bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved Anemia, Organomegaly and Sustained Fetal Hemoglobin Induction within Aging TR2/TR4 Overexpressing UAB Sickle Cell Mice
Authors: Campbell Andrew , Bradley Kori , Amachong Ama , Addo Jennifer , Urbonya Rebekah , Bonsu Kwaku Osei , Harro David , Giacherio David , Shi Lihong , Cui Shuaiying , Tanabe Osamu 
Published: October 2019

Abstract
Abstract 3248

Background:
Fetal Hemoglobin (Hgb F)has been shown to improve morbidity and mortality with SCD patients. We recently published that forced expression of TR2/TR4 resulted in 2.5 fold induction of Fetal Hemoglobin and improved phenotypes within adult 2–3 month humanized sickle cell mice (PNAS Campbell and Cui et al 2011). We wanted to determine the if the Hgb F expression remained durable by HPLC within > 6 m/o mice within SCD:TgTR2/TR4 UAB Mice when compared to 2–3 m/o SCD:TgTR2/TR4 UAB Mice. Further, we wanted to determine if the increased Hgb F expression and improved phenotype observed previously persisted within the 6–8m/o SCD:TgTR2/TR4 mice when compared to the 6–8 m/o SCD UAB Mice.

Methods:
Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate SCD-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We compared the hemoglobin F levels(HPLC), CBC, and % body weight (liver, spleen, kidney) of 6–8 m/o SCD:TgTR2/TR4(n=5) mice to 2–3 m/o SCD:TgTR2/TR4(n=5) mice. We also analyzed 6–8m/o SCD mice to the TR2/TR4 SCD Mice. Tail PCR genotyping of all SCD Mice were confirmed by HPLC Hemoglobin electrophoresis.

Results:
Fetal Hemoglobin Expression remained elevated in the 6–8 m/o SCD:TR2/TR4 Mice at 16.7% (+/− 3.94) compared to 18.5% (+/−2.91) in 2–3 month old SCD:TR2/TR4 mice. %BW of spleen (3.7% vs 3.4%) liver (7.29% v 7.8%), kidney (0.98% vs 1.1%), remained similarly lower in the 6–8 m/o compared to the 2–3 m/o mice of the SCD:TR2/TR4 Mice. Table 1 shows increased Hgb F and overall improved anemia and %BW of organs within the older SCD:TR2/TR4 SCD Mice.

Conclusions:
TR2/TR4 overexpression within >6 m/o humanized SS mouse model resulted in continued >3 fold induction of fetal hemoglobin based on HPLC compared control 6–8 m/o SCD Mice. Further, increased TR2/TR4 overexpression improved anemia, organomegaly, within sickle cell disease mice. Expression of Fetal Hemoglobin by TR2/TR4 remains sustained within older sickle cell mice with improved organomegaly with reversion phenotypically to the wild type phenotype.

Disclosures:
No relevant conflicts of interest to declare.
Initial Genomic Analysis of a Pure Erythroid Leukemia Developing in Association with Hydroyurea Treatment for Sickle Cell Anemia
Authors: Wang Zhengyuan , Darbari Deepika S. , Melenhorst J. Joseph , McIver Zachariah A. , Maric Irina , Diaw Lena , Song Young , Johansson Peter , He Jianbin , Weiyin Zhou , Jacobs Kevin B. , Wei Jun , Barrett A. John , Khan Javed , Taylor James G. 
Published: October 2019

