bluuebunny/crossref_metadata_2025 huggingface dataset:
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 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.
Retention of Slow-Cycling CD34+ cells During Imatinib Treatment and Rapid Decline After 2nd ABL-TKI Treatment in Ph+ Leukemia Cells
Authors: Minami Yosuke , Abe Akihiro , Minami Miho , Kuwatsuka Yachiyo , Fukushima Nobuaki , Kitamura Kunio , Hiraga Junji , Yamamoto Kazuhito , Jamieson Catriona , Naoe Tomoki
Published: October 2019

Abstract 641

Several mathematical models and ex-vivo examinations suggested that imatinib (IM) therapy does not eradicate BCR-ABL-positive leukemia stem cells (LSCs). We investigated 47 CML-chronic phase cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations, while a gradual reduction was observed during the first 12 months in all populations. Moreover, the discrepancy of minimum residual diseases (MRD) between HSC and other populations became greater (about 2 log difference) in patients after longer IM therapy. We also prospectively investigated BCR-ABL transcripts in each population of 27 IM-resistant or -intolerant cases during treatment with the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months for IM-intolerant cases, and there was no statistical difference of MRD among each population in optimal responders to 2nd TKI therapy. In order to examine mechanisms of Ph+ LSC resistance to IM, Ph+ ALL patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice and cells derived from leukemic NOG mice were then co-cultured with S17-stromal cells using previously reported methods (Minami et al., PNAS, 2008). Despite complete dephosphorylations of BCR-ABL and its substrate CrkL, slow-cycling (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to IM, which indicated that addiction of BCR-ABL activity is lower for survival in such quiescent cells. On the other hand, treatment with lower doses of 2nd TKIs induced not only growth inhibition but also substantial cell death including slow-cycling CD34+ population. More detailed biomarkers during pro-apoptotic status by 2nd TKI treatment are also under investigation. From comprehensive drug screening of small compounds in this co-culturing system, we also found several candidates involved with survival pathways in the cells. These results imply that 2nd TKI therapy can be a more promising approach than IM treatment for early reduction of Ph+ LSCs.

Disclosures:

Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Research Funding; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
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 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 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 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 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 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.
Implication of Telomerase in Functions Besides Telomere Elongation in Human Cells
Authors: Gazzaniga Francesca , Lin Jue , Blackburn Elizabeth
Published: October 2019

Abstract SCI-40

The ribonucleoprotein enzyme telomerase, which counteracts telomere shortening by adding telomeric DNA repeats to the 3’ ends of chromosomes, contains a core protein reverse transcriptase (called hTERT in humans), the essential template-containing RNA (hTER) and associated factors. Thus, telomerase is capable of circumventing the limit on cell division imposed by telomere attrition. Telomerase enzymatic activity is readily detectable in stem and progenitor cell populations, but is largely downregulated in most adult somatic human cells. In contrast, telomerase is highly active in the large majority (∼80%–90%) of cancer cells. While telomerase is essential for telomere maintenance in cells in vitro and in vivo, several studies now also indicate that telomerase may have telomere-independent functions. We previously showed that lowering the level of the telomerase ribonucleoprotein (RNP) enzyme complex causes rapid changes in cell cycle program in human and mouse cancer cells: partial reduction of telomerase induced by directly lowering telomerase RNP component levels causes altered transcriptional profiles and reduced glucose metabolism in human cancer cells, yet bulk telomere shortening or detectable telomere uncapping are not required for these effects. Work by others has shown that reduction of telomerase core protein component level reduced Wnt signaling and caused Wnt-mutant like developmental defects in Xenopus and mouse and aberrant hematopoiesis in zebrafish embryos. We acutely depleted telomerase RNA components in cultured T cells from healthy human adults using shRNAs targeting both the telomerase protein and the RNA component of telomerase (hTR), delivered immediately after in vitro stimulation. Cell proliferation and telomerase activity levels were quantified through the first and second stimulation cycles. Effects of such telomerase RNP reduction on cell proliferation were observed in time frames too short to be accounted for by bulk telomere attrition. Models to account for these findings will be discussed.

