bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of Multimerin 1 with Collagens: Role in Platelet Adhesion
Authors: Tasneem Subia , Pawlikowska Monika , Bihan Dominique , Farndale Richard W. , Hayward Catherine P. M. 
Published: October 2019

Abstract
Abstract 2207
Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein stored in platelets and endothelium that binds to activated platelets, endothelial cells and the extracellular matrix after agonist stimulation. MMRN1 supports platelet adhesion by von Willebrand factor (VWF) dependent and independent mechanisms, and also increases platelet adhesion to Horm collagen. Mice deficient in Mmrn1/Snca (α-synuclein) showed defective platelet adhesion to collagen in vitro and in vivo which was corrected by MMRN1. The ability of MMRN1 to support platelet adhesion in vivo and enhance platelet adhesion to collagen ex vivo, led us to explore the molecular basis of MMRN1 interactions with collagen. Solid phase binding assays were used to test MMRN1 binding to human fibril forming collagen (types I, II, III and V) and to types IV and VI collagen. Binding assays were also used to map the MMRN1 binding sites on collagen using collagen peptide toolkits III and II (which has significant similarity to type I collagen). Static adhesion assays were used to test selected collagen peptides for platelet adhesion. Platelet adhesion assays at high shear rates 1500s−1, were used to test the adhesion of washed platelets from normal and VWF-deficient subjects to type I collagen pre-treated or in fluid phase with: BSA (negative control), MMRN1, VWF or their combination. Human collagens types I, II, III and VI (p-values <0.001) but not types IV or V (p-values = 0.84 and 0.09, respectively) supported MMRN1 binding. Peptide toolkits binding studies indicated that MMRN1 bound to a single site on collagen III (peptide III-38) and to two sites on collagen II, with peptide II-9 showing much stronger binding than peptide II-44. Like the VWF binding peptide III-23 (which did not overlap the MMRN1 binding site), peptide III-38 supported platelet adhesion in combination with GFOGER, the peptide with high affinity for platelet α2β1. The possibility that MMRN1 binds to collagen at sites distinct from VWF was supported by the observations that pre-treatment of collagen I matrices, with the combination of MMRN1 and VWF, increased platelet adhesion more than MMRN1 or VWF alone (p-values< 0.001). Moreover, adhesion deficit of VWF-deficient platelets on collagen type I matrix pre-treated with a combination of MMRN1 and VWF was corrected by adding fluid phase VWF but not MMRN1 (p-values < 0.0001). Taken together, our data indicates that MMRN1 binds to different forms of human collagen that support platelet adhesion. As MMRN1 binds to sites on collagen distinct from VWF or integrin α2β1, it may be important for maximizing platelet adhesion at sites of vascular injury.

Disclosures:
No relevant conflicts of interest to declare.
Biochip Array Analysis of Various Mediators of Inflammation in Disseminated Intravascular Coagulation
Authors: Rahman Saud , Hoppensteadt Debra , Cunanan Josephine , Davis Rachael , Sadeghi Nasir , Kaul Inder , Fareed Jawed 
Published: October 2019

Abstract
Abstract 2302
Disseminated intravascular coagulation is a polypathologic syndrome which involves blood, endothelial and target organ dysfunction resulting in the generation of various mediators of vascular dysfunction, hemostatic aberrations and hemodynamic disturbances. In addition, various disorders of target organs such as kidney, liver, heart and brain are observed. Uncontrolled protease generation results in the formation of various mediators of inflammation such as neuron specific enolase (NSE), neutrophil gelatinase associated lipocalin (NGal) and soluble tumor necrosis factor receptor 1 (TNF R1). Endothelial damage results in the generation of thrombomodulin (TM) and endogenous coagulation/fibrinolysis results in D- Dimer formation. In order to study the circulating levels of these mediators, a Biochip array method (Randox, Oceanside, CA) was utilized with samples obtained from clinically diagnosed DIC (n=100) and normal individuals (n=10). The results are summarized in the following table. Circulating levels of these mediators were markedly increased in DIC patients in comparison to normals. The most striking increase was noted in NGal (4–6 fold) and TNF R1 (10 fold). Other mediators were also increased in the DIC group, however the data was broadly scattered. These results indicate that beside the aberration in the hemostatic system NGal and TNF R1 along with other inflammatory mediators may play a major role in the pathophysiology of DIC.MarkerNormal ControlsDIC BaselineCRP (ug/ml)3.47 + 0.913.11 + 0.2*NSE (ng/ml)6.11 + 8.610.78 + 1.6*NGAL(ng/ml)306.45 + 25.51376.56 + 48.5*TNFR1 (ng/ml)0.35 + 0.013.89 + 0.3*DD (ng/ml)232.8 + 74.41318.94 + 80.5*TM (ng/ml)3.4 + 0.33.16 + 0.2
P=<0.05

Disclosures:
No relevant conflicts of interest to declare.
Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells
Authors: Hirata Shinji , Jono-Ohnishi Ryoko , Nishimura Satoshi , Takayama Naoya , Nakamura Sou , Nakauchi Hiromitsu , Murata Takahiko , Eto Koji 
Published: October 2019

Abstract
Abstract 2323
Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs.

