bluuebunny/crossref_metadata_2025_split huggingface dataset:
Outpatient Treatment In Patients with Acute Pulmonary Embolism: The Hestia Study
Authors: Zondag Wendy , Mos Inge , Creemers Dina , Hoogerbrugge Lidia , Dekkers Olaf , Dolsma Anneke , Eijsvogel Michiel , Faber Laura , Hofstee Herman MA , Hovens Marcel , Jonkers Ge , Kralingen Klaas , Kruip Marieke , Vlasveld Tom , Vreede Marielle , Huisman Menno 
Published: October 2019

Abstract
Abstract LBA-1

Introduction:
Patients with pulmonary embolism (PE) are initially treated in the hospital with low molecular weight heparin (LMWH). The most recent guideline of the American College of Chest Physicians on Antithrombotic therapy 2008 reports some small studies on outpatient treatment in patients with pulmonary embolism, which suggest outpatient treatment in selected patients with PE is potentially effective and safe but firm recommendations for clinical practice are lacking. Clinicians urgently need reliable, easy-to-use selection criteria for selection of patients with pulmonary embolism eligible for outpatient treatment.

Objective:
To evaluate the efficacy and safety of outpatient treatment according to predefined criteria (Hestia criteria) in patients with acute PE.

Patients and Methods:
Open-label, single-arm, multicenter clinical trial of patients with objectively proven acute pulmonary embolism, conducted in twelve hospitals in the Netherlands from 2008 to 2010. Follow-up was completed in September 2010. Patients with acute PE were triaged with the predefined Hestia criteria for eligibility for outpatient treatment starting with therapeutic weight adjusted doses of LMWH (Nadroparin), followed by vitamin K antagonists. All patients eligible for outpatient treatment according to the Hestia criteria, were sent home either immediately or within 24 hours after PE was objectively diagnosed.

Outcome:
Outpatient treatment was evaluated with respect to recurrent venous thromboembolism (VTE), including PE or deep venous thrombosis (DVT), major haemorrhage and total mortality during initial LMWH treatment and 3 months follow up. We considered outpatient treatment to be effective if the upper limit of the 95% confidence interval of the incidence of recurrent VTE would not exceed 7%.

Results:
Of 297 included patients, who all completed follow-up, 6 patients (2.0%; 95% confidence interval [CI], 0.8–4.3) had recurrent VTE (5 PE (1.7%), 1 DVT (0.3 %)). Three patients (1.0%, 95% CI 0.2–2.9) died during three months follow-up, but none as a result of fatal PE. One patient died of fatal intracerebral haemorrhage, the other two patients died of progressive malignancy. In addition to the patient with intracranial bleeding, one other patient had a major bleeding event (0.7 %, 95% CI 0.08%-2.4%).

Conclusion:
Outpatient anticoagulant treatment is effective and safe for patients with pulmonary embolism who have been selected with the Hestia criteria. (Dutch Trial Register NTR1319).

Disclosures:
Huisman: GSK: Research Funding; Actelion: Research Funding; Bayer: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Pfizer: Speakers Bureau.
Micromanipulating Hematological Malignancies and Other Cancers
Authors: Lieberman Judy 
Published: October 2019

Abstract
Abstract SCI-33
MicroRNAs regulate the response of the cell to environmental changes and developmental cues. microRNA expression is dysregulated in cancer – microRNA expression is generally reduced in cancer cells compared to normal tissue and individual microRNAs, termed oncomirs, that act as tumor suppressor genes or oncogenes are frequently aberrantly expressed in cancer and their expression can be linked to prognosis and response to therapy. Because microRNAs regulate cancer cell differentiation, proliferation, survival, and metastasis, manipulating microRNA function, either by mimicking or inhibiting miRNAs implicated in cancer, could provide a powerful therapeutic strategy to interfere with key pathways for cancer progression. This talk will explore some of the opportunities and obstacles to harnessing microRNA biology for cancer therapy.

Disclosures:
No relevant conflicts of interest to declare.
Isolation and Functional Characterization of Human Erythroid Progenitors: BFU-E and CFU-E
Authors: Bhagia Pooja , Mohandas Narla , An Xiuli 
Published: October 2019

Abstract
Abstract 1028
The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes.

