bluuebunny/crossref_metadata_2025_split huggingface dataset:
p53-Inducible Micrornas 192 and 215 Regulate p53 Expression and IGF1 Axis in Multiple Myeloma.
Authors: Pichiorri Flavia , Suh Sung-Suk , De Luca Luciana , Taccioli Cristian , Benson Don , Hofmeister Craig , Aqeilan Rami , Croce Carlo M. 
Published: October 2019

Abstract
Abstract 1973
Poster Board I-996
Multiple myeloma (MM) is an incurable neoplastic disease of B cell origin, affecting 50,000 patients in the United States, occurring in approximately 16,000 new individuals each year. In MM, mutation or deletion of p53 is rarely detected at diagnosis, although both become more frequent in advanced disease. Therapeutic activation of p53 might therefore be particularly suitable for the treatment of MM. The discovery of microRNAs (miRNAs) has revealed a new level of regulation of protein expression. We have shown that an altered miRNA expression profile plays a role in the malignant transformation of plasma cells (PCs); we recently published the first comprehensive global microRNA expression profiles of MM, monoclonal gammopathy of undetermined significance (MGUS) and contrasted these expression patterns with that of normal plasma cells (PCs). Now we have used nutlin-3a, a recently developed small-molecule activator of p53, that efficiently disrupts the p53-MDM2 interaction, to profile a p53 miRNA activation pathway in the MM1s cell line. Results of the analysis have shown differential expression of 46 individual miRNAs after p53 activation, 14 showing up-regulation and 32 showing down-regulation. We confirmed our data by qRT-PCR in p53 wild type and mutated MM cell lines and in primary MM cells. Our results confirm published results; after p53 stabilization we found up-regulation of the miR-34a, but we also detected strong up-regulation of several other miRNAs. In fact we found that p53 induces two additional, mutually related clusters of miRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same miRNAs were detected at high levels in normal plasma cells (n.3) but were severely reduced in many untreated MM samples (n.22). Here we characterize the promoter and the p53 activation site for miR-192-194 cluster on chromosome 11(q13.1) but we have also confirmed the previous published p53 consensus site for miR-215-194 cluster on chromosome 1(q41) in multiple myeloma cells. Furthermore we found that miR-192 and its cousin miR-215 but not miR-194 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest in multiple myeloma cells acting directly on p53 regulation. On the other hand, we define that IGF-1 and IGFR are direct targets of miR-192-215. In fact the re-expression of these miRNAs in multiple myeloma cells blocks their migration through both endothelial barriers and associated bone marrow stroma. Our data suggest that downstream signaling in the p53 pathway is functional in MM cells and able to induce miRNAs that play a pivotal role in cell cycle progression, mobility and invasive properties of these cells. These studies provide the basis for the development of new miRNA-targeted therapies for MM.