Disclosures:
No relevant conflicts of interest to declare.
Use of Genomics for Transfusion Therapy Management in Patients with Sickle Cell Disease
Authors: Westhoff Connie M , Chou Stella T , Smith-Whitley Kim , Friedman David 
Published: October 2019

Abstract
Abstract 2324
A genomic approach to blood group typing is now possible and high-throughput automated platforms have been developed to test for numerous blood group antigens in a single assay. These methods are reproducible and highly correlated with RBC serologic phenotype. We routinely perform a complete RBC phenotype for clinically significant minor red cell antigens on pre-transfusion samples from patients with sickle cell disease, and we antigen match patients for C, E, and K for transfusion.
In this study we compared the historic serologic typing with that predicted from DNA testing for clinically significant antigens in 114 samples from chronically transfused patients with SCD to determine concordance and to evaluate the clinical utility of genotyping for the management of transfusion therapy.
Serologic typing was performed by standard methods with licensed commercial reagents. DNA was isolated from WBCs, and minor antigen genotyping was performed with HEA (human erythrocyte antigen) BeadChip (BioArray, Inc). RH genotyping was by a combination of methods including PCR-RFLP, AS-PCR, exon-specific amplification and sequencing, and, for some, Rh-cDNA amplification and sequencing.
Comparison of serologic typing with DNA-based testing for thirteen blood group antigens, CcEe, Fya/b, K, Jka/b, MN and Ss, in 114 samples found 8 discrepancies in 1,482 antigens analyzed, for 99.5 % concordance. Discrepancies were in several systems (C, Fy, Ss, and M), and at least one has been confirmed to be a serologic recording error. All are under investigation.
DNA-based testing for RH found 54 of 114 patients inherited variant RHD alleles; many also had conventional RHD in trans. Sixteen patients had made anti-D, despite typing as D+. Ten of 35 patients (∼30%) whose RBCs typed as C+ had a hybrid allele encoding variant C antigen. Five had made anti-C, which prompted us to change our protocol so patients with variant C by DNA testing are transfused on a C- protocol. DNA testing found a large amount of diversity in ce-alleles in this population. Seventy-two of 114 patients carried at least one of nine different variant ce-alleles. Ten patients had made anti-e, despite typing as e+, and were homozygous for variant ce-alleles. In total, 49/114 patients with SCD were homozygous for variant RH alleles and were not truly Rh matched for D, C and e antigens by serology.
Similar to the way in which HLA typing by DNA has revolutionized bone marrow transplantation by providing a superior alternative to serological testing, we find that minor blood group antigen typing by DNA improves efficiency, reduces cost, and expands antigen matching, especially in the Rh system. Continuing studies are needed to identify more precisely which variant alleles are associated with clinically significant antibody production to improve antigen matching for patients with sickle cell disease.

Disclosures:
No relevant conflicts of interest to declare.
ADAMTS13 Helps Distinguish Thrombotic Thrombocytopenic Purpura From Other Microangiopathies and Guide Necessity of Plasma Exchange Therapy
Authors: Shah Neil , Matevosyan Karen , Burner James , Sarode Ravindra 
Published: October 2019

Abstract
Abstract 2328

Introduction:
Congenital thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13, a metalloproteinase that cleaves ultra-large VWF multimers to generate normal sized multimers present in circulation. However, acquired deficiency of ADAMTS13 due to auto-antibodies is not universally accepted as diagnostic of idiopathic TTP. Distinguishing idiopathic TTP from other thrombotic microangiopatheis (TMA) is important in guiding treatment as plasma exchange (PE) is only beneficial in TTP and select cases of other microangiopathies such as atypical HUS. Since January 2006 we routinely obtain ADAMTS13 activity performed by FRET assay from The Blood Center of Wisconsin in all patients with TMA. We report the diagnostic utility of ADAMTS13 to distinguish TTP from other forms of TMAs.

