bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.
Authors: Staber Janice M. , Burnight Erin R , Korsakov Pavel , Kaminski Joseph , Craig Nancy L , McCray Paul B 
Published: October 2019

Abstract
Abstract 1477
Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein.

Disclosures:
Staber: Bayer Healthcare: Research Funding.
Improvement In Survival of Patients with Multiple Myeloma: An Update of Population Based Survival Trends
Authors: Pulte Dianne , Gondos Adam , Brenner Hermann 
Published: October 2019

Abstract
Abstract 1510

Background:
Treatment options for multiple myeloma have changed significantly over the past decade with new treatment options demonstrating improved survival in multiple clinical trials. We have previously published data demonstrating that survival has improved on the population level (Brenner et al Blood 2008) and projections of survival expectations for patients recently diagnosed with multiple myeloma which predicted continued improvement in survival for patients diagnosed in the years 2006-10 (Brenner et al Haematologica 2009). Here, we provide updated estimates of survival in myeloma and compare the results to projected survival.

Methods:
Period analysis was used to calculate 5- and 10-year relative survival for patients diagnosed with multiple myeloma from 1997-01 and 2002-06, using data from the Surveillance, Epidemiology, and End Results (SEER) database. Survival estimates for single years were calculated as well to determine whether changes in survival were ongoing throughout the 2002-06 period.

Results:
Five and 10-year relative survival in 2002-06 was significantly higher than in earlier time periods and was similar to survival predicted for patients diagnosed 2006–2010 (see table). When individual years between 2002 and 2006 were evaluated, survival was higher in 2005 and 2006 as compared to 2002-4. Survival was higher for all age groups as compared to survival previously calculated by period analysis for 1997–2001 (see table).

Conclusions:
Survival for patients diagnosed with multiple myeloma is improving for all age groups at a rate faster than predicted by previous models of survival. This implies a greater than linear increase in survival over the early part of the 21st century.

Disclosures:
No relevant conflicts of interest to declare.
A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.
Authors: Shibayama Hirohiko , Saito Yuri , Tanimura Akira , Tanaka Hirokazu , Matsumura Itaru , Kanakura Yuzuru 
Published: October 2019

Abstract
Abstract 1552

[Background]
Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated.

[Methods & Results]
In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells.

[Conclusion]
P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions.

Disclosures:
No relevant conflicts of interest to declare.
FLT3ITD Expression Confers the Leukemogenic Properties of Growth Factor Independence and Enhanced Self-Renewal to Yolk Sac Progenitors.
Authors: Xiao Nan , Jani Kaushal , Yang Yuxia , Raffel Glen D 
Published: October 2019

Abstract
Abstract 1576
Congenital leukemias are clinically and biologically distinct from those manifesting in later childhood and in adults. Derivation of the leukemic cell of origin from the primitive hematopoietic elements of the yolk sac compartment rather than definitive hematopoietic precursors may explain the unique features of this class of leukemias. We hypothesized that in the cellular context of the yolk sac precursor, common leukemogenic mutations such as the FLT3 receptor internal tandem duplication (FLT3ITD), induce characteristics allowing expansion or propagation of this developmentally self-limited cell population. Yolk sacs were dissected from pregnant C57BL/6 mice at E9.5, disaggregated and then infected with an MSCV-FLT3ITD-GFP or an MSCV-GFP control virus. To assess growth factor independence, infected yolk sac cells were plated in methylcellulose lacking growth factors. Colony numbers were scored after 7–10 days and demonstrated FLT3ITD was capable of establishing growth factor independence in yolk sac precursors, similar to results obtained using transduced fetal liver and adult bone marrow. To determine whether FLT3ITD expression in yolk sac progenitors enhanced self-renewal, the ability to serially replate was tested. Yolk sac cells were infected as above and plated in methylcellulose containing Il-3, Il-6, Scf and Epo. Colonies were scored and the cells were replated every 7–10 days. In contrast to infected fetal liver or adult bone marrow, FLT3ITD-expressing yolk sac cells were capable of replating beyond the 4th round. Analysis of derived FLT3ITD-expressing yolk sac colonies demonstrated a CD41+CD34+c-Kit+ population with an undifferentiated morphology similar to primary yolk sac progenitors. In summary, aberrant expression of the FLT3ITD mutation in yolk sac cells results in the acquisition of growth factor independence and enhanced self-renewal, characteristics essential for leukemogenesis. Although FLT3ITD expression in fetal liver and bone marrow allows growth factor independence, the distinctive ability to promote enhanced self-renewal as well in yolk sac cells suggests the yolk sac is a uniquely vulnerable target for leukemic initiation during fetal development.

Disclosures:
No relevant conflicts of interest to declare.
Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.
Authors: Nakamura Lanelle V. , Mukherjee Malini , Goodell Margaret A. , Pati Debananda 
Published: October 2019

Abstract
Abstract 1593

Introduction:
Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure.

