bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preclinical Safety of Baxter's Recombinant Factor VIIa
Authors: Dietrich Barbara , Kubik Susan , Auer Wilfried , Reipert Birgit , Horling Frank , Ehrlich Hartmut J. , Scheiflinger Friedrich , Schwarz Hans Peter , Muchitsch Eva-Maria 
Published: October 2019

Abstract
Abstract 1417
Baxter has developed a recombinant FVIIa (rFVIIa) product for the treatment of patients with hemophilia A and hemophilia B with inhibitors. Baxter's new rFVIIa is produced by a genetically engineered Chinese hamster ovary (CHO) cell line in a cell culture medium free from any animal or human proteins. The objective of this preclinical study-program was to evaluate the safety of Baxter's rFVIIa in different species.
To assess the anaphylactoid and thrombogenic potential of rFVIIa and its effects on blood pressure, cardiac and respiratory function, and the coagulation system the preclinical program included studies in established models of safety pharmacology in telemetered cynomolgus monkeys, rabbits, normal and spontaneously hypertensive rats, and guinea pigs. Furthermore, studies on single and repeated dose toxicity (hemophilia A mouse, rat, cynomolgus monkey), local tolerance (rabbit), and comparative immunogenicity (mouse) were conducted. Commercially available licensed rFVIIa served as active reference item.
Treatment with rFVIIa did not result in any anaphylactoid reaction, and had no adverse effect on the cardiovascular or respiratory system or on hematology parameters. Thrombogenicity studies showed no greater thrombogenic potential of Baxter's rFVIIa than the commercially available rFVIIa product. Single dose toxicity studies in mice, and repeated administration of Baxter's rFVIIa in rats and cynomolgus monkeys, administered at doses of 2.7 mg/kg, confirmed the safety of the new product. As expected, due to the pharmacological action of rFVIIa, high doses of either recombinant FVIIa product resulted in exaggerated pharmacological effects leading to thrombus formation in some organs, which are well- known effects for this class of compound. Furthermore, Baxter's rFVIIa was well tolerated at the injection site after intravenous injection. Results obtained in comparative immunogenicity studies demonstrate that Baxter's new rFVIIa and the commercially available rFVIIa product have a similar immunogenicity profile.
In summary, Baxter's rFVIIa has a safety profile that supports the evidence necessary for proceeding with human trials.

Disclosures:
Dietrich: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Auer:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
Inhibitor Eradication In Acquired Factor VIII Inhibitor: A Case Series
Authors: Badari Ambuga , Bernstein Zale P , Logue Gerald L. , Patel Anush , Miller James , Tran Tan , Keshavamurthy Chandana 
Published: October 2019

Abstract
Abstract 1423

Acquired factor VIII (FVIII) inhibitor is an uncommon condition, with an incidence of ≂f1.5 cases/million/year, as reported by Collins et al. (Blood 2007. 109: p. 1870–1877) in a two year observational study in the UK. However, this is a serious condition, characterized by auto-antibodies against circulating factor VIII and has high mortality from bleeding. Controlling the acute bleeding event with FVIII inhibitor bypassing agents like recombinant activated factor VII or activated prothrombin complex concentrates is of utmost importance. Equally important is the eradication of inhibitor, in order to restore normal hemostasis. There are several approaches for inhibitor eradication, including, but not limited to, the use of Corticosteroids, Cyclophosphamide, Azathioprine, Vincristine, IVIG, Cyclosporine, Mycophenolate and, recently, Rituximab, either singly, or, in different combinations. However, there is no single standard approach. We report 4 cases of acquired FVIII inhibitor from our center that were initially treated with factor VIII bypassing agents. Inhibitor eradication was achieved by using a combination of Rituximab, Prednisone and Cyclophosphamide.
Complete and sustained remission was achieved in all 4 patients (100%).All 4 patients tolerated the therapy well. None of the patients had significant adverse effects like neutropenia, hemorrhagic cystitis or other complications, either immediately or during follow up. Thus, the combined use of Cyclophosphamide, Rituximab and Prednisone seems to be an effective and safe regimen for inhibitor eradication in patients with acquired FVIII inhibitor.

