bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unravelling an HLA-DR Association in Childhood Acute Lymphoblastic Leukemia
Authors: Dorak M. Tevfik , Lawson Tom , Machulla Helmut K.G. , Darke Chris , Mills Ken I. , Burnett Alan K. 
Published: October 2019

AbstractGenetic and environmental factors play an interactive role in the development of childhood acute lymphoblastic leukemia (ALL). Since the demonstration of a major histocompatibility complex (MHC) influence on mouse leukemia in 1964, an HLA association has been considered as a possible genetic risk factor. Despite extensive efforts, however, no strong evidence comparable to the H-2k influence on mouse leukemia has been shown. The number of negative serological studies resulted in a loss of interest and consequently, no molecular HLA-DR association study has been published to date. We reconsidered the HLA-DR association in childhood ALL in 114 patients from a single center and 325 local newborn controls by polymerase chain reaction (PCR) analysis of the HLA-DRB1/3/4/5 loci. With conventional analysis, there was a moderate allelic association with the most common allele in the HLA-DR53 group, HLA-DRB1*04, in the whole group that was stronger in males (P = .0005, odds ratio = 2.9). When the other expressed HLA-DRB loci were examined, homozygosity for HLA-DRB4*01, encoding the HLA-DR53 specificity, was increased in patients (21.1%v 8.3%; odds ratio = 2.9, P = .0005). Consideration of gender showed that all of these associations were reflections of a male-specific increase in homozygosity for HLA-DRB4*01 (32.8% v 4.0%; odds ratio = 11.7, 95% confidence interval [CI] = 4.9 to 28.0; P = 3 × 10−8). This highly significant result provided the long-suspected evidence for the HLA-DR influence on the development of childhood ALL while confirming the recessive nature of the MHC influence on human leukemogenesis as in experimental models. The cross-reactivity between HLA-DR53 and H-2Ek, extensive mimicry of the immunodominant epitope of HLA-DR53 by several carcinogenic viruses, and the extra amount of DNA in the vicinity of the HLA-DRB4 gene argue for the case that HLA-DRB4*01 may be one of the genetic risk factors for childhood ALL.
Somatic ATM Mutations Indicate a Pathogenic Role of ATM in B-Cell Chronic Lymphocytic Leukemia
Authors: Schaffner Claudia , Stilgenbauer Stephan , Rappold Gudrun A. , Döhner Hartmut , Lichter Peter 
Published: October 2019

Abstract
Deletion in chromosome bands 11q22-q23 is one of the most common chromosome aberrations in B-cell chronic lymphocytic leukemia (B-CLL). It is associated with extensive lymph node involvement and poor survival. The minimal consensus deletion comprises a segment, which contains the ATM gene presenting an interesting candidate gene, as mutations in ATM predispose A-T patients to lymphoid malignancies. To investigate a potential pathogenic role of ATM in B-cell tumorigenesis, we performed mutation analysis of ATM in 29 malignant lymphomas of B-cell origin (B-CLL = 27; mantle cell lymphoma, [MCL] = 2). Twenty-three of these carried an 11q22-q23 deletion. In five B-CLLs and one MCL with deletion of one ATMallele, a point mutation in the remaining allele was detected, which resulted in aberrant transcript splicing, alteration, or truncation of the protein. In addition, mutation analysis identified point mutations in three cases without 11q deletion: two B-CLLs with one altered allele and one MCL with both alleles mutated. In four cases analyzed, theATM alterations were not present in the germ line indicating a somatic origin of the mutations. Our study demonstrates somatic disruption of both alleles of the ATM gene by deletion or point mutation and thus its pathogenic role in sporadic B-cell lineage tumors.
High Frequency of Adhesion Defects in B-Lineage Acute Lymphoblastic Leukemia
Authors: Geijtenbeek Teunis B.H. , van Kooyk Yvette , van Vliet Sandra J. , Renes Maurits H. , Raymakers Reinier A.P. , Figdor Carl G. 
Published: October 2019

AbstractAberrant proliferation, differentiation, and/or migration of progenitors observed in various hematological malignancies may be caused by defects in expression and/or function of integrins. In this study, we have developed a new fluorescent beads adhesion assay that facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)– and very late activation antigen-4 (VLA-4)–mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10− and CD10+ (leukemic) cell population within one blood or bone marrow sample. Surprisingly, of the 20 B-lineage ALL patients investigated, 17 contained a leukemic cell population with LFA-1– and/or VLA-4–mediated adhesion defects. Five patients contained CD10+ cells that did not exhibit any LFA-1–mediated adhesion due to the lack of LFA-1 surface expression. The CD10+ cells from 10 ALL patients expressed LFA-1 that could not be activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), whereas the CD10− cells expressed a functional LFA-1. Seven patients contained CD10+ cells that expressed a PMA-unresponsive form of VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4 expressed by the CD10+ cells may be due to mutations in the integrins itself, in protein kinases, or in other intracellular molecules involved in integrin adhesion. These data clearly demonstrate the importance of investigating integrin function in addition to integrin surface expression. The strikingly high frequency (85%) of adhesion defects in ALL could suggest a causal relationship between integrin-mediated adhesion and B-lineage ALL.
The Potential of Iron Chelators of the Pyridoxal Isonicotinoyl Hydrazone Class as Effective Antiproliferative Agents III: The Effect of the Ligands on Molecular Targets Involved in Proliferation
Authors: Darnell G. , Richardson D.R. 
Published: October 2019