Methods:
A retrospective analysis was performed on consecutive patients with TMA who had ADAMTS13 assayed from January 2006 to October 2010. Demographics, presenting clinical and laboratory features are given in the Table. Responses to therapeutic plasma exchange (TPE), diagnoses at discharge, and other underlying clinical conditions were also recorded. Relevant statistical analysis was performed using unpaired t-test to compare means and Fisher's exact method for contingency tables.

Results:
We divided our cases based on severe ADAMTS13 deficiency (<10%) as TTP and non-severe deficiency (>20%) as TMA. TMA causes included quinine induced HUS (1), gemcitabine related HUS (1), malignant hypertension/pre-eclampsia/HEELP (3), sepsis (2), MCTD (5), malignancy (3), ITP (1), HIV/opportunistic infection (5), drugs (1), and multiple (5).

Conclusions:
In our experience severe ADAMTS13 deficiency appears to distinguish TTP from TMA. TMA had better mortality despite either not initiating or discontinuing PE based on ADAMTS13 levels. However, if we had continued PE in TMA then these patients would have been considered as NON-ADAMTS13 deficient TTP who responded well to PE.

Disclosures:
No relevant conflicts of interest to declare.
NAC Improves the Differentiation of IPS Cells Into Hematopoietic Progenitors
Authors: Berniakovich Ina , Laricchia-Robbio Leopoldo , Izpisua Juan Carlos 
Published: October 2019

Abstract
Abstract 2349
In vitro differentiation of human induced pluripotent stem (iPS) cells is a new way to obtain donor material for blood transplantation. However, this is a long process in the course of which cells are exposed to alien environmental factors, capable to change cellular properties and decrease cellular viability. Indeed, cells in vitro are exposed to mechanical stress, artificial growth surface, unnatural gas composition etc. This fact could partially explain why cells obtained during directed iPS cells differentiation have different qualities in comparison with the analogous population from in vivo.
It is supposed that oxidative stress is the major underlying mechanism of negative influence of various in vitro environmental factors on cells. N-acetylcysteine (NAC) is a powerful antioxidant. Due to free radical scavenging ability, the principal function of NAC is rendered to the inhibition of cellular damage and cell death in response to reactive oxygen species. Cytoprotective function of NAC is well demonstrated both in vitro and in vivo.
We have established a protocol to obtain hematopoietic stem cell-like cells from human iPS cells with a pick of their production at three weeks of differentiation. We analyzed how intracellular accumulation of reactive oxygen species and NO, as well as cell viability, apoptosis, stress resistance, mitochondrial membrane potential were changed during this process by comparing status of cultures at 1 and at 3 weeks of differentiation. We found that during differentiation cells progressively accumulated intracellular reactive oxygen species and increased production of NO. The level of apoptosis in culture was significantly higher at 3 weeks of differentiation than at 1 week. Cell viability, on the contrary, decreased from 1 week till 3 weeks of differentiation. Stress resistance quantified through the amount of cells resistant to the H2O2 treatment was also decreased in 3 weeks old cultures. We also demonstrated that during in vitro culture the mitochondrial membrane potential of the cells under basal conditions and upon stimulation with carbonyl cyanide m-chlorophenylhydrazone was decreased at 3 weeks of differentiation in comparison with that at 1 week. All these phenomena were reversed by NAC supplementation. Remarkably, NAC administration also improved the hematopoietic differentiation of human iPS cells in terms of production of CD34, CD45, CD43 positive cells, that showed normal functionality in colony forming unit assay. CD34+ cells obtained from NAC treated cultures also increased their migration towards SDF1, therefore showing an increased ability of our CD34+ cells to home into bone marrow.
Our results suggest that supplementation with NAC is beneficial for the improvement of hematopoietic differentiation of human iPS cells.

Disclosures:
No relevant conflicts of interest to declare.
Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells
Authors: de Bruin Alexander M. , Hooibrink Berend , Nolte Martijn A. 
Published: October 2019

Abstract
Abstract 2351
Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells.
To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo.
Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced.

Disclosures:
No relevant conflicts of interest to declare.
Activation of a Mir-181-Targeting HOXA-PBX3 Homeobox Gene Signature Is Associated with Adverse Prognosis of Cytogenetically Abnormal Acute Myeloid Leukemia
Authors: Li Zejuan , Huang Hao , Li Yuanyuan , Jiang Xi , Chen Ping , Arnovitz Stephen , Radmacher Michael D. , Maharry Kati , Elkahloun Abdel , Yang Xinan , He Chunjiang , Price Colles O. , Dohner Konstanze , Zhang Zhiyu , Shen Chen , Neilly Mary Beth , Lussier Yves A , Zhang Yanming , Larson Richard A. , Le Beau Michelle M. , Caligiuri Michael A , Bullinger Lars , Valk Peter , Delwel Ruud , Lowenberg Bob , Liu Paul P. , Marcucci Guido , Bloomfield Clara D. , Rowley Janet D. , Chen Jianjun 
Published: October 2019

