bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombosis and shock induced by activating antiplatelet antibodies in human FcγRIIA transgenic mice: the interplay among antibody, spleen, and Fc receptor
Authors: Taylor Scott M. , Reilly Michael P. , Schreiber Alan D. , Chien Paul , Tuckosh Joseph R. , McKenzie Steven E.
Published: October 2019

Transgenic mouse lines were created that express FcγRIIA on platelets and macrophages at human physiologic levels, and they were used to explore the consequences in vivo of activating antiplatelet antibodies. Anti-CD9 antibody activated platelets of FcγRIIA transgenic (tg) mice and, following injection in vivo, caused more rapid severe thrombocytopenia than nonactivating antiplatelet antibody. Anti-CD9 injected into FcγRIIA tg crossed with FcR γ-chain knockout (γ-KO) mice caused thrombosis and shock in all mice, and death in 16 of 18 mice. The shock depended on platelet Fc receptor density and antibody dose. On histologic examination, the lung vasculature of anti-CD9–treated FcγRIIA tg × γ-KO mice contained extensive platelet-fibrin thrombi. Thrombosis and shock in FcγRIIA tg mice in the context of the FcR γ-chain knockout suggested the importance of the interplay of intravascular platelet activation and splenic clearance. Reduction of splenic clearance surgically (splenectomy) or functionally (monoclonal antibody treatment) also facilitated anti-CD9–mediated shock in FcγRIIA tg mice. The spleen, which clears nonactivating antibody-coated platelets leading to thrombocytopenia, appears to play a protective role in the thrombosis and shock observed with activating antiplatelet antibody. The data indicate that antibodies, which activate platelets in an FcγRIIA-dependent manner, can lead to thrombosis, shock, and death. Furthermore, antibody titer, platelet Fc receptor density, and splenic clearance are likely important determinants of the outcome.
Interferon α prevents spontaneous apoptosis of clonal Th2 cells associated with chronic hypereosinophilia
Authors: Schandené Liliane , Roufosse Florence , de Lavareille Aurore , Stordeur Patrick , Efira André , Kennès Bernard , Cogan Elie , Goldman Michel
Published: October 2019

A recent study identified a clonal expansion of CD3−CD4+cells secreting Th2-type cytokines in 4 patients with chronic hypereosinophilia. Because interferon α (IFN-α) is used in the therapy of the idiopathic hypereosinophilic syndrome, the effects of this cytokine on the survival of clonal Th2 cells isolated from the blood of 2 patients were determined. First, these cells displayed a high rate of spontaneous apoptosis on culture in cytokine-free medium and were also sensitive to Fas-mediated apoptosis induced by soluble Fas ligand. Addition of IFN-α or interleukin-2 (IL-2) to culture medium resulted in significant protection against spontaneous but not Fas-induced apoptosis. Although spontaneous apoptosis of the clonal Th2 cells was clearly associated with down-regulation of both bcl-2 and bcl-xL levels, IFN-α had no significant effect on the expression of these antiapoptotic proteins, whereas addition of IL-2 resulted in higher levels of bcl-2. On the other hand, IFN-α decreased the numbers of cells with disrupted mitochondrial transmembrane potential both during spontaneous apoptosis and after exposure to protoporphyrin IX. Thus, IFN-α might promote the survival of clonal Th2 cells, an effect that could be relevant to the therapeutic approach for patients with chronic hypereosinophilia caused by clonal expansion of Th2-type cells.
Keratinocyte growth factor facilitates alloengraftment and ameliorates graft-versus-host disease in mice by a mechanism independent of repair of conditioning-induced tissue injury
Authors: Panoskaltsis-Mortari Angela , Taylor Patricia A. , Rubin Jeffrey S. , Uren Aykut , Welniak Lisbeth A. , Murphy William J. , Farrell Catherine L. , Lacey David L. , Blazar Bruce R.
Published: October 2019