Disclosures:
Off Label Use: Rituximab.For th purpose of auto-antibody supression.
Relevance of Intact Von Willebrand Factor (VWF) Triplet Structure for VWF Function.
Authors: Schwartz Bruce A. , Fuchs Birte , Kannicht Christoph , Solecka Barbera , Kröning Mario 
Published: October 2019

Abstract
Abstract 1425

Introduction:
The characteristic multimer pattern of plasmatic von Willebrand factor (VWF) results from asymmetric cleavage by the processing metalloprotease ADAMTS13 between Y1605/M1606 within the VWF A2 domain. In normal plasma, characteristic species of various multimeric sizes with flanking satellite bands (triplets) encircling the major band on VWF multimer gels are present. The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Even though the distribution of VWF satellite bands is significantly altered in some types of von Willebrand disease (VWD) and several commercial VWF concentrates, the impact of triplet structure on VWF function has not been investigated so far.

Methods:
Four commercially available VWF concentrates were analyzed with respect to ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by the fluorescence resonance energy transfer (FRET) assay. VWF zymogram gels were used to test for ADAMTS13 activity. Samples composed of different VWF triplet distribution but comparable VWF multimers were obtained by fractionation of plasmatic VWF using heparin affinity chromatography. VWF affinity to collagen was measured by surface plasmon resonance (SPR).

Results:
VWF concentrates markedly differed in their content of ADAMTS13 antigen and activity. A higher ADAMTS13 content correlated with an increased portion of the proteolyzed faster migrating VWF triplet band. The degree of VWF proteolysis, i.e. lack of an additional N-terminal fragment, correlated with a decreased collagen binding level measured by SPR.

Conclusion:
Proteolytic cleavage of N-terminal domains of VWF resulting in a higher content of faster migrating satellite bands affects the function of VWF. The impact of VWF N-terminal domains on collagen binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated.

Disclosures:
Schwartz: Octapharma: Employment. Fuchs:Octapharma: Employment. Kannicht:Octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment.
The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.
Authors: Wang Jianhui , Pan Ruimin , Nardi Michael A. , Li Zongdong 
Published: October 2019

Abstract
Abstract 1434
We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation.

Disclosures:
No relevant conflicts of interest to declare.
IgG Antibodies Directed towards a Single Antigenic Surface In the Spacer Domain of ADAMTS13 Predominate In Plasma of Patients with Acquired TTP.
Authors: Pos Wouter , Fijnheer Rob , Vidarsson Gestur , Voorberg Jan 
Published: October 2019

Abstract
Abstract 1438

Background:
The majority of patients diagnosed with thrombotic thrombocytopenic purpura (TTP) have primarily autoantibodies directed towards the spacer domain of ADAMTS13. Previously, we have shown that residues Arg660, Tyr661 and Tyr665 are involved in binding of anti-ADAMTS13 antibodies. Inspection of the three-dimensional structure of the spacer domain reveals that Arg658 and Phe592 are in close proximity to Arg660, Tyr661 and Tyr665.

Objective:
In this study we explored whether Arg658 and Phe592 contribute to a previously identified antigenic surface in the spacer domain. We also addressed Ig class distribution as well as the contribution of antibodies directed towards the carboxy-terminal TSP2-8 and CUB1-2 domains in a large cohort of patients.

Methods:
The epitope specificity of anti-spacer domain directed antibodies was examined using two recombinant anti-spacer domain directed antibodies I-9 and II-1 and plasma of 48 patients with acute acquired TTP by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Reactivity of patient-derived antibodies directed towards the TSP2-8 and CUB1-2 domains was also addressed by immunoprecipitation. Quantitative Ig class analysis was performed using human monoclonal antibody II-1 as an internal reference.

Results:
Plasma from all 48 patients included in this study contained anti-ADAMTS13 antibodies of subclass IgG; plasma of 5 patients contained anti-ADAMTS13 IgM and in plasma of 9 patients had anti-ADAMTS13 antibodies of subclass IgA1. No anti-ADAMTS13 antibodies of subclass IgA2 were observed. Plasma of some patients contained anti-TSP2-8 (∼17%) and anti-CUB1-2 (∼35%) domain directed antibodies. Analysis of anti-spacer domain antibodies revealed that residues Arg568 and Phe592 contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost the reactivity towards the spacer domain of ADAMTS13 through the introduction of multiple alanine substitutions at positions Arg568, Phe592, Arg660, Tyr661 and Tyr665.

Conclusions:
IgG antibodies directed towards a single antigenic surface in the spacer domain of ADAMTS13 predominate in plasma of patients with acquired TTP.