AbstractWe have identified specific iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class that are far more effective ligands than desferrioxamine (DFO; Richardson et al, Blood 86:4295, 1995; Richardson and Milnes, Blood 89:3025, 1997). In the present study, we have compared the effect of DFO and one of the most active chelators (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 311) on molecular targets involved in proliferation. This was performed to further understand the mechanisms involved in the antitumor activity of Fe chelators. Ligand 311 was far more active than DFO at increasing Fe release from SK-N-MC neuroepithelioma and BE-2 neuroblastoma cells and preventing Fe uptake from transferrin. Like DFO, 311 increased the RNA-binding activity of the iron-regulatory proteins (IRPs). However, despite the far greater Fe chelation efficacy of 311 compared with DFO, a similar increase in IRP-RNA binding activity occurred after 2 to 4 hours of incubation with either chelator, and the binding activity was not inhibited by cycloheximide. These results suggest that, irrespective of the Fe chelation efficacy of a ligand, an increase IRP-RNA binding activity occurred via a time-dependent step that did not require protein synthesis. Further studies examined the effect of 311 and DFO on the expression of p53-transactivated genes that are crucial for cell cycle control and DNA repair, namely WAF1,GADD45, and mdm-2. Incubation of 3 different cell lines with DFO or 311 caused a pronounced concentration- and time-dependent increase in the expression of WAF1 and GADD45 mRNA, but not mdm-2 mRNA. In accordance with the distinct differences in Fe chelation efficacy and antiproliferative activity of DFO and 311, much higher concentrations of DFO (150 μmol/L) than 311 (2.5 to 5 μmol/L) were required to markedly increase GADD45 and WAF1 mRNA levels. The increase in GADD45 and WAF1 mRNA expression was seen only after 20 hours of incubation with the chelators and was reversible after removal of the ligands. In contrast to the chelators, the Fe(III) complexes of DFO and 311 had no effect on increasing GADD45 and WAF1 mRNA levels, suggesting that Fe chelation was required. Finally, the increase in GADD45 and WAF1 mRNAs appeared to occur by a p53-independent pathway in SK-N-MC and K562 cells, because these cell lines lack functional p53. Our results suggest that GADD45 and WAF1 may play important roles in the cell cycle arrest observed after exposure to these chelators.
Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions
Authors: Kuwahara Mitsuhiro , Sugimoto Mitsuhiko , Tsuji Shizuko , Miyata Shigeki , Yoshioka Akira 
Published: October 2019

AbstractRecent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.
Low Levels of Hepatitis C Virus RNA in Serum, Plasma, and Peripheral Blood Mononuclear Cells of Injecting Drug Users During Long Antibody-Undetectable Periods Before Seroconversion
Authors: Beld Marcel , Penning Maarten , van Putten Marieke , van den Hoek Anneke , Damen Marjolein , Klein Michèl R. , Goudsmit Jaap 
Published: October 2019

Abstract
Screening of antibodies to hepatitis C virus (HCV) is widely used for monitoring the prevalence of HCV infections and to assess HCV infectivity. Among HCV-infected individuals in the general population, the interval between the detection of HCV RNA and the development of HCV antibodies is usually 5 to 6 weeks, but in rare cases, seroconversion may be prolonged up to 6 to 9 months. In this study, we tested for the presence of HCV RNA during the antibody-undetectable period of 19 drug-injecting HCV seroconverters to gain insight into the antibody-negative carrier status in this population. HCV seroconversion status was determined by testing the first and last serum samples obtained from each subject, using third-generation antibody screening and confirmation assays. Serial samples were tested for HCV-specific antibodies to establish the moment of seroconversion and HCV RNA by single reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA assay (bDNA) in serum. Plasma and peripheral blood mononuclear cells (PBMCs) were independently collected and tested for HCV RNA. HCV RNA-positivity was confirmed by Southern blot hybridization and sequencing of serial samples. The 19 HCV seroconverters had a mean follow-up of 5 years (range, 1 to 8 years). Of the 19, 4 were human immunodeficiency virus (HIV)-infected before HCV seroconversion. HCV RNA was detected in serum before seroconversion in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status. In 7 of these 12, the antibody-undetectable period was relatively short (2 to 10 months). The other 5, who were all HIV-negative before HCV seroconversion, had intermittent low levels of HCV RNA before seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent repeats of the experiments confirmed the presence of HCV RNA in serum, and in 3 of these individuals, HCV-positivity was confirmed in independently collected plasma and PBMC samples. Low levels of HCV RNA may be present during prolonged antibody-undetectable periods before seroconversion in a number of injecting drug users. Independent of HIV status, their immune system appears to be unable to respond to these low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV infection may not always elicit the rapid emergence of HCV antibodies and suggests that persistent low levels of HCV RNA (regardless of the genotype) may not elicit at all or delay antibody responses for prolonged periods of time.
Primitive Myeloid Cells Express High Levels of Phospholipase A2 Activity in the Absence of Leukotriene Release: Selective Regulation by Stem Cell Factor Involving the MAP Kinase Pathway
Authors: Roberts Pamela J. , Mollapour Elahe , Watts Michael J. , Linch David C. 
Published: October 2019