We have previously shown that pretreatment of mice with keratinocyte growth factor (KGF), an epithelial tissue repair factor, can ameliorate graft-versus-host disease (GVHD) after intensive chemoradiotherapeutic conditioning and allogeneic bone marrow transplantation (BMT). To determine whether this effect was dependent on a KGF-mediated mechanism affecting repair of conditioning-induced epithelial cell injury, we studied GVHD in the absence of conditioning using BALB/c severe combined immune-deficient (SCID) recipients given C57BL/6 T cells. KGF (5 mg/kg per day, subcutaneously) given either before or after T-cell transfer enhanced body weights and extended survival. KGF-treated recipients had elevated serum levels of the Th2 cytokine interleukin 13 (IL-13) on day 6 after T-cell transfer concomitant with reduced levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon gamma (IFN-γ). A 3-day KGF pretreatment also depressed the secondary in vitro mixed lymphocyte response (MLR) of C57BL/6 splenocytes taken 7 days after in vivo alloimmunization with irradiated BALB/c spleen cells. To determine whether KGF would inhibit host-antidonor–mediated BM rejection, pan-T-cell–depleted BALB/c BM cells were infused into sublethally irradiated C57BL/6 mice and administered KGF either before or before and after BMT. Surprisingly, all KGF schedules tested actually resulted in enhanced alloengraftment. The presence of KGF receptor on donor antihost alloreactive T cells could not be detected by binding studies with radiolabeled KGF, reverse transcriptase–polymerase chain reaction, and Western blotting. Therefore, the mechanism of action of KGF on inhibiting T-cell–mediated immune effects may not be due to a direct effect of KGF on T cells. These studies demonstrate that KGF, by mechanisms independent of repair of conditioning-induced injury, has great potential as an anti-GVHD therapeutic agent with the added benefit of inhibiting the rejection of pan-T-cell–depleted donor BM allografts.
Stimulation of γδ T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma
Authors: Kunzmann Volker , Bauer Eva , Feurle Juliane , Tony Florian Weißinger, Hans-Peter , Wilhelm Martin
Published: October 2019

Bisphosphonates are well-known inhibitors of osteoclastic bone resorption, but recent clinical reports support the possibility of direct or indirect antitumor effects by these compounds. Because bisphosphonates share structural homologies with recently identified γδ T-cell ligands, we examined the stimulatory capacity of bisphosphonates to γδ T cells and determined whether γδ T-cell stimulation by bisphosphonates could be exploited to generate antiplasma cell activity in multiple myeloma (MM). All tested aminobisphosphonates (alendronate, ibandronate, and pamidronate) induced significant expansion of γδ T cells (Vγ9Vδ2 subset) in peripheral blood mononuclear cell cultures of healthy donors at clinically relevant concentrations (half-maximal activity, 0.9-4 μmol/L). The proliferative response of γδ T cells to aminobisphosphonates was IL-2 dependent, whereas activation of γδ T cells (up-regulation of CD25 and CD69) occurred in the absence of exogenous cytokines. Pamidronate-activated γδ T cells produced cytokines (ie, interferon [IFN]-γ) and exhibited specific cytotoxicity against lymphoma (Daudi) and myeloma cell lines (RPMI 8226, U266). Pamidronate-treated bone marrow (BM) cultures of 24 patients with MM showed significantly reduced plasma cell survival compared with untreated cultures, especially in cultures in which activation of BM-γδ T cells was evident (14 of 24 patients with MM). γδ T-cell depletion from BM cultures completely abrogated the cytoreductive effect on myeloma cells in 2 of 3 tested patients with MM. These results show that aminobisphosphonates stimulating γδ T cells have pronounced effects on the immune system, which might contribute to the antitumor effects of these drugs.
A new transacting factor that modulates hypoxia-induced expression of the erythropoietin gene
Authors: Gupta Madhu , Mungai Paul T. , Goldwasser Eugene
Published: October 2019