Disclosures:

Lin: Telome Health, Inc.: Consultancy, Equity Ownership. Blackburn:Telome Health, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.
Challenges in Design and Development of PAR Inhibitors
Authors: Andrade-Gordon Patricia
Published: October 2019

Abstract SCI-45

Inappropriate and sustained activation of platelets is a major cause of vascular occlusive diseases such as angina, myocardial infarction, and stroke. The development of thrombi within blood vessels results from the formation of platelet aggregates and fibrin deposits, and heavily depends on the actions of α-thrombin. It is now well recognized that human platelet responses to α-thrombin are mediated by the protease-activated receptors PAR1 and PAR4. Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a “tethered” activating ligand. Since the discovery of PAR1 as the major contributor to human platelet aggregation, there has been a keen interest to develop antagonist as potential antithrombotics. However, there have been many issues and challenges in this endeavor. One crucial challenge to the discovery of potent antagonists is the strong entropy advantage offered by the intramolecular binding mechanism of PARs activation, which presents a great disadvantage to a circulating small molecule antagonist. To be an effective therapeutic agent, a PAR1 antagonist not only should bind tightly to the receptor but also possess suitable binding kinetics. Another issue for the discovery of PAR1 antagonists is the preclinical logistics associated with species variability of PAR1 on platelets. Despite these challenges, PAR1 has been an attractive drug target and there has been considerable activity and progress in the discovery and development of PAR1 antagonist as therapeutic agents. The promise of these novel therapeutics is reflected by two antiplatelet PAR1 antagonists in advanced clinical trials. The clinical benefits derived from these agents will be determined by the tight balance between delivering efficacy in the context of thrombotic disease and controlling the bleeding risk. The discussion will focus on the challenges from discovery to development of these great potential opportunities in the prevention of atherothrombotic disease.

Disclosures:

Andrade-Gordon: Johnson & Johnson: Employment.
Genetics of Intestinal Graft-Versus-Host Disease
Authors: van den Brink Marcel R.M.
Published: October 2019

Abstract SCI-51

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important therapy with curative potential for a variety of malignant and nonmalignant diseases. Despite standard prophylactic regimens, graft-versus-host disease (GVHD) continues to limit the success of outcomes in allo-HSCT patients. Gastrointestinal (GI) GVHD is the predominant contributor to acute GVHD-related mortality. The pathophysiology of GI GVHD has unique features that are related to the important interactions between the donor allograft (especially donor alloreactive T cells), mucosal adaptive and innate intestinal immunity, intestinal epithelial homeostasis, and the intestinal microbial flora. The pathophysiology of intestinal GVHD has many similarities with inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis (UC). At last count, 99 susceptibility loci/genes for Crohn’s and UC have been published. Multiple genes are involved in IL-23/Th17 signaling, defective processing of intracellular bacteria involving autophagy and innate immunity (including NOD2 and ATG16L1), and defects in barrier function and shared susceptibility with other autoimmune diseases (such as type 1 and 2 DM). Early studies in mice and in patients suggested a link between an individual’s intestinal microbial flora and his/her propensity for GVHD resulting in varying protocols for gut decontamination, which continue at some centers to this date. We will discuss the latest data regarding the role in intestinal GVHD of genetic changes in the before-mentioned pathways and genes in the donor and host, as well as its interactions with the intestinal flora.

Disclosures:

No relevant conflicts of interest to declare.
Hydroxyurea Is Associated with Lower Prevalence of Albuminuria in Adults with Sickle Cell Disease
Authors: Laurin Louis-Philippe , Nachman Patrick H. , Desai Payal C. , Ataga Kenneth I. , Derebail Vimal K.
Published: October 2019

Abstract 3211

Background:

Albuminuria is an early manifestation of sickle cell disease (SCD) nephropathy, denoting glomerular injury. Minimal clinical data exist on the association of hydroxyurea (HU) use with albuminuria in adults with SCD.

