bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNMT1 Cxxc Domain Can Functionally Substitute In An MLL Fusion Protein
Authors: Risner Laurie E. , Kuntimaddi Aravinda , Bushweller John H. , Zeleznik-Le Nancy J. 
Published: October 2019

Abstract
Abstract 4193
The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties.
We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia.
The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties.

Disclosures:
No relevant conflicts of interest to declare.
Candidate Gene Polymorphisms and the Risk for Pregnancy Related Venous Thrombosis
Authors: Dahm Anders E. A. , Bezemer Irene D. , Bergrem Astrid , Jacobsen Anne Flem , Jacobsen Eva Marie , Skretting Grethe , Rosendaal Frits , Sandset Per Morten 
Published: October 2019

Abstract
Abstract 4203

Background:
Venous thrombosis (VT) is the leading cause of maternal death in the western world. Genetic causes of pregnancy related VT are insufficiently understood.
Objectives: To investigate if recently discovered associations between single nucleotide polymorphisms (SNPs) and thrombotic disease influenced the risk for pregnancy related VT. To investigate candidate genes for new associations between SNPs and pregnancy related VT.

Methods:
A hospital based case control study. Cases were women with objectively verified VT during pregnancy or puerperium, controls were women giving birth without having VT. We selected 49 candidate genes involved in coagulation (35 genes), inflammation (4 genes), and hormonal metabolism (10 genes). In these genes, 463 tag-SNPs were selected from the CEU population in Hapmap and analyzed in 313 cases and 353 controls. Odds ratios for VT were calculated with the most common genotype as the reference genotype for each SNP. P-values were adjusted for multiple testing to false discovery rates.

Results:
Of the SNPs previously shown by others to be associated to thrombotic disease, rs2289252 in the FXI gene (F11), rs3917643 in the tissue factor gene (F3), rs1613662 in the glycoprotein VI gene, rs2001490 in NAT8B, rs4524 in the FV gene (F5), and rs3087505 in the prekallikrein gene were associated to pregnancy related VT. After analyzing all 463 tag-SNPs, 13 SNPs were associated to total pregnancy related VT with a false discovery rate ≤ 0.20. These SNPs belonged to F5, SELP (coding for P-selectin), F3, F8 (coding for factor VIII), and F11. When we studied antenatal and postnatal VT separately, 17 SNPs were associated with antenatal VT, while only 1 SNP was associated with postnatal VT. Thirteen SNPs in F5, SELP, or the gene for E-selectin were associated with thrombosis. After excluding carriers of factor V Leiden, 4 of these SNPs remained associated. Two SNPs in the gene for epidermal growth factor receptor (EGFR) were associated to antenatal thrombosis.

Conclusion:
Genetic variation seems more important for antenatal than for postnatal VT. We confirmed the importance of some SNPs for the risk for pregnancy related VT, in particular rs2289252 in F11. The EGFR-gene appears to be a new genetic locus associated with pregnancy related VT.

Disclosures:
No relevant conflicts of interest to declare.
Sub-Optimal Inhibition of Thrombus Formation by Aspirin (as measured by RTTP analysis) In Primary Thrombocythemia
Authors: Stephens Gillian , Tauscher Jan , Siegel Fabian , Phillips David R. , Andre Patrick , Petrides Petro E. 
Published: October 2019

Abstract
Abstract 4218
Primary thrombocythemia (PT) is a myeloproliferative disease with a high incidence of thrombotic complications. First-line therapy is the inhibition of platelet aggregation with aspirin (via COX-1 inhibition) for the prevention of thromboembolic complications. However, this does not account for the potential for COX-2 expression in newly synthesized platelets. The purpose of the present study was 1) to determine whether aspirin reduces the thrombotic potential of PT patients who did not undergo cytoreductive therapy to the levels of those achieved in healthy volunteers (HV), and 2) to determine whether the presence of the JAK2V617F mutation is responsible for the elevated thrombotic potential in PT patients.
We determined the thrombotic potential of a cohort of 16 PT patients and compared these values to those of 10 healthy volunteers all treated with 100mg aspirin QD. Thrombotic potential was determined using a custom built Real Time Thrombosis Profiler (RTTP). Whole blood anticoagulated with a Factor Xa inhibitor (to preserve physiological Ca++ concentration) with Rhodamine 6G-labeled platelets was perfused over a fibrillar collagen surface at shear rates approximating those in veins (100s-1), arteries (600 s-1) and moderately stenosed arteries (1600s-1). The deposition of fluorescently labeled platelets on the collagen surface was monitored in real time by fluorescence microscopy in the RTTP. Endpoint thrombosis (size of thrombi at t=300sec expressed as Mean Fluorescent Intensity/Area of coverage) and rate of thrombus growth (initial rate of platelet deposition) were recorded for each individual as measures of thrombotic potential and data are expressed as mean ± SEM (statistics performed using GraphPad Prism v4.03 using unpaired, 2-tailed Students t-test).
Despite aspirin therapy, PT patients were characterized by significant increases in thrombotic potential at venous and arterial shear rates (Table 1) with a greater than 2-fold increase in the rate of platelet deposition. Interestingly only the size of thrombi formed but not the rate of formation was elevated in PT patients at moderately stenosed shear, likely a reflection of the increasing contribution of shear and decreasing dependence on absolute platelet count and other individual cellular factors in whole blood. The