Disclosures:
No relevant conflicts of interest to declare.
Sphingomyelinase-Induced Remodeling of the Erythrocyte Membrane
Authors: Dinkla Sip , Wessels Katharina , Verdurmen Wouter P.R. , Joosten Irma , Brock Roland E. , Bosman Giel J.C.G.M. 
Published: October 2019

Abstract
Abstract 1032
Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, a major lipid component of cell membranes, generating phosphocholine and ceramide. Inflammation arising from various diseases including sepsis is known to enhance SMase secretion in the circulation. Also, chronic inflammation typically induces anemia through reduced production and enhanced clearance of red cells, which is often associated with poor disease outcome. Incubation of red cells with plasma of septic patients with enhanced SMase activity has been described to result in ceramide formation and exposure of phosphatidylserine (PS). Therefore, we studied the effect of SMase on various pathophysiologically relevant parameters of red cell homeostasis in fresh and stored erythrocytes.
Using time-lapse confocal microscopy we observed a typical sequence of events during SMase treatment of erythrocytes. The cells first lost their discoid shape, PS became externalized, and ultimately there was a sudden loss of cytoplasmic content. Additional confocal microscopy, flow cytometry and immunoblot analyses showed that SMase treatment resulted in ceramide-induced alterations in red cell membrane-cytoskeleton interaction and organization, including lipid raft formation. These changes probably underlie the increase in osmotic fragility, membrane vesiculation and invagination, as well as large hemoglobin aggregates that we also observed. The membrane lipid scrambling, altered morphology, enhanced fragility and presence of rigid membrane patches may all contribute to enhanced removal of sphingomyelinase-exposed red cells.
Generation of very low ceramide levels in the membrane by SMase, as detected by Positive-Ion MALDI-TOF MS, already affected red cell shape, membrane rearrangement and osmotic responsiveness. Combined with the observation that 24 hour incubation of red cells with pathological SMase levels induced shape change and PS exposure, these data suggest that red cell homeostasis could well be disturbed by SMase during chronic inflammation. Of note, red cell storage greatly increased the sensitivity to SMase-induced changes. This may well have implications for red cell transfusion in critically ill patients.

Disclosures:
No relevant conflicts of interest to declare.
The Lymphoid Chemokine CCL21 Enhances the Migration- and CTL-Inducing Functions of Dendritic Cells
Authors: Hong Cheol Yi , Kalinski Pawel , Kim Hyeoung-Joon , Lee Je-Jung 
Published: October 2019

Abstract
Abstract 1127
The migration of dendritic cells (DCs) to secondary lymphoid organs is very important to elicit an adaptive immune response in cancer immunotherapy. Here, we show the effect of lymphoid cytokine on the ability of maturing DCs to migrate in response to the lymph node-associated chemokines. The secondary-lymphoid organ chemokine (SLC/CCL21) during DC maturation dramatically enhanced DC migratory capacity responding to CCL21 and CCL19, and, moreover, produced strongly enhanced cytotoxic T cells, although it did not affect the expression of cell surface markers such as CD80, CD83, CD86, and CCR7 and the production of cytokines such as IL-12p70, IL-10, and IL-23. Mature DCs (mDCs) exposed by chemokine produced higher levels of CXCL10 (IP-10) that is one of the chemokines involved in Th1 attraction, but did not affect the production of Th2-attracting cytokine CCL22, compared with unstimulated mDCs. CCL21-exposed DCs induced strongly enhanced numbers of the interferon-g (IFN-g)-expressing antigen-specific CD8+ T cells against tumor-specific antigens in an CXCL10-dependent manner. Cytotoxic CD8+ T cells stimulated with CCL21-exposed DCs expressed higher level of IFN-g than those stimulated with control mDCs. Interestingly, generation of cytotoxic T cells (CTLs) stimulated by TNFa/IL-1b/IL-6/PGE2-treated DCs (sDCs) supplemented with IP-10 produced strong cytotoxic T cells expressing higher level of IFN-g. Tetramer assay showed that CCL21-treated DCs enhanced generation of antigen-specific CTLs. Taken together, our data suggest that mDCs pre-stimulated by chemokine CCL21 enhanced migratory capacity to secondary lymphoid organs and produced strong cytotoxic T cells via IP-10 signaling pathway.

Disclosures:
No relevant conflicts of interest to declare.
Waveform Analysis Extends the Capabilities of aPTT and PT Clotting Assays for Detecting Factor Deficiency and Monitoring Procoagulant Drug Therapy
Authors: Leong Lilley , Jiang Xiaoqiao , Cifrese Jill , Murphy John E. , Haaning Jesper , Myles Timothy 
Published: October 2019