Hypoxia is a strong stimulus for the transcription of a set of genes, including erythropoietin and vascular endothelial growth factor. Here we report on the cloning, functional significance, and expression of a complementary DNA (cDNA) that is involved in hypoxia-mediated expression of these 2 genes. The full-length cDNA encodes a predicted protein of 806 amino acids that contains a leucine zipper motif. This protein, termed HAF for hypoxia-associated factor, binds to a 17-base pair (bp) region of the erythropoietin promoter, which was shown earlier to participate in hypoxia-induced expression of the erythropoietin gene. In Hep3B cells, clones modified to express HAF antisense RNA showed an attenuated response to hypoxia-mediated induction of both erythropoietin and vascular endothelial growth factor transcription. HAF showed sequence-specific interaction with a DNA element in the 5′ untranslated region ofVEGF gene. The HAF 2.6-kilobase (kb) messenger RNA (mRNA) is expressed in most adult tissues. The highest expression occurs in fetal liver and the least in adult liver. HAF is the murine homolog of Sart-1, a 125-kd human protein expressed in the nuclei of normal and malignant cells.
Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors
Authors: Giri Tusar Kanti , Yamazaki Tomio , Sala Núria , Dahlbäck Björn , de Frutos Pablo Garcı́a
Published: October 2019

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.
Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain
Authors: van den Brink Edward N. , Turenhout Ellen A. M. , Bank Christine M. C. , Fijnvandraat Karin , Peters Marjolein , Voorberg Jan
Published: October 2019

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The VHdomain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg484–Ile508 , a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The VH domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp712–Ala736 in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp712–Ala736for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor.
The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation
Authors: Franco Rendrik F. , de Jonge Evert , Dekkers Pascale E. P. , Timmerman Janneke J. , Spek C. Arnold , van Deventer Sander J. H. , van Deursen Peter , van Kerkhoff Liesbeth , van Gemen Bob , ten Cate Hugo , van der Poll Tom , Reitsma Pieter H.
Published: October 2019

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean ± SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 ± 0.02. A progressive and significant (P < .0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 ± 0.1, +1 hour: 1.3 ± 0.9, +2 hours: 4.1 ± 0.9), peaking at +3 hours (10 ± 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge.
A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion
Authors: Allen Simon , Abuzenadah Adel M. , Hinks Joanna , Blagg Joanna L. , Gursel Turkiz , Ingerslev Jørgen , Goodeve Anne C. , Peake Ian R. , Daly Martina E.
Published: October 2019

In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.
Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3ζ
Authors: Munday Adam D. , Berndt Michael C. , Mitchell Christina A.
Published: October 2019

The binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have revealed that the signaling protein 14-3-3ζ binds directly to the cytoplasmic domain of GP Ib. In this study, the dynamic association of 14-3-3ζ with GP Ib-IX, the phosphoinositide 3-kinase (PI 3-kinase), or both, was investigated in resting, thrombin, or vWF and botrocetin-stimulated platelets by analysis of discrete subcellular fractions. Results of this study demonstrate maximal coimmunoprecipitation of 14-3-3ζ with GP Ib-IX in the nonstimulated cytosolic fraction and in the actin cytoskeletal fraction of thrombin- or vWF-stimulated human platelets. Immunoprecipitated 14-3-3ζ or GP Ib from cytosolic fractions contained PI 3-kinase enzyme activity and an 85-kd polypeptide recognized by antibodies to the p85 subunit of PI 3-kinase. After platelet activation, the level of association between these species decreased in the cytosolic fraction. However, increased complex formation between 14-3-3ζ and GP Ib-IX and between PI 3-kinase and GP Ib-IX was detected in actin cytoskeletal fractions derived from thrombin- or vWF-stimulated platelets. Recombinant glutathione S-transferase-14-3-3ζ fusion protein (14-3-3ζ–GST) inhibited affinity-captured PI 3-kinase enzyme activity up to 70% at 2 μmol/L 14-3-3ζ–GST. However, increasing concentrations up to 5 μmol/L 14-3-3ζ–GST resulted in the 3-fold enhancement of PI 3-kinase enzyme activity. We propose that the association between PI 3-kinase and 14-3-3ζ with GP Ib-IX serves to promote the rapid translocation of these signaling proteins to the activated cytoskeleton, thereby regulating the formation of 3-position phosphoinositide-signaling molecules in this subcellular compartment.
Characterization of Grb4, an adapter protein interacting with Bcr-Abl
Authors: Coutinho Sunita , Jahn Thomas , Lewitzky Marc , Feller Stephan , Hutzler Peter , Peschel Christian , Duyster Justus
Published: October 2019

We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2–mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl–induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624)
Altered lymphoid development in mice deficient for the mAF4 proto-oncogene
Authors: Isnard Patricia , Coré Nathalie , Naquet Philippe , Djabali Malek
Published: October 2019

