bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thrombin Generation Mediators and Markers in Sepsis Associated Coagulapathy,
Authors: Hoppensteadt Debra , Cunanan Josephine , Khan Hussein , Litinas Evangelos , Sharma Gautam , Kaul Inder , Fareed Jawed 
Published: October 2019

Abstract
Abstract 3341
Thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), fibrinopeptide A (FPA) and thrombin antithrombin complex (TAT), along with tissue factor (TF), microparticles (MP), D-dimer (DD) and antithrombin (AT). Baseline plasma samples from one hundred patients diagnosed with sepsis associated DIC were analyzed for various parameters by using ELISA methods. The MP levels were measured by using a functional method utilizing annexing trapping whereas the antithrombin was measured using both the functional and immunologic methods. Plasma samples from 50 normal male and female individuals comprised the control group. The results are tabulated in the following table. Both TF and MP levels were markedly elevated in comparison to the control group. All of the other markers of thrombin generation showed elevated levels F1.2 (3.4 fold), FPA (4.3 fold) and TAT (4.0 fold). Marked elevation of D-dimer was also noted (5.0 fold). Both the immunologic and functional levels of AT were also decreased. While the data was widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes such as the antithrombin, thrombomodulin and TFPI may play an important regulatory role in the pathogenesis of this syndrome.MarkerNormal ControlsDIC BaselineF 1.2 (pM)108.6+12.3473.7+320FPA (ng/ml)1.6+0.86.8+3.1MP (nM)8.47+1.124.56+2.1AT (% NHP)94.2+2.780.4+3.1DD (ng/ml)415.4+65.23685+112TAT (ng/ml)3.4+0.317.0+3.1

Disclosures:
No relevant conflicts of interest to declare.
Non-Muscle Myosin II Mediates Microparticle Release From Endothelial Cells Activated by Antiphospholipid Antibodies,
Authors: Betapudi Venkaiah , Hsi Linda , Allen Kristi L , Willard Belinda , McCrae Keith R. 
Published: October 2019

Abstract
Abstract 3343
The antiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLA). The majority of pathogenic APLA are actually directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. Microparticles are small phospholipid-rich vesicles that may express tissue factor and are thought to play an important role in thrombosis. Increased release of microparticles may be stimulated by cellular activation or apoptosis, and previous work from our laboratory and others has demonstrated increased levels of circulating microparticles in patients with APS, even far removed from thrombotic events. Though several mechanisms have been proposed for mediating release of microparticles from cells, this process is poorly understood and there is little information available concerning the mechanisms of microparticle release from endothelial cells stimulated by APLA/anti-β2GPI antibodies. To address this issue we assessed the role of non-muscle myosin II motor protein in mediating the release of microparticles from endothelial cells activated by anti-β2GPI antibodies. We observed that incubation of endothelial cells with β2GPI and anti-β2GPI antibodies caused significant increases in the release of microparticles compared to cells treated with β2GPI and control antibodies. Proteomic and biochemical studies demonstrated that anti-β2GPI antibodies caused increased phosphorylation of endothelial cell Myosin II light chain (MLC), an essential step in the activation of Myosin II motor protein. Moreover, examination of endothelial cells stimulated by anti-β2GPI antibodies using confocal microscopy revealed a dramatic increase in the formation of actin-myosin II filaments. To assess the effects of these changes on microparticle release, we inhibited MLC phosphorylation in anti-β2GPI-treated endothelial cells using ML-7, a highly specific inhibitor of MLC. ML-7 caused a significant decrease in the release of microparticles from anti-β2GPI-treated cells. In conclusion, our results demonstrate that non-muscle myosin II motor protein activation by anti-β2GPI antibodies is necessary for the increased release of microparticles from activated cells. These microparticles may play important roles in the propagation of thrombosis and other clinical sequelae observed in patients with APS.