Abstract
Abstract 1205
Coagulation assays, such as activated partial thromboplastin time (aPTT) and prothrombin time (PT), are important for the diagnosis and treatment of bleeding disorders. Waveform analysis (WA) of the optical data generated in performing coagulation assays may provide additional information that will allow further understanding of coagulation disorders and their treatment (Hemophilia. 2006;12(suppl 3):76). Since WA employs assays currently in use in the clinical laboratory, it has the potential to be easily adapted to the clinical setting. To assess the utility of WA in procoagulant drug discovery for hemophilia, we determined its ability to: 1) discriminate between different factor deficiencies by factor-specific coagulation assays and by standard aPTT; and 2) discriminate between hemophilic plasma (with and without inhibitors) spiked with therapeutic proteins used to treat hemophilia. The ultimate goal of the study was to determine the feasibility of WA in monitoring hemophilia therapy in the clinic.
WA of plasma from factor (F)-deficient (FVIII, von Willebrand factor [type III vWD], FIX, and FVII) donors showed both absorbance and first-derivative waveform differences. Differences in first-derivative curves for severe hemophilia A plasma relative to vWD type III plasma were observed. The hemophilic plasma had a longer lag time (113 vs 93 seconds, respectively) and lower peak amplitude (29.7 vs 54.7 AU) than the vWD type III plasma. To determine whether WA might be useful for assessing the procoagulant activity of FVIII and FVIII bypass therapeutic agents, we compared waveforms of plasma-derived FVIII (WHO 8th) with pegylated FVIII (BAY 94–9027) in hemophilia A plasma or of rFVIIa variant and BAY 86–6150 in hemophilia A plasma containing 112 BU inhibitor. The waveforms obtained with WHO 8th and BAY 94–9027 were almost identical by ellagic acid aPTT with the first derivative giving lag times of approximately 31 seconds for both 5% WHO 8th and 5% BAY 94–9027. WA was able to detect rFVIIa and BAY 86–6150–mediated coagulation at 1.4 nM by first-derivative waveform in silica aPTT. Comparison of rFVIIa with BAY 86–6150 at this concentration indicated that BAY 86–6150 was more procoagulant than rFVIIa, with a shorter lag time of 75.9 seconds vs 97.9 seconds (measured by taking the first derivative of the waveform). Therefore, in addition to clot time or activity levels currently available by standard aPTT/PT, WA allows further insight into the kinetics of coagulation. In conclusion, WA holds promise as a global hemostasis assay that will extend the current utility of optical coagulation analyzers used in the clinic.

Disclosures:
Leong: Bayer HealthCare: Employment. Jiang:Bayer HealthCare: Employment. Cifrese:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment. Haaning:Bayer HealthCare: Employment. Myles:Bayer HealthCare: Employment.
Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin
Authors: Hoppensteadt Debra , Jeske Walter , Gray Angel , Walenga Jeanine M. , Wahi Rakesh , Thethi Indermohan , Fareed Jawed 
Published: October 2019

Abstract
Abstract 1251
Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence.

Disclosures:
No relevant conflicts of interest to declare.
A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors
Authors: Pulecio Julian , Laricchia-Robbio Leopoldo , Izpisua Juan Carlos , Barragan Montserrat , Vitaloni Marianna , Martinez Ignacio Sancho , Nivet Emmanuel 
Published: October 2019

Abstract
Abstract 1290
After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors.

Disclosures:
No relevant conflicts of interest to declare.
Enhanced Generation of Human Hematopoietic Stem/Progenitor Cells From ES and Patient Derived IPS Cells Using a Novel Compound Screen
Authors: Rönn Roger Emanuel , Moraghebi Roksana , Guibentif Carolina , Woods Niels-Bjarne 
Published: October 2019

Abstract
Abstract 1294
The ability to generate hematopoietic stem and progenitor cells from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using a small molecule compound library with our optimized iPS-2-blood lineage differentiation protocol, we have identified two novel chemical compounds that specifically enhance the generation of phenotypic adult hematopoietic stem cells. Compound A and compound B both enable increases of CD45/43+CD34+CD38-CD90+CD45RA- cells by 183+/−53%, n=5 and 275%, n=1, respectively. This is in comparison to DMSO carrier control wells, where the percentage of blood cells (CD45/43+) produced per total cells for these experiments was 73+/−2.9%, n=5. The increase in hematopoietic cell output using compound A was highly significant for the adult phenotypical hematopoietic stem cell fraction with a P-value of 0.03, n=5 (see Figure). Hematopoietic progenitor, CD45/43+ CD34+, counts were also slightly increased for compound A and B at 129+/−32%, n=5 and at 211%, n=1, respectively. Interestingly, no statistically significant increase in the number of total blood CD45/43+ cells was detected at the time of harvest, with either compound; at 108% +/− 8.4%, n=5, for compound A, and 164%, n=1, for compound B. In addition to the improvements, as measured by FACS, compounds A and B both increased the numbers of clonogenic progenitors as measured by CFU-assay, allowing for a 241+/−67%, n=4, and 443%, n=1, increase in total hematopoietic colony counts, respectively. These results identify 2 novel compounds with the ability to expand the more primitive fractions of hematopoietic cells with preferential expansion of the most primitive fraction of hematopoietic cells derived from human ES and iPS lines. We are currently performing transplantation experiments with cells generated using these compounds to assess their repopulating potential. Further studies are being performed to investigate the molecular mechanisms of these compounds for hematopoietic stem and progenitor cell generation from ES and iPS cells.

Disclosures:
No relevant conflicts of interest to declare.