Abstract
Abstract 3254
The antimetabolite hydroxyurea (HU) is the only widely available therapy for reducing complications of sickle cell anemia. It is not widely prescribed due to concerns about adverse effects, including cancer. Insufficient data currently exist regarding its leukemogenic potential. HU associated leukemia in sickle cell anemia is exceptionally rare, with only 6 literature reports. Whether such cases are coincidental or related to HU remains unanswered. We identified a 33 year old male with pure erythroid leukemia (formerly M6 AML) and a unique combination of sickle cell anemia, HU treatment, and short treatment latency. Cytogenetics showed a complex karyotype with 5q rearrangement, 7q deletion, losses of chromosomes 15–22 and Y, suggesting a therapy related etiology. To date, no sickle cell anemia AML cases have undergone in-depth genomic analysis to determine if HU has an etiologic role in susceptibility to therapy related leukemogenesis. High density SNP genotyping was performed with 2.5 million markers to understand progressive chromosomal disease changes. Comparison of germline DNA prior to HU and tumor DNA identified 10 mosaic aneuploidy events present at AML diagnosis including losses at 5q, 7q and 17p and gains at 19p. Whole exome sequencing was also performed on the patient's germline, tumor and relapsed tumor DNA. We identified 33 putative somatic missense mutations including a validated somatic TP53 mutation, however somatic mutations were not present in commonly mutated AML genes like NPM1, RUNX1 or IDH1. Genome-wide cytogenetic band enrichment analysis of exome sequence data using the Gene Set Analysis Toolkit suggests the presence of a significant enrichment of mutations along chromosome 19p, and the same analysis of peripheral blood gene expression from an unrelated cohort of 10 sickle cell anemia subjects with brisk hematopoietic activity also suggested association of chromosome 19p with stress erythropoiesis. Together, the mosaic aneuploidy and exome sequence analysis converged on a region of 19p potentially important to the pathogenesis of pure erythroid leukemia and possibly stress erythropoiesis. This region extends at least 1.9 Mb and includes 58 genes. We are validating mutations in this region and exploring the role of 19p during stress erythropoiesis to further determine the significance of this finding. Specifically, several 19p mutations have been introduced into the BaF3 cell line to investigate their role in myeloid cell growth. Investigation of this rare leukemia case offers a unique opportunity to elucidate the pathogenesis of erythroleukemia and possibly to determine if HU therapy is associated with leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Erythropoietin-Associated Increase in the Proportion of Kupffer Cells
Authors: Ohana Yasmin Haim , Deshet Naamit , Souroujon Miriam , Gassmann Max , Mittelman Moshe , Neumann Drorit 
Published: October 2019

Abstract
Abstract 3266
Erythropoietin (EPO), the hormone produced mainly by the kidney, promotes the survival, proliferation and differentiation of erythroid precursor cells. Hence, recombinant human EPO (rHuEPO) is one of the key therapies for the treatment of anemia in patients with end-stage renal disease and in cancer patients, suffering from chemotherapy-induced anemia. The past decade has revealed extra-hematopoietic sites of EPO production along with abundance of EPO receptors (EPO-Rs) in various tissues and cell lines, suggesting that this hormone may have pleiotropic activities. Indeed, previous studies in our laboratory have shown that murine bone marrow-derived macrophages express EPO-R and EPO affects their cellular phenotype.
Kupffer cells are liver macrophages that play a key role in the body's defense system against invading pathogens. These cells affect normal and pathological conditions of the liver either directly or through their influence on other cells, such as stellate cells and are important components in iron metabolism. In this study, we found that transgenic mice, overexpressing human EPO (tg6 mice), have a larger population of F4/80 positive liver macrophages compared to wild type (wt) mice. In addition, C57bl/6 wt mice, injected 3 times a week with 180 U/ml of rHuEPO, displayed a 2 fold increase in the percentage of F4/80 expressing cells compared to their untreated littermates. This increase seems to stem from EPO's effect on a subpopulation of cells expressing both F4/80 and CD11b. As for pathological conditions, we demonstrate the same effect of EPO in an experimental murine model of 5T33 multiple myeloma (MM). 5T33 MM mice that were treated with rHuEPO (10 consecutive days of 30 U/ml followed by 3 weekly injections of 30 U/ml for 2 weeks), presented a 3 fold increase in the percentage of cells double positive for F4/80 and CD11b. In addition, these mice displayed a 20% increase in the mean fluorescence intensity of CD11b, suggesting that EPO causes not only an increase in the proportion of Kupffer cells but also induces the activation of CD11b positive cells, as reflected by the elevated expression of this marker per cell.
Our findings thus contribute to the understanding of EPO's effects on the immune system in general, and on Kupffer cells in particular. These findings may have clinical significance in those cases where activation of liver macrophages is desired, and EPO treatment for anemia is recommended.

