bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Phenotype of Endothelium in Children with ALL Might Be a Predictor of the Outcome – a Pilot Study
Authors: Doroszko Adrian , Niedzielska Ewa , Porwolik Julita , Szahidewicz- Krupska Ewa , Chybicka Alicja , Szuba Andrzej 
Published: October 2019

Abstract
Abstract 5314

Background:
Activation of endothelium has been demonstrated to be associated with an increased risk of mortality in severe diseases. In this study we hypothesize that ALL might be also associated with endothelial dysfunction (ED) and that children with concomitant ED might be at higher risk for death.

Methods:
N=18 children at age of 4–18 years with ALL treated with the ALLIC protocol were investigated. Plasma levels of markers of ED (VCAM-1, ICAM-1, E-selectin, P-selectin and PAI-1), lipid peroxidation (malonyldialdehyde – MDA), angiogenesis (VEGF) as well as routine blood tests were analyzed at baseline – the day of diagnosis of ALL, next during the 33rd day of the protocol (steroid therapy, evaluation of remission) and subsequently at the 78th day of therapy (beginning of the protocol M). The control group constituted of N=15, age-matched healthy children.

Results:
Children with ALL were characterized with significantly worse kidney and liver function at baseline as compared to the control. Similarly, the fasting glycemia was higher in the investigated group. Markers of endothelial proagreagattory and procoagulative function (P-selectin and PAI-1) were similar in both groups at baseline. However, vascular inflammation was significantly more pronounced in children with ALL at baseline as compared to the healthy control (ICAM-1= 467.1±72.8 vs. 206.3±21.7 ng/ml, p<0.05). High baseline plasma level of E-selectin and VEGF (as compared to the healthy control) were positive predictors for death in children with ALL (E-selectin: 56.1±10.9 vs. 28.1±10.0 ng/ml, respectively; VEGF: 366.1±61.6 pg/ml vs. 190.1±71.4 pg/ml, respectively, p<0.05).

Conclusion:
Endothelial inflammatory activation is a common phenomenon in children with ALL. High baseline level of VEGF and E-selectin are associated with higher mortality in ALL.

Disclosures:
No relevant conflicts of interest to declare.
Aspirin After Oral Anticoagulants for Prevention of Recurrence in Patients with Unprovoked Venous Thromboembolism. the Warfasa STUDY
Authors: Becattini Cecilia , Agnelli Giancarlo , Poggio Renzo , Eichinger Sabine , Bucherini Eugenio , Silingardi Mauro , Bianchi Marina , Moia Marco , Ageno Walter , Vandelli Maria Rita , Grandone Elvira , Siragusa Sergio , Salvi Rosamaria , Ria Luigi , Zanatta Nello , Poli Daniela , Camporese Giuseppe , Salvi Aldo , Santi Roberto , Cimminiello Claudio , Scannapieco Gianluigi , Barillari Giovanni , De Gaudenzi Egidio , Cappelli Roberto , di Minno Giovanni , Frausini Gabriele , Bova Carlo , Prandoni Paolo 
Published: October 2019

Abstract
Abstract 543

Background
A recurrence occurs in 15–20% of patients with unprovoked venous thromboembolism (VTE) in the two years after the withdrawal of oral anticoagulant treatment. Extending anticoagulant treatment is effective but associated with increased bleeding risk. We assessed the efficacy and safety of aspirin for the prevention of VTE recurrence after a conventional course of oral anticoagulation.

Methods
Warfasa was an investigator-initiated double-blind randomized placebo-controlled event-driven study. Patients with a first-ever unprovoked VTE who had completed 6–12 months of oral anticoagulant treatment were randomized to receive aspirin, 100 mg daily, or placebo for at least two years. The primary efficacy outcome was objectively confirmed recurrent symptomatic VTE and VTE-related death. Clinically relevant (major and non-major) bleeding were the main safety outcome. All outcome events were blindly adjudicated by an independent committee.

