bluuebunny/crossref_metadata_2025 huggingface dataset:
Trafficking Kinesin Binding Protein Is Essential for Human Erythropoiesis
Authors: Noh Seung-Jae , Lee Y.Terry , Byrnes Colleen , Rabel Antoinette , Miller Jeffery L.
Published: October 2019

Abstract 683

Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that <1% of the CD36+ progenitors cells differentiated into glycophorin A erythroblasts compared with >90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis.

Disclosures:

No relevant conflicts of interest to declare.
ENU Mutagenesis in the Mouse for Identification of Genes Regulating Erythropoiesis: a Mouse Mutant with An Activating Mutation of the KCl Cotransporter, KCC1 Causing Dehydrated Red Cells
Authors: Curtis David J. , Brown Fiona , Wiley James , Ho Shwen , Hilton Douglas J. , Kile Benjamin T. , Jane Stephen M
Published: October 2019

Abstract 684

Genome-wide phenotype-driven studies have emerged as a powerful tool for the identification of novel genes in a biological process of interest, new functions for existing genes and the establishment of mouse models of human disease. Recent advances in exomic and whole genome sequencing has simplified mutation detection, making genetic screens in the mouse more facile. We have employed a large-scale mutagenesis screen in mice using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to identify genes regulating erythropoiesis. In this dominant screen, G1 progeny were screened at 7 weeks of age by automated hematological analysis for isolated abnormalities of red cell indices (HCT, RCC and MCV) defined as greater than three standard deviations from the wild type population. Here, we describe the RBC10 mouse line, in which homozygotes had mild anemia with microcytosis, target cells, spherocytes, reticulocytosis and increased resistance to osmotic stress. A combination of SSLP and SNP mapping isolated the genetic mutation to a 3.65 Mb region on chromosome 8. Whole genome Next Generation Sequencing of the mapped interval identified an A to T nucleotide substitution within the coding region of the potassium chloride (KCl) co-transporter, Slc12a4 (KCC1), resulting in a non-conservative amino acid substitution from methionine to lysine at position 935 within the C-terminal cytoplasmic domain. KCC1 and KCC3 function are the major erythroid KCl co-transporters that export KCl in response to hypotonic swelling. We hypothesized that the M935K was an activating mutation of KCC1, based upon the microcytosis and resistance to osmotic stress, which contrasts with the macrocytic phenotype of red cells lacking both KCC1 and KCC3. 86Rb efflux assay confirmed increased KCC1 activity of RBC mutant red cells. We propose that the M935K mutation increases activity of KCC1 by preventing phosphorylation of the nearby threonine reside, a critical regulatory target of the serine-threonine kinase, WNK1.

Disclosures:

No relevant conflicts of interest to declare.
Reciprocal Interaction Between Osteoclasts and T Cells: Osteoclasts Inhibit T Cell Proliferation by IDO Produced in Response to T Cell-Derived IFN-γ and CD40 Ligand
Authors: Li Haiyan , Lu Yong , Hong Sungyoul , Qian Jianfei , Liu Zhiqiang , Zheng Yuhuan , Lan Yongsheng , He Jin , Yang Jing , Yi Qing
Published: October 2019

Abstract 694

Osteoclasts (OCs), the responsible cells for bone resorption, are derived from monocytic precursor cells, with the stimulation of macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor κb ligand (RANKL). The formation and activity of OCs can be either promoted by activated T cell derived RANKL, IL-17, or suppressed by T cell derived IFN-γ, IL-10, and IL-4. On the other hand, OCs express MHC, and costimulatory molecules, secrete IL-10, TGF-β, TNF-α and IL-6 and can act as antigen presenting cells to activate T cells, which indicates that OCs can be considered as immune cells. However, the immune function of OCs is largely unknown, and whether activated T cells can regulate the immune function of OCs is also unclear. In this study, we investigated the effect of OCs on T cell responses, and the cross regulation between activated T cells and immune regulatory OCs. Results showed that autologous OCs could inhibit the proliferation of CD4+ T cells activated by allogeneic antigen, tetanus toxin, staphylococcal enterotoxin B, and anti-CD3/CD28 antibodies. The inhibitory rate range varied from 63% to 88%. To identify the mechanism of OC-mediated T cell suppression, we blocked or inhibited TGF-β, IL-10, (prostaglandin E2) PGE-2, and indoleamine 2,3-dioxygenase (IDO) with neutralizing antibodies or specific inhibitors during the coculture. Results showed that only 1-methyl-DL-tryptophan (1-MT, an IDO inhibitor) could rescue the T cell proliferation, which suggested that OCs mediated the T cell suppression through IDO. To confirm this result, we knocked down IDO expression in OCs with siRNA and found that T cell proliferation was restored completely. As normal OCs didn't express IDO, next we investigated which molecules induced IDO expression in OCs, when cocultured with activated T cells. Results showed that blocking IFN-γ and CD40 ligand (CD40L) could inhibit IDO expression in OCs and rescue the T cell proliferation, and recombinant IFN-γ and soluble CD40L could induce IDO expression in OCs, synergistically. In conclusion, our study identified that OCs can function as immune regulatory cells to suppress T cell proliferation through IDO, which is induced by activated T cell derived IFN-γ and CD40L. This study provides new insight into the reciprocal interaction between OCs and T cells and may be helpful to develop novel therapeutic strategies for diseases involved in both bone and immune systems, such as bone-invasive tumors and autoimmune arthritis.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Structure and Biological Activity of NPP-4, An Endothelial Cell Surface Pyrophosphatase/ Phosphodiesterase That Stimulates Platelet Aggregation and Secretion Via Liberation of ADP Upon Hydrolysis of Diadenosine Triphosphate
Authors: Ornstein Deborah L. , Albright Ronald , Chang William C. , Robert Donna , Cao Wenxiang , De La Cruz Enrique , Braddock Demetrios
Published: October 2019