Disclosures:
No relevant conflicts of interest to declare.
Gas6-Induced Tissue Factor Expression in Endothelial Cells Is Mediated Through Caveolin-1-Enriched Domains
Authors: Laurance Sandrine , Lemarie Catherine A , Blostein Mark D 
Published: October 2019

Abstract
Abstract 3308
Gas6 is the ligand for the TAM family of receptors, which are composed of three members namely Tyro3, Axl and Mer. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Our laboratory has identified important intracellular signaling pathways important in gas6-Axl mediated protection of endothelial cells from apoptosis. However, as both gas6 and Axl null mice are protected from lethal thromboembolism, we sought to explore novel gas6-Axl intracellular signaling pathways regulating thrombosis. Caveolae have been shown to play a crucial role in the activation of signaling cascades following ligand binding to receptor tyrosine kinases. Caveolae are formed from lipid rafts by polymerization of caveolins. Caveolin-1 is the most abundant protein found in caveolae. Caveolin-1-enriched microdomains are well known to play a role as a docking platform for receptor tyrosine kinases and intracellular adaptor signaling proteins. Axl association with these highly specialized domains of the plasma membrane has not been previously elucidated. In the present study, we investigated the role of caveolin-1-enriched microdomains in gas6/Axl signaling in endothelial cells. First, we demonstrated that gas6-induced Akt and Erk1/2 phosphorylation required the presence of a functional Axl receptor as shown by Axl siRNA knockdown experiments in human umbilical vein endothelial cells. Then, caveolin-1 fractions, enriched by a detergent-free cell lysis followed by sucrose gradient ultra centrifugation, were studied by western blot analysis. After 5 and 10 min of gas6 treatment, Axl colocalized with caveolin-1 suggesting Axl recruitment into caveolin-1-enriched cell fractions. We found that c-Src, a signaling molecule known to behave as a transient docking platform in lipid rafts, also moved in caveolin-1-enriched cell fractions after gas6 stimulation. Caveolin-1 siRNA abolished gas6-induced Akt, Erk1/2 and c-Src phosphorylation suggesting that caveolin-1 enriched fractions are required for gas6-Axl signaling. Interestingly, we have shown that gas6-induced Akt phosphorylation required c-Src activation using c-Src siRNA and the pharmacological inhibitor (PP2). However, gas6-induced Erk1/2 phosphorylation was independent of c-Src. Finally, we found that gas6 increased tissue factor expression through the Axl-c-Src-Akt signaling cascade. Taken together, our results demonstrate that caveolin-1-enriched domains are required for gas6-Axl signaling and lead to the upregulation of tissue factor expression by gas6 in endothelial cells. These results highlight new insights of gas6-Axl signaling and function in endothelial cells.

Disclosures:
No relevant conflicts of interest to declare.
Human and Murine Immune Thrombocytopenia (ITP) Is Associated with a Peripheral Deficiency of CD4- T Regulatory Cells (Tc-regs)
Authors: Semple John W. , Ma Daoxin , Aslam Rukhsana , Hou Ming , Ni Heyu , Hu Yu 
Published: October 2019

Abstract
Abstract 3333
CD8+ T suppressor cells were originally described by Gershon on Kondo more than 40 years ago, however, because of difficulties in their characterization, they fell out of favor for decades. T suppression was resurrected by the discovery of CD4+ CD25+ FoxP3+ T regulatory cells as being the critical cell in the maintenance of self tolerance. Recently, a non-conventional CD4-CD25+FoxP3+ T cell subset termed Tc regulatory cells (Tc-regs) has been described that are also important in inducing tolerance against self. A peripheral CD4+ Treg deficiency has been suggested to be the potential cause of ITP, however, the role of this newly described CD4- T cell population has not been determined. To address the role of these cells, we studied lymphocyte responses in 36 patients with ITP (18 newly-diagnosed and 18 in remission) and a murine model of ITP. Peripheral blood mononuclear cells from patients or spleens and thymi from mice were isolated and FoxP3 expressing Tc-regs were evaluated by flow cytometry. Results show that compared with healthy control subjects, the percentages of peripheral Tc-regs in newly diagnosed patients with ITP were significantly decreased. The Tc-reg deficiency was rescued in those patients in remission due to therapy. Similar results were observed in an active model of murine ITP. Additionally, the murine model of ITP revealed that the CD4- deficient population expressed CD8. These results suggest that, like CD4+ T regs, there is a significant reduction in peripheral Tc-regs in both human and murine ITP and disease remission correlates with rescuing the deficiency.