Results
A VTE recurrence occurred in 27 of the 205 patients who received aspirin and 42 of the 197 patients who received placebo (6.3% versus 11.0% patient-years; hazard ratio, 0.57, 95% CI 0.35 to 0.93) during the study period (mean 24 months) (Figure A). While on study treatment, 22 and 38 patients who received aspirin or placebo, respectively had a recurrence (5.7% versus 10.7% patient-years; hazard ratio, 0.54, 95% CI 0.32 to 0.91) (mean on-treatment period 22 months) (Figure B). One patient in each treatment group had a major bleeding, with a similar incidence of clinically relevant non-major bleeding.

Conclusions
Aspirin reduces by about 40% the risk of recurrence in patients with unprovoked VTE without increasing bleeding, when given after a 6–12 month anticoagulant treatment. For its safety, practicality and low cost, aspirin is a valid alternative to oral anticoagulants in the extended treatment of VTE.

Disclosures:
No relevant conflicts of interest to declare.
Polymeric Immunoglobulin A1 Controls Erythroblast Proliferation and Accelerates Erythropoiesis Recovery in Anemia
Authors: Coulon Séverine , Dussiot Michael , Grapton Damien , Maciel Thiago , Wang Pamella Huey Mei , Callens Celine , Arnulf Bertrand , Mayeux Patrick , Marc Benhamou , Cogné Michel , Monteiro Renato , Hermine Olivier , Moura Ivan Cruz 
Published: October 2019

Abstract
Abstract 679
Erythropoietin (Epo) is the main cytokine controlling terminal erythropoiesis. Twenty years ago a model of erythropoiesis regulation has been described suggesting that the circulating Epo level is suboptimal and only a fraction of erythroid progenitors survives to produce erythrocytes (Koury and Bondurant; Science, 1990). Therefore differential Epo sensitivity would control erythroblasts survival. However, cell autonomous and/or environmental factors controlling this process remain elusive. Erythroblasts highly express the transferrin receptor 1 (TfR1), which has been shown to be essential for iron uptake allowing hemoglobin synthesis. We have previously identified TfR1 as an unsuspected polymeric (p)IgA1 receptor. Here we show that TfR1-bound pIgA1 (but not monomeric (m)IgA1) rescued the growth and clonogenic potential of human erythroblasts under suboptimal Epo concentrations. In a humanized mouse model (α1KI mice) IgA1 polymers increased spleen homeostatic erythropoietic activity and accelerated stress erythropoiesis in several models of anemia (phenylhydrazine and autoimmune hemolytic anemia; 5-fluorouracyl-induced central anemia). By contrast, mice lacking the immunoglobulin joining-(J) chain (α1KI/J-chain−/−), which are devoid of pIgA1 demonstrated a delayed recovery from anemia. Moreover, IgA deficiency patients presented increased Epo levels. Upon hypoxia, pIgA1 levels increased in both humans and mice. Likewise, α1KI mice submitted to chronic intermittent hypoxia presented an enhanced erythroblasts expansion relative to their LT controls. Therefore, hypoxia regulates pIgA1 production and, in turn, pIgA1/TfR1 interaction modulates erythroblast sensitivity to growth factors by decreasing cell activation threshold. We also showed that the natural TfR1 ligand, iron-loaded transferrin (Fe-Tf), also accelerated recovery from acute anemia. At the molecular level, pIgA1 and Fe-Tf converged for Epo-dependent erythroblasts proliferation. TfR1 engagement increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling pathways. These cellular responses were mediated by the TfR1-internalization motif, YXXΦ. Therefore, pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. This study shed light on a new role of TfR1 as a signaling competent receptor. Targeting this pathway could provide alternative approaches to treat anemia, in particular in Epo hypo-responsive patients and dyserythropoiesis.