Some chromosomal translocations in acute leukemias involve the fusion of the trithorax-related protein Mll (also called HRX, All1, Htrx,) with a variety of heterologous proteins. In acute lymphoblastic leukemia associated with the t(4;11)(q21;q23) translocation, the4q21 gene that fuses with Mll is AF4. To gain insight into the potential role of AF4 in leukemogenesis and development, this gene was inactivated by homologous recombination in mice. As expected from the tissue distribution of the AF4 transcript, development of both B and T cells is affected in AF4 mutant mice. A severe reduction of the thymic double positive CD4/CD8 (CD4+/CD8+) population was observed; in addition most double- and single-positive cells expressed lower levels of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the double-positive compartment by expansion of the double-negative cell compartment was severely impaired in these mutant mice. In the bone marrow pre-B and mature B-cell numbers are reduced. These results demonstrate that the function of the mAF4 gene is critical for normal lymphocyte development. This raises the possibility that the disruption of the normal AF4 gene or its association with Mll function by translocation may orient the oncogenic process toward the lymphoid lineage. This represents the first functional study using a knock-out strategy on one of the Mll partner genes in translocation-associated leukemias.
A novel endoproteolytic processing activity in mitochondria of erythroid cells and the role in heme synthesis
Authors: Dzikaite Vijole , Kanopka Arvydas , Brock Jeremy H. , Kazlauskas Arunas , Melefors Öjar
Published: October 2019

The erythroid isoform of aminolevulinate synthase (eALAS) protein is a major control point in erythroid heme synthesis and hemoglobin formation. Erythroid cells were extracted from mouse blood and bone marrow and metabolically labeled with 35S-methionine. This was followed by immunoprecipitation of eALAS protein products. The results show that the N-terminus of the expected full-length 59-kd form of the eALAS protein is truncated in bone marrow erythroid cells by approximately 7 kd. More differentiated erythroid cells in the peripheral blood exhibit very little of this protein truncation. Erythroid cells from the bone marrow were isolated using monoclonal antibody TER-119 and were shown to contain a unique endoprotease activity that could cleave the eALAS protein to the shorter form in vitro. With or without the mitochondrial signal sequence, the eALAS protein could serve as a substrate for the cleavage. This cleavage renders a functional eALAS protein and only removes a domain of unclear function, which has previously been reported to vary in size as a result of alternative RNA splicing. The protease activity was enriched from the membranes of mitochondria from bone marrow cells and was shown to be different from mitochondrial processing peptidase, medullasin, and other known proteases. Apart from the mitochondrial processing peptidase that cleaves the import signal sequence, this is the first description of a mitochondrially located site-specific processing protease activity.
Homozygous truncation of the fibrinogen Aα chain within the coiled coil causes congenital afibrinogenemia
Authors: Fellowes Andrew P. , Brennan Stephen O. , Holme Randi , Stormorken Helge , Brosstad Frank R. , George Peter M.
Published: October 2019

The molecular basis of a novel congenital afibrinogenemia has been determined. The proposita, the only affected member in a consanguineous Norwegian family, suffers from a moderate to severe bleeding disorder due to the total absence of any detectable fibrinogen. Dot blots of solubilized platelets revealed a small amount of γ chain but no A or Bβ chains, whereas no chains were detected in plasma dot blots. DNA sequencing of the A chain gene revealed a homozygous C→T transversion 557 nucleotides from the transcription initiation site. This nucleotide change predicts the nonsense mutation A 149 Arg (CGA)→stop (TGA). Early truncation of the A chain appears to result in defective assembly or secretion of fibrinogen, probably due to the removal of the C-terminal disulfide ring residues that are critically required for the formation of a stable 3-chained half molecule.
Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein
Authors: Kelly Patrick F. , Vandergriff Jody , Nathwani Amit , Nienhuis Arthur W. , Vanin Elio F.
Published: October 2019

Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector–mediated gene transfer. Human hematopoietic cell lines and cord blood–derived CD34+ and CD34+, CD38− cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34+ cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells.
A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis
Authors: Zandstra Peter W. , Lauffenburger Douglas A. , Eaves Connie J.
Published: October 2019