Abstract
Abstract 236
Altered expression of microRNAs (miRNAs, a class of small regulatory RNAs) is associated with various types of cancers, including acute myeloid leukemia (AML). We showed previously that increased expression of miR-181a with or without miR-181b was associated with a favorable prognosis for patients with cytogenetically normal AML (CN-AML). However, the prognostic value of miR-181 expression in cytogenetically abnormal AML (CA-AML) remains elusive, even though CA-AML represents the majority of human AML. To investigate the association of expression signatures of miR-181 family members and of their potential target genes with outcome in patients with primary CA-AML, we employed two independent sets of 86 CA-AML patients to investigate the association of expression signatures of miR-181 family members with outcome. We also used four independent sets of 454 CA-AML patients to identify and validate a prognostic signature of miR-181 targets. In addition, we investigated the biological functions of miR-181a/b and target(s) in leukemia cell lines and in a leukemia mouse model. As with CN-AML, we found that both miR-181a and miR-181b expression signatures are significantly (P<.05) associated with favorable overall survival (OS) of patients, but only the miR-181b signature is an independent predictor in multivariable model tests. An overexpressing gene signature of potential targets of miR-181a/b, HOXA7, HOXA9, HOXA11, and PBX 3, derived from a meta-analysis of three independent sets of 183 patients, was an independent predictor of adverse OS, which was confirmed in a validation set of 271 patients. Ectopic expression of miR-181b significantly (P<.05) promoted apoptosis and decreased viability of MONOMAC-6/t(9;11), THP-1/t(9;11), and KOCL-48/t(4;11), and delayed leukemogenesis in our mouse model; such effects could be reversed by forced expression of PBX3. Our data suggest that the silencing of miR-181b and thereby the activation of the four homeobox genes likely contributes to the poor prognosis of adverse CA-AML patients. Thus, restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways in poor prognosis AML patients may provide new strategies to improve outcome substantially.

Disclosures:
No relevant conflicts of interest to declare.
Cooperating Oncogenes and Their Targets in LMO2-Induced T-Cell Leukemia
Authors: Smith Stephen B. , Tripathi Rati , Cleveland Susan , Mathias Elizabeth , Yi Andrew , Mullighan Charles G , Downing James R. , Copeland Neal G. , Jenkins Nancy A , Dave Utpal P. 
Published: October 2019

Abstract
Abstract 2458
LIM domain Only 2 (LMO2) is an important driver in acute T-cell lymphoblastic leukemia. Enforced expression of LMO2 in mouse models induces T-ALL with long latency suggesting that it requires cooperating oncogenic mutations for leukemia to fully manifest. To identify cooperating oncogenes, we analyzed spontaneous T-ALLs induced by retroviral insertional mutation, and T-ALLs that developed in B6.CD2-Lmo2 transgenic mice. First, we noted that the CD2-Lmo2 transgenic T-ALLs exclusively upregulated the class II basic helix-loop-helix transcription factor, Lyl1, whereas the retrovirally-induced T-ALLs showed upregulation of Tal1. We attributed this difference to the stage at which the Lmo2 activating mutations occurred. Nevertheless, we found that LYL1 protein upregulation is functionally important, as it is part of a macromolecular complex containing LMO2 that binds tandem E boxes as described for TAL1. LMO2 and LYL1 are known to be co-expressed in human T-ALL, but we found that they are part of a larger gene expression signature comprised of NMYC, HHEX, MEF2C, and BCL2 genes. The latter genes are remarkable because they are oncogenic when overexpressed; and, Mef2c and Nmyc are concordant insertions in independent tumors driven by Lmo2 or Hhex in retroviral insertional mutagenesis models. In fact, insertion site analysis suggests that Lmo2 and Hhex may actually operate in the same pathway and that Hhex may be a target of Lmo2. We tested this genetic result by chromatin immunoprecipitation and reporter assays and discovered that Lmo2 and its binding partners, Ldb1 and Lyl1, occupy an enhancer in the first intron of Hhex in T-ALL cells. Furthermore, LMO2 or LYL1 knockdown downregulated expression of HHEX in T-ALL. HHEX activation has significant functional consequences in T-ALL. We found that its upregulation is associated with T-ALL treatment resistance, clinical latency, and differential activation of Notch1 targets. These data offer important insight into the pathogenesis of Lmo2-induced T-ALL.

Disclosures:
No relevant conflicts of interest to declare.