Disclosures:
No relevant conflicts of interest to declare.
Differentiation of Mature Eosinophils From Human Embryonic and Induced Pluripotent Stem Cells.
Authors: Yang Wenyu , Ma Feng , Matsumoto Kenji , Nishihama Natsumi , Sagara Hiroshi , Ebihara Yasuhiro , Gu Yanzheng , Saito Hirohisa , Zhu Xiaofan , Nakauchi Hiromitsu , Tsuji Kohichiro 
Published: October 2019

Abstract
Abstract 1594
Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes. As the major effectors, eosinophils function in a variety of biological responses, allergic diseases and helminth infections. It is generally accepted human eosinophils develop through a pathway initially sharing common feature with basophils. However, there lacks a clear chart for early development of human eosinophils, such as during embryonic or fetal stages. We recently established an efficient method for producing eosinophils from human embryonic and induced pluripotent stem cells (hESC/iPSCs). By a two-step induction, we first generated multipotential hematopoietic progenitors by co-culturing hESC/iPSCs with mouse AGM-derived stromal cells for 2 weeks. Then, total co-culture cells were transferred into suspension culture favoring eosinophil development with addition of IL-3 and other factors (SCF, IL-6, TPO, Flt-3 ligand) . The maturation of hESC/iPSC -derived eosinophils was shown in a time-dependent manner, first co-expressing eosinophil-and basophil-specific markers [eosinophil peroxidase (EPO), and 2D7, respectively], then the portion of eosinophil markers gradually increased while that of basophil markers decreased (EPO+ cells from 56.4% at day 7 to 94.4% at day 21, while 2D7+ cells from 62.8% to 25.7%, respectively), typically mimicking the development of eosinophils from human adult hematopoietic progenitors. By flowcytometric analysis, an eosinophil-specific surface marker, Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependent manner (from 10.8% at day 7 to 91.3% at day 21), paralleling to those with EPO. The expression of eosinophil-specific granule cationic proteins (EPO, MBP, ECP, EDN) and IL-5 receptor mRNA was also detected by RT-PCR. Furthermore, transmission electron microscopy (TEM) observation confirmed the eosinophil property. Eosinophils derived from hiPSCs hold similar characteristics as those from hESCs. The function of hES/hiPSC-derived eosinophils is being under investigation. Our study provides an experimental model for exploring early genesis of eosinophils, especially in uncovering the mechanisms controlling the development of the initial innate immune system of human being in normal and diseased individuals.

Disclosures:
No relevant conflicts of interest to declare.
Low Molecular Weight Heparin Reduces sVCAM-1 and Lung Congestion In a Murine Model of Sickle Cell Disease
Authors: Guo YiHe H. , Field Joshua , Foster Thomas D. , Scott J. Paul , Wandersee Nancy , Hillery Cheryl A. 
Published: October 2019

Abstract
Abstract 1635
There is evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in individuals with sickle cell disease. Both thrombin and tissue factor also have potent proinflammatory effects that likely further contribute to the vascular dysfunction and organ injury observed in sickle cell disease. In this study, we examine the effect of the LMW heparin, enoxaparin, on markers of vascular injury and inflammation in the Berkeley mouse model of severe sickle cell disease (HbSS mice). HbSS mice or control HbAA mice (that exclusively express normal human HbA) were treated with continuous subcutaneous infusion of enoxaparin (10 mg/kg/day) or saline for 14 days using a surgically implanted Alzet pump (n=12-14 mice per group). Blood was collected at baseline and following 14 days enoxaparin therapy. Anesthetized animals were perfused with PBS at constant physiologic pressure (80 mmg Hg) and lung and liver collected for pathologic analyses. Enoxaparin-treated mice had steady state LMW heparin levels of 0.45 ± 0.17 anti-Xa U/mL compared to <0.1 anti-Xa U/mL in saline treated animals. Enoxaparin reduced d-dimer levels by approximately 35% in both HbSS and HbAA mice compared to saline-treated mice (p=0.03 and p=0.02, respectively), showing a functional reduction in coagulation and fibrinolytic activity. The hemoglobin level did not change and no obvious hemorrhage was noted in enoxaparin-treated mice suggesting no major bleeding complications. sVCAM-1 is a measure of endothelial injury and is increased at baseline in HbSS mice compared to HbAA mice (p<0.001). sVCAM-1 levels were reduced in HbSS mice after 14 days of enoxaparin therapy (1424 ± 83 ng/mL baseline, 1314 ± 85 ng/mL post enoxaparin, p=0.02), but not in saline treated HbSS mice (1462 ± 69 ng/mL baseline, 1392 ± 85 ng/mL post saline, p=0.3). Furthermore, the sVCAM-1 level negatively correlated with the steady state heparin level (r=-0.48, p=0.02) suggesting a modest dose-dependent effect of heparin on sVCAM-1 level. There was no effect of enoxaparin or saline therapy on sVCAM-1 levels in control HbAA mice. Preliminary histological analysis revealed a reduced number of congested pulmonary arteries in enoxaparin-treated HbSS mice compared to saline treated HbSS mice (p=0.01). These data suggest that enoxaparin therapy may improve endothelial injury as measured by sVCAM-1 and pulmonary vascular congestion in a mouse model of severe sickle cell disease. The data also suggest that heparin-based anticoagulant therapy may be a novel therapeutic approach to improve vascular health in individuals who suffer from sickle cell disease.