Disclosures:
No relevant conflicts of interest to declare.
Mycophenolate Mofetil Therapy Is a Steroid Sparing In Childhood and Young Adult Chronic ITP and Evans Syndrome.
Authors: Naji Zaher , Rajpurkar Madhvi , Savasan Sureyya , Chu Roland , Chitlur Meera , Callaghan Michael 
Published: October 2019

Abstract
Abstract 1441
Chronic ITP and Evans syndrome are diseases characterized by autoantibody formation with resultant destruction of platelets or both platelets and red blood cells on neutrophils, respectively. Affected patients are at risk of life threatening bleeding complications and/or life threatening anemia. Conventional therapies are often ineffective or transiently effective and have significant toxicity. One of the most commonly used therapeutic strategies employs chronic corticosteroid administration with the attendant weight gain, hypertension, hyperglycemia, bone loss, infection risk, mood changes and protean other undesirable side effects. Mycophenolate Mofetil (MMF) is an immunosuppressive agent with a favorable side effect profile. It is converted to the active metabolite, mycophenolic acid, and interferes with purine metabolism in T-lymphocytes, effectively killing many of these cells and down-regulating autoimmune phenomena. With approval of our local IRB /HIC we retrospectively reviewed the charts of 11 chronic ITP/Evans syndrome patients who had received MMF, all such patients treated at a large urban Children's Hospital. Clinical variables included age, sex, duration of disease, steroid use, IVIG use, Anti-D use, platelet counts, hemoglobin concentrations and reticulocyte percentages. These data were analyzed using paired t-tests, one-sample t-test and descriptive statistics. The 11 patients ranged in age from 9–22 years old, with a mean age of 15 years. The mean time from diagnosis of disease was 41.8 months with a range of 6–95 months. There were 5 female subjects, 6 Evans syndrome patients and 5 with chronic ITP alone. The median platelet count over the 6 months prior to MMF was 70×109/L, (18-223), with a median of 90×109/L with (27-145) during the first 6 months of MMF therapy (p=0.4). In the Evans syndrome group, the mean hemoglobin prior to MMF was 10.9 g/dL, (9.1-14.6), with a mean of 12.1 g/dL, (6.9-16.7) on MMF (p=0.54). Similarly, the mean reticulocyte percentage was 2.7%, (0.5-14) prior to MMF, with a mean of 2.3%, (0.3-8.9) during MMF therapy. The mean total dose of steroids used in the 6 months prior to MMF was 84.2 mg/kg (prednisone equivalent), (11.2-170), compared to 62.2 mg/kg, (0-193.8) on MMF. 9 of 11 patients had reductions in steroid requirement by an average of 49.3% (p=0.0013). During the first 6 months of MMF both IVIG and anti-D usage decreased from total doses for the entire group of 17 and 3 doses to 8 and 1 dose, respectively. None of the patients experienced severe bleeding episodes or side effects more serious than a transient rash while on MMF. These data suggest that MMF may have a role as a steroid sparing therapy in the treatment of chronic ITP and Evans syndrome.

Disclosures:
Off Label Use: mycophenolate mofetil for ITP Evans syndrome.
Evaluation of Platelet Dysfunction In Children: Improved Detection and Efficiency
Authors: Nugent Diane J. , Roberts Ryan , Nakagawa Peggy 
Published: October 2019

Abstract
Abstract 1446
Currently, there is no single assay that will detect platelet function abnormalities in all individuals. We prospectively studied 369 patients with a strong history of bruising and mucosal membrane bleeding for possible platelet dysfunction following documentation of normal Von Willebrand antigen and activity levels. In an effort to evaluate platelet function under more diverse conditions we chose to simultaneously evaluate 1) aggregation using platelet rich plasma and light transmission aggregometry (LTA), 2) adhesion under high shear using the Platelet Function Analyzer (PFA-100, ADP-collagen, and Epinephrine-collagen cartridges) and 3) platelet initiated clot formation using heparinized whole blood and the two standard mapping agonists, arachadonic acid (AA) and ADP (Hemascope Thromboelastograph Analyzer).
Of the 369 platelet evaluations performed, 87 patients (24%) were found to be normal in all three assays with all agonists. On repeated assays with increased attention to medication and food history, an additional 152 patients were felt to have a transient or acquired dysfunction which improved or normalized on further testing. Leaving 130 patients with persistent bleeding and documented abnormalities on one or more of the assays used. There were no patients with only Collagen (CN) or arachadonic acid (AA) aggregation alone on LTA. Using LTA, there was one patient with combined CN and ADP aggregation defect only, another with AA and EPI absent aggregation only, and one with only AA and Thrombin receptor agonist peptide (TRAP) aggregation abnormalities. A summary of the remaining 127 patients are displayed in the table below:Abnormal AssayLTA EPILTA ADPLTA ADP + EPILTA ADP, EPI CNLTA ADP, EPI, AALTA ADP, EPI CN, AAPFA and PLT Mapping ONLYNumber201240841825M/F3M/17F6M/6F16M/24F1M/7F1M/3F8M/10F12M/13FPFA1M/2F4M7M/4F01M01M/3FPlt Mapping2M/2F1M/2F3F1F009M/7FBoth PFA and Mapping1M0001F2M/9F2M/3F
Summary: By using a combination of three assays, we were able to identify 25 additional patients with significant platelet dysfunction detected with abnormal platelet mapping or PFA-100 despite normal light transmission aggregometry. Patients with the most abnormalities on aggregation, also demonstrated abnormal adhesion and platelet initiated clot formation. However, the use of PFA-100 and/or platelet mapping alone would miss the majority of patients with aggregation defects. In the future, the unique combination of platelet function defects as measured by these assays, and future technologies, will not only improve detection, but also facilitate phenotype to genotype associations and expedite mutational analysis.