Abstract 701

Diadenosine triphosphate (Ap3A) is a component of platelet dense granules, and is released into the extracellular space during the second wave of platelet aggregation following platelet activation by ADP and other agonists. Ap3A has long been thought to stimulate platelet aggregation after release into the extracellular space by providing a prolonged source of ADP via hydrolysis by a slow extracellular enzyme present in human plasma. Here, we identify NPP-4, a member of the nucleotide pyrophosphatase/phosphodiesterase enzyme family present on endothelial cell surfaces, as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion at nanomolar concentrations. We performed lumiaggregometry with citrated platelet-rich plasma in the presence of Ap3A with or without increasing nanomolar concentrations of NPP-4. In these experiments we determined that Ap3A alone in concentrations up to 80 uM initiated a primary wave of platelet aggregation that was followed by rapid disaggregation. In the presence of nanomolar concentrations of NPP-4, however, the primary and secondary waves of platelet aggregation and dense granule release are strongly induced in an enzyme concentration-dependent fashion. In contrast, mutant NPP-4, catalytically inactivated by an active site threonine to alanine mutation, had no effect on platelet aggregation and dense granule release under identical conditions. In order to clarify the structural basis of the enzymatic mechanism, we determined the high-resolution structure of NPP4 in the presence and absence of the enzymatic product, AMP. In aggregate, our studies define the biological, enzymatic, and molecular basis for NPP-4 and Ap3A activity in platelet aggregation and secretion in vitro and suggest that NPP-4 may play a role in hemostasis in vivo by augmenting platelet aggregation and release of granule contents at the site of vascular injury. With these studies we have thus established the molecular foundation for the rational development of a new class of therapeutics capable of modulating vascular thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
ENDOTHELIAL GAS6 Contributes to Thrombogenesis and Promotes TISSUE FACTOR Upregulation DURING Vessel Injury
Authors: Robins Richard , Lemarie Catherine A , Wu Jianqiu , Blostein Mark
Published: October 2019

Abstract 702

Gas6 is a vitamin K-dependent, secreted protein that amplifies platelet aggregation and secretion in response to platelet agonists. Gas6−/− mice are protected from experimentally induced lethal venous and arterial thromboembolism. This protection has been attributed to defective aggregation in platelets from gas6−/− mice. However, this platelet phenotype was only observed when platelets were challenged by only one agonist, ADP, and only at a concentrations of 5.0μM. This subtle platelet abnormality resulting in a rather dramatic clinical phenotype raise the possibility that gas6 from a source other than platelets contributes to thrombus formation. We hypothesize that gas6 derived from the endothelium plays a role in venous thrombus formation. Gas6−/− mice are protected against venous thrombosis induced by 0.37 M FeCl3 in the inferior vena cava (IVC). Bone marrow transplantation experiments generating mice with selective ablations of gas6 from either the hematopoietic or non-hematopoietic compartments demonstrate an approximately equal contribution by gas6 from both compartments to thrombus formation. Platelet depletion in wild type or gas6−/− mice followed by reconstitution with platelets from either WT or gas6−/− mice confirm that gas6 from compartments other than the platelet contribute to thrombosis development. Furthermore, gas6−/− mice are hyporesponsive to FeCl3 mediated tissue factor induction in venous endothelium, as observed by immunofluorescence staining and later validated by a functional assay. In addition, in vitro, gas6−/− endothelial cells are hyporesponsive to thrombin mediated tissue factor mRNA induction. Taken together, these results suggest that non-hematopoietic gas6, possibly from the endothelium, contributes to thrombus formation in vivo and can be explained by the ability of gas6 to promote endothelial tissue factor induction. These findings support the notion that endothelial gas6 may play a pathophysiologic role in venous thromboembolism.