Methods:

A cross-sectional study was performed to evaluate the association of HU with the prevalence of albuminuria among adults with SCD using clinical data collected from 2000 to 2011. HU exposure was defined as ≥3 months of therapy prior to assessment of albuminuria. Albuminuria was defined as a urinary albumin-to-creatinine ratio (UACR) ≥30 mg/g and/or ≥1+ proteinuria on two dipstick measurements at least 2 months apart. A multivariate logistic regression model was constructed from univariate analyses and with covariates previously identified to be associated with albuminuria in SCD, including history of acute chest syndrome, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACEi/ARB) use, age, and elevated tricuspid regurgitation jet velocity (TRV). Backward elimination of covariates was used to produce the most parsimonious model.

Results:

149 adult patients (mean age 38±13 years; 113 with HbSS, 10 HbSß0, 7 HbSß1, 18 HbSC, and 1 HbSD) were included. The prevalence of albuminuria was lower among patients on HU therapy than those not on HU [26/75 (34.7%) vs. 41/74 (55.4%); p=0.01]. Among 112 patients with a measured UACR, median albumin excretion was lower in patients on HU [17.9 (6.0–53.0) vs. 40.5 (7.0–204.9) mg/g; p=0.04]. In univariate analyses, ACEi/ARBuse, hemoglobin level and percent reticulocytes were also related to albuminuria. By multivariate logistic regression model, HU use was associated with a lower risk of albuminuria (odds ratio 0.31, 95% CI 0.12 to 0.81; p=0.02), adjusting for age, ACEi/ARB use, TRV ≥2.5 m/s, systemic hypertension and history of acute chest syndrome.

Conclusion:

The use of HU was associated with lower prevalence of albuminuria, after controlling for age, ACEi/ARB use, TRV ≥2.5 m/s, systemic hypertension and history of acute chest syndrome. Based upon these findings, the potential of HU to prevent overt nephropathy or the progression of SCD nephropathy to end-stage renal disease merits further investigation.

Disclosures:

No relevant conflicts of interest to declare.
An Elevated TRV Is Associated with Venous Thromboembolism in Sickle Cell Disease
Authors: Adams Rachel M , Kopel Lianne S , Bland Demedrick Anton , Klings Elizabeth S
Published: October 2019

Abstract 3222

Objective:

An elevated tricuspid regurgitant jet velocity (TRV) by echocardiography, used to screen for pulmonary hypertension (PH) and mortality in sickle cell disease (SCD), likely has a multi-factorial etiology. SCD is a hyper-coagulable state, yet, the contribution of venous thromboembolic disease (VTE) to an elevated TRV is unknown. Based on the known association between VTE and PH, we hypothesized that SCD patients with an elevated TRV will have an increased prevalence of VTE.

Methods:

We reviewed data collected prospectively as part of the PH in SCD study conducted at Boston University/Boston Medical Center from 2004–2010. Subjects were included if they underwent both echocardiography and testing for VTE. An elevated TRV was defined as > 2.5 m/sec or the presence of PH on right heart catheterization. A history of VTE was defined by a positive CT/angiogram, ventilation/perfusion scan or Duplex ultrasound of an extremity (not catheter-related). Data were analyzed using a Chi-Square test and an odds ratio for VTE was calculated.

Results:

We reviewed the records of 162 patients enrolled in the PH of SCD study; 97 underwent both echocardiography and an evaluation for VTE.5/53 patients (9.4%) with normal echocardiography had a history of VTE, compared with 13/44 (29.5%) in the elevated TRV group. SCD patients with an elevated TRV were four times more likely to have a history of VTE compared to those with a normal echocardiogram (OR: 4.03, 95% CI 1.31–12.41, p=0.011). There was no significant difference in age, gender, history of asthma, hemoglobin genotype, hematologic profiles or renal function between patient groups.

Conclusion:

An elevated TRV and PH are associated with a history of VTE in SCD patients. This suggests a role for thrombosis in disease modulation and underscores the need for a complete evaluation for VTE in SCD patients with an elevated TRV.

