bluuebunny/crossref_metadata_2025 huggingface dataset:
New Insights Into the Negative Regulation of Hematopoiesis by Chemokine Platelet Factor 4 and Related Peptides
Authors: Lecomte-Raclet Laurence , Alemany Mònica , Grand Anabelle Sequeira-Le , Amiral Jean , Quentin Gérard , Vissac Anne Marie , Caen Jacques P. , Han Zhong Chao
Published: October 2019

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit–granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34+ marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.
Evidence That Amyloidogenic Light Chains Undergo Antigen-Driven Selection
Authors: Perfetti Vittorio , Ubbiali Paola , Vignarelli Maurizio Colli , Diegoli Marta , Fasani Roberta , Stoppini Monica , Lisa Antonella , Mangione Palma , Obici Laura , Arbustini Eloisa , Merlini Giampaolo
Published: October 2019

AL amyloidosis is characterized by fibrillar tissue deposits (amyloid) composed of monoclonal light chains secreted by small numbers of indolent bone marrow plasma cells whose ontogenesis is unknown. To address this issue and to provide insights into the processes that accompanied pathogenic light chain formation, we isolated the complete variable (V) regions of 14 light (VL) and 3 heavy (VH) chains secreted by amyloid clones at diagnosis (10 Bence Jones and 4 with complete Igs, 9 λ and 5 κ) by using an inverse polymerase chain reaction-based approach free of primer-induced biases. Amyloid V regions were found to be highly mutated compared with the closest germline genes in the databases or those isolated from the patients' DNA, and mutations were not associated with intraclonal diversification. Apparently high usage of the λIII family germline gene V λIII.1 was observed (4 of 9 λ light chains). Analysis of the nature and distribution of somatic mutations in amyloid V regions showed that there was statistical evidence of antigen selection in 8 of 14 clones (7 in VL and 1 in VH). These results indicate that a substantial proportion of the amyloid clones developed from B cells selected for improved antigen binding properties and that pathogenic light chains show evidence of this selection.
Role of p53 in Hematopoietic Recovery After Cytotoxic Treatment
Authors: Wlodarski Pawel , Wasik Mariusz , Ratajczak Mariusz Z. , Sevignani Cinzia , Hoser Grazyna , Kawiak Jerzy , Gewirtz Alan M. , Calabretta Bruno , Skorski Thomas
Published: October 2019

Prompt reconstitution of hematopoiesis after cytoreductive therapy is essential for patient recovery and may have a positive impact on long-term prognosis. We examined the role of the p53 tumor suppressor gene in hematopoietic recovery in vivo after treatment with the cytotoxic drug 5-fluorouracil (5-FU). We used p53 knock-out (p53−/−) and wild-type (p53+/+) mice injected with 5-FU as the experimental model. Analysis of the repopulation ability and clonogenic activity of hematopoietic stem cells (HSCs) and their lineage-committed descendants showed a greater number of HSCs responsible for reconstitution of lethally irradiated recipients in p53−/− bone marrow cells (BMCs) recovering after 5-FU treatment than in the corresponding p53+/+ BMCs. In post–5-FU recovering BMCs, the percentage of HSC-enriched Lin− Sca-1+c-Kit+ cells was about threefold higher in p53−/− than in p53+/+ cells. Although the percentage of the most primitive HSCs (Lin− Sca-1+ c-Kit+CD34low/−) did not depend on p53, the percentage of multipotential HSCs and committed progenitors (Lin−Sca-1+ c-Kit+ CD34high/+) was almost fourfold higher in post–5-FU recovering p53−/− BMCs than in their p53+/+ counterparts. The pool of HSCs from 5-FU–treated p53−/− BMCs was exhausted more slowly than that from the p53+/+ population as shown in vivo using pre–spleen colony-forming unit (CFU-S) assay and in vitro using long-term culture-initiating cells (LTC-ICs) and methylcellulose replating assays. Clonogenic activity of various lineage-specific descendants was significantly higher in post–5-FU regenerating p53−/− BMCs than in p53+/+ BMCs, probably because of their increased sensitivity to growth factors. Despite all these changes and the dramatic difference in sensitivity of p53−/− and p53+/+ BMCs to 5-FU–induced apoptosis, lineage commitment and differentiation of hematopoietic progenitors appeared to be independent of p53 status. These studies suggest that suppression of p53 function facilitates hematopoietic reconstitution after cytoreductive therapy by: (1) delaying the exhaustion of the most primitive HSC pool, (2) stimulating the production of multipotential HSCs, (3) increasing the sensitivity of hematopoietic cells to growth factors, and (4) decreasing the sensitivity to apoptosis.
Membrane Phospholipid Asymmetry in Human Thalassemia
Authors: Kuypers Frans A. , Yuan Jie , Lewis Rachel A. , Snyder L. Michael , Kiefer Charles R. , Bunyaratvej Ahnond , Fucharoen Suthat , Ma Lisa , Styles Lori , de Jong Kitty , Schrier Stanley L.
Published: October 2019

Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe β-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the α- or β-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of β-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound α-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.
Adaptive Response of Iron Absorption to Anemia, Increased Erythropoiesis, Iron Deficiency, and Iron Loading in β2-Microglobulin Knockout Mice
Authors: Santos Manuela , Clevers Hans , de Sousa Maria , Marx J.J.M.
Published: October 2019

Recently, a novel gene of the major histocompatibility complex (MHC) class I family, HFE (HLA-H), has been found to be mutated in a large proportion of hereditary hemochromatosis (HH) patients. Further support for a causative role of HFE in this disease comes from the observation that β2-microglobulin knockout (β2m−/−) mice, that fail to express MHC class I products, develop iron overload. We have now used this animal model of HH to examine the capacity to adapt iron absorption in response to altered iron metabolism in the absence of β2m-dependent molecule(s). Mucosal uptake, mucosal transfer and retention of iron were measured in control and β2m−/−mice with altered iron metabolism. Mucosal uptake of Fe(III), but not of Fe(II), by the mutant mice was significantly higher when compared with B6 control mice. Mucosal transfer in the β2m−/−mice was higher, independent of the iron form tested. No significant differences were found in iron absorption between control and β2m−/− mice when anemia was induced either by repetitive bleeding or by hemolysis through phenylhydrazine treatment. However, iron absorption in mice made anemic by dietary deprivation of iron was significantly higher in the mutant mice. Furthermore, the β2m−/− mice manifested an impaired capacity to downmodulate iron absorption when dietary or parenterally iron-loaded. The expression of the defect in iron absorption in the β2m−/− mice is quantitative, with iron absorption being excessively high for the size of body iron stores. The higher iron absorption capacity in the β2m−/− mice may involve the initial step of ferric mucosal uptake and the subsequent step of mucosal transfer of iron to the plasma.
Risks of Developing Epstein-Barr Virus–Related Lymphoproliferative Disorders After T-Cell–Depleted Marrow Transplants
Authors: Hale Geoff , Waldmann Herman , Users CAMPATH
Published: October 2019

T-cell depletion of bone marrow for allogeneic transplantation is known to increase the risks of Epstein-Barr virus–driven lymphoproliferative disorders that may result in fatal lymphoma, especially with transplants from unrelated or mismatched donors. Over the past 15 years, we have monitored the outcome of 2,582 transplants using CAMPATH-1 (CD52) antibodies to deplete lymphocytes from donor and/or recipient to prevent graft-versus-host disease or rejection. Unlike many other methods of T-cell depletion, CAMPATH-1 antibodies also deplete B lymphocytes. The actuarial risk of lymphoproliferative disease using CAMPATH-1 for depletion of donor lymphocytes was up to 1.3%, hardly different from reported figures for conventional nondepleted transplants. In contrast, the risk in a small group of patients transplanted from unrelated donors using E-rosette depletion was as high as 29%, comparable to other reports of specific T-cell depletion. We conclude that the additional depletion of B cells is beneficial, possibly because it reduces either the virus load or the virus target until the time when T cells begin to regenerate.
Cancer Procoagulant and Tissue Factor Are Differently Modulated by All-trans-Retinoic Acid in Acute Promyelocytic Leukemia Cells
Authors: Falanga A. , Consonni R. , Marchetti M. , Locatelli G. , Garattini E. , Passerini C. Gambacorti , Gordon S.G. , Barbui T.
Published: October 2019