Disclosures:

No relevant conflicts of interest to declare.
Polymorphisms in Antitrombin (SERPINC1) and Tissue Factor Pathway Inhibitor (TFPI) Genes Are Associated with Recurrent Pregnancy Loss
Authors: Guerra-Shinohara Elvira Maria , Bertinato Juliano Felix , da Silva Giusti Kelma Cordeiro , Seino Fernanda Midori , Bueno Carolina Tosin , de Carvalho Mario Henrique Burlacchini , Amorim Filho Antônio Gomes , Zugaib Marcelo , Chiattone Carlos S
Published: October 2019

Abstract 711

Background:

Recurrent pregnancy loss (RPL) is a multifactorial condition. The effects of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) gene polymorphisms in the risk of having RPL are unknown.

Aims:

To determine the effects of SERPINC1, PROC, THBD and TFPI gene polymorphisms in the risk of having RPL.

Methods:

One hundred and fifteen non-pregnant women with three consecutive losses prior to 20 weeks of pregnancy without carrying a fetus to viability (primary RPL) and 253 healthy fertile non-pregnant women who had at least two children and no known pregnancy losses were studied. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292 and rs8176592) gene polymorphisms were detected respectively by Real Time PCR.

Results:

No difference was observed among the genotypes frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T and TFPI T-33C polymorphisms in two groups (Table 1, P>0.05). However, the carriers of 140T allele (CT + TT genotypes) for SERPINC1 C+140T polymorphism had 1.73 (95% CI: 1.00 – 2.99) times risk of having RPL. Furthermore, the carriers of TC + CC genotypes for TFPI T-287C polymorphism had a protection effect of having RPL (OR: 0.48, 95% CI 0.27– 0.86).

Conclusions:

The variant of SERPINC1 C+140T polymorphism is associated with higher risk of having RPL while the variant of TFPI T-287C polymorphism is associated with a protection effect.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Stem Cell Transplantation in Thalassemia Major. Report from the EBMT Hemoglobinopathy Registry
Authors: Baronciani Donatella , Pilo Federica , Lyon-Caen Sarah , Poetschger Ulrike , Dini Giorgio , Peters Christina
Published: October 2019

Abstract 905

Background.

Hemopoietic allogeneic stem cell transplantation (HSCT) still remains the only available curative option for β-thalassaemia major. At today more than 3000 transplants have been performed worldwide and registered. We analyze transplant activity and outcome after 20 years of the European Hemoglobinopathy Bone Marrow Transplant (EBMT) Registry.

Methods.

Data have been analyzed from promise MED-A form. Survival data have been reported with the method of Kaplan-Meier in patients with complete survival data.

Results.

One hundred and 34 centres from 28 different countries reported their data to the registry. Three thousands and 821 (3821) consecutive first HSCT for patients affected by a hemoglobinopathy have been registered. There were only 364 (10.5%) patients aged 18 years or older. Further analyses were restricted to patients with thalassemia major and to transplants performed after January 1st, 2000. In this latter period 1493 transplants have been performed (median age 7.2 years, range 0.3–45.1) with 133 (9.8%) patients aged 18 years or older.

During the last decade 70% of the transplants have been performed in Europe and 30% in Asia and Africa with a trend to an increase activity in these countries compared to previous decades.

Of the 1493 transplants performed after January 2000, 1061 (71%) have been performed from an HLA identical sibling donors, 129 (8.6%) from a matched other relative, 57 (3.8%) from a mismatched relative, 210 (14%) from an unrelated donor and in 36 (2.4%) cases donor information were missing. Source of stem cells was bone marrow in 1011 patients (67.7%), peripheral blood in 303 patients (20.3%), cord blood in 60 patients (4%), mixed in 82 patients (5.5%). Source of stem cell was missing in 37 (2.5%).

In matched sibling donor transplant recipients overall survival (OS) and thalassemia free survival (TFS) plotted at 91 ± 0.01% and 83 ± 0.01%, respectively. Significant differences have been reported in OS and TFS when related to different age groups as reported in Table 1 (P < 0.001).

Conclusion.

This analysis confirms allogeneic transplantation is a worldwide diffuse high success curative procedure for β-thalassaemia major. Bone marrow still remains the principal source of stem cells. The large majority of transplants have been performed from a matched sibling donor with a marginal role of other donors. In addition these data demonstrate the trend to not transplant adult patients. In the univariate analyses 14 years old appears as threshold age for best transplant strategy.

Disclosures:

No relevant conflicts of interest to declare.
Primary T Cell Immunodeficiencies and Autoimmunity
Authors: Fischer Alain
Published: October 2019

Abstract SCI-21

There are a variety of primary T cell immunodeficiencies that can impair T cell differentiation or T cell activation. The latter include CD38δ deficiency, ZAP70 deficiency, Ca++ influx deficiency (ORAI1 and Stim1 deficiencies), and ITK deficiency as well. A new T cell activation deficiency as observed in a patient with defective T cell receptor triggered T cell activation and low CD4 T cell counts will be reported. A remarkable and quasi constant feature shared by all those T cell activation defects is the occurrence of autoimmune diseases, mostly related to autoantibodies, and inflammation such as colitis or panniculitis. Several mechanisms can account for these findings that include defective regulatory T cell development or function, defective negative selection impaired intrinsic feedback mechanism as well as non TCR-mediated T cell activation ultimately leading to proinflammatory cytokines release and autoantibody production by B cells. Another new form of primary T cell immunodeficiency with autosomal recessive inheritance observed in four patients from two families will be described. It is characterized by defective survival of naïve T cells. There again, autoimmunity appeared to be a significant component of the phenotype. Collectively, these results indicate that further insight into the role of key molecules in T cell activation/survival is provided by the analysis of new primary immunodeficiency phenotypes. In addition, the occurrence of autoimmunity in these settings stresses on one hand the role of T cells in the control of reactivity to self and, on the other hand, should be considered in the therapeutic strategy of these conditions.