Disclosures:
No relevant conflicts of interest to declare.
Addressing the Issue of the Pathogenesis of Deep Vein Thrombosis In Cancer
Authors: Thomas Grace M , Brill Alexander , Schatzberg Daphne , Wagner Denisa D. 
Published: October 2019

Abstract
Abstract 4220
The risk of thrombotic complications during tumor development has been the focus of several clinical studies over the past decades. Thrombotic disease is a common cause of death in cancer patients and almost one in five of all symptomatic cases of Deep Vein Thrombosis (DVT) are thought to be cancer related. Tumor cells generate and profit from a local and systemic hypercoagulable state, which promotes tumor growth, angiogenesis, and metastasis. The resulting procoagulant state is responsible for the heightened risk for thrombotic complications and the etiology of the resulting DVT event. However, despite the fact that some molecules, such as tissue factor, have recently been described as potential modulators of clot formation in DVT, the true trigger of DVT in cancer patients remains unknown. To determine whether the presence of cancer cells can trigger formation of a thrombus in a deep vein in vivo, we combined a murine model of Trousseau's Syndrome with a model of DVT. First, we induced the development of a tumor by subcutaneous injection of syngeneic pancreatic tumor cells in C57BL/6 mice. It was previously demonstrated that these mice have significantly reduced tail bleeding time and time to occlusion in injured mesenteric arteries and veins compared to control vessels. After 3 hours of partial flow restriction of the inferior vena cava, we observed that all tumor-bearing mice developed an occlusive thrombus (n=8) while only 29% of the control mice (n=7) formed occlusive thrombi. Interestingly, perfusion of cancer-derived microparticles in mice also induced thrombus formation in this DVT model, reproducing the tumor-bearing mice phenotype. This new approach showed that microparticles shed by cancer cells have a key role in the pathogenesis of cancer-associated DVT, and they may become a reasonable pharmaceutical target to prevent thrombosis associated with cancer development.

Disclosures:
No relevant conflicts of interest to declare.
Molecular Analysis of the SEC23B Gene In Patients Affected by Congenital Dyserythropoietic Anemia Type II (CDAII)
Authors: Bianchi Paola , Schwarz Klaus , Fermo Elisa , Heinrich Katja , Vercellati Cristina , Marcello Anna Paola Maria Luisa , van Wijk Richard , Zwieten Rob van , Banov Laura , Sau Antonella , Wuillemin Walter A. , Opat Stephen , Barcellini Wilma , Heimpel Hermann , Zanella Alberto 
Published: October 2019

Abstract
Abstract 4227
CDAII, the most frequent type of congenital dyserythropoietic anemia, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblast morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Last year we and others identified SEC23B as the gene responsible for CDAII (Schwarz et al, 2009, Bianchi et al, 2009). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles from the endoplasmic reticulum to the Golgi apparatus. The gene, localized on chromosome 20p11, is split in 20 exons and codifies for a 767 aa protein. The aim of the study was to characterize the molecular defect in a large series of CDAII patients of Caucasian origin. Fifty-one CDAII patients from 49 unrelated families (17 Italians, 20 German or Swiss, 4 Dutch, 2 British, 2 Czech and 1 Greek, Turkish, Bulgarian and Irish origin) were analyzed by direct exon sequencing. We identified 36 different mutations, 25 of which were not described before. Eighteen were disruptive and 18 were missense mutations. The patients' molecular data are summarized in the Figure (novel mutations are reported in bold). All the missense mutations affected highly conserved aminoacids and were not found in 200 normal alleles examined. The c.325G>A mutation was identified in 15 homozygous patients and in two cases in combination with other mutations. The change c.40C>T was detected in 16 unrelated patients as heterozygous mutation and, for the first time, at the homozygous level in one patient only. Considering the entire series of patients (including previously published cases) characterized by our groups (86 CDAII patients from 77 unrelated families) c.325G>A and c.40C>T mutations account for 49% (76/154) of the unrelated mutated alleles and therefore should be firstly screened during molecular diagnosis of CDAII. A c.325G>A mutation usually results in a mild to moderate clinical picture at homozygous level, whereas it may cause a very severe clinical pictures when combined with other mutations (Fermo, et al 2010). Despite the sequencing of all exons and flanking intronic regions, 5 patients displayed only one mutation, suggesting the possibility that mutations could be located in regulatory regions or that a second gene could be involved in the pathogenesis of CDAII.
P. Bianchi and K. Schwarz contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.
Single ROI Versus Multislice T2* MRI Approach for the Quantification of Hepatic Iron Overload
Authors: Meloni Antonella , Luciani Antongiulio , Positano Vincenzo , Marchi Daniele De , Valeri Gianluca , Restaino Gennaro , Roccamo Gaetano , Armari Sabrina , Dell'Amico Maria Chiara , Favilli Brunella , Lippi Alma , Lombardi Massimo , Pepe Alessia 
Published: October 2019

