bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythropoietin-Associated CFU-E Expansion: A Novel Mechanism Responsible for Erythroid Recovery Following Sublethal Radiation.
Authors: Peslak Scott , Wenger Jesse , Kingsley Paul , Koniski Anne , Bemis Jeffrey , McGrath Kathleen E. , Palis James 
Published: October 2019

Abstract
Abstract 1483
Poster Board I-506
Although the synthesis of two million red blood cells every second is essential for the maintenance of steady state levels of blood cells as well as our well-being, very little is known about the sensitivity of the erythroid lineage to injury or its kinetics and mechanisms of recovery. A novel multispectral imaging flow cytometry analysis utilizing the AMNIS ImageStream and functional colony assays were utilized to study the response of the erythroid lineage in C57Bl/6 mice following 4 Gray whole body irradiation. BrdU cell cycle analysis and erythropoietin ELISA assays were also utilized to investigate erythroid recovery after radiation injury. Two days post-radiation, all erythroid progenitors and precursors in the marrow were severely depleted. Over the next week, early-stage progenitors (BFU-E) only made a partial recovery. In marked contrast, late-stage progenitors (CFU-E) dramatically expanded in numbers at 5-6 days post-radiation to greater than 200% of normal. These CFU-E subsequently generated a wave of erythroid precursors that culminated in the emergence of circulating reticulocytes beginning at 8-9 days post-radiation. Rapid expansion of the CFU-E compartment was associated with an increase in cell cycling. CFU-E expansion was also temporally associated with an acute 20-fold increase in circulating levels of erythropoietin (EPO). Additionally, exogenous EPO administration at 4 days post-radiation resulted in further expansion of CFU-E 2 days later. Interestingly, erythroid progenitors and precursors were not found in the spleen after radiation, indicating that recovery of the erythroid lineage after direct marrow injury is not associated with extramedullary erythropoiesis. We conclude that erythroid progenitors and precursors, unlike circulating erythrocytes, are exquisitely sensitive to clastogenic insults such as radiation. Furthermore, we conclude that the acute recovery of erythropoiesis following sublethal radiation injury is centered on profound expansion and robust cycling of CFU-E. The temporal association of this CFU-E expansion both with the endogenous upregulation and with the exogenous administration of EPO suggests that CFU-E expansion is due primarily to erythropoietin stimulation. We hypothesize CFU-E expansion in the marrow may represent a novel adaptive mechanism of the erythroid lineage to acute injury. Overall, a more thorough understanding of radiation-induced erythroid injury and recovery will ultimately lead to new treatments to protect and mitigate the hematopoietic system from clastogenic agents such as radiation and chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
TIMP–1 Plays a Functional Role in CD34. + Hematopoietic Stem and Progenitor Cells.
Authors: Wilk C. Matthias , Czibere Akos G. , Cadeddu Ron-Patrick , Buest Sebastian , Schildberg Frank A. , Lauterbach Marcel A. , Westphal Volker , Hell Stefan W. , Haas Rainer , Bruns Ingmar 
Published: October 2019

Abstract
Abstract 1487
Poster Board I-510
TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34+ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs.
All experiments were carried out using highly enriched CD34+ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34+ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34+ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34+ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples.
In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34+ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34+ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34+ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model.

Disclosures:
No relevant conflicts of interest to declare.
The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.
Authors: Milsom Michael D. , Yabuuchi Akiko , Daley George Q. , Williams David A. 
Published: October 2019

Abstract
Abstract 1494
Poster Board I-517

Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1−/− ES lines from blastomeres resulting from the mating of Rac1+/− mice. Rac1−/− ES lines were produced in normal Mendelian ratios (4 Rac1+/+: 9 Rac1+/−: 3 Rac1−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41+/c-Kit+ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper.

Disclosures:
No relevant conflicts of interest to declare.
The California Sickle Cell Database.
Authors: Wun Ted , Brunson Ann , Brown Monica 
Published: October 2019

Abstract
Abstract 1530
Poster Board I-553

INTRODUCTION
The Cooperative Study of Sickle Cell Disease (CSSCD) was a landmark prospective observational study that helped to define the natural history of sickle cell disease. Although this study provided (and is still yielding) a wealth of robust data, there are still gaps in knowledge on long term complications, especially in adults. In addition, the CSSCD was conducted by investigators at specialized centers, and it is possible findings might be different in a population based cohort. Therefore, we wished to use available administrative datasets to study Californians with sickle cell disease (SCD) and create a California Sickle Cell Database (CSCD).

METHODS
We conducted a retrospective study with which to determine complications and health care utilization amongst patients with sickle cell disease. The California Office of Statewide Health Planning and Development (OSHPD) Hospital Discharge Dataset (HDD) collects data on all hospital discharges in California (exclusive of federal facilities) since 1991. Since 2005, all non-federal emergency department (ED) visits have also been reported to the Emergency Department Utilization (EDU) system of OSHPD. Data elements include basic demographics and diagnoses. Patients can be tracked using social security numbers; therefore, longitudinal data on individual patient hospitalizations and ED utilization can be generated. The ICD-9 codes used were 282.6X.