A major limitation to the widespread use of hematopoietic stem cells (HSC) is the relatively crude level of our knowledge of how to maintain these cells in vitro without loss of the long-term multilineage growth and differentiation properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of HSC stimulation by exogenous cytokines would thus be useful. An emerging theme from recent HSC expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by extracellular matrix- or cell-bound cytokine presentation. According to such a model, when the relevant ligand-receptor interaction falls below a critical level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Thus, in addition to the identity of a particular receptor-ligand interaction being important to the regulation of stem cell responses, the quantitative nature of this interaction, as well as the dynamics of receptor expression, internalization, and signaling, may have a significant influence on stem cell fate decisions. This review uses examples from hematopoiesis and other tissue systems to examine existing evidence for a role of receptor activation thresholds in regulating hematopoietic stem cell self-renewal versus differentiation events.
How I treat patients with thrombotic thrombocytopenic purpura–hemolytic uremic syndrome
Authors: George James N.
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are, in adults, clinically and pathologically indistinguishable except for the severity of renal failure. They are best described as a single disorder, TTP-HUS, because the diagnostic evaluation and initial management are the same. Treatment with plasma exchange, available for more than 20 years, has dramatically altered the course of disease in adults with TTP-HUS. Plasma exchange has improved survival rates from 10% to between 75% and 92%, creating urgency for the initiation of treatment. This has resulted in decreased stringency of diagnostic criteria, which in turn has resulted in a broader spectrum of disorders for which the diagnosis of TTP-HUS is considered. Long-term follow-up has revealed increasing frequencies of relapse and of chronic renal failure. Although the increased survival rate is dramatic and recent advances in understanding the pathogenesis of these syndromes are remarkable, clinical decisions remain empirical. Therefore, the management decisions for patients with suspected TTP-HUS rely on individual experience and opinion, resulting in many different practice patterns. Multipractice clinical trials are required to define optimal management.
Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage– colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses
Authors: Stripecke Renata , Cardoso Angelo A. , Pepper Karen A. , Skelton Dianne C. , Yu Xiao-Jin , Mascarenhas Leo , Weinberg Kenneth I. , Nadler Lee M. , Kohn Donald B.
Published: October 2019

Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.
Recombinant staphylokinase variants with reduced antigenicity due to elimination of B-lymphocyte epitopes
Authors: Laroche Yves , Heymans Stephane , Capaert Sophie , De Cock Frans , Demarsin Eddy , Collen Désiré
Published: October 2019

Site directed mutagenesis (350 variants) of recombinant staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activity or reduced binding of human staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, which absorbed down to less than a third of SakSTAR-specific antibodies. Intra-arterial administration in 61 patients with peripheral arterial occlusion caused no significant allergic reactions. Median neutralizing antibody titers (with 15 to 85 percentiles), expressed as microgram (μg) compound neutralized per milliliter plasma, were 4.4 (0.3 to 49) for the variants, compared with 12 (4 to 100) in 70 patients given wild-type SakSTAR (P = .002 by Mann-Whitney rank sum test). Overt neutralizing antibody induction (more than 5 μg compound neutralized per milliliter plasma) was observed in 57 of 70 patients (81%) given wild-type SakSTAR, but only in 28 of 60 patients (47%) treated with variants (P < .0001 by Fisher exact test). On the basis of this study, the variant SakSTAR (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R) (code SY155) has been selected for further clinical development.
Platelet adhesion to collagen in healthy volunteers is influenced by variation of both α2β1 density and von Willebrand factor
Authors: Roest Mark , Sixma Jan J. , Wu Ya-Ping , Ijsseldijk Martin J. W. , Tempelman Mariëlle , Slootweg Pieter J. , de Groot Philip G. , van Zanten G. Henrita
Published: October 2019