Abstract
Abstract 4263

Introduction.
Precise and effective measurements of iron overload in the liver, where iron deposition seems to be primarily noticeable (Noetzli LJ et al, Blood 2008), are important for the early diagnosis, treatment and follow-up of patients with hemochromatosis or hemosiderosi. T2* Magnetic resonance imaging (MRI) represents the most available noninvasive technique to assess hepatic iron content and shows a good correlation with biopsy results (Wood JC et al, Blood 2005). In the clinical practice, a single hepatic slice is acquired and T2* measurement is performed in a single region of interest (ROI) of the parenchyma (Pepe A et al, Eur J Haematol 2006). This approach may mask an heterogeneous iron distribution. Thus, the aims of this study were to set up a MRI acquisition technique for the detection of the iron burden in the whole liver and to evaluate the effectiveness of the single ROI technique.

Methods.
Five transverse hepatic slices were acquired by a T2* gradient-echo sequence in 101 thalassemia major (TM) patients (48 males, mean age 29 ± 8 years, mean serum ferritin level 1413 ± 1209 ng/mL, mean pre-transfusion hemoglobin 9 ± 1 g/dl.) and 20 healthy subjects. The T2* value was calculated in a single ROI defined in the medium-hepatic slice. Moreover, the T2* value was extracted on each of the eight ROIs defined in the functionally independent segments according to Couinaud classification. The mean hepatic T2* value was obtained by averaging all segmental values.

Results.
For patients the mean T2* values over segments VII and VIII were significantly lower than the other segments (P<0.0001) (figure 1). This pattern was substantially preserved in the two groups identified considering the T2* normal cut-off (Ramazzotti A. et al J Magn Reson Imaging 2009) (group A: single ROI T2* < 15.8 ms and group B: single ROI T2* ≥ 15.8 ms). All mean segmental T2* values as well as the mean hepatic T2* value were strongly correlated with the single ROI T2* value. After the application of a correction map based on T2* fluctuations in the healthy subjects, no significant differences were found in the segmental T2* values for the whole patient population (P=0.251), for both the previously defined groups (Group A: P = 0.073 and Group B: P=0.476) as well as for the Group A patients (N = 23) with chronic hepatitis (P=0.111).

Conclusions.
Hepatic T2* variations are low and due to artifacts and measurement variability. The single ROI approach can be adopted in the clinical arena, taking care to avoid the susceptibility artifacts, occurring mainly in segments VII and VIII.

Disclosures:
No relevant conflicts of interest to declare.
Maturation of Monocyte-Derived Dendritic Cells Induced by Dual Targeting of Endocytosis Receptor and CD40
Authors: Staudinger Matthias , Catenhusen Inga Rebekka , Peipp Matthias , Kellner Christian , Repp Roland , Gramatzki Martin 
Published: October 2019