All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4.306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] α), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARβ) and CD437 (selective for RARγ), both lacking this capability. Cells were treated with either ATRA or the analogues (10−6 to 10−8 mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARα agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARβ agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both α and β nuclear retinoid receptors.
Transcriptional Inhibition of p53 by the MLL/MEN Chimeric Protein Found in Myeloid Leukemia
Authors: Maki Kazuhiro , Mitani Kinuko , Yamagata Tetsuya , Kurokawa Mineo , Kanda Yoshinobu , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

The t(11;19)(q23;p13.1) translocation is frequently found in adult myeloid leukemia. In the MLL/MEN fusion protein generated by this translocation, most of the coding region of the MEN protein, an RNA polymerase II elongation factor, is fused to the N-terminal third of the MLL protein, a possible transcriptional regulator. However, the molecular mechanism of leukemogenesis by the fusion protein remains unclear. We investigated the effects of the fusion protein on p53 function using luciferase assays. Overexpression of the fusion protein suppressed the transactivation ability of p53. This negative effect of the fusion protein on p53 function was dependent on the region derived from MEN. Moreover, p53 coimmunoprecipitated with MLL/MEN as well as MEN, suggesting that the fusion protein binds to p53 through the MEN region. We found that MEN binding to p53 was mediated by its N-terminal region and repression of p53 transcriptional activity was mediated by its C-terminal region. We also found that these two functional regions were essential for the transformation of Rat1 cells mediated by MEN. Although we could not demonstrate a functional difference between MLL/MEN and MEN in this study, these data suggest that the MLL/MEN chimeric transcriptional regulator may exert its oncogenic activity by inhibiting the function of the p53 tumor-suppressor protein by binding to it. Our findings provide a novel insight into the leukemogenic mechanism exerted by the t(11;19)(q23;p13.1) translocation.
Chemotactic Response Toward Chemokines and Its Regulation by Transforming Growth Factor-β1 of Murine Bone Marrow Hematopoietic Progenitor Cell-Derived Different Subset of Dendritic Cells
Authors: Ogata Masafumi , Zhang Yi , Wang Yong , Itakura Meiji , Zhang Yan-yun , Harada Akihisa , Hashimoto Shin-ichi , Matsushima Kouji
Published: October 2019

Dendritic cells (DCs) are highly specialized antigen-presenting cells that distribute widely in all organs. DCs initiate the primary immune response and activate naive T cells and B cells responsible for the acquired immunity. In this study, CCR7 mRNA was proved to be expressed in DCs and their precursors derived from murine bone marrow-derived hematopoietic progenitor cells (HPCs), whereas CCR1 mRNA was expressed in both CD11b−/dullCD11c+ and CD11b+hiCD11c+ DC precursors. CCR6 mRNA was not detected in any murine DC populations. In agreement with the chemokine receptor mRNA expression by each population in the DC differentiation pathway, SLC (also termed as MIP-3β), one of the ligands for CCR7, strongly and selectively chemoattracted both CD11b−/dullCD11c+ and CD11b+hiCD11c+ DC precursors (days 6 to 7) and more mature DCs (days 13 to 14). We have recently found that transforming growth factor-β1 (TGF-β1), a cytokine that is essential for the appearance of Langerhans cells in the skin, polarizes murine HPCs to generate Langerhans-like cells through monocyte/macrophage differentiation pathway. We observed here that TGF-β1 not only inhibited the expression of CCR7 in DCs and DC precursors derived from HPCs, but also inhibited the migration of these cells in response to SLC. This is the first report describing the chemokine and chemokine receptors responsible for murine DC migration and downregulation of DC migration by TGF-β1.
Regulated Expression and Functional Role of the Transcription Factor CHOP (GADD153) in Erythroid Growth and Differentiation
Authors: Coutts Margaret , Cui Kunyuan , Davis Kerry L. , Keutzer Joan Cleves , Sytkowski Arthur J.
Published: October 2019