Disclosures:

No relevant conflicts of interest to declare.
Functions of Platelet Microparticles in Inflammatory Arthritis
Authors: Cloutier Nathalie , Farndale Richard W. , Brisson Alain , Boilard Eric
Published: October 2019

Abstract SCI-35

Besides their pivotal role in thrombosis and wound repair, platelets can participate in inflammatory responses via a broad arsenal that includes CD40L and CD40, cyclooxygenase-1 mediated prostaglandins, leukotrienes, IL-1, RANTES, serotonin, platelet factor 4, matrix metalloproteinases -2 and -9, P-selectin, adenosine diphosphate, and reactive oxygen species. Rheumatoid arthritis (RA) is amongst the most common autoimmune chronic inflammation that affects the joints. Interestingly, we found copious amounts of submicron particles that harbor platelet integrins in the synovial fluid of patients suffering from RA, pointing to evidence of platelet activation in this inflammatory autoimmune disease. Importantly, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. In addition to the transcellular collaboration between platelets and fibroblast-like synoviocytes in generation of pro-inflammatory prostacyclin (1), we found that platelets contribute to synovitis by production of IL-1-rich platelet microparticles (2). These recent advances in understanding of the platelet activities in inflammation notwithstanding, the mechanisms by which platelet microparticles invade the diseased joint in RA remain obscure. Using synovial biopsies from patients suffering from RA in addition to in vivo imaging strategies in a murine model of arthritis, our current work aspires to dissect the means of transportation of platelet microparticles during RA. Given their pro-inflammatory properties, to understand the process by which microparticles invade the synovial joint is of great clinical interest.

Disclosures:

No relevant conflicts of interest to declare.
The Interplay of Inflammation and Cancer
Authors: Dranoff Glenn
Published: October 2019

Abstract SCI-4

Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in some advanced cancer patients. The detailed analysis of patients achieving sustained clinical benefits from these therapies illustrated the importance of a coordinated cellular and humoral anti-tumor immune response. Indeed, the extent of tumor necrosis in posttreatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs. Moreover, therapy induced a potent antibody response to several key tumor cell surface and secreted proteins; these antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine-binding protein MFG-E8 in antigen-presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. While blockade of MFG-E8 can enhance therapeutic immunity in the context of cancer vaccines, loss of Treg homeostasis with MFG-E8 deficiency can lead to chronic inflammation. Mice deficient in GM-CSF and IFN-g develop at high frequency germinal center derived lymphomas and non-small cell lung carcinomas. The pathogenesis of these tumors reflects a combination of tumor-promoting inflammation and a loss of protective tumor immunity. Taken together, these findings highlight dual roles for immunity in cancer pathogenesis and therapy.

Disclosures:

No relevant conflicts of interest to declare.
Cost Effectiveness of Rivaroxaban Versus Warfarin for the Prevention of Recurrent Venous Thromboembolism.
Authors: Seaman Craig D. , Ragni Margaret V. , Smith Kenneth J.
Published: October 2019

Abstract 3177

Background:

Rivaroxaban is an oral anticoagulant administered once daily that works by direct inhibition of factor Xa. It has been found to be noninferior to warfarin in the prevention of recurrent venous thromboembolism (VTE). Whether rivaroxaban is cost effective in the prevention of recurrent VTE, however, is not known.

Methods:

To assess the cost effectiveness of rivaroxaban compared with warfarin in the prevention of recurrent VTE, we built a Markov state transition model over a 10-year time horizon. The base case analysis consisted of a hypothetical cohort of 60-year-old patients who were diagnosed with their first VTE and received secondary prophylaxis with either rivaroxaban or warfarin for 6 months. Cost estimates were derived from the Healthcare and Utilization Project (HCUP) and other sources. Probabilities for VTE recurrence and mortality and for major and minor bleeding were based on the EINSTEIN PE trial (NEJM 2012; 366: 1287–97). Outcomes included costs in 2011 United States dollars, quality adjusted life years (QALYs), and incremental cost effectiveness ratios (ICERs) over 10 years from a societal perspective.

Results:

Base case results are shown in the Table, showing total medication and other medical costs associated with each strategy. Compared with warfarin, the rivaroxaban strategy cost less ($4057 vs $7004) and was more effective (9.30 QALYs vs 9.16 QALYs). Thus, warfarin was dominated by rivaroxaban. In sensitivity analyses, results were most sensitive to changes in the probability of major bleeding with rivaroxaban. As shown in the Figure, when major bleeding with rivaroxaban (base case 1.1%) was less than 4.5%, the ICRE was within the $100,000 per QALY gained acceptability threshold range. Results favoring rivaroxaban were robust to individual variation of other parameter values.