Abstract
Abstract 4293
Dendritic cells (DC) are professional antigen presenting cells, which can induce and regulate adaptive immune responses. Hence, DC are attractive candidates for anti-tumor vaccination strategies. The aim of this study was to create recombinant bispecific Fab to target and activate DC, by using derivates of monovalent agonistic CD40 scFv and endocytosis receptors FcγRI (CD64), FcαR (CD89), DEC205 (CD205), as well as DC-SIGN (CD209), respectively. The recombinant bispecific molecules were expressed in the eukaryotic cell line Hek 293T, enriched to homogeneity by affinity chromatography and analyzed for specific binding to the targeted receptors. Functional analysis of these molecules indicated, that concurrent engagement of CD40 and endocytosis receptors expressed by immature IL-4/GM-CSF cultured monocytes-derived DC induced maturation of these DC. This was suggested by changes of DC phenotype, especially enhanced surface expression of co-stimulatory molecules like CD80 and CD86, and gain of CD83 expression as measured by flow cytometry. To particularly analyze the T cell activation properties of these DC, MACS enriched T cells of autologous donors were co-cultivated with antigen-loaded and subsequently maturated DC for seven days. After restimulation of T cells, flow cytometric detection of intracellular IFN-γ levels and surface expression of CD69 used as markers for T cell activation implies, that these DC exhibit elevated properties to activate antigen specific T cells. In contrast, a bispecific [CD64scFv × CD40scFv] Fab used as control failed to induce maturation of DC lacking CD64 expression, which may be due to abrogation of bivalent binding of this molecule and therefore CD40 cross-linking. Moreover, dual targeting with these novel immunoconstructs when linked to peptides of a tumor-associated antigen may allow direct antigen delivery to DC in combination with DC activation, and thus anti-tumor vaccination in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Endothelial Cell Tube Formation by ADAMTS13 Occurs Via Interaction with VEGF
Authors: Rodansky Eva S. , Smith Justin K. , Fryer Hugh J.L. , Greenfield Robert S. , Rodgers George M. 
Published: October 2019

Abstract
Abstract 4307
The von Willebrand factor-cleaving protease, also known as ADAMTS13, is synthesized, in part, by endothelial cells (EC). We previously reported that proliferating EC secreted ∼3-fold more ADAMTS13 (antigen and activity) than confluent EC, and that this synthesis was transcriptionally regulated (SJ Kling et al, Pathophysiol Haemost Thromb 2008; 36: 233.) Thrombospondin domains, a defining feature of the ADAMTS protease family, in other ADAMTS family members mediate inhibition of angiogenesis. In particular, ADAMTS1 inhibits angiogenesis by sequestering vascular endothelial growth factor (VEGF) (A Luque et al, J Biol Chem, 2003; 278: 23656). Since ADAMTS13 also has thrombospondin domains, we hypothesized that ADAMTS13 might also mediate anti-angiogenic activity. Human umbilical vein EC angiogenesis was quantified by Angioquant analysis of fluorescence microscopy tube images in a Matrigel assay. Treatment of EC with inhibitors of angiogenesis (anti-VEGF, vasostatin) did not alter EC production of ADAMTS13. However, exogenous recombinant ADAMTS13 inhibited angiogenesis in a dose-dependent manner (Figure 1), in media containing VEGF. In VEGF-free media, ADAMTS13 had no effect on EC tube formation. Preincubation of ADAMTS13 with an antibody to the ADAMTS13 thrombospondin domains 5–7 partially reversed the inhibition of tube formation, implicating these domains in the anti-angiogenic interaction (Figure 2). We also found that VEGF co-immunoprecipitates with ADAMTS13, providing strong evidence that these two proteins interact. When EC lysates were crosslinked with DTSSP prior to immunoprecipitation, anti-ADAMTS13 immunoprecipitated as much VEGF as did anti-ADAMTS1, while an antibody to ADAM17, a similar protein that lacks thrombospondin domains, failed to co-immunoprecipitate VEGF (Figure 3). These data indicate that as with other ADAMTS family members, ADAMTS13 inhibits tubule formation - a parameter of angiogenesis – through its interaction with VEGF, an effect likely mediated by its thrombospondin domains. Inhibition of angiogenesis adds to the expanding roles of ADAMTS13 in down-regulation of thrombosis and inflammation.
Fig. 1. Fig. 1.
Fig. 2. Fig. 2.
Fig. 3. An antibody to ADAMTS13 immunoprecipitates VEGF in similar amounts as anti-ADAMTS1 when proteins from HUVEC lysates are crosslinked prior to co-immunoprecipitation. Nonspecific IgG fails to immunoprecipitate VEGF, as does anti-ADAM17. (A1 = ADAMTS1; A13 = ADAMTS13) Fig. 3. An antibody to ADAMTS13 immunoprecipitates VEGF in similar amounts as anti-ADAMTS1 when proteins from HUVEC lysates are crosslinked prior to co-immunoprecipitation. Nonspecific IgG fails to immunoprecipitate VEGF, as does anti-ADAM17. (A1 = ADAMTS1; A13 = ADAMTS13)

Disclosures:
Fryer: American Diagnostica Inc: Employment. Greenfield:American Diagnostica Inc: Employment.