Disclosures:
No relevant conflicts of interest to declare.
Compound Deficiency of Nrf2 and Selenoproteins in Hematopoietic Cells Leads to Severe Defects in Erythroid and Lymphoid Lineage Development.
Authors: Kawatani Yukie , Suzuki Takafumi , Shimizu Ritsuko , Kelly Vincent , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 1993
Poster Board I-1015
Precise regulation of reactive oxygen species (ROS) level is essential for normal cellular homeostasis. Defect of ROS scavenging system results in excess accumulation of ROS in cells, leading to the cellular aging and senescence. Nrf2 (NF-E2-related factor 2) is a basic leucine zipper-type transcription factor, which regulates expression of a set of genes related to the ROS scavenging and detoxification. While accumulating lines of evidence support the significance of Nrf2 for the cellular protection in many types of cells, Nrf2-deficient mice live out their natural life span under the homeostatic condition, and show no significant hematological abnormality except for anemia with aging. We previously found that the Nrf2 activity rose in the macrophages and liver cells derived from conditional knockout (CKO) mice of the selenocystein tRNA (Trsp) gene. The Trsp gene is essential for production of selenoproteins, including glutathione peroxidase and thioredoxin reductase. Thus, there are two prevalent ROS scavenging systems based on different molecular mechanisms; Nrf2 and selenoproteins seemingly act compensatory and cooperatively for maintenance of hematopoietic homeostasis. To explore the roles Nrf2 and selenoproteins play in hematopoietic cells, we conducted conditional knockout of the Trsp gene by crossing Trsp-floxed mice with the interferon-inducible Mx1-Cre transgenic mice in combination with Nrf2 gene knockout (KO) in germline. The Trsp-CKO mice in the background of Nrf2 wild type (Trsp single KO mice) showed thymic atrophy with massive reduction of CD4/CD8 double-positive cells in thymus. This T-lymphocytic phenotype was not changed in the Trsp-CKO mice under the circumstance of Nrf2-null condition (Trsp:Nrf2 double KO mice), suggesting that selenoproteins were prerequisite for the T-cell development. The Trsp single KO mice suffered from mild anemia with increased number of erythroid progenitors in bone marrow and spleen, and importantly this erythroid phenotype was dramatically exacerbated in the Trsp:Nrf2 double KO mice. A large number of poikilocytes and teardrop-shaped cells were observed in the blood smear of the Trsp:Nrf2 double KO mice, whereas these deformed red blood cells were vaguely found in the Trsp single KO mice. Intracellular ROS level was significantly elevated in erythroid progenitors recovered from the Trsp:Nrf2 double KO mice, although that of the Trsp single KO mice were maintained within normal level, indicating that Nrf2 compensates for the defect of ROS scavenging function caused by the loss of selenoproteins in erythroid cells. These results thus demonstrate for the first time that the ROS scavenging conducted by Nrf2 and selenoproteins is essential for the homeostatic maintenance in erythroid and lymphoid cells in a lineage-specific manner.

Disclosures:
No relevant conflicts of interest to declare.
Both Nramp1 and Dmt1 Are Necessary for Efficient Macrophage Iron Recycling.
Authors: Soe-Lin Shan , Apte Sameer S , Mikhael Marc R , Kayembe Lidia , Nie Guangjun , Ponka Prem 
Published: October 2019

Abstract
Abstract 1994
Poster Board I-1016
Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage.

Disclosures:
No relevant conflicts of interest to declare.
Deciphering Mitochondrial Iron Metabolism Using a Knockout Mouse.
Authors: Huang Michael , Becker Erika , Whitnall Megan , Rahmanto Yohan Suryo , Ponka Prem , Richardson Des R. 
Published: October 2019

Abstract
Abstract 1995
Poster Board I-1017
We utilized the muscle creatine kinase conditional frataxin knockout mouse to elucidate how frataxin-deficiency alters iron metabolism. This is of significance since frataxin-deficiency leads to the neuro- and cardio-degenerative disease, Friedreich's ataxia. Using cardiac tissues, we demonstrate that frataxin-deficiency leads to down-regulation of key molecules involved in three mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase, Nfs1); mitochondrial-iron storage (mitochondrial ferritin); and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase and ferrochelatase). This marked decrease in mitochondrial-iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excess mitochondrial-iron observed. Indeed, this effect is compounded by increased iron availability for mitochondrial uptake through: (1) transferrin receptor1 up-regulation that increases iron uptake from transferrin; (2) decreased ferroportin1 expression, limiting iron export; (3) increased expression of the heme catabolism enzyme, heme oxygenase1, and down-regulation of ferritin-H and —L, both of which likely lead to increased “free iron” for mitochondrial uptake; and (4) increased expression of the mammalian exocyst protein, Sec15l1, and the mitochondrial-iron importer, mitoferrin-2 (Mfrn2), that facilitate cellular iron uptake and mitochondrial-iron influx, respectively. This study enables construction of a model explaining the cytosolic iron-deficiency and mitochondrial-iron-loading in the absence of frataxin that is important for understanding the pathogenesis of Friedreich's ataxia.