Conclusion:

Prophylactic anticoagulation with rivaroxaban appears to be a cost effective, and perhaps cost saving, alternative to warfarin for the prevention of recurrent VTE.

Disclosures:

Off Label Use: Rivaroxaban - Prevention of recurrent venous thromboembolism.
Missense Mutations in the ABCB6 Transporter Cause Dominant Familial Pseudohyperkalemia
Authors: Andolfo Immacolata , Alper Seth , Delaunay Jean , Auriemma Carla , Russo Roberta , Esposito Maria Rosaria , Sharma Alok K. , Shmukler Boris E , Vandorpe David H , Brugnara Carlo , De Franceschi Lucia , Iolascon Achille
Published: October 2019

Abstract 3184

Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced slow ‘passive leak’ of red cell K+ into plasma, first described in a large Scottish family from Edinburgh (Stewart GW, et al., 1979). Although in freshly obtained blood samples plasma [K+] was normal, it was increased when measured in blood stored at or below room temperature. This trait was unaccompanied by clinical symptoms or signs except for mild abnormalities of red cell shape. FP Lille was later described in a large Flemish kindred with morphologically normal red cells (Dagher G, et al., 1989; Vantyghem MC, et al., 1991). In this family, red cell K+ efflux measured in the presence of ouabain and bumetanide was normal at 37°C, but greatly increased at 22°C and 9°C. FP Lille mapped to 2q35-q36 (Carella M, et al., 2004), whereas FP Edinburgh mapped to 16q23-qter (Iolascon A, et al., 1999). Subsequently, asymptomatic cases FP Chiswick and FP Falkirk with remarkable increased MCV were reported (Haines PG, et al., 2001).

Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval in three multigenerational FP families with 20 affected individuals identified two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that in erythrocyte membranes bears the Langereis blood group antigen system (Krishnamurthy PC, et al., 2006; Helias V, et al., 2012). Structural modeling predicts subtle changes in protein structure associated with either mutation.

ABCB6 mRNA and protein levels increased during erythroid differentiation of CD34+ erythroid precursors (at 7 and 14 days of EPO induced differentiation), and of HEL and K562 erythroleukemia cells. However, the ABCB6 R375Q mutation altered neither levels of ABCB6 mRNA or protein, nor protein localization in mature erythrocytes or erythroid precursor cells. These data strongly suggest that missense mutations in residue 375 of the ABCB6 polypeptide either mediate the cold-induced K+ leak of chromosome 2-linked FP, or activate an independent, cold-induced cation permeability pathway of the red cell.

Disclosures:

No relevant conflicts of interest to declare.
The R1306W Type 2B Natural Mutation of Von Willebrand Factor Dramatically Enhances the Multimer Sensitivity to Shear Stress
Authors: De Cristofaro Raimondo , Scaglione Giovanni Luca , Lancellotti Stefano , Pagliari Maria Teresa , Papi Massimiliano , De Spirito Marco , Maiorana Alessandro , Baronciani Luciano , Arcovito Alessandro , Stasio Enrico Di , Peyvandi Flora
Published: October 2019

Abstract 3306

Background.

Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13.

Methods.

In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR).

Results.

The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values <10dyn/cm2 the R1306W mutant, at variance with WT vWF, already showed an initial self-aggregation with formation of a network characterized by a roughness significantly higher than that of WT vWF. Mechanical stretching experiments, performed using the AFM cantilever, showed that R1306W vWF needs 30% less energy per length unit to be stretcted compared to WT protein (1.6 vs 2.2×10−11J/m)

Conclusions.

These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb.

Disclosures:

No relevant conflicts of interest to declare.
Skewing towards Decreased Fc-Fucosylation of Platelet-Alloantibodies in Pregnancy
Authors: Kapur Rick , Vestrheim Anne , Koeleman Carolien AM , Porcelijn Leendert , Jackson Dave , Kumpel Belinda , Deelder André M , Michaelsen Terje E , de Haas Masja , Van der Schoot C. Ellen , Wuhrer Manfred , Vidarsson Gestur
Published: October 2019