Disclosures:
No relevant conflicts of interest to declare.
Zebrafish Growth Factor Independence Transcription Factors Establish a New Paradigm for Regulation of Primitive and Definitive Hematopoietic Lineages,
Authors: Cooney Jeffrey D. , Shafizadeh Ebrahim , McBride Paul F. , Carroll Kelli J. , Anderson Heidi , Ganis Jared J. , North Trista E. , Paw Barry H. 
Published: October 2019

Abstract
Abstract 3379
The Growth Factor Independence (Gfi) zinc finger transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic lineage specification. In mammals, Gfi1 regulates hematopoietic stem cell (HSC) and lymphoid populations, while Gfi1b is required for megakaryocyte and erythroid development (van der Meer, et al. 2010 Leukemia 11:1834–43). In zebrafish, gfi1.1 plays an essential role in primitive hematopoiesis, preserving primitive HSC populations and regulating the erythroid-myeloid balance (Wei, et al. 2008 Cell Res. 6:677–85). However, little is known about the role of gfi1.1 in definitive hematopoiesis or about the role of additional hematopoietic gfi family members in zebrafish. Here, we report the isolation and characterization of an additional zebrafish gfi family transcription factor, gfi1.2b. We compare and contrast gfi1.1 and gfi1.2b, showing that they are highly expressed in the intermediate cell mass (ICM) and aorta-gonad-mesonephros (AGM), the respective sites of primitive and definitive hematopoiesis in zebrafish. Using antisense morpholino oligos (MO), whole mount in situ hybridization (WISH) and fluorescent activated cell sorting (FACS) of transgenic reporter fish, we demonstrate that gfi1.1 and gfi1.2b have distinct, essential roles in preserving primitive and definitive HSC populations, respectively. Loss of gfi1.1 specifically silences expression of scl and gata-1, markers of primitive HSC and erythroid progenitors. Conversely, loss of gfi1.2b silences expression of Tg(cd41:eGFPlo) cells, indicating an essential role for gfi1.2b in preserving definitive hematopoietic progenitors (Ma, et al. 2011 Blood 118:289–297). Consistent with the discrete roles of gfi1.1 and gfi1.2b in primitive and definitive lineages, knockdown of gfi1.2b silences lymphocyte rag-1 expression in the developing thymus, while knockdown of gfi1.1 has no effect on the thymic lymphocyte population. gfi1.1 and gfi1.2b have overlapping roles in erythropoiesis, as loss of either gfi factor reduces erythrocyte populations, while loss of both gfi paralogs results in a more profound silencing of erythrocytes. We further demonstrate that loss of gata-1 reduces gfi1.1 expression and silences gfi1.2b, suggesting that gata-1 plays an essential role in regulating the transcription of both genes. Together, these studies demonstrate that gfi1.1 and gfi1.2b have distinct and overlapping roles in zebrafish hematopoiesis and establish a new paradigm for the regulation of primitive and definitive hematopoietic lineages by gfi transcription factors.

Disclosures:
No relevant conflicts of interest to declare.
Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,
Authors: Zhao Xinyang , Vu Ly P. , Perna Fabiana , Liu Fan , Xu Hao , Nimer Stephen 
Published: October 2019

Abstract
Abstract 3403
RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases.

Disclosures:
No relevant conflicts of interest to declare.
Membrane-Bound Human Stem Cell Factor Promotes Differentiation of Human Granulocytes and Mast Cells In Vivo,
Authors: Takagi Shinsuke , Saito Yoriko , Hijikata Atsushi , Tanaka Satoshi , Watanabe Takashi , Koseki Haruhiko , Ohara Osamu , Saito Takashi , Taniguchi Shuichi , Shultz Leonard , Ishikawa Fumihiko 
Published: October 2019