Disclosures:
No relevant conflicts of interest to declare.
Hemochromatosis in Italy in the Last 30 Years. Role of Genetic and Acquired Factors.
Authors: Fracanzani Anna Ludovica , Piperno Alberto , Valenti Luca , Fraquelli Mirella , Coletti Sabina , Maraschi Alessandra , Consonni Dario , Coviello Enzo , Conte Dario , Fargion Silvia 
Published: October 2019

Abstract
Abstract 2003
Poster Board I-1025
Background & Aims Clinical presentation of hereditary hemochromatosis markedly changed in the recent years. The aim of the study was to analyze a large series of consecutive Italian patients with hemochromatosis diagnosed between 1976 and 2007 to define whether the genetic background and the presence of acquired risk factors influenced the severity of iron overload and the natural history of the disease across the years. Methods: A cohort of 452 Italian patients with iron overload, of whom 338 HFE-related (C282Y homozygotes or compound C82Y/H63D heterozygotes, and 114 non-HFE-related, prospectively followed for a median of 112 months. Results: Alcohol intake, smoking habits and iron removed to depletion were similar in patients with and without HFE-related iron overload. HBV (4% and 10% p=0.03) and HCV (9% and 17% p=0.02) infections were more frequent in patients with non-HFE-related iron overload. Seventy-three percent and 61% of the patients with HFE and non-HFE-related disease had no acquired risk factor. Cirrhosis was significantly more frequent in non-HFE patients, independently of the presence of acquired risk factors (p=0.02). Gender, alcohol intake, prevalence of smokers, HCV infection, glucose, lipids, iron-related parameters and prevalence of C282Y/H63D significantly differed across the years. At enrolment cirrhosis was present in 145 cases, being significantly more frequent in the first decade (80%, 47% and 13%, p=0.001). Survival did not differ across the decades in cirrhotic patients, HCC occurring similarly in HFE and non-HFE patients. Conclusion: Patients with HFE and non-HFE related iron overload have comparable iron overload and similar clinical history. Patients, unless cirrhotics at enrolment, diagnosed during the last 10 years have less severe disease and lower prevalence of acquired risk factors, independently of genetic background.

Disclosures:
No relevant conflicts of interest to declare.
Severe Thalassemia Intermedia Resulting From Coinheritance of IVSI-110 G>A Beta Gene Mutation and Duplication of Alpha-Globin Gene Cluster in Homozygosis.
Authors: Refaldi Chiara , Barcellini Wilma , Cassinerio Elena , Graziadei Giovanna , Cappellini Maria Domenica 
Published: October 2019

Abstract
Abstract 2011
Poster Board I-1033

Introduction
The clinical severity of thalassemia intermedia depends on the degree of a/non-a-chains iimbalance. Among the molecular mechanisms responsible for thalassemia intermedia is the coinheritance of excessive a-globin gene production with a defective beta-globin gene.

Materials and Methods:
we describe an Italian family where thalassemia intermedia apparently segregates as a dominant form but it tourned out to be due to the coinheritance of a beta-globin mutation and a duplication of the alpha-globin gene cluster. The father (aged 51yrs) showed a well tolerated severe chronic hemolytic anemia (Hb 7.5-8.5 g/dL) not transfusion dependent, jaundice, splenomegaly and leg ulcers:The mother (aged 46yrs) has a completely normal hematological and hemoglobin pattern. Two sons (19 and 14 yrs) showed more severe clinical manifestations than the father. They underwent splenectomy at 12 and 13 years respectively without any benefit and afterwards they become transfusion dependent.

Results:
The hemoglobin analysis revealed that the father and the sons were heterozygotes for the beta mutation IVSI-110 G>A. MLPA analysis of the alpha-globin gene cluster disclosed a full duplication of the alpha-globin locus, spanning a 175 kb from the telomere to the 3'HVR downstream of the alpha-globin gene and including the upstream regulatory element HS-40. This rearrangement increases the number of the active alpha-globin genes in cis from 2 to 4.Surprisengly it was found in heterozygosis in both parents and in homozygosis in both sons. The hematological and molecular data of the family are reported in the table.
In the father the 6 alpha-globin genes led to increased synthesis of alpha-chains; the coinheritance with a beta-thalassemia mutation causes a moderate/severe thalassemia intermedia phenotype. The presence of 8 alpha-globin genes in the sons raises further the degree of globin-chains imbalance and exacerbates the clinical phenotype. It is important to note that splenectomy worsened the clinical course.in the 2 homozygotes for the alpha duplication.