Abstract 3331

IgG-antibody responses during pregnancy against human platelet antigens (HPA) of the fetus can result in fetal or neonatal alloimmune thrombocytopenia (FNAIT) with various clinical scenarios, as patients can be asymptomatic, suffer from petechiae, major organ bleedings, or even intracranial hemorrhages. Various biological activities of IgG including antibody-dependent cellular cytotoxicity (ADCC) can be modulated by the structural features of the N-glycans in the Fc-part. The extent of sialylation, galactosylation, and fucosylation have been described to affect the binding of IgG to Fc-receptors (FcγR). Fucosylation is particularly important, as a lack of IgG-Fc-fucose has >50 times greater affinity and associated activity through FcγRIIIa, as compared to fucosylated IgG, which normally comprises >90% of normal serum IgG. By extensive analysis of the Fc-glycosylation using mass spectrometry, we found slightly increased levels of galactosylation (p<0.0001) but markedly decreased levels of core-fucosylation (p<0.0001) of the pathogenic IgG (anti-HPA1a) formed in FNAIT compared to the total IgG1 in the same patients. This unique response led us to question whether lack of fucose was specific for antibodies formed against platelets or due to the immune response during pregnancy. Analyses of anti-HLA antibodies formed after platelet transfusion (refractory thrombocytopenia) unveiled a similar increase in anti-platelet IgG galactosylation (p: 0.0002) as seen in FNAIT, but without any change in IgG fucosylation. Also when analyzing autoantibodies to platelet-glycoprotein IIb/IIIa in auto-immune thrombocytopenia (ITP), we could not detect any changes in IgG-glycosylation including fucosylation. We therefore conclude that the atypical fucosylation seen in the FNAIT-anti-platelet response is related to the immune milieu defined by pregnancy. This infuences IgG effector functions, including ADCC activity and may have a profound effect on disease severity and prognosis of alloimmune-mediated reactions against fetal cells and tissues.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Safety of Baxter's Recombinant ADAMTS13
Authors: Piskernik Christina , Dietrich Barbara , Kubik Susan , Ruthsatz Tanja , Scheiflinger Friedrich , Schwarz Hans Peter , Muchitsch Eva-Maria
Published: October 2019

Abstract 3381

Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for treatment, and routine prophylaxis of acute episodes of thrombotic thrombocytopenic purpura (TTP) in patients with ADAMTS13 deficiency. TTP is a rare, life-threatening microvascular disease characterized by single or recurrent episodes of thrombocytopenia, microangiopathic haemolytic anaemia and widespread microvascular thrombosis, which leads to the ischemic damage of multiple organs.

To characterize the safety and the no observed adverse effect level (NOAEL) of Baxter's rADAMTS13, a 1-month repeat dose toxicity study in rats, an escalating dose and pilot 4-week repeat dose toxicity study in cynomolgus monkeys, and a 1-month repeat dose toxicity study including cardiovascular/pulmonary safety pharmacology in cynomolgus monkeys was conducted. Species suitability was shown by demonstrating that human rADAMTS13 can cleave monkey and rat VWF in vitro and in vivo under native conditions to present a worst case scenario for repeat dose toxicity studies. A local tolerance study was conducted in rabbits.

Baxter's rADAMTS13 was well tolerated in rats at doses of 80, 400 and 800 FRETS-U/kg administered every third day for one month. Thus, the NOAEL in rats was the highest dose of 800 FRETS-U/kg. In cynomolgus monkeys, no adverse effects were detected at 800 FRETS-U/kg after intravenous treatment with rADAMTS13 once a week for 5 weeks (pilot repeat dose study). The NOAEL for the escalating dose phase of the study was 1790 FRETS-U/kg. Treatment of cynomolgus monkeys with bolus injection of Baxter's rADAMTS13 at doses of 80, 200, and 400 FRETS-U/kg once a week for 29 days did not reveal adverse findings. Thus, the NOAEL for this study was the highest dose of 400 FRETS-U/kg. As expected for a heterologous human protein drug, repeat doses of rADAMTS13 resulted in the formation of anti-drug antibodies specific for human ADAMTS13 and in neutralising human ADAMTS13 activity in animal models. No injection site reactions were observed in animals of either species. rADAMTS13 was well tolerated after intravenous (intended clinical administration route), intraarterial and paravenous administration in rabbits.

In conclusion, Baxter's rADAMTS13 was well tolerated in rats, monkeys and rabbits, with no toxicity observed even at the highest dose levels.

Disclosures:

Piskernik: Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Ruthsatz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
Pharmacokinetic Evaluation of a Newly Introduced Von Willebrand Factor Concentrate At Two London Haemophilia Centres
Authors: Hegener Oliver , Wiles Natasha M , Wareing Anne , Madan Bella , Austin Steve K
Published: October 2019