The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA ofchop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp β was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression ofchop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)–derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)–derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.
Characterization of HOX Gene Expression During Myelopoiesis: Role of HOX A5 in Lineage Commitment and Maturation
Authors: Fuller John F. , McAdara Jeanne , Yaron Yifah , Sakaguchi Mark , Fraser John K. , Gasson Judith C.
Published: October 2019

During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.
A Novel Function for Transforming Growth Factor-β1: Upregulation of the Expression and the IgE-Independent Extracellular Release of a Mucosal Mast Cell Granule-Specific β-Chymase, Mouse Mast Cell Protease-1
Authors: Miller Hugh R.P. , Wright Steven H. , Knight Pamela A. , Thornton Elisabeth M.
Published: October 2019

Intestinal mucosal mast cells (IMMC) express granule neutral proteases that are regulated by T-cell–derived cytokines, including interleukin-3 (IL-3) and IL-9, and by stem cell factor (SCF). The IMMC-specific chymase, mouse mast cell protease-1 (mMCP-1), is released in substantial quantities into the blood stream during gastrointestinal allergic responses. We used cultured bone marrow-derived mast cells (mBMMC) to identify cytokines that regulate the expression and extracellular release of mMCP-1. When grown in IL-3–rich WEHI (15% vol/vol) and 50 ng/mL recombinant rat SCF (rrSCF) bone marrow cells supplemented with IL-9 (5 ng/mL) differentiated into mBMMC that expressed a maximum of less than 250 ng mMCP-1/106 cells and 189 ng mMCP-1/mL of culture supernatant. Supplementation of the same three cytokines with transforming growth factor-β1(TGF-β1; 1 ng/mL) resulted in substantially enhanced expression (6 μg/106 mBMMC) and extracellular release (2 μg/mL of culture supernatant) of mMCP-1. The response to TGF-β1 was dose-dependent, with maximal effect at 1 ng/mL, and was associated with immunohistochemical and ultrastructural changes in the secretory granules. IL-9–induced expression of mMCP-1 may be due to endogenously expressed TGF-β1, because it was blocked by anti–TGF-β antibodies. In conclusion, the expression and extracellular release of the IMMC-specific chymase, mMCP-1, is strictly regulated by TGF-β1.
Persistence of BCR-ABL genomic rearrangement in chronic myeloid leukemia patients in complete and sustained cytogenetic remission after interferon-α therapy or allogeneic bone marrow transplantation
Authors: Chomel Jean-Claude , Brizard Françoise , Veinstein Anne , Rivet Jérôme , Sadoun Alain , Kitzis Alain , Guilhot François , Brizard André
Published: October 2019

In recent years, the prognosis of chronic myeloid leukemia (CML) has been greatly improved either with interferon- (IFN-) therapy or allogeneic bone marrow transplantation (BMT). In the present study, minimal residual disease was evaluated in 21 patients in complete cytogenetic response (CCR) after such treatments. Samples from bone marrow aspirates or peripheral blood or both were analyzed by conventional cytogenetics, Southern blot, interphase fluorescent in situ hybridization (FISH), and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). In all patients, FISH detected 1% to 12% nuclei with a BCR-ABL fusion gene, whereas Q-RT-PCR experiments were negative or weakly positive. Based on these results, we hypothesize that the BCR-ABL genomic rearrangement persists unexpressed in nonproliferating cells whatever the treatment (IFN- or BMT). These data point to the need for follow-up of CML patients in CCR over an extensive period at the DNA level (FISH) to evaluate the residual disease and at the RNA level (Q-RT-PCR) to estimate the risk of relapse.
Relationship between selectin-mediated rolling of hematopoietic stem and progenitor cells and progression in hematopoietic development
Authors: Greenberg Adam W. , Kerr William G. , Hammer Daniel A.
Published: October 2019