Disclosures:
No relevant conflicts of interest to declare.
“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion
Authors: Verma Rakesh , Leu Karen , Young Peter R. , Schatz Peter J , Green Jennifer M , Wojchowski Don M. 
Published: October 2019

Abstract
Abstract 682
Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion.

Disclosures:
Leu: Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.
A High Through-Put Screen Identifies MCP-1 (CCL2) As a Novel Regulator of Iron Homeostasis and a Modifier of Hereditary Hemochromatosis Disease Severity
Authors: Muckenthaler Martina U. , Spasic Maja Vujic , Mleczko-Sanecka Katarzyna , Zhu Mingang , Pepperkok Rainer , Runz Heiko , Maus Ulrich , Gomes Joana , Pinto Jorge P , Lawless Matthew , Kulozik Andreas E , Porto Graf¸ca 
Published: October 2019

Abstract
Abstract 685
To identify genes that modify the severity of human iron disorders we pre-selected 74 genes from gene expression profiles of cells and tissues with altered iron levels and assessed whether siRNA-mediated knock-down of these genes affects uptake of transferrin, a key cellular process to acquire iron. This screen identifies the monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, as a critical suppressor of transferrin receptor mRNA expression in human cells. We next analyzed CCL2-deficient mice and demonstrate profound alterations of parameters of systemic iron homeostasis. Specifically, CCL2 knock-out mice show decreased serum iron levels and transferrin saturation, strong iron-overload in the spleen and duodenum as well as mild iron accumulation in the liver. Iron imbalance in CCL2−/− mice is unlikely explained by an impairment of the major control system of systemic iron homeostasis, the hepcidin/ferroportin regulatory system: hepcidin mRNA expression is unaltered and splenic ferroportin protein expression is strongly increased in CCL2−/− mice, as would be expected as a consequence of splenic iron overload. We speculate that increased iron absorption from the plasma, possibly mediated by inappropriately high levels of TfR1 in the spleen, duodenum and liver, may be responsible for tissue iron overload.
It is of note that CCL2 levels are strongly decreased in Hfe-deficient mice and patients with Hfe-associated Hereditary Hemochromatosis (HH). We therefore asked whether CCL2 levels could modify disease severity of HH. Analysis of 51 HH patients, all homozygous for the C282Y HFE mutation, confirms significantly lower MCP-1 levels in the serum compared to a group of 23 sex- and age-matched normal controls. Importantly, CCL2 levels in HH patients show a significant negative correlation with liver iron overload at the time point of diagnosis. Furthermore, low CCL2 concentrations are significantly associated with the HLA-A3 genotype and the CD8+ T lymphocyte phenotype, both traits previously shown to correlate with iron overload in HH patients. These patient data and the data from CCL2-deficient mice suggest that appropriate CCL2 expression is required to prevent iron overload.
Taken together our data demonstrate the power of siRNA screens to identify novel regulators of iron metabolism in human cells that are critically involved in maintaining systemic iron homeostasis in the mouse and that play a role in modifying hepatic iron overload in the frequent iron overload disorder Hereditary Hemochromatosis.

Disclosures:
No relevant conflicts of interest to declare.
Cited2 Regulates Hematopoietic Stem Cell Quiescence Through HIF-1α Dependent and Independent Pathways
Authors: Du Jinwei , Chen Yu , Li Qiang , Wang Zhengqi , Dunwoodie Sally , Bunting Kevin D. , Yang Yu-Chung 
Published: October 2019

