bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.
Successful Engrafment After HLA Identical Granulocyte Transfusion As Support Therapy in a Septic Shock Patient with Acute Lymphoblastic Leukemia
Authors: Ovilla Roberto , Barrera-Carmona Claudia , Guzman-Bouilloud Nicolas , Buganza-Torio Elizabeth , Jimenez-Alvarado Rosa , Monreal-Alcantara Claudia 
Published: October 2019

Abstract
Abstract 4507
A 53 years old male started in February 2010 with ecchymosis, petechiae and spontaneous gum bleeding. He was diagnosed with acute lymphoblastic leukemia, and was started on HYPER-CVAD, in combination with anti-tumoral lysis syndrome measures and antimicrobial, antiviral y antifungal prophylaxis. After finishing HYPER-CVAD phase A, he developed severe myelosuppression even with the use of G-CSF, 72 hours later he presented with abdominal cramping, fever up to 38.2°C and hypotension, with a high clinical suspicion of neutropenic colitis; his mean arterial pressure average was 45 mmHg, he was started on intravenous colloids, dobutamine and norepinephrine drips.
Because of severe myelosuppression, septic shock and myocardial depression, a granulocyte transfusion without previous mobilization was performed with an identical sibling donor with concomitant use of granulocytic colony stimulation factor. Severe myelosuppression was maintained during 7 days, however shock state was reversed some hours after performing granulocyte transfusion, without infectious signs, 36 hours later a mononuclear cell infusion was performed from the same donor, with previous 2 days G-CSF mobilization. A gradual increase in leukocyte number appeared, with 400, 900 and finally 1900.
A new bone marrow aspiration was performed, where hematopoietic recovery was confirmed from his sister's cells with confirmation by karyotype and microsatellites. He was then considered bone marrow grafted HLA compatible.
On April 2010 he presented with acute diarrheic syndrome secondary to a CMV infection, he was started on ganciclovir and intravenous immunoglobulin. On May he presented an Aspergillus pneumonia that was treated both clinically with antifungal therapy and surgically with thoracoscopy to remove the fungi lesion. On October 2010 he started with graft versus host manifestations on the skin and in the liver. He started immunosuppressive treatment with Prednisone and Sirolimus. On November he developed anogenital herpes zoster infection. After this complication, every GVHD manifestation ceded. Now, 28 months post-bone marrow transplant he presents full remission with no evidence of leukemia.
Granulocyte transfusion is an uncommon technique. Preferably it should be done with and identical donor. In this case report, the justification of the procedure was to be able to achieve a remission from a potentially irreversible septic shock; this was successfully done, in an unexpected clinical scenario with severe life threatening, and in a casual manner, the patient achieved a successfully bone marrow transplant, and now 30 months post-granulocyte infusion the patient is free from any evidence of disease.

Disclosures:
No relevant conflicts of interest to declare.