Abstract 3384

Introducing new factor concentrates in VWD patients is complicated by VWD subtype and PK variability. Wilate, a VWF concentrate with a 1:1 VWF: FVIII ratio and has not yet been widely used in the UK. We conducted PK studies with Wilate, to determine its efficacy, peak VWF activity and FVIII levels, and clearance in patients with VWD. Where feasible, we compared the data with previously used concentrate (Haemate-P) handling. 17 VWD patients (4 type 1, 6 type 2, 7 type 3) from two London Haemophilia centres were evaluated. The median age was 36 yrs (range 12– 67 yrs); sex M:F 6:11; mean body weight 73 kg (range 39–122 kg). Seven were blood group O, 1 group AB, 4 group A and 4 group B. The mean dose of Wilate administered was 44 iu/kg VWF: RiCoF (range 17–61). VWF activity, VWF antigen and FVIII levels were measured pre- and post for up to 8 hrs. The mean dose of Wilate required to reach target VWF:RiCof activity level of >65 iu/dl in the type 3 VWD was 49.6 iu/kg, and 38.7 iu/kg in type 2 VWD. Of the type 1 patients mean dosing was 43.0 iu/kg. The median peak VWF:RiCoF activity was 82 iu/dl in type 3 and 115 iu/dl in type 2, and 92 iu/dl in type 1. VWF Antigen levels were 116 iu/dl in type 3, and 164 iu/dl in type 2 and 135 iu/dl in type 1. VWF handling in some individuals was suggestive of increased VWF clearance with a half-life below the suggested half-life of 12 hours. This observation indicates the importance of prolonged PK studies in individual cases. As expected, peak FVIII levels were generally higher with Wilate compared to previously used concentrate (94 iu/dl in type 3 and 82 iu/dl in type 2) but the PK profiles between the products were similar, with some inter-individual variability. In conclusion, Wilate achieves adequate VWF activity and FVIII levels and has similar PK properties to previously used concentrate. However, based on the results of this study prolonged PK assessment appears important in selected cases.

Disclosures:

Hegener: Octapharma AG: Employment. Austin:Baxter: Advisory Board Other; Pfizer: Advisory Board, Advisory Board Other.
Effects of Preoperative Vitamin K On the INR in Bridging Therapy
Authors: Bayewitz Ashrei , Scorziello Barbara , Maala Clarice , Giannattasio Emily , Billett Henny Heisler
Published: October 2019

Abstract 3407

Patients who require interruption of their warfarin therapy before surgery need a safe and effective bridging protocol.

Objective:

We investigated whether routine incorporation of oral Vitamin K three days before surgery would improve perioperative management.

Patients/Methods:

Patients on chronic warfarin therapy requiring perioperative bridging therapy were the participants in this observational study. Results from two bridging protocols, 30 cases each, with and without oral Vitamin K, were analyzed. Day of Surgery was considered Day 0. The first cohort (control arm) only received oral Vitamin K preoperatively if their INR was >1.5 on Day -1. The intervention cohort (Vitamin K group) routinely received 2.5mg of oral Vitamin K on Day -3. Primary outcome was INR on Day -1. Secondary outcomes were number of patients with INRs ≥1.5 on Day -1 or Day 0, number of surgeries delayed, number of postoperative bleeding or thrombotic incidents and INR on Day 4. Results: On Day -1, the INR for the Vitamin K group was 1.16, compared with 1.28 for the control group (p=0.037). On re-bridging, Day 4 INR was similar in the two groups; 1.37 in the Vitamin K group, and 1.35 in the control group but there was an increase in the dose needed to achieve these levels for the Vitamin K group (24.2% over usual dose for the Vitamin K group vs 7.2% for the control). Only the control group had patients with INRs ≥1.5 on Day -1 (n=5), INRs ≥1.5 on Day 0 (n=2), or had postoperative bleeding incidents (n=2). Neither group had delayed surgeries or postoperative thrombosis.

Conclusions:

Adding Vitamin K on Day -3 into bridging protocols leads to a safe preoperative INR and may limit other complications, but this may come at the expense of increased dosing requirements postoperatively.

Disclosures:

No relevant conflicts of interest to declare.
A Defect in the Bone Marrow Microenvironment of ERdj4GT/GT Mice Impacts B Cell Development
Authors: Fritz Jill M , Weaver Timothy E
Published: October 2019

Abstract 3463

ERdj4 is a critical component of the unfolded protein response (UPR) that facilitates the removal of unfolded/misfolded substrates from the ER lumen for proteasomal degradation. Since previous studies have linked the UPR to B lymphopoiesis [Reimold, A.M. et al., Nature, 2001; Zhang, K. et al., JCI, 2005], we hypothesized that a deficiency in ERdj4 would impact B cell development. The ERdj4 allele was disrupted by a gene trap leading to hypomorphic expression of ERdj4 in mice homozygous for the gene trap allele (ERdj4GT/GT). Pro-B, pre-B, immature, and mature B cell populations were significantly reduced in the bone marrow of ERdj4GT/GT mice, which was associated with an increase in pro-B cell death. ERdj4GT/GT donor cells transplanted into ERdj4+/+ recipients rescued all stages of B cell development, implicating a defect in the bone marrow microenvironment. Osteoblasts support B cell development through VCAM-1, SDF-1 and IL-7 signaling [Visnjic, D. et al., Blood, 2004; Zhu, J. et al., Blood, 2007]; coincidently, the UPR is essential for osteoblast differentiation [Tohmonda, T. et al., EMBO, 2011; Wei, J. et al., J Cell Physiol, 2008]. ERdj4 deficiency in primary bone cells was associated with increased ER stress. Osteoblast-specific markers and the B cell growth factor SDF-1 was also significantly reduced in primary ERdj4GT/GT bone cells. Importantly, osteoblasts were reduced in femur tissue sections of ERdj4GT/GT mice. Collectively, these data suggest that the loss of ERdj4 in osteoblasts is associated with a defect in B lymphopoiesis. This research was supported by the NHLBI (HL103923 and HL086492).