Disclosures:
Off Label Use: Rituximab to treat ITP.
HDAC11 Inhibits T-Cell Response to Alloantigens and Reduces Gvhd In Mice.
Authors: Wang Dapeng , Cheng Fengdong , Yu Yu , Semple Kenrick , Peng Lirong , Telles Elphine , Luetteke Noreen , Seto Edward , Anasetti Claudio , Sotomayor Eduardo M. , Yu Xue-Zhong 
Published: October 2019

Abstract
Abstract 1466

Background:
Histone acetyltransferases and histone deacetylases (HDAC) regulate gene expression through acetylation-deacetylation of histones. HDACs are the target of a family of compounds known as HDAC inhibitors, which have been shown to suppress pro-inflammatory cytokines and reduce acute graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT) in mice. However, the role of individual HDAC members in the development of GVHD is not clear. Recently, HDAC11, the newest member of the HDAC family has emerged as an important transcriptional regulator of inflammatory responses in antigen-presenting cells (APCs)1. Here, we evaluated the role of HDAC11 on APCs and T cells in the allogeneic BMT setting in mice with genetic disruption of HDAC11.

Method:
Proliferation of wild-type (WT) and HDAC11 knock-out (KO) T cells in response to allogeneic antigens was compared by [H3] thymidine incorporation assay. Using the same method, we also tested the antigen presentation ability of WT and HDAC11 KO APCs. For in vivo studies, we used a clinical relevant mouse model of BMT: C57BL/6 (B6) ® BALB/c. To evaluate the role of HDAC11 in the function of T cells and APCs, WT and KO mice on B6 background were used as donors and recipients, respectively. Recipient survival was monitored daily and GVHD symptom was evaluated at least twice a week. HDAC11 KO mice were supplied by Merck and Co., Inc.

Results:
In vitro, HDAC11 KO T cells proliferated stronger than WT T cells under the stimulation of allogeneic APCs. Recipients of HDAC11 KO T cells lost significantly more body weight (p < 0.05), and died significantly sooner than those of WT T cells (p < 0.01). The pathologic score of KO recipients was higher than that of WT recipients in each of GVHD target organs including lung, liver, small intestine and colon. Mechanistically, we found that there were significantly more total and IFNγ-producing donor T cells in the recipients of KO cells than those of WT cells (p < 0.05). Collectively, HDAC11 KO T cells have higher activity in response to alloantigens in vitro and induced more severe GVHD in vivo compared to WT T cells. In contrast, KO and WT APCs had a similar ability to stimulate allogeneic T cells in vitro, and no significant difference in GVHD development was observed in WT or KO recipients after allogeneic BMT.

Conclusion:
HDAC11 negatively regulates T-cell function, but has no significant effect on APC function. This finding provides a rationale to promote T-cell immunity or tolerance by inhibiting or enhancing HDAC11, respectively.
1 Villagra et al. Nature Immunology, 10:92-100, 2009.

Disclosures:
No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).
Authors: Aerbajinai Wulin , Chin Kyung , Lee Hyun Woo , Zhu Jianqiong , Rodgers Griffin P. 
Published: October 2019

Abstract
Abstract 1482
Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease.

Disclosures:
No relevant conflicts of interest to declare.