Disclosures:
Field: Novartis: Honoraria.
Overexpression of IL-3R alpha on CD34+CD38- Stem Cells Defines Leukemia-Initiating Cells In FA AML.
Authors: Du Wei , Sipple Jared , Pang Qishen 
Published: October 2019

Abstract
Abstract 1667
Patients with Fanconi anemia (FA) have high risk of developing acute myeloid leukemia (AML). In this study, we attempted to identify cell surface markers for leukemia-initiating cells (LICs) in FA AML patients. We found that interleukin-3 receptor alpha (IL-3R alpha) is a promising candidate as an LIC-specific antigen for FA AML. While IL-3R alpha expression is undetectable on normal CD34+CD38- hematopoietic stem cells (HSCs), it is overexpressed on CD34+CD38- cells from FA patients with AML. We have examined whether the LIC activity of IL-3R alpha-positive FA AML cells in a “humanized” FA xenotransplant model, in which we separated AML cells into IL-3R alpha-positive and IL-3R alpha-negative CD34 fractions and transplanted them into irradiated recipient mice. In all three FA AML samples, only IL-3R alpha-positive cells showed significant levels of engraftment and developed leukemia in the recipient mice. The FA CD34+IL-3R alpha+ blasts isolated from leukemic mice exhibited hypersensitivity to IL-3 deprivation and JAK2-STAT5 overactivation following IL-3 treatment. Finally, pretreatment of FA CD34+IL-3R alpha+ blasts with the IL-3Ra neutrolizing antibody inhibits IL-3-mediated proliferation and STAT5 activation. These results demonstrate that IL-3Ra is a cell surface marker present on FA AML-LIC and may serve a valuable therapeutic target.

Disclosures:
No relevant conflicts of interest to declare.
Influence of Residual Normal Metaphases In Patients with Monosomal Karyotype.
Authors: Xie Bin , Othus Megan , Medeiros Bruno C. , Fang Min , Appelbaum Frederick R. , Estey Elihu H. 
Published: October 2019

Abstract
Abstract 1671
Breems et al.(J Clin Oncol 2008;26:4791-97) observed that a monosomal karyotype(MK: ≥ 3 cells with ≥2 autosomal monosomies or with 1 autosomal monosomy plus a structural abnormality) conveyed a worse prognosis in untreated AML than a complex karyotype (CC ≥ 4 distinct clonal abnormalities),although there was considerable overlap between MK and CC. The SWOG (Medeiros et al. Blood e pub June, 2010) has confirmed these findings and extended them to patients age 60 or above. Previous data (Estey et al. Haematologica 2000;85:246-49) suggested that the presence of residual normal cells improved prognosis in patients with monosomy 7, prompting us to see if the same was true in patients with MK. Our analysis focused on survival and used the same 176 MK patients identified by Mederios et al. from 1,344 patients enrolled in one of 10 successive SWOG clinical trials. The MK patients had a median age of 61 years and 94% had a CC (≥ 3 distinct clonal abnormalities); 68% received induction with standard dose ara-C, 14% with other ara-C doses, and 18% without ara-C. A univariate proportional hazards model indicated that survival improved as the % of normal cells increased and accordingly as the % of abnormal cells (MK and non MK) decreased. Further investigation indicated that this reflected the number of normal, rather than the number of abnormal, cells. Thus having one or more normal cells was associated with a hazard ratio (HR) of 0.95 (p = 0.02), while the presence of 5 or more normal cells decreased the HR to 0.76. In contrast, there was no association between the number of abnormal MK cells and survival (p=0.15). Accounting for age and treatment (as noted above) did not alter these conclusions (HR for having 1 or more normal cells 0.95, p = 0.03).Despite the p-values these findings have limited medical significance for MK patients; since even the aforementioned decrease in HR to 0.76 would result in a median survival of only 5.3 months vs. the median of 4 months for all 176 MK patients. Nonetheless the results indicate that it might be worthwhile to examine the prognostic effect of residual normal metaphases in patients with better prognosis karyotypes.

Disclosures:
No relevant conflicts of interest to declare.