AbstractThe activation of phospholipase A2 (PLA2) with release of eicosanoids and prostanoids in mature myeloid cells and the augmentation (priming) of this activity by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) are central to the inflammatory process. Yet, there are few data concerning PLA2 activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA2activity of mobilized human CD34 antigen-positive (CD34+) stem cells by quantitation of the extracellular release of3H-arachidonate. The PLA2 activity of CD34+ cells stimulated with calcium ionophore (A23187) was of similar magnitude to that of mature neutrophils and monocytes. Preincubation of CD34+ cells with stem cell factor (SCF) before A23187-stimulation resulted in primed PLA2 activity, whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor  had no significant effect. When CD34+ cells were induced to differentiate, PLA2 activity remained responsive to SCF for several days, but after 8 days, at which stage morphological and functional evidence of maturation was occurring, priming of PLA2 by SCF could no longer be elicited, whereas responses to GM-CSF and IL-3 had developed. The further metabolism of arachidonic acid to eicosanoids by CD34+ cells was not detected by either thin-layer chromatography, enzyme immunoassay, or differential spectroscopy. SCF stimulated the rapid but transient activation of ERK2 (p42 MAP kinase) in CD34+ cells, and we used the MAP kinase kinase inhibitor, PD 098059, which at 30 μmol/L blocks ERK2 activation in CD34+ cells, to investigate whether SCF-mediated priming of arachidonate release was mediated by this kinase. PD 098059 only partially inhibited A23187-stimulated PLA2 activity primed by SCF, suggesting the involvement of ERK2 and possibly a further signal transduction pathway. Methyl arachidonyl fluorophosphonate (5 μmol/L), a dual inhibitor of i and cPLA2 isoforms, completely inhibited arachidonate release without affecting ERK2 activation, demonstrating the lack of cellular toxicity. These data provide the first evidence that primitive myeloid cells have the capacity to release arachidonate, which is regulated by an early acting hematopoietic growth factor important for the growth and survival of these cells.
Increased Clearance Explains Lower Plasma Levels of Tissue-Type Plasminogen Activator by Estradiol: Evidence for Potently Enhanced Mannose Receptor Expression in Mice
Authors: Lansink Mirian , Jong Miek , Bijsterbosch Martin , Bekkers Marian , Toet Karin , Havekes Louis , Emeis Jef , Kooistra Teake 
Published: October 2019

AbstractSeveral clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0.32 mL/min in controls; P &lt; .01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL · min in treated animals v 31.9 ng/mL · min in controls; P &lt; .05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P &lt; .001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P &lt; .005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.
Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation
Authors: Wolf Beni B. , Goldstein Joshua C. , Stennicke Henning R. , Beere Helen , Amarante-Mendes Gustavo P. , Salvesen Guy S. , Green Douglas R. 
Published: October 2019

AbstractApoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.
Unique Antiplatelet Effects of a Novel S-Nitrosoderivative of a Recombinant Fragment of von Willebrand Factor, AR545C: In Vitro and Ex Vivo Inhibition of Platelet Function
Authors: Inbal Aida , Gurevitz Osnat , Tamarin Ilia , Eskaraev Regina , Chetrit Angela , Novicov Ilia , Feldman Monica , Varon David , Eldar Michael , Loscalzo Joseph 
Published: October 2019

AbstractThe recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC50 = 0.02 ± 0.006 μmol/L v 0.1 ± 0.03 μmol/L, P = .001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate–induced platelet aggregation (IC50 = 0.018 ± 0.002 μmol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 μmol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P ≤ .004 for each parameter by ANOVA). In an ex vivo rabbit model, we also found that S-nitroso-AR545C had a more marked and more durable inhibitory effect on botrocetin-induced platelet aggregation than did AR545C, and these differences were also reflected in the extent and duration of effect on the prolongation of the bleeding time in these animals. These data show that S-nitroso-AR545C has significant and unique antiplatelet effects, inhibiting both adhesion and aggregation, by blocking platelet GPIb receptor through the AR545C moiety and elevating platelet cyclic 3′,5′-guanosine monophosphate through the -SNO moiety. These observations suggest that this NO-modified fragment of vWF may have potential therapeutic benefits as a unique antithrombotic agent.