Current understanding of the adhesion molecules and mechanisms regulating hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow is limited. In contrast, the process by which mature leukocytes are able to home to and extravasate out of blood vessels at sites of inflammation has been well characterized and invites comparison. We studied the interaction of human HSPC from adult bone marrow (ABM) and fetal liver (FL) with E-, P-, and L-selectin immobilized in a flow chamber. CD34+ HSPC from both ABM and FL rolled avidly on E-, P-, and L-selectin across a range of physiologic shear rates, indicating the presence of ligands for all three selectins on HSPC. Results indicate that CD34+ ABM and FL cells roll more efficiently (to a greater extent and more slowly) than more differentiated CD34− cells, especially on P- and L-selectin. In a similar fashion, increased rolling efficiency was seen with CD34+CD38− ABM cells when compared with committed progenitor cells of the CD34+CD38+ phenotype. Rolling of CD34+ ABM cells on P-selectin could be partially inhibited by monoclonal antibody (mAb) against PSGL-1, and was not inhibited by a mAb against CD34, suggesting that HSPC have unique carbohydrate repertoires that facilitate selectin-mediated rolling. Our results provide direct evidence of selectin ligands on HSPC under physiologic flow conditions and are the first to show a correlation between the maturity of HSPC during development and rolling efficiency on selectins, suggesting a mechanism by which HSPC subsets may differentially home to the extravascular spaces of the bone marrow.
Interleukin-4-induced transcriptional activation by Stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of Stat6
Authors: Pesu Marko , Takaluoma Kati , Aittomäki Saara , Lagerstedt Anssi , Saksela Kalle , Kovanen Panu E. , Silvennoinen Olli
Published: October 2019

Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3′-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway.
Cytoplasmic domains of GpIbα and GpIbβ regulate 14-3-3ζ binding to GpIb/IX/V
Authors: Feng Shuju , Christodoulides Nicolaos , Reséndiz Julio C. , Berndt Michael C. , Kroll Michael H.
Published: October 2019

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIb binds directly to a dimeric 14-3-3 adapter protein ζ isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3ζ, the authors examined 14-3-3ζ interactions with GpIb and GpIbβ in heterologous cells and platelets. Truncations of GpIb at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3ζ. Deletion of GpIb from Trp570 to Ser590 eliminated 14-3-3ζ binding, and deletion of the sequence from Arg542-Trp570 enhanced binding of 14-3-3ζ to GpIb. All GpIb mutations that eliminated GpIb binding to the GST-14-3-3ζ fusion protein also eliminated GpIbβ binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIb/β/IX resulted in the phosphorylation of GpIbβ associated with enhanced binding of GpIbβ to GST-14-3-3ζ fusion protein and increased 14-3-3ζ coimmunoprecipitated with GpIb. When intact human platelets aggregated in response to 90 dynes/cm2 shear stress, 14-3-3ζ disassociated from GpIb. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3ζ from GpIb. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIb regulate 14-3-3ζ binding to GpIb/β/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbβ enhances 14-3-3ζ binding to the GpIb/IX/V complex in human platelets.
Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes
Authors: van den Brink Edward N. , Turenhout Ellen A. M. , Davies Julian , Bovenschen Niels , Fijnvandraat Karin , Ouwehand Willem H. , Peters Marjolein , Voorberg Jan
Published: October 2019

A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by VH domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the VH1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study.
Identification and characterization of a thrombomodulin gene mutation coding for an elongated protein with reduced expression in a kindred with myocardial infarction
Authors: Kunz Gabriella , Ireland Helen A. , Stubbs Peter J. , Kahan Mel , Coulton Gary C. , Lane David A.
Published: October 2019

Thrombomodulin is an endothelial cell receptor for thrombin. It functions as a natural anticoagulant by greatly accelerating activation of protein C by thrombin. Using a direct gene screening strategy we identified a frameshift insertion mutation, insT 1689, in the thrombomodulin gene of a patient with myocardial infarction. The mutation predicts an elongated gene product because of substitution of the 12 C-terminal amino acids by 61 abnormal residues. Pedigree analysis showed that the mutation was also likely to have been present in a sibling who had had fatal myocardial infarction. Carriers of the mutant allele express significantly lower amounts of thrombomodulin on the surface of their monocytes detected by flow cytometry and have lower levels of soluble thrombomodulin in plasma. Wild type and the mutant thrombomodulin were expressed in COS-7 cells. Cellular distribution of the expressed proteins was evaluated by immunofluorescence microscopy, which showed reduced cell surface expression and intense juxtanuclear localization of the abnormal protein. This suggests impaired translocation through the endoplasmic reticulum/Golgi apparatus. Cells expressing abnormal thrombomodulin had reduced ability (∼2.5-fold) to accelerate the thrombin mediated activation of protein C. This is the first demonstration of reduced expression arising from a natural thrombomodulin gene mutation. The results provide support for the suggestion that gene mutation of thrombomodulin may be important in the pathogenesis of some cases of occlusive thrombotic disease.
The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia
Authors: Jones Dan , Benjamin Richard J. , Shahsafaei Aliakbar , Dorfman David M.
Published: October 2019

