6 antibodies did not increase proliferative responses to these stimuli. However, anti-CD6 stimulation was found to protect against anti-IgM–induced apoptosis in B-CLL. baxα upregulation and bcl-2 downregulation were found in anti-IgM– and glucocorticoid (GCC)-induced apoptotic cells, respectively. Furthermore, CD6 cross-linking downregulated baxα mRNA levels in anti-IgM–treated cells, resulting in an increased bcl-2/baxα ratio. CD6 activation also prevented bcl-2 mRNA downregulation and apoptosis induced by GCC in one of six GCC-sensitive patients. These data suggest that an interaction between CD6 and its ligand might contribute to B-CLL survival through the modulation of the Bcl-2/Bax ratio.
A Synthetic CD4-CDR3 Peptide Analog Enhances Bone Marrow Engraftment Across Major Histocompatibility Barriers
Authors: Koch Ute , Korngold Robert 
Published: October 2019

AbstractThe efficacy of a synthetic peptide analog mimicking the CDR3-D1 domain of the CD4 molecule was investigated in murine models of allogeneic bone marrow engraftment after transplantation across major histocompatibility complex (MHC) barriers. A single dose of a CD4-CDR3 peptide analog was administered at the time of marrow transplantation to three different allogeneic mouse strain combinations after appropriate sublethal total body irradiation: (1) B10.BR → C57BL/6J (B6), a full allogeneic MHC difference; (2) (B6xDBA/2)F1 → (B6xCBA)F1 , a haploidentical MHC combination; and (3) B6.C-H2bm12 → B6-Ly5.2, involving only a MHC class II difference. Donor-host chimerism was assessed after 1 and 2 months posttransplantation by flow cytometric analysis of spleen and/or lymph node cells. Peptide-treated animals in all three strain combinations exhibited significantly enhanced donor lymphoid engraftment, which was similarly reflected in the total lymphocyte compartment and its T-cell (CD4+, CD8+) and B-cell subsets. In addition, peptide-treated mice in the haploidentical and MHC class II-mismatched strain combinations exhibited prolonged tolerance of both donor and syngeneic host-type tail skin grafts while rejecting third-party allogeneic grafts, thus supporting the reconstitution of immunocompetence in these chimeras. Lymphocytes from the peptide-treated haploidentical chimeric mice also displayed donor-specific tolerance upon stimulation in a one-way mixed lymphocyte reaction. In a 6-day colony-forming unit–granulocyte-macrophage (CFU-GM) assay to quantitate the level of hematopoietic cell engraftment in both the haploidentical and class II-disparate strain combinations, bone marrow cells from the peptide-treated mice exhibited significant increases in CFU-GM compared with the saline-treated control groups. Finally, early multiple treatments with the peptide after transplantation significantly enhanced donor chimerism in donor-presensitized recipient mice across the MHC class II barrier and proved to be significantly more effective than anti-CD4 monoclonal antibody treatment. These results indicate that the structure-based CD4-CDR3 peptide analog may represent a valuable approach to the inhibition of graft rejection after MHC-mismatched bone marrow transplantation.
Hyaluronate-CD44 Interactions Can Induce Murine B-Cell Activation
Authors: Rafi Asimah , Nagarkatti Mitzi , Nagarkatti Prakash S. 
Published: October 2019