Conclusions:
Based also on previous experience we suggest that splenectomy in patients with a real excess of alphaa chain production is unconvenient since a large amount of circulating red cells with precipitated alpha chains may be responsible for increased hemolysis as well as increased risk of thrombosis This family moreover raises concerns regarding genetic counselling, suggesting that whenever one of the partner is affected by TI it is advisable a complete molecular screening of the couple in order to exclude any possible alpha gene defects interaction

Disclosures:
No relevant conflicts of interest to declare.
Generation of Autologous Pre-B Acute Lymphocytic Leukemia (ALL) Reactive T Cell Lines Using ALL Blasts as Antigen Presenting Cells.
Authors: Zhong Rui-kun , Lane Thomas A. , Ball Edward D 
Published: October 2019

Abstract 2035
Poster Board II-12
Although hematologic remissions can be achieved in the majority of patients with adult acute lymphocytic leukemia (ALL) by chemotherapy, long term survival is only 30–40%. The inability of the immune system to recognize and eliminate residual malignant leukemia cells may be an important mechanism contributing to relapse. In this study, we investigated the possibility of using pre-B-ALL cells as antigen presenting cells in an in vitro culture to induce autologous ALL reactive T cells. After 7 day culture of Pre-B-ALL peripheral blood mononuclear cells (CD19+ 93±4%) in 96 well culture plates in culture medium supplemented with a cytokine combination of IL-2/IL-3/IL-4/IL-7/GM-CSF, CD80, CD86, CD83, CD54, HLA-Dr and CD40 expression was analyzed. Significant enhancement of CD80, CD86, CD83 and CD40 on ALL cells was observed (n=8, P≦0.001-0.02). Addition of lipopolysaccharide (LPS) to the cytokine combination further increased CD80, CD86 or CD40 expression by 3 of 8 ALL samples above the baseline enhancement by cytokines (Figure), while CD40 ligand (CD40L) enhanced expression of the co-stimulatory molecules in 4 of 8 cases. Autologous T cells remaining in the culture after day 7 were then expanded with high dose IL-2 and autologous ALL reactive T cell lines were identified by IFN-g release by T cells in response to autologous ALL cells. Autologous ALL reactive T cells were generated from 5 of 8 pre-B ALL samples studied. The data from 3 experiments demonstrated that although the cytokine combination plus LPS or CD40L could effectively induce ALL cell expression of co-stimulatory molecules, the presence of CD40L and LPS in the culture induced significantly greater activation of autologous ALL-reactive T cells than did the cytokine combination plus LPS alone, as assessed by average IFN-g release of 24-48 culture wells (P≦0.004). ALL-reactive T cell lines selected by high IFN-g release response showed effective elimination of autologous ALL cells in a 2 day co-culture assay with an E:T ratio of 1:1 by flow cytometry analysis. Average residual CD19+ cells was 2.0±1.4% for highly reactive T cell lines (n=17) vs 16.5±25.9% for the less reactive control T cell lines (n=9) (P<0.02). Conclusion: Autologous ALL-reactive T cells can be induced from most ALL patients in an in vitro culture with a cytokine combination. Adoptive transfer of these anti-ALL CTL to patients is a possible therapeutic approach for further study.

Disclosures:
No relevant conflicts of interest to declare.
Gender-Based Differences in Hemostatic Responses. Implications in Effective Anticoagulant Management.
Authors: Iqbal Omer , Sadeghi Nasir , Bakhos Fadi , Hoppensteadt Debra , Fareed Jawed 
Published: October 2019