Abstract
Abstract 912
Hematopoietic stem cells (HSCs) are thought to be localized in hypoxic microenvironment of the bone marrow (BM) and can remain quiescent or differentiate into multiple blood cell lineages. A number of factors have been found to regulate HSC quiescence in either cell-intrinsic or cell-extrinsic manner. Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2), a member of a newly identified transcriptional modulator, is a cytokine inducible gene and plays various roles during mouse development. In particular, Cited2 is essential for fetal liver hematopoiesis. In this study, we used conditional knockout strategy to delete Cited2 in order to further investigate its function in adult hematopoiesis. Sequential injection of poly(I)-poly(C) (pI-pC) efficiently deleted the Cited2 gene in Cited2fl/fl;Mx1-Cre mice. In this mouse model (Cited2−/− mice), the white blood cell (WBC) count, BM cellularity and Lin−Sca-1+c-Kit+ (LSK) cell number were within the normal range. However, the long-term HSC (LT-HSC; defined as Flt3−CD34−LSK or CD48−CD150+LSK) frequency was significantly decreased. Cited2−/− mice also exhibited increased apoptosis in both LSK and CD34−LSK cells. In addition, Cited2 deficiency led to loss of HSC quiescence evidenced by cell cycle analysis and BrdU incorporation assay. HSC reconstitution capacity was significantly impaired assessed by 5-fluorouracil (5-FU) treatment and transplantation experiments. Transcriptional profiling revealed that multiple HSC quiescence and hypoxia related genes were affected, including p57, Hes1 and Egr1. Recent studies have shown that both HIF-1α-deficient and HIF-1α-stabilized HSCs result in impaired hematopoietic reconstitution, suggesting that precise regulation of HIF-1α level is essential for maintaining HSC quiescence and transplantation activity. Cited2 has been shown to be a negative regulator for HIF-1α through competitive binding to CBP/p300 with higher affinity. In addition, we previously showed that HIF-1α haploinsufficiency (HIF-1α+/−) partially rescues the heart defects in Cited2−/− embryos and HIF-1α deletion (HIF-1α−/−) rescues aberrant vasculature in Cited2−/− embryonic lens. These findings prompted us to explore whether defects of Cited2−/− HSC in adult mice are mediated by dysregulated HIF-1 activity by generating Cited2fl/flHIF-1αfl/fl;Mx1-Cre mice. Additional deletion of HIF-1α partially rescued impaired HSC quiescence and reconstitution capacity caused by Cited2 deficiency. Cited2−/− HIF-1α−/− HSCs displayed comparable apoptosis to Cited2−/− counterparts. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to a normal level. Taken together, these results suggest that Cited2 regulates HSC quiescence through HIF-1 dependent and independent pathways.

Disclosures:
No relevant conflicts of interest to declare.
From Hematopoietic Stem Cell to Erythroblast: Regulation of Red Cell Production At Multiple Levels
Authors: Lodish Harvey F. 
Published: October 2019

Abstract SCI-1
Hematopoietic stem cells (HSC) undergo self-renewal and also generate all types of blood cells, including red cells, myeloid cells, and all immune system cells. Formation of red cells from HSC involves multiple cellular stages, including early erythroid-specific progenitors (burst-forming unit-erythroid [BFU-E]) that respond to several growth factors and an erythropoietin (Epo)-responsive progenitor, and the colony-forming unit-erythroid (CFU-E) that in 3 days generates ∼30 reticulocytes. In adult animals most CFU-E cells undergo apoptosis. A short-term need for red cells is met by increased Epo production by the kidney, which rescues increasing numbers of CFU-Es from apoptosis and increases red cell production over a few days. Chronic stress, such as in certain types of anemias and inflammatory diseases, leads to a marked increase in numbers of HSC and many types of progenitor cells. I will focus most of my talk on stress-triggered BFU-E proliferation and formation of CFU-Es, since our very recent work showed how glucocorticoids – an important class of stress hormones – as well as HIF-1α stimulate red cell production. Acting synergistically these allow ∼300 times more CFU-Es and erythroblasts to be formed from each BFU-E. More specifically, these molecules stimulate limited self-renewal of BFU-Es during cell division, thus maintaining progenitor immaturity and allowing over time increased numbers of CFU-E cells and thus mature red cells to be formed. This explains why certain corticosteroids are useful in treating some non-Epo responsive anemias, and I will present a molecular dissection of glucocorticoid and HIF-1α action on BFU-E progenitors that has led to the identification of genes critical for self-renewal and that suggest other possible avenues for treatment of chronic anemias.