AbstractCD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. In the current study, we investigated whether HA or monoclonal antibodies (MoAbs) against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude, but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 MoAbs. Furthermore, purified B cells, but not T cells, were found to respond to HA. HA was unable to stimulate T cells even in the presence of antigen presenting cells (APC) and was unable to act as a costimulus in the presence of mitogenic or submitogenic concentrations of anti-CD3 MoAbs. In contrast, stimulation of B cells with HA in vitro, led to B-cell differentiation as measured by production of IgM antibodies in addition to increased expression of CD44 and decreased levels of CD45R. The fact that the B cells were responding directly to HA through its binding to CD44 and not to any contaminants or endotoxins was demonstrated by the fact that F(ab)2 fragments of anti-CD44 MoAbs or soluble CD44 fusion proteins could significantly inhibit the HA-induced proliferation of B cells. Also, HA-induced proliferation of B cells was not affected by the addition of polymixin B, and B cells from lipopolysaccharide (LPS)-unresponsive C3H/HeJ strain responded strongly to stimulation with HA. Furthermore, HA, but not chondroitin-sulfate, another major component of the ECM, induced B-cell activation. It was also noted that injection of HA intraperitoneally, triggered splenic B cell proliferation in vivo. Together, the current study demonstrates that interaction between HA and CD44 can regulate murine B-cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells.
Identification of the Major Casein Kinase I Phosphorylation Sites on Erythrocyte Band 3
Authors: Wang Cheng C. , Tao Mariano , Wei Tiequan , Low Philip S. 
Published: October 2019

AbstractHuman erythrocyte band 3 is a major substrate of two red blood cell protein kinases, casein kinase I and p72syk protein tyrosine kinase. Although the phosphorylation sites and physiologic consequences of p72syk phosphorylation have been characterized, little is known regarding casein kinase I phosphorylation. In this report, we identify the major phosphorylation site of casein kinase I. Using isolated components, casein kinase I was found to phosphorylate the cytoplasmic domain of band 3 (CDB3), primarily on Thr residues. Classical peptide mapping narrowed the major phosphorylation site to a peptide encompassing residues 24-91. Computer-assisted evaluation of this sequence not only showed two consensus casein kinase I phosphorylation sites, but also provided information on how to proteolytically separate and isolate the candidate sites. Following the suggested protocols, a heptapeptide containing the major phosphorylation site was isolated, subjected to amino acid sequencing, and found to be phosphorylated on Thr 42. A minor phosphorylation site was similarly identified as Ser 303. Because Thr 42 is situated near the binding sites on CDB3 of ankyrin, protein 4.1, protein 4.2, and the glycolytic enzymes, phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or function.
Lack of Expression of Thy-1 (CD90) on Acute Myeloid Leukemia Cells With Long-Term Proliferative Ability In Vitro and In Vivo
Authors: Blair A. , Hogge D.E. , Ailles L.E. , Lansdorp P.M. , Sutherland H.J. 
Published: October 2019

AbstractAcute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo, we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1−. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1− phenotype. However, significant engraftment was achieved using both CD34+/Thy-1− and CD34− subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients, the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1.
CIS, a Cytokine Inducible SH2 Protein, Is a Target of the JAK-STAT5 Pathway and Modulates STAT5 Activation
Authors: Matsumoto Akira , Masuhara Masaaki , Mitsui Kaoru , Yokouchi Masahiro , Ohtsubo Motoaki , Misawa Hiroyuki , Miyajima Atsushi , Yoshimura Akihiko 
Published: October 2019

Abstract
We searched for immediate early cytokine responsive genes and isolated a novel gene, CIS (Cytokine Inducible SH2 containing protein) that is induced in hematopoietic cells by a subset of cytokines including interleukin-2 (IL-2), IL-3, and erythropoietin (EPO). The mutant IL-2 receptor that fails to activate STAT5 could not induce CIS, suggesting that STAT5 is involved in the cytokine-inducible expression of CIS. We cloned the 5′-flanking region of the CIS gene and found that about 200 bases upstream of the transcription-initiation site contain four potential STAT5 binding sites (MGF boxes). Luciferase reporter assays showed that these MGF boxes were essential for EPO-dependent promoter activity. Expression of STAT5 and the EPO receptor in HEK293 cells conferred EPO-dependent activation of the CIS promoter. These data indicate that CIS is a target of the JAK-STAT5 pathway of cytokine receptors. CIS contains an SH2 domain and binds to tyrosine-phosphorylated EPO and IL-3 receptors. In HEK293 cells expressing STAT5 and the EPO receptor, EPO-dependent tyrosine phosphorylation of STAT5, as well as EPO-dependent CIS-promoter activation, was suppressed when CIS was coexpressed. Moreover, the induction of oncostatin M, another STAT5 target, as well as the tyrosine-phosphorylation of STAT5, were partially suppressed by CIS expression in Ba/F3 cells. Thus, CIS is a feedback modulator of STAT5; its expression is induced by STAT5 and it negatively modulates STAT5 activation.
Expression of the Id Family Helix-Loop-Helix Regulators During Growth and Development in the Hematopoietic System
Authors: Cooper Cathleen L. , Brady Gerard , Bilia Fillio , Iscove Norman N. , Quesenberry Peter J. 
Published: October 2019