Abstract
Abstract 2103
Poster Board II-80
Abstract: Recent reports from the National Heart, Lung and Blood Institute indicate that as many as 3 million women (particularly young) in the United States suffer from a form of heart disease fundamentally different from that in men, characterized by more even plaque development inside major and smaller blood vessels, posing diagnostic and treatment challenges leading to increased morbidity and mortality. It is hypothesized that women have variable but attenuated hemostatic responses to anticoagulant drugs when compared to men. In order to validate this hypothesis the hemostatic responses in healthy males (n=10) and females (n=10) were evaluated by performing the global clotting assays, fibrinokinetic assays and thrombin generation assays in the presence of Rivaroxaban, an oral Factor Xa inhibitor likely to replace warfarin, Enoxaparin, a low molecular weight heparin and saline as a control. Blood (20 ml) was drawn from healthy volunteers, males (n=10) and females (n=10) and placed into citrated tubes with one part of 3.2% sodium citrate to 9 parts of blood. The citrated whole blood was supplemented with Rivaroxaban (FC=0.3mg/ml), Enoxaparin (FC=5mg/ml) and saline as a control. The samples were analyzed to determine the whole blood APTT and Heptest clotting assays. The remaining citrated blood was centrifuged at 3000 rpm to obtain platelet poor plasma that was aliquoted and kept frozen at -70°C until further analysis. The plasma was then thawed and supplemented with saline, rivaroxaban (FC=0.3mg/ml) and Enoxaparin (FC=5mg/ml). A statistically significant difference between males and females was noted in APTT (p=0.0442)) and Heptest (p=0.0345) assays in the saline control values. However, the anticoagulant response to supplementation of the plasma samples with rivaroxaban at a final concentration of 0.3ug/ml and Enoxaparin at 5 ug/ml showed a statistically significant difference between males and females in the Heptest (P=0.0423) while the APTT assay felt a little short of statistical significance (P=0.0511).
Fibrinokinetics was performed and absorbance recorded (405 nm) at every minute for the next 30 minutes. There are gender-based differences in fibrinokinetic responses to anticoagulant drugs with females showing faster fibrin formation than males. The attenuated hemostatic responses observed in women compared to men may interfere in achieving adequate and effective anticoagulation leading to thrombotic complications.
Time (min) 0 30 Gender Male ODs Female ODs Male - ODs Female - ODs Male - ODs Female - ODs Saline control 0.857±0.31 0.611±0.22 1.367±0.28 1.214±0.28 1.377±0.26 1.24±0.28 Rivaroxaban (0.3ug/ml) 0.853±0.32 0.602±0.21 1.353±0.27 1.221±0.18 1.363±0.27 1.23±0.18 Enoxaparin (5ug/ml) 0.713±0.35 0.507±0.24 0.794±0.33 0.621±0.23 0.802±0.33 0.629±0.23

Disclosures:
No relevant conflicts of interest to declare.
Platelet Senescence and Phosphatidylserine Exposure.
Authors: Dasgupta Swapan Kumar , Abdel-Monem Hanan , Prakasam Anthony , Thiagarajan Perumal 
Published: October 2019

Abstract
Abstract 2117
Poster Board II-94
The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of membrane bilayer occurs during platelet activation and is essential for platelet procoagulant activity. Expression of phosphatidylserine also occurs in platelets stored more than 5 days in the blood bank. We have examined the role of mitochondrial pathways in activation-induced platelet procoagulant activity and during in vitro senescence. Collagen and thrombin-induced exposure of phosphatidylserine is associated with a decrease in mitochondrial membrane potential (MMP). Cyclosporin A, a known inhibitor of cyclophilin D, inhibited phosphatidylserine exposure and the loss of MMP. Consistent with this, platelets from CypD deficient mice had decreased loss of MMP and impaired phosphatidylserine exposure when treated with a combination of thrombin and collagen. However, there is no release of cytochrome c into the cytosol. Also there is no activation of caspase-3 or Rho associated kinase I (ROCK1). In sharp contrast to this, in platelets stored for more than 5 days, phosphatidylserine exposure is found to be associated with caspase-3 and ROCK1 activation. To delineate the mechanism we used ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis via Bcl-2 family of proteins. ABT737-induced phosphatidylserine exposure was also associated with cytochrome c release, caspase-3 and ROCKI activation and it is unaffected in platelets isolated from CypD knock out mice or in platelets treated with cyclosporine A prior to activation. Caspase-3 inhibitor (z-DVED-fmk and ROCK1 inhibitor Y-27632 reduced ABT-737 induced phosphatidylserine expression.
Our results also show that activation-induced phosphatidylserine exposure is dependent on mitochondrial membrane depolarization and CypD but is independent of cytochrome c release or caspase-3 or ROCK1 activation. In contrast, the phosphatidylserine exposure in stored platelets occurs via mitochondrial cytochrome c release, caspase-3 activation and ROCKI activation and does not depend on mitochondrial membrane depolarization or CypD.

Disclosures:
No relevant conflicts of interest to declare.