Platelet thrombus formation on collagen is initiated by platelet GPIb interaction with von Willebrand factor (vWF) bound to collagen, followed by firm attachment of the platelet to collagen by the integrin α2β1. Platelet and plasma vWF levels and α2β1 density on the platelet surface are highly variable among normal subjects; however, little is known about the consequences of this variability on platelet adhesion to collagen. A population of 32 normal subjects was studied to evaluate the relation between genetic and phenotypic variations of α2β1 density on the platelet surface, plasma vWF levels, platelet vWF levels, and adenosine diphosphate and adenosine triphosphate concentrations on the one hand and platelet adhesion to collagen under flow on the other hand. Platelet adhesion to collagen types I and III under flow was correlated with plasma levels of vWF (r2 = 0.45 and 0.42, respectively) and α2β1 density on the platelet surface (r2 = 0.35 and 0.17, not significant). Platelet adhesion to collagen type IV under flow was significantly correlated with platelet vWF levels (r2 = 0.34) and α2β1 density on the platelet surface (r2 = 0.42). Platelet adhesion to collagen types I and III depends on both plasma levels of vWF and α2β1 density on the platelet surface, whereas platelet adhesion to collagen type IV is mediated by both platelet vWF levels and α2β1 density on the platelet surface.
Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family
Authors: Castoldi Elisabetta , Simioni Paolo , Kalafatis Michael , Lunghi Barbara , Tormene Daniela , Girelli Domenico , Girolami Antonio , Bernardi Francesco
Published: October 2019

The study of the molecular bases of thrombophilia in a large family with 4 symptomatic members is reported. Three thrombophilic genetic components (FV R506Q, FV H1299R, and PT 20210G/A), all affecting the activity of the prothrombinase complex, were detected alone and in combination in various family members. In addition, a newly identified missense mutation (factor V [FV] Y1702C), causing FV deficiency, was also present in the family and appeared to enhance activated protein C (APC) resistance in carriers of FV R506Q or FV H1299R by abolishing the expression of the counterpart FV allele. The relationships between complex genotypes, coagulation laboratory findings, and clinical phenotypes were analyzed in the family. All symptomatic family members were carriers of combined defects and showed APC resistance and elevated F1 + 2 values. Evidence for the causative role of the FV Y1702C mutation, which affects a residue absolutely conserved in all 3 A domains of FV, factor VIII, and ceruloplasmin, relies on (1) the absolute cosegregation between the mutation and FV deficiency, both in the family and in the general population; (2) FV antigen and immunoblot studies indicating the absence of Y1702C FV molecules in plasma of carriers of the mutation, despite normal levels of the FV Y1702C messenger RNA; and (3) molecular modeling data that support a crucial role of the mutated residue in the A domain structure. These findings help to interpret the variable penetrance of thrombosis in thrombophilic families and to define the molecular bases of FV deficiency.
PML/RARα fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype
Authors: Grignani Francesco , Valtieri Mauro , Gabbianelli Marco , Gelmetti Vania , Botta Rosanna , Luchetti Luisella , Masella Barbara , Morsilli Ornella , Pelosi Elvira , Samoggia Paola , Pelicci Pier Giuseppe , Peschle Cesare
Published: October 2019

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RARα fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RARα mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RARα alters the early hematopoietic program through N-CoR–dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells.
Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, is identical to p36, a protein associated to lupus inclusion formation in response to α-interferon
Authors: Amici Adolfo , Emanuelli Monica , Raffaelli Nadia , Ruggieri Silverio , Saccucci Franca , Magni Giulio
Published: October 2019

Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an α-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins.
Immune tolerance induction in hemophilia patients with inhibitors: costly can be cheaper
Authors: Colowick Alan B. , Bohn Rhonda L. , Avorn Jerry , Ewenstein Bruce M.
Published: October 2019

The development of inhibitory antibodies to factor VIII (FVIII) occurs in approximately 30% to 40% of patients with severe hemophilia A. Management options for patients with inhibitor include eradicating it via immune tolerance induction (ITI) or treating bleeding episodes with large quantities of hemostatic agents. ITI is costly, approaching $1 million for the average 5-year-old, but if successful results in improved clinical outcomes. We constructed a decision analysis using the Markov process to model expected clinical outcomes and costs over a lifetime for a typical 5-year-old hemophiliac with high inhibitor levels. Estimates of relevant variables were based on a thorough review of the medical literature. Outcomes modeled included total lifetime costs as well as life expectancy. The decision analytic model revealed that the ITI strategy was associated with an increase in projected life expectancy of 4.6 years. Total estimated lifetime costs for the ITI strategy were approximately $1.7 million less per patient. Sensitivity analyses over clinically and economically reasonable ranges did not change these findings. The insight that ITI can achieve an improved clinical outcome while being cost-saving is not reflected in many current treatment regimens. This example also illustrates that expensive therapy for patients with a chronic disease may be cost effective when analyzed from a societal perspective over the patient's lifetime. This finding has important policy implications for medical decision makers at many levels and reinforces the need to undertake pharmacoeconomic analyses and choose therapies from a long-term, societal perspective.
Impact of the patient population on the risk for heparin-induced thrombocytopenia
Authors: Warkentin Theodore E. , Sheppard Jo-Ann I. , Horsewood Peter , Simpson Patricia J. , Moore Jane C. , Kelton John G.
Published: October 2019