RESULTS
There were 11,351 individual SCD patients identified in the OSHPD HDD (1991-2007) and EDU (2005-2007) data systems. Analysis to date has yielded some basic demographic data on utilization. Females comprised 59.3% of the cohort. As anticipated most patients (81.9%) were non-Hispanic Black. However, 7.9% were reported as non-Hispanic White and 6% as Hispanic. The majority was less than 60 years of age; however, 744 were between 60-69 years and 406 were 70-79 years of age. There were 124,455 admissions of patients with SCD from 1991 to 2007 (mean ± SD =7321 ± 473 per year). 2135 ± 168 patients per year accounted for these hospitalizations with mean hospitalizations per patient of 3.4 with SD's from 3.6 to 4.6. In the three years 2005-2007 inclusive a mean of 1,871 ± 127 patients a year utilized the ED. The mean number of ED encounters per year per patient was 5 with SD's of 13 to 22.

CONCLUSIONS
Limitations include those inherent with any retrospective study dependent on administrative data, including inaccurate coding. In addition, true prevalence could be underestimated due to the acquisition bias of the requirement for hospitalization and/or ED utilization to be counted. Longitudinal data may be affected by migration to other states. Nonetheless, the large number of identified cases in the CSCD will allow determination of rates for many complications of interest that commonly require hospitalization and/or ED utilization, and provide important information on the individual and societal burden of disease.

Disclosures
No relevant conflicts of interest to declare.
Chronic Kidney Disease and Mild Elevations in Pulmonary Arterial Systolic Pressure Are Associated with Increased Mortality in Adult Patients with Sickle Cell Disease.
Authors: Lorch Daniel , Spevack Daniel , Little Jane 
Published: October 2019

Abstract
Abstract 1539
Poster Board I-562

Background
Pulmonary arterial hypertension (PAH) has recently been shown to be an important predictor of prognosis in sickle cell disease (SCD). We studied this association in a large population of adults with SCD.

Methods and Results
215 patients with sickle cell syndromes in whom echoes had been performed, 113 as outpatients and 102 as inpatients, were identified from our database. Clinical characteristics were analyzed, and survival data were extracted from a national mortality registry. At the time of examination, subjects were 39 ± 14 years old, with hematocrits (Hcts) of 23.6 ± 4.4. 78 out of 213 patients who had undergone echoes had PAH (PASP ≥ 40 mmHg) on echocardiogram. Those patients were older (41 ± 15 vs 37 ± 13 years, p=0.03), had higher creatinine values (1.3 vs 0.9 mg/dL, p=0.01) higher serum alkaline phosphatase levels (172.2 ± 23.2 vs 104.5 ± 5.5 U/L, p=.0058), and were more often taking anti-hypertensive medications (38 vs 17%, p<0.001). During a median follow-up of 7.8 years [interquartile range (IR); 3.9, 10.0] there were 30 deaths. In a multivariate Cox proportional hazards model, PAH (hazard ratio (HR) 11.3; 95% CI 2.7, 47.9), chronic kidney disease (CKD) (HR 17.0; 95% CI 3.7, 79.1, defined as an estimated glomerular filtration rate, eGFR, of <60 cc/min/1.73 m2) and hyperbilirubinemia (HR 13.1; 95% CI 2.0, 83.9) independently predicted mortality after adjustment for inpatient/outpatient status, age, sex, blood counts, measurement of chamber size and ventricular function on echocardiogram, oxygen saturation, history of transfusion and background medical therapy. The association of PAH and mortality was not modified by inpatient/outpatient status (p=0.6).

Conclusion
In a community-based academic center, we found that mild elevation in PASP on echocardiogram, whenever obtained (see figure), depressions in eGFR (see figure), and hyperbilirubinemia were highly predictive for mortality in sickle cell disease. It is unclear whether these relationships are causal or an epiphenomenon of severe underlying disease. Hemolysis (lower Hcts, higher Lactate dehydrogenase levels), implicated in other studies, was not prominent in the PAH cohort.

Disclosures
No relevant conflicts of interest to declare.
Increased Efflux of Oxidized Glutathione (GSSG) Causes Glutathione Depletion in Sickle Erythrocytes.
Authors: Nur Erfan , de Waart Rudi , Verwijs Mirjam , Otten Hans-Martin , Brandjes Dees P. , Elferink Ronald P.J. Oude , Biemond Bart J. 
Published: October 2019

Abstract
Abstract 1542
Poster Board I-565

Introduction
Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes.

Materials and methods
Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H2O2) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H2O2 stimulation.

Results
While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H2O2 stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD.

Conclusion
GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD.