Disclosures:

No relevant conflicts of interest to declare.
Protective Effect of Fetal Hemoglobin On Inflammation-Induced Expression of Hypoxia-Inducible Factor-1α (HIF-1α) in Sickle Mice: Microvascular Implications
Authors: Kaul Dhananjay K. , Fabry Mary E. , Suzuka Sandra M , Shah Janki
Published: October 2019

Abstract 3250

Chronic inflammation is a salient feature of human sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mouse model. Although tissue ischemia is the primary instigator of hypoxia-inducible factor (HIF) activation, a number of inflammatory factors/pathways and oxidative stress can potentially induce expression of HIF-1α. Increased oxidative stress and inflammation are implicated in the activation of HIF-1α under normoxic conditions. HIF can trigger transcription of genes for vasoactive molecules such as vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1) and endothelin, which are implicated in the pathophysiology of SCD. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and βS- globins, and evaluated the effect of HbF in BERK mice (i.e., <1.0%, 20% and 40% HbF). We have previously shown that HbF exerts anti-sickling and anti-inflammatory effects (Kaul et al. J Clin Invest, 2004; Dasgupta et al. Am J Physiol, 2010). Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions (i.e., normal hemoglobin oxygen saturation levels). In BERK mice expressing HbF, HIF-1α expression decreased concomitantly with increasing HbF, commensurately with increased NO bioavailability, and showed a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression in BERK mice expressing HbF was associated with decreased HO-1 and VEGF expression, and reduced serum endothelin-1 (ET-1) levels, which are among the target vasoactive molecules of HIF-1α. Furthermore, the commensurate decrease in HIF-1α expression with increase in HbF levels in BERK mice was accompanied by a distinct decrease in soluble (s) forms of endothelial activation markers such as sP-selectin and vascular cell adhesion molecule-1 (sVCAM-1). Notably, arteriolar dilation, enhanced volumetric blood flow and low blood pressure in normoxic BERK mice all showed a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α expression as well as ET-1 levels in normoxic BERK mice, supporting a role of decreased NO bioavailability in HIF-1α activation. The present in vivo studies show that reduced inflammation and increased NO production in normoxic BERK mice (expressing HbF or treated with arginine) are distinctly associated with suppression of HIF-1α activation and inhibition of vasodilators, resulting in improved microvascular and hemodynamic parameters in the BERK model of sickle cell disease. The unique feature of inflammation in SCD is that it can be ameliorated by increased HbF, thereby coupling HbS polymerization/sickling to NO depletion, HIF-1α expression and inflammation in this disease.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of the Two Populations of NK-Like CD56+ T Cells and KIR+ T Cells in Human
Authors: Chan Wing Keung , Rujkijyanont Piya , Neale Geoffrey A. , Yang Jie , Bari Rafijul , Gupta Neha Das , Holladay Martha , Rooney Barbara , Wendt Susanne , Leung Wing
Published: October 2019

Abstract 3292

Previous studies have demonstrated that many T cell subsets possess NK-like features, including the CD56+ and KIR+ populations. Collectively, these studies showed that these NK-like T cells are predominantly αβ+ CD8+ with memory phenotype and could recognize HLA-E associated viral peptides after expansion upon TCR engagement. However, their clonality, transcriptome, regulation, specificity, and memory response in human have not been fully elucidated. We hypothesize that these NK-like T cells are phenotypically and functionally distinct from conventional T and NK cells and they play unique roles in virus and cancer control. Herein, we extensively characterized the CD56+ T cells and the KIR+ T subset by analysis of TREC, TCRVβ spectrum, telomere length, surface biomarkers, genome-wide transcriptome, multi-analyte cytokine profiling, cancer cell susceptibility, tetramer staining, and real-time response to CMV reactivation in stem cell transplant recipients. In contrast to CD56– T cells, CD56+ T cells are limited in TREC, TCRVβ, telomere length, cytokine secretion, transcription of metabolic genes stx6, nnt, galnt2, hvcn1, tyms, rpa1 tmf1, ecop, and tspan3, and are mostly KIR+, CD8+, DNAM1+, NKG2D+, CD44+, NKp46– and CD25–. Compared to KIR– CD56+ T cells, KIR+ CD56+ T cells are even more limited in TREC, TCRVβ, telomere length, and cytokine secretion, but have elevated transcription of NK cytotoxicity-related genes arrb1, ppp3cc, and lamc3, higher degranulation after activation by IL-2/IL-15 and CD3/CD28 antibodies, better killing of cancer cells after cytokine priming, and are mostly KIR2DL2/3+, NKG2D+, NKp46+, CD16+, NKG2C+, CD57+, and 2B4+. Importantly during CMV reactivation after stem cell transplantation, the percentage of KIR+ CD56+ T cells in the patient's blood increased dramatically and was significantly higher (p=0.0021) than in those without viral reactivation. Ex vivo, KIR+ CD56+ T cells demonstrate CMVpp65 tetramer staining, memory response to CMV peptides, and potent lysis of CMVpp65-pulsed target cells dependent on both KIR and TCR specificity. Furthermore, we identified for the first time that KIR– CD56+ T cells are Rorc+ IL-13-secretor. In conclusion, both CD56+ and KIR+ NK-like T-cell subsets are unique in biological and clinical properties and have distinct roles in cancer and infection control.