The frequency of immune heparin-induced thrombocytopenia (HIT) varies among prospective studies. It is unknown whether this is caused by differences in the heparin preparations, the patient populations, or the types of serologic assay used to confirm the diagnosis. Seven hundred forty-four patients were studied from 3 different clinical treatment settings, as follows: unfractionated heparin (UFH) during or after cardiac surgery (n = 100), UFH after orthopedic surgery (n = 205), and low-molecular-weight heparin (LMWH) after orthopedic surgery (n = 439). Both an activation assay and an antigen assay were used to detect heparin-dependent IgG (HIT-IgG) antibodies. By activation assay, the frequency of HIT-IgG formation ranged from a low of 3.2% in orthopedic patients receiving LMWH to a high of 20% in cardiac patients receiving UFH; by antigen assay, the corresponding frequencies ranged from 7.5% to 50%. Both UFH use (P = .002) and cardiac surgery (P = .01) were more likely to be associated with HIT-IgG formation. However, among patients in whom HIT-IgG formed and who were administered UFH, the probability for HIT was higher among orthopedic patients than among cardiac patients (by activation assay: 52.6% compared with 5%; odds ratio, 21.1 [95% CI, 2.2-962.8]; P = .001; by antigen assay: 34.5% compared with 2.0%; odds ratio, 25.8 [95% CI, 3.2-1141]; P < .001). It is concluded that there is an unexpected dissociation between the frequency of HIT-IgG formation and the risk for HIT that is dependent on the patient population. HIT-IgG antibodies are more likely to form in patients who undergo cardiac surgery than in orthopedic patients, but among patients in whom antibodies do form, orthopedic patients are more likely to develop HIT.
Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro
Authors: Bryder David , Jacobsen Sten E. W.
Published: October 2019

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily
Authors: Jandrot-Perrus Martine , Busfield Samantha , Lagrue Anne-Helène , Xiong Ximing , Debili Najet , Chickering Troy , Couedic Jean-Pierre Le , Goodearl Andrew , Dussault Barry , Fraser Christopher , Vainchenker William , Villeval Jean-Luc
Published: October 2019

Injuries to the vessel wall and subsequent exposure of collagen from the subendothelial matrix result in thrombus formation. In physiological conditions, the platelet plug limits blood loss. However, in pathologic conditions, such as rupture of atherosclerotic plaques, platelet–collagen interactions are associated with cardiovascular and cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets, and people who are deficient in GPVI suffer from bleeding disorders. Based on the fact that GPVI is coupled to the Fc receptor (FcR)-γ chain and thus should share homology with the FcR chains, the genes encoding human and mouse GPVI were identified. They belong to the immunoglobulin (Ig) superfamily and share 64% homology at the protein level. Functional evidence demonstrating the identity of the recombinant protein with GPVI was shown by binding to its natural ligand collagen; binding to convulxin (Cvx), a GPVI-specific ligand from snake venom; binding of anti-GPVI IgG isolated from a patient; and association to the FcR-γ chain. The study also demonstrated that the soluble protein blocks Cvx and collagen-induced platelet aggregation and that GPVI expression is restricted to megakaryocytes and platelets. Finally, human GPVI was mapped to chromosome 19, long arm, region 1, band 3 (19q13), in the same region as multiple members of the Ig superfamily. This work offers the opportunity to explore the involvement of GPVI in thrombotic disease, to develop alternative antithrombotic compounds, and to characterize the mechanism involved in GPVI genetic deficiencies.