Abstract
Abstract 3419
Recently, advances in xenograft models for human hemamtopoietic stem cells (HSCs), or the humanized mice, have begun to allow investigators to examine the differentiation of human hematopoietic and immune cells in vivo. However, lymphoid-skewed human hematopoietic development in the mouse bone marrow is one of the remaining limitations in the humanized mouse models. The inefficient human myeloid development could at least partly be attributed to the mouse microenvironment not fully supporting differentiation and maturation of human myeloid lineage. To overcome this problem, we focused on the role of membrane-bound human stem cell factor in supporting the maintenance of human HSCs and inducing the development of human myeloid cells and created human stem cell factor transgenic NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. Transplantation of 5000–50000 cord blood-derived Lin-CD34+CD38- cells resulted in significantly higher engraftment of human CD45+ leukocytes at 3–6 months post-transplantation in the bone marrow, spleen, and peripheral blood of hSCF Tg NSG recipients compared with those of non-transgenic NSG recipients. The enhanced human CD45+ engraftment was most prominent in the bone marrow (hSCF Tg recipients: 98.0 +/− 1.3%, n= 15, non-Tg NSG controls: 75.3 +/− 7.3%, n=7). In the bone marrow, the frequency of human CD33+ myeloid cells within the total human CD45+ population was significantly higher in the hSCF Tg NSG recipients than in the non-Tg NSG recipients and constituted the majority of human hematopoietic cells (hSCF Tg recipients: 54.6 +/− 4.5%, n=15 and non-Tg NSG controls: 29.3 +/− 4.0%, n=7). Flow cytometric analysis demonstrated that the majority of engrafted human myeloid cells in the hSCF Tg recipient bone marrow were side-scatter high, HLA-DR negative granulocytes. Reflecting the effect of human SCF on the development of human mast cells, human c-Kit+CD203c+ mast cells were identified in the bone marrow, spleen, and gastrointestinal tracts of the hSCF Tg NSG recipients. Altogether, the in vivo humanized mouse model demonstrates the essential role of membrane-bound SCF in human myeloid development. The hSCF Tg NSG humanized mice may facilitate the in vivo investigation of human HSCs, myeloid progenitors and mature myeloid lineage.

Disclosures:
No relevant conflicts of interest to declare.
Outcomes in Children with Severe Aplastic Anemia Receiving Bone Marrow Transplantation from an HLA-Matched Family Donor or Intensive Immunosuppressive Therapy As First-Line Treatment,
Authors: Yoshida Nao , Kikuchi Akira , Kobayashi Ryoji , Yabe Hiromasa , Kosaka Yoshiyuki , Muramatsu Hideki , Takahashi Yoshiyuki , Kato Koji , Suzuki Ritsuro , Ohara Akira , Kojima Seiji 
Published: October 2019

Abstract
Abstract 3421
Bone marrow transplantation (BMT) from an HLA-matched family donor (MFD) is the treatment of choice for severe aplastic anemia (SAA) in children. For children without an MFD, immunosuppressive therapy (IST) with a combination of antithymocyte globulin and cyclosporine has been successful. However, this treatment approach is based on the results of comparative studies between these therapies conducted in the 1980s, and the outcomes of both BMT and IST have improved markedly over the past three decades. Therefore, updated evidence for treatment decisions in pediatric SAA is required. In the present study, we compared the outcomes of children with SAA who received IST (subjects enrolled in the prospective multicenter trials of IST conducted by the Japan Childhood Aplastic Anemia Study Group) or BMT from an MFD (subjects registered in the Transplant Registry Unified Management Program conducted by the Japan Society for Hematopoietic Cell Transplantation). The influence of potential risk factors on overall survival (OS) and failure-free survival (FFS) was assessed according to first-line treatment, time period of treatment (1992–1999 and 2000–2009), age and other variables related to each treatment. FFS was defined as survival with treatment response. Death, primary or secondary graft failure, relapse and secondary malignancy were considered treatment failures in patients who received BMT. Death, relapse, disease progression requiring stem cell transplantation from an alternative donor or 2nd IST, clonal evolution and evolution to paroxysmal nocturnal hemoglobinuria were considered treatment failures in patients who received IST. Between 1992 and 2009, 599 children with SAA younger than 17 years received BMT from an MFD (n=213) or IST (n=386) as first-line treatment. While the OS did not differ between patients receiving IST and BMT (88±2% vs. 90±2% at 15 years), FFS was significantly inferior in patients receiving IST as compared to those receiving BMT (54±3% vs. 84±3% at 15 years, P<0.0001). There was no significant improvement in outcomes over the two time periods; OS and FFS at 10 years in 1992–1999 vs. 2000–2009 were 87±2% vs. 93±2% and 66±3% vs. 67±3%, respectively. On multivariate analysis, age <10 years was identified as a favorable factor for OS (P=0.007) and choice of first-line IST was the only unfavorable factor for FFS (P<0.0001). These updated data support the current algorithm for treatment decisions, which recommends BMT when an MFD is available.