Disclosures:

No relevant conflicts of interest to declare.
Role of PDGF/PDGFR in Regulation of Thrombopoiesis: A Possible Explanation for Imatinib Mesylate-Induced Thrombocytopenia in the Treatment of CML
Authors: Yang Mo , Meng Fanyi , Ye Jie yu , Xu Yue , Xiao Bin , Chong Beng
Published: October 2019

Abstract 3348

Platelet-derived growth factor (PDGF), a platelet alpha-granule molecule, imply their potential effect in the regulation of megakaryocytopoiesis and thrombopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on human megakaryocytes and platelets (Yang et al, Thromb Haemastasis, 1997) and CD34+ cells, and their ability to mediate a mitogenic response. PDGF promoted the ex vivo expansion of human hematopoietic stem (CD34+) and progenitor (CD41+ CD61+) cells. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. PDGF stimulated in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also showed a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF activated the PI3k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors with subsequent activation of the PI3k/Akt pathway. The study provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia in the treatment of CML.

Disclosures:

No relevant conflicts of interest to declare.
Development of a Factor VIII-Specific Immunotherapy for Hemophilia A
Authors: Naumann Anja , Kahle Jörg , Reiss Nadia , Schwabe Dirk , Heller Christine , Klingebiel Thomas , Königs Christoph
Published: October 2019

Abstract 3361

The development of neutralizing allo-antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. Non-hemophiliacs can spontaneously develop inhibitory auto-antibodies to FVIII, which results in a condition called acquired hemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of an anti-idiotypic immune response. Immune tolerance induction (ITI) by frequent administration of high doses of FVIII is successful in most patients. The remaining up to 20% failing ITI face increased morbidity and mortality.

To elucidate the capacity of single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors), scFvs that specifically interact with strong inhibitory murine monoclonal anti-human FVIII antibodies (anti-hFVIII mAbs) were selected by screening synthetic scFv phage displayed libraries. Since the majority of inhibitory anti-hFVIII mAbs are directed against the FVIII A2 or C2 domain, selection of scFvs was performed for two anti-A2 and two anti-C2 mAbs. Affinity selection identified several specific, potential anti-idiotypic scFvs for each anti-hFVIII mAb, which were further analyzed. ScFvs expressed as IgG fusion protein bind to mAbs with affinities up to 0,1nM. Binding of mAbs to immobilized FVIII was inhibited via specific scFvs by more than 90%. In the functional Bethesda Assay FVIII activity was fully restored when corresponding anti-idiotypic scFvs were added to plasma spiked with FVIII-specific mAbs. In addition, the cross-reactivity of scFvs with heterologous mAbs specific for A2 and C2 was tested and confirmed the exclusive interaction of the selected scFvs with their respective mAb.

Overall, anti-idiotypic scFvs specific for anti-hFVIII mAbs can be successfully selected from phage displayed libraries and efficiently neutralize FVIII inhibitors. ScFv anti-idiotypes may therefore facilitate the development of specific immunotherapies for hemophilia patients with inhibitors.

Disclosures:

No relevant conflicts of interest to declare.