Chemotaxis in leukocytes is mediated through binding of soluble chemokines to transmembrane G-protein coupled receptors. The chemokine receptor CXCR3 has been previously shown to be widely expressed on activated T cells and to mediate T-cell chemotaxis on binding to various ligands, including Mig, IP-10, and ITAC. By using immunohistochemical and flow cytometric analysis, we report that CXCR3 is also expressed on a subset of peripheral blood B cells and in distinct subtypes of B-cell lymphoma. CXCR3 immunohistochemical or flow cytometric expression was seen in 37 of 39 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (diffusely positive in 33 cases), whereas mantle cell lymphoma (30 cases), follicular lymphoma (27 cases), and small noncleaved cell lymphoma (8 cases) were negative in all but 2 cases. Strong CXCR3 expression was also seen in splenic marginal zone lymphoma (14 of 14 cases) and in the monocytoid and plasmacytic cells in extranodal marginal zone lymphoma (15 of 16 cases). This differential expression of CXCR3 in B-cell tumors contrasts with that of another B-cell–associated chemokine receptor, BLR1/CXCR5, which we show here is expressed on all types of B-cell lymphoma tested. We also report that the CXCR3 ligand, Mig, is coexpressed on tumor cells in many cases of CLL/SLL (10 of 13 cases examined) with Mig expression less frequently seen in other B-cell lymphoma subtypes. Coexpression of CXCR3 and its ligand, Mig, may be an important functional interaction in B-CLL, as well as a useful diagnostic marker for the differential diagnosis of small cell lymphomas.
CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines
Authors: Teoh G. , Tai Y.-T. , Urashima M. , Shirahama S. , Matsuzaki M. , Chauhan D. , Treon S. P. , Raje N. , Hideshima T. , Shima Y. , Anderson K. C.
Published: October 2019

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.
Polytrauma induces increased expression of pyruvate kinase in neutrophils
Authors: Oehler Rudolf , Weingartmann Gertrude , Manhart Nicole , Salzer Ulrich , Meissner Michael , Schlegel Werner , Spittler Andreas , Bergmann Michael , Kandioler Daniela , Oismüller Christiane , Struse Heidi M. , Roth Erich
Published: October 2019

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate–polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.
Mucosa-associated lymphoid tissue lymphoma is a disseminated disease in one third of 158 patients analyzed
Authors: Thieblemont Catherine , Berger Françoise , Dumontet Charles , Moullet Isabelle , Bouafia Fadhela , Felman Pascale , Salles Gilles , Coiffier Bertrand
Published: October 2019

Mucosa-associated lymphoid tissue–derived lymphoma (MALT lymphoma) is usually a very indolent lymphoma, described as localized at diagnosis and remaining localized for a prolonged period; dissemination occurs only after a long course of evolution. In our database, out of 158 patients with MALT lymphoma, 54 patients presented with a disseminated disease at diagnosis. Of these 54 patients, 17 patients (30%) presented with multiple involved mucosal sites; 37 patients (70%) presented with 1 involved mucosal site, but in 23 of these patients (44%), dissemination of the disease was due to bone marrow involvement; 12 patients (22%) had multiple lymph node involvement; and 2 patients (4%) had nonmucosal site involvement. No significant difference in clinical characteristics (sex, age, performance status, B symptoms) and biological parameters (hemoglobin [Hb] and lactate dehydrogenase levels) was observed between localized or disseminated MALT-lymphoma patients. Only β2-microglobulin level was significantly more elevated in disseminated disease patients than in localized disease patients. Complete response after the first treatment was achieved in 74% of the patients, and there was no difference between the 2 groups. With a median follow-up of 4 years, the estimated 5- and 10-year overall survival rates were similar in the 2 groups, 86% and 80%, respectively. The median freedom-from-progression survival was 5.6 years for all patients, surprisingly without any difference between localized and disseminated MALT-lymphoma patients. In conclusion, MALT lymphoma is an indolent disease but presents as a disseminated disease in one-third of the cases at diagnosis. The dissemination does not change the outcome of the patients.
Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells
Authors: Gustafsson Åsa , Levitsky Victor , Zou Jie-Zhi , Frisan Teresa , Dalianis Tina , Ljungman Per , Ringden Olle , Winiarski Jacek , Ernberg Ingemar , Masucci Maria G.
Published: October 2019