Disclosures:
No relevant conflicts of interest to declare.
Factor V Inhibits PAR2-Mediated Lethal Inflammation
Authors: Kerschen Edward J , Liang Hai-Po Helena , Fernandez Jose A. , Toso Raffaella , Ruf Wolfram , Camire Rodney M. , Griffin John H. , Weiler Hartmut 
Published: October 2019

Abstract
Abstract 3360
The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioactivity of purified, plasma-derived or recombinant fV was not expressed by the fV Leiden variant. Thus, in heterozygous fV Leiden individuals, aPC levels are increased by fV Leiden, while wild type fV enhances aPC's beneficial actions on innate immune cells. This explains why heterozygous carriers are protected from sepsis mortality by aPC, whereas homozygous Leiden carriers do not respond to endogenous or therapeutically administered aPC. FV Leiden hence emerges as a unique example of a balancing and evolutionary selectable polymorphism, whose balanced nature is the consequence of beneficial, cooperative interactions of the variant fV Leiden allele that augments generation of aPC and the normal fV allele that is necessary for the protective effects of aPC.

Disclosures:
Weiler: BloodCenter of Wisconsin: Patents & Royalties.
FXII Promotes Coagulation in a FXI and FIX Independent Manner
Authors: Puy Cristina , Wong Zoë C , Tucker Erik I , Gruber Andras , Gailani David , Smith Stephanie A. , Choi Sharon H. , Morrissey James H. , McCarty Owen J.T. 
Published: October 2019

Abstract
Abstract 3362
Activation of coagulation factors (F) XII and XI support thrombogenesis through multiple pathways. FXII-deficient mice are more resistant to FeCl3-induced arterial occlusion than either FIX or FXI deficient mice, suggesting that the resistance of FXII-deficient mice to experimental thrombosis is not completely explained by the FXII-FXI-FIX pathway, suggesting the existence of a pathological FXII by-pass, in vivo.
The APTT of FXII deficient plasma is longer than the APTT of FXI, FIX, or FX deficient plasmas. We found that addition of 150 nM activated FXII (FXIIa) decreased the recalcification time of FXI or FIX-deficient plasma by up to 85%. In a purified system FXIIa could activate prothrombin but not FX. Addition of rivaroxaban, a FXa inhibitor, to FXI or FIX-deficient plasma blocked the observed procoagulant effect of FXIIa, suggesting that FXIIa promotes the activation of FX independent of FXI or FIX, but the ability of FXIIa alone to induce coagulation is insufficient in plasma, in vitro.
Addition of long polyphosphate (polyP), typically found in bacteria, but not short polyP, which is secreted by activated platelets, decreased the recalcification time of FXI or FIX-deficient plasma. The presence of either corn trypsin inhibitor (CTI), that inhibits FXIIa, or rivaroxaban blocked the procoagulant effect of long polyP, suggesting that the activation of FXII by long polyP promotes coagulation in an FXI- and FIX-independent manner.
Addition of CTI or an antibody that inhibits FIX activation by FXIa, but not addition of an antibody that inhibits activation of FXI by FXIIa, increased the time of occlusive thrombus formation in recalcified human blood that was driven through collagen and tissue factor (TF)-coated capillary tubes, consistent with the thrombogenic roles of FXIIa and feedback activation of FXI. Only CTI inhibited the prothrombotic effect of long polyP, also suggesting that FXIIa could be thrombogenic independent of FXI and FIX.
In summary, we propose that pathological FXII activation, e.g., by foreign surfaces or long polyP, is thrombogenic both in FXI/FIX-dependent and -independent manners. Provided that FXII has no significant physiological function in humans, our data support the hypothesis that inhibition of FXII activity or activation may have safe antithrombotic effects.