AbstractTo better understand the molecular mechanism(s) by which growth and differentiation of the primitive hematopoietic stem cell is initiated, as well as the means by which the maturing cell can commit to development along a specific cell lineage, we elected to study the Id family of helix-loop-helix (HLH) transcriptional regulators. Some members of the HLH family are expressed in a stage-specific manner during hematopoietic development and can regulate the ability of immature hematopoietic cells to terminally differentiate. None of the four Id family genes were detected in the most primitive progenitors. Id-1 was widely expressed in proliferating bi- and unipotential progenitors, but its expression was downregulated in cells of increasing maturity; conversely, Id-2 and, to a limited extent, Id-3 gene expression increased as cells matured and lost proliferative capacity. Id-2 expression ran counter to that of Id-1 not only during maturation, but during periods of cell growth and arrest as well. This is quite distinct from the nonhematopoietic tissues, in which these two factors are coordinately expressed and suggests that Id-1 and Id-2 might be regulating very different events during hematopoiesis than they regulate in other cell types.
Activation of the Coagulation Cascade in C1-Inhibitor Deficiencies
Authors: Cugno Massimo , Cicardi Marco , Bottasso Bianca , Coppola Raffaella , Paonessa Raffaella , Mannucci Pier Mannuccio , Agostoni Angelo 
Published: October 2019

AbstractActivation of the contact and complement systems in C1-inhibitor deficiencies is thought to contribute to the pathogenesis of angioedema attacks by releasing kinins. Trigger stimuli of attacks may also activate coagulation. This is particularly important because experimental data suggest that thrombin, the main enzyme of the coagulation cascade, increases vascular permeability and can thus influence edema formation. We have studied 19 patients with hereditary angioedema (HAE) during remission, 5 HAE patients during acute attacks, and 6 patients with acquired angioedema (AAE) during remission and during seven attacks. Thirty normal subjects, matched for sex and age, served as controls. Generation of thrombin was measured by enzyme-linked immunosorbent assay (ELISA) as plasma levels of the prothrombin fragment 1 + 2 (F1 + 2); the initiators of the tissue factor and contact coagulation pathways were investigated by measuring plasma levels of activated factor VII (FVIIa) coagulometrically and activated factor XII (FXIIa) by ELISA. Cleavage of high molecular weight kininogen (HK) was evaluated by immunoblotting analysis. F1 + 2 was slightly increased during remission and further significantly increased during attacks in both HAE (P = .0115) and AAE. FVIIa and FXIIa, normal during remission, increased strikingly during attacks in both HAE (P = .0022 and P = .0044) and AAE. During remission, cleaved HK was normal in HAE and high in AAE; during attacks it increased in HAE (P = .0008) and remained elevated in AAE. Our data indicate that in C1-inhibitor deficient patients there is increased generation of thrombin during attacks, with signs of activation of both the contact and tissue factor coagulation pathways. In conclusion, C1-inhibitor deficiency, whether hereditary or acquired, has demonstrable activation of the coagulation and kinin-forming cascades during attacks and that thrombin should be considered a possible contributing factor in the pathogenesis of edema in HAE and AAE.