Disclosures
No relevant conflicts of interest to declare.
Association of Janus Kinase 2(JAK2) A830G Polymorphism with Acute Leukemia Susceptibility.
Authors: Chen Bao-An , Zhong Yue-Jiao , Feng Ji-feng , Li Yu-feng , Ding Jia-Hua , Gao Chong , Cheng Jian , Xia Guo-Hua , Sun Ning , Chen Ning-Na , Zhang Xiao-ping , Xu Pei-pei , Pei Xiao-Ping , Qian Jun , Cheng Lu , Lu Zu-hong 
Published: October 2019

Abstract
Abstract 1573
Poster Board I-599

Background
Aberrant activation of the The Janus kinase(JAK)/signal transducer and activator of transcription (STAT) pathway may predispose to leukemia cells due to deregulation of proliferation, differentiation or apoptosis. One of the members of this family, JAK2, plays a very important role in metabolizing carcinogens and medications. In this study, we aimed to determine whether any association exists between genetic polymorphism in JAK2 A830G and individual susceptibility to acute leukemia.

Method
We carried out a case-control study using a China sample set with 243 acute leukemia cases and 282 controls matched by age, ethnicity. 68 of the acute leukemia patients were diagnosed with acute lymphoblastic leukemia (ALL) and 175 patients with acute myeloid leukemia (AML). Genomic DNA was isolated from peripheral blood and genomic DNA samples were assayed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in the A830G on JAK2 gene. The data were analyzed statistically employing chi-square and logistic regression analyses.

Result
The frequencies of G/G genotype (wild type) were 4%, 8% and 31.4% in ALL, AML and control groups, respectively. The frequencies of polymorphic G/A genotype (heterozygous variant) were found to be 69% in ALL patients, 71% in AML patients and 48.6% in controls. The A/A genotype (homozygous variant) were existed to be 69% in ALL patients, 20% in AML patients and 20% in controls. Logistic regression analyses showed a significant correlation between the JAK2 A830G polymorphism (G/G) and acute leukemia patients (OR = 1.309, 95% CI =0.839-2.043, p<0.001).

Conclusion
Our findings indicate that G/G genotype may be an important genetic determinant for acute leukemias. According to our knowledge, this is the first report of an association between acute leukemia cases and the JAK2 A830G polymorphism.

Disclosures
No relevant conflicts of interest to declare.
Molecular Characterization of a Novel 3' Mutation in RUNX1 in a New Pedigree with Familial Platelet Disorder.
Authors: Churpek Jane E , Garcia Jacqueline S , Madzo Jozef , Jackson Sarah , Onel Kenan , Godley Lucy A 
Published: October 2019

Abstract
Abstract 1626
Poster Board I-652

Introduction
Familial Platelet Disorder with Propensity to Acute Myelogenous Leukemia (FPD/AML; OMIM 601399) is a rare disorder with an autosomal dominant inheritance pattern characterized by varying degrees of thrombocytopenia, clinical bleeding due to platelet dysfunction, and an increased risk of developing myeloid malignancy. To date, twenty-six families with this disorder have been described and all carry germline RUNX1 mutations as the causative genetic abnormality. The spectrum of RUNX1 mutations includes point mutations within the RUNT domain and frameshift and termination mutations throughout the body of the gene. Here we report identification of a new family with FPD with a novel nonsense mutation resulting in premature protein termination at amino acid 388.

Patients and Methods
Our four generation pedigree includes a mother (II:4) diagnosed with dysplasia and normal karyotype acute myeloid leukemia now in remission after a matched sibling allogeneic stem cell transplant, and her daughter (III:2) with thrombocytopenia since childhood, excessive bleeding with childbirth, and 5q- syndrome diagnosed at 37 years old. Genomic DNA was obtained from all available family members, and RUNX1 cDNA (transcription variants a through c) was sequenced. In addition, RUNX1 cDNA was analyzed for second mutations in bone marrow samples from both patients at the time of diagnosis of their initial bone marrow malignancy.

Results
RUNX1 sequencing of germline DNA revealed heterozygosity for a novel nonsense mutation in exon 8 (c.1163C>A), which is predicted to result in premature protein truncation (p.Ser388X). Full sequencing of RUNX1 cDNA from II:4's AML does not show any secondary mutations. Our current efforts include full sequencing of RUNX1 cDNA from III:2's bone marrow malignancy as well as functional studies of the truncated protein.

Conclusions
We have identified a novel 3' RUNX1 mutation within exon 8, which is predicted to result in premature protein truncation at amino acid 388. To date, this is the most distal mutation identified in an FPD/AML pedigree. The identification of this mutation suggests that the last 100 amino acids, which are known to contain the RUNX1 inhibition domain, contribute an essential function. Further characterization of this RUNX1 mutation and its encoded truncated protein may yield insight into RUNX1's role in leukemogenesis in FPD and de novo AML.

Disclosures
No relevant conflicts of interest to declare.