Disclosures:
No relevant conflicts of interest to declare.
Transcriptional Regulation in Normal and Malignant Hematopoiesis
Authors: Akashi Koichi 
Published: October 2019

Abstract SCI-28
Lineage commitment should involve selective and temporally-regulated expression of essential genes. In multi- or oligo-potent progenitors, the expression of oligo-lineage-affiliated genes is primed: oligo-lineage genes are co-expressed prior to the commitment at the single cell level, and once lineage fate is decided, genes of irrelevant lineages are immediately downregulated. For example, single common myeloid progenitors (CMP) co-express both granulocyte/monocyte-affiliated and megakaryocyte/erythroid-affiliated genes. The priming of these lineage-restricted genes could be dependent also upon the priming of lineage-specific transcription factors. Consistent with this hypothesis, a population with potent CMP activity that co-express both PU.1 (myeloid) and GATA-1 (erythroid) transcription factors was newly identified by using mice having PU.1 and GATA-1 reporters. In downstream of such “priming” stage, the order of expression as well as the level of expression of multiple transcription factors plays a critical role in reading-out specific lineages. The precise regulation of transcription factors should be critical to maintain hematopoietic homeostasis, and the deregulation of transcription factor expression could induce leukemic transformation. To understand the regulation machinery upstream of transcription factors, we are currently attempting to model an epigenetic landscape in hematopoietic development. Genome-wide analysis of histone positioning revealed that a histone variant marks hematopoietic transcription factors and other lineage-related genes prior to commitment, and therefore the variant can predict the actively-transcribed region in a later stage of hematopoiesis. For example, in hematopoietic stem cells, the histone marking was observed broadly in genes-related to myelo-erythroid and lymphoid genes, while in committed progenitors, it was restricted to specific lineages such as myeloid, erythroid and/or lymphoid genes. These results suggest that the genome marking by a histone variant should be a primary event for initiating lineage commitment.

Disclosures:
No relevant conflicts of interest to declare.
Gene Regulatory Networks Governing Hematopoietic Stem Cell Development and Identity
Authors: Enver Tariq 
Published: October 2019

Abstract
Abstract SCI-30
Several studies have addressed questions about transcriptional regulation within particular hematopoietic cell compartments. Few, however, have attempted to capture the transcriptional changes that occur during the dynamic transition from one compartment to another. We have profiled gene expression as multipotential progenitors underwent commitment and differentiation to two alternative lineages, focusing on the first 3 days of differentiation when the majority of decisions about cell fate are made. We have combined this with genome-wide identification of the targets of three key transcription factors before and after differentiation; GATA-2, usually associated with the stem/progenitor compartment; GATA-1 (erythroid); and PU.1 (myeloid). These data have been compiled into a custom-made queryable database, designed to be intuitive to use and to provide tools to interrogate the data on many levels. We used correlation analyses to associate transcription factor binding with particular modules of co-expressed genes, alongside detailed sequence analysis of bound regions. These approaches have informed our understanding of GATA factor switching, and highlighted novel roles for both GATA-2 and Pu.1 in erythroid cells. Overall, the data reveal greater degree of complexity in the interplay between these three factors in regulating hematopoiesis than has hitherto been described, and highlights the importance of a genome-wide approach to understanding complex regulatory systems. A significant challenge in the field is how to relate these types of population-based data to the action of transcriptional regulators within single cells where cell fate decisions ultimately are affected. As a step toward this, we have generated single cell profiles of gene expression for a limited set of transcriptional regulators in self-renewing and committed blood cells and used these data to build a stochastic computational model, which affords exploration of commitment scenarios in silico.

Disclosures:
No relevant conflicts of interest to declare.