A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-cell–depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLs)/m2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.
Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse
Authors: Tobal K. , Newton J. , Macheta M. , Chang J. , Morgenstern G. , Evans P. A. S. , Morgan G. , Lucas G. S. , Yin J. A. Liu
Published: October 2019

One of the most common translocations in acute myeloid leukemia (AML) is the t(8;21), which produces the fusion gene AML1-MTG8. We have developed a sensitive competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay forAML1-MTG8 transcripts, coupled with a competitive RT-PCR for the ABL transcript as a control to accurately estimate the level of amplifiable RNA. We have shown that AML1-MTG8 andABL transcripts have equal degradation rates. Thus, this method is useful for multicenter studies. We studied 25 patients with t(8;21) AML by means of serial analysis done on bone marrow (BM) and peripheral blood (PB) samples from 21 patients. Our analysis showed that, in general, a successful induction chemotherapy produces a reduction of 2 to 3 log in the level of AML1-MTG8, followed by a further 2 to 3 log after consolidation/intensification chemotherapy. Levels up to 1 × 103 and 1 × 102 molecules/μg of RNA in BM and PB, respectively, were compatible with durable remission. On the other hand, 5 patients with levels of 0.71 × 105to 2.27 × 105 molecules/μg of RNA in BM and 2.27 × 103 to 2.27 × 104 molecules/μg of RNA in PB had hematologic relapse within 3 to 6 months. Our data indicate that serial quantitation of AML1-MTG8 transcripts is useful in identifying patients at high risk of relapse and may offer an opportunity for clinical intervention to prevent hematologic relapse. This approach was applied successfully in a patient who had an allogeneic BM transplantation. We also suggest that PB may be used an alternative to BM for quantitating AML1-MTG8 transcripts.
Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor
Authors: Azim A. C. , Barkalow K. , Chou J. , Hartwig J. H.
Published: October 2019

Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1–mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.
Switch in the protein tyrosine phosphatase associated with human CD100 semaphorin at terminal B-cell differentiation stage
Authors: Billard Christian , Delaire Stéphanie , Raffoux Emmanuel , Bensussan Armand , Boumsell Laurence
Published: October 2019

Human CD100, the first semaphorin identified in the immune system, is a transmembrane protein involved in T-cell activation. In the present study, we showed that activation of peripheral blood or tonsillar B lymphocytes induced the expression of CD100 in CD38+CD138− cell populations, including in CD148+ subpopulations, thus expressing a memory B-cell–like phenotype. Using an in vitro enzymatic assay, we found that protein tyrosine phosphatase (PTP) activities were immunoprecipitated with CD100 in these cell populations, which were isolated by cell sorting, as well as in most B-cell lines representing various stages of B-cell differentiation. Immunodepletion and Western blotting experiments demonstrated that CD45 was the PTP associated with CD100 in cell lines displaying pre-B, activated B, and pre-plasma cell phenotypes. CD45 also accounted for PTP activity immunoprecipitated with CD100 in CD38+CD138− cells sorted after activation of peripheral blood or tonsillar B lymphocytes. In contrast, no CD100-CD45 association was observed in plasma cell lines corresponding to the terminal B-cell differentiation stage. CD148, the other transmembrane PTP known to be implicated in lymphocyte signaling pathways, was either only partly involved in the CD100-associated PTP activity or not expressed in plasma cell lines, indicating the association of CD100 with another main PTP. Our data show that CD100 is differentially expressed and can functionally associate with distinct PTPs in B cells depending on their activation and maturation state. They also provide evidence for a switch in the CD100-associated PTP at terminal stage of B-cell differentiation.