Disclosures:
No relevant conflicts of interest to declare.
Beneficial Effects of Nucleoside Treatment in Zebrafish Models of Diamond Blackfan Anemia Deficient in Ribosomal Proteins L11 or S19,
Authors: Danilova Nadia , Sakamoto Kathleen M , Lin Shuo 
Published: October 2019

Abstract
Abstract 3439
Deficiency of ribosomal protein (RPs) is associated with Diamond Blackfan Anemia, (DBA) a congenital syndrome with bone marrow failure and variable malformations. Recent studies of ours and other labs established p53 network activation as a major contributor to DBA. Therefore, modulation of p53-dependent pathways emerges as a most promising approach to DBA treatment. Direct suppression of 53 may, however, lead to increased risk of malignant transformation. Search for more physiological modulators of p53 pathways is therefore warranted. p53 has been shown both to respond to metabolic alterations and to change metabolism. Specifically, nutrients can influence p53 status through mTOR and other pathways. Recently, Ortega et al reported that treatment of Caco-2 cells with exogenous nucleosides modulated expression and activity of many transcription factors including p53 (J Nutr Biochem, 22:595, 2011). We applied treatment with exogenous nucleosides to RP-deficient zebrafish and found positive effects. As a DBA model, we use zebrafish mutant for ribosomal protein RPL11; we also employ morpholino nucleotides to create deficiency of various ribosomal proteins including RPS19. Treatment of both models resulted in inhibition of p53-dependent pathways. Specifically, expression of genes associated with cell cycle arrest such as p21 and p53 targets inducing apoptosis such as puma, was decreased by nucleoside treatment. We hypothesize that nucleoside treatment affects p53 by modulating the activity of mTOR and MEK/Erk pathways. Previously, Gu et al. showed that nucleotide depletion strongly inhibited mTOR and activated MEK/Erk. We also study how nucleoside treatment influences other stress-response pathways and affects interaction of p53 and mTOR pathways. Our data indicate that nucleosides may be effective in modulation of p53-dependent signal transduction pathways and suggest their usefulness for treatment of p53-associated disorders.

Disclosures:
Sakamoto: Abbott: Research Funding; Genentech: Research Funding.
Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation
Authors: Santos Daniel Garcia , Schranzhofer Matthias , Chies José Artur Bogo , Ponka Prem 
Published: October 2019

Abstract
Abstract 344
Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Disclosures:
No relevant conflicts of interest to declare.
Haploinsufficiency of Dnmt1 Impairs Leukemia Stem Cell Function Through Derepression of Bivalent Chromatin Domains,
Authors: Trowbridge Jennifer J. , Sinha Amit U. , Armstrong Scott A. , Orkin Stuart H. 
Published: October 2019

Abstract
Abstract 3459
Leukemia stem cells (LSCs) are an attractive target in treatment of many types of blood cancers. There remains an incomplete understanding of the epigenetic mechanisms driving LSC formation and maintenance, and how this compares to the epigenetic regulation of normal hematopoietic stem cells (HSCs). One of the major epigenetic modifications, DNA methylation, is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed decreased expression of Dnmt3a and Dnmt3b in LSCs isolated from a model of MLL-AF9-induced acute myeloid leukemia (AML) compared to normal HSCs. In contrast, expression of Dnmt1 was maintained in LSCs compared to HSCs, suggesting that Dnmt1 may have a critical function in the formation and maintenance of LSCs. Supporting this hypothesis, we found that conditional knockout of Dnmt1 fully ablates the development of AML. Furthermore, haploinsufficiency of Dnmt1 (Dnmt1fl/+ Mx-Cre) was sufficient to delay progression of leukemogenesis and impair LSC self-renewal. Strikingly, haploinsufficiency of Dnmt1 did not functionally alter normal hematopoiesis or HSCs, suggesting an enhanced dependence of LSCs on DNA methylation. Mechanistically, we observed that haploinsufficiency of Dnmt1 in LSCs resulted in derepression of genes that had been silenced by MLL-AF9-mediated transformation and marked by bivalent H3K27me3/H3K4me3 chromatin domains. These results suggest that the formation and maintenance of LSCs depends not only upon activation of a leukemogenic program, but also upon silencing of a specific gene signature that is active in HSCs through crosstalk between two epigenetic mechanisms, polycomb-mediated repression and DNA methylation-mediated repression. This silenced gene signature includes known and candidate tumor suppressor genes as well as genes involved in lineage restriction. These studies present evidence that distinct epigenetic regulatory mechanisms are dominant in LSCs compared to HSCs and provide novel gene candidates for targeted reactivation in AML therapy.

Disclosures:
Armstrong: Epizyme: Consultancy.