Disclosures:

No relevant conflicts of interest to declare.
The Role of OX40 and Its Ligand in Acute Myeloid Leukemia: Expression, Function and Modulation of NK Cell Anti-Leukemia Reactivity
Authors: Schumacher Carla E , Nuebling Tina , Hofmann Martin , Schmiedel Benjamin J , Kanz Lothar , Jung Gundram , Salih Helmut R
Published: October 2019

Abstract 3548

The TNF/TNF receptor (TNFR) family comprises various molecules that substantially influence cellular functions of both tumor and immune effector cells. The TNFR family member OX40 has been shown to influence proliferation and differentiation of T cells in autoimmune diseases. Here we studied the yet unknown role of OX40 in acute myeloid leukemia (AML). Substantial surface expression of OX40 was detected on malignant cells of AML patients in 24 of 60 (40%) investigated cases. Expression of OX40 mRNA and protein by leukemic cells was confirmed by analysis of AML cells lines, which displayed substantial surface levels in 6 of 7 investigated cases. Induction of OX40 signaling into AML cells by recombinant OX40L or agonistic antibodies lead to the release of cytokines like IL-10 and TNF which contribute to AML pathophysiology and stimulated metabolic activity (WST test) of the leukemia cells. Moreover, we found that NK cells, which play an important role in anti-tumor immunity and largely contribute to the clinical success of allogeneic stem cell transplantation (SCT) in AML, express OX40 ligand (OX40L) following activation, and OX40L triggering stimulated NK cell reactivity. Functional analyses with OX40 transfectants and OX40-negative controls confirmed that OX40L signaling promotes NK cell cytotoxicity and IFN-γ production. Furthermore, disruption of OX40-OX40L interaction by blocking OX40 F(ab)2 fragments resulted in reduced cytotoxicity and cytokine production of allogenic NK cells, thereby further confirming the stimulatory effect of OX40L on NK cell anti-leukemia reactivity when interacting with its AML-expressed counterpart. Our data suggest that OX40 is involved in disease pathophysiology of AML and identify OX40-OX40L interaction as previously unknown modulator of NK cell immunosurveillance in AML.

Disclosures:

No relevant conflicts of interest to declare.
Early Absolute Lymphocyte Count Is a Strong Predictor of Long Term Improved Survival in Childhood Acute Myeloid Leukaemia
Authors: Keenan Russell , Williamson Rebecca , Caswell Mark , Salim Rahuman , Campbell Helen Jane
Published: October 2019

Abstract 3553

The absolute lymphocyte count (ALC) has been shown to be a predictor of event free survival in the post transplant setting in many diseases including lymphoma, myeloma and leukaemia. (Porrata et al). Studies carried out at Alder Hey Childrens' Hospital, Liverpool in 2003 was the first one of its kind in looking at the ALC as a prognostic tool in the non-bone marrow transplantation setting in childhood leukaemia (Lomas et al, Hudson et al). The studies in acute myeloid leukaemia were small with short follow up. We present here the largest study of the prognostic significance of ALC in paediatric Acute Myeloid Leukaemia.

We perfomed a retrospective single centre study involving 59 paediatric patients diagnosed with Acute Myeloid Leukaemia between 1985 and 2010 and treated using Medical Research Council AML 10, AML 12 and AML 15 protocols. Data was analysed for event free and overall survival. All 28 deaths occurred within 3 years. Follow up was 11 months to 27 years in the 31 survivors.

A clear relationship was observed between patients with higher absolute lymphocyte counts and overall and event-free survival At 28 days post course 1 chemotherapy, using an ALC threshold value of 1.35 × 109/L, overall survival is 82% v 47% (p = 0.0336). The effect of the ALC diminishes with subsequent chemotherapy courses. At 28 days post course 2 chemotherapy using an ALC threshold value of 1.22 × 109/L, overall survival is 65% v 36% (p = 0.2952). At 28 days post course 4 chemotherapy using an ALC threshold of 0.67 × 109/L, overall survival is 69% v 50% p = 0.5983.

We hypothesise that the higher ALC in the event-free patients, is due to tumour specific lymphocyte proliferation, causing clearance of low levels of leukaemia after chemotherapy. The chemotherapy acts to cause leukaemia cell death and antigen processing through macrophages. Chemotherapy causes tissue damage leading to upregulation of immunological danger signals with T and B lymphocyte expansion with specificity against the residual leukaemia cells. The reduced effect of the ALC predicting survival with each subsequent course of therapy may be a result of less leukaemia cells being present prior to each course. Less leukaemia cell death with each course results in less leukaemia antigen processing, presentation and lymphocyte expansion. If this hypothesis is confirmed it may be possible to carry out harvesting of tumour specific lymphocytes prior to chemotherapy with post chemotherapy add back to enhance the curative effect.

Disclosures:

No relevant conflicts of interest to declare.