Disclosures:
Morrissey: No organization, but the speaker is co-inventor on pending patent applications on the medical uses of polyphosphate: Patents & Royalties.
Circulating Nucleosomes and Neutrophil Activation As Risk Factors for Deep Vein Thrombosis
Authors: van Montfoort Maurits L , Stephan Femke , Lauw Mandy N , Hutten Barbara A , van Mierlo Gerard J , Solati Shabnam , Middeldorp Saskia , Meijers Joost CM , Zeerleder Sacha S 
Published: October 2019

Abstract
Abstract 3387

Objective:
The formation of neutrophil extracellular traps (NETs) and the exposure of nucleosomes on these NETs contribute to coagulation activation and the propagation of deep vein thrombosis (DVT) in animal models. However, no data is available on the role of NETs or nucleosomes in patients with thrombosis.

Methods and results:
We conducted a case-control study, in which levels of circulating nucleosomes and neutrophil elastase–α1-antitrypsin (EA) complexes were assessed in plasma from 150 patients with objectified symptomatic DVT (cases) and compared with 195 patients with a clinical suspicion of DVT, but in whom DVT was excluded (controls). We explored the association between both nucleosomes and EA complexes, and the presence of DVT by calculating the odds ratio (OR) with corresponding 95% confidence intervals (CI). Elevated levels of both circulating nucleosomes and EA complexes were associated with a 3-fold risk of DVT, and the associations remained similar after adjustment for potential confounders (malignancy, smoking, recent immobilization, recent hospitalization). The risk increased with higher nucleosome and EA complex levels, suggesting a dose-dependent relationship between circulating nucleosomes, activated neutrophils and DVT.

Conclusions:
Circulating nucleosomes and activated neutrophils contribute to the development of DVT in humans and might have implications for treatment and prevention.

Disclosures:
No relevant conflicts of interest to declare.
Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic STEM and Progenitor CELL Mutagenesis in Response to Irradiation
Authors: de Laval Bérengère , Pawlikowska Patrycja , Roch Benoit , Petit-Cocault Laurence , Bilhou-Nabera Chrystele , Souyri Michele , Gaudry Murielle , Pouzoulet Frédéric , Porteu Francoise 
Published: October 2019

Abstract
Abstract 3447
Radiation-induced double-strand breaks (DSBs) represent a serious threat to the preservation of genetic information when it reaches hematopoietic stem cells (HSCs). Residual loss of HSC functions and increased risk of developing hematopoietic malignancies are two concerning complications of anti-cancer radiotherapy. Management of acute myelosuppression following radio- or chemotherapy has been significantly improved in recent years by the use of growth factors. However, how cytokine/environmental signals integrate the DNA damage responses in HSCs and regulate the long-term residual HSC defects following radio-or chemotherapy is unknown. Notably, the contribution of cytokines regulating HSC functions to HSC intrinsic DNA damage repair processes remains to be delineated. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal, survival and expansion posttransplantation. In this study, we uncover an unknown and unique function for TPO/Mpl in the regulation the DNA damage response. We show that DSB repair, measured by both γH2Ax foci resolution and neutral comet assays, following γ-irradiation (IR) or topoisomerase II inhibitor treatments, is defective in Mpl−/− and Mpl+/− HS and progenitor cells (HSPCs). Similar defects were found in wild-type cells treated in the absence of TPO. This indicates that the impaired DNA repair of Mpl−/− and Mpl+/− cells results from a specific loss of TPO-mediated DNA damage response signaling at the time of IR rather than from intrinsic constitutive differences. TPO stimulates DNA repair by increasing IR-induced DNA-PK phosphorylation at Ser2056 and Thr2609 and non-homologous end joining (NHEJ) efficiency in both HSPCs and the human UT7-Mpl cell line. This is to our knowledge the first demonstration that a cytokine involved in the homeostatic maintenance of HSCs may also regulate their response to external DNA damaging insults by controlling the DSB repair machinery. Short TPO treatment in vitro or single TPO injection to TPO/Mpl proficient mice prior to sublethal total body IR reduced IR-induced HSC genomic instability and loss of long-term reconstitution ability. This may open new avenues for administration of TPO agonists before radiotherapy to minimize radiation-induced HSC injury and mutagenesis. In addition, since Mpl is haploinsufficient in the regulation of DNA damage repair, these data suggest that Mpl might also act as a tumor suppressor in response to radiotherapy.

Disclosures:
No relevant conflicts of interest to declare.