bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endothelial Microparticles Improve Primary and Secondary Hemostasis in Patients with Von-Willebrand Disease
Authors: Trummer Arne , Birschmann Ingvild , Werwitzke Sonja , Ganser Arnold , Tiede Andreas 
Published: October 2019

Abstract
Abstract 2287
The bleeding phenotype in patients with von Willebrand disease (vWD) type 1 and 3 can usually be correlated to the amount and function of von Willebrand factor (vWF:Ag, vWF:RCo) but also to the impairment of functional coagulation assays: the ristocetin-induced platelet aggregation (RIPA) for primary hemostasis and the parameters of thrombin generation in a calibrated automated thrombogram (CAT, Thrombinoscope®) for secondary hemostasis. We therefore evaluated whether addition and depletion of endothelial microparticles (EMP) might influence these assays for samples of vWD patients.
EMP were obtained from stimulated endothelial cell lines, isolated either by ultracentrifugation or magnetic beads selection for Annexin V and detected and quantified by flow cytometry after labelling for Annexin V and vWF. For CAT, EMP concentration was adjusted to 3×103/μl, for RIPA to 2×104/μl. vWD patient samples were diluted 3:1 or 2:1 with control buffer (HEPES/CaCl) or EMP solution.
For thrombin generation, there was a significant shortening of lag time (6.8min vs 16.5min, p=.029) and time-to-peak (13.6min vs. 23.0min, p=.049) in vWD type 3 plasma (n=4) after addition of EMP using no reagent or MP reagent while there was no significant effect on these parameters in normal plasma. After addition of a tissue factor blocking antibody, the EMP effect could be largely reversed. For RIPA, we found a median maximum aggregation of 4% in vWD patients (n=6, vWF-Ag <40%). After addition of EMP, aggregation levels increased to 17.3%, which was significantly higher compared to control buffer (9.8%, p=.028), but lower compared to 250 IU/ml of a plasma-derived vWF product (Humate P®, 88.7%). To evaluate if significant amounts of vWF are bound to circulating microparticles in human plasma, we used magnetic bead selection to reduce MP counts in plasma of DDAVP stimulated vWD patients (n=10), because depletion of microparticles by 0.2μm filtration showed significant disruption of large vWF multimers. After MP reduction (1464/μl vs. 1863/μl, p=.036), both vWF:Ag (134.7% vs 161.1%, p=.005) and vWF:RCo (131.8% vs. 167.2%, p=.017) were significantly decreased.
In summary, we could show that in vitro generated endothelial microparticles are able to improve thrombin generation and platelet aggregation in vWD patients and that significant amounts of vWF are bound to circulating microparticles in human plasma after DDAVP stimulation. Thus, in vitro generated EMP might have the potential to improve replacement therapy for vWD patients.

Disclosures:
No relevant conflicts of interest to declare.
Transfusion of Stored Autologous Blood Alters Reactive Hyperemia and Circulating Nitrite Levels in Healthy Volunteers
Authors: Berra Lorenzo , Dzik Walter Sunny , Coppadoro Andrea , Bloch Kenneth D. , Yu Binglan , Spagnolli Ester , Steinbicker Andrea U. , Feelisch Martin , Fernandez Bernadette , Stowell Christopher , Warren Shaw H , Lin Tian , Zapol Warren M. 
Published: October 2019

Abstract
Abstract 2327

Background:
Transfusion of human blood stored for over 2 weeks is associated with increased mortality and morbidity. During storage, packed red blood cells (PRBC) progressively release hemoglobin, which avidly binds nitric oxide (NO). We hypothesized that the NO mediated hyperemic response following ischemia would be reduced after transfusion of PRBC stored for 40 days.

Methods and results:
We conducted a cross-over randomized interventional study, enrolling ten healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced PRBC, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index (RHI) measurements were performed before and 10 minutes, 1 hour, 2 hours, and 4 hours after transfusion. The RHI was lower (13% mean post-transfusion difference) after transfusion of 40-day as compared to 3-day stored PRBC (p=0.01). Plasma cell-free hemoglobin and bilirubin levels were higher after transfusion of 40-day than after 3-day stored PRBC (p=0.02 and 0.001, respectively). Plasma levels of potassium, LDH, haptoglobin, cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored PRBC, but not after transfusion of 3-day stored PRBC (p=0.01).

Conclusions:
Transfusion of autologous PRBC stored for 40 days is associated with increased hemolysis and reduced RHI, possibly due to enhanced NO scavenging by cell-free oxyhemoglobin.

Disclosures:
Bloch: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.
“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis
Authors: Komeno Yukiko , Yan Ming , Matsuura Shinobu , Lo Miao-Chia , Downing James R. , Zhang Dong-Er 
Published: October 2019

Abstract
Abstract 2329
Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications.

Disclosures:
No relevant conflicts of interest to declare.
C/EBPα-Induced MicroRNA 30c Inactivates Notch1 During Granulopoiesis and Is Downregulated In Acute Myeloid Leukemia
Authors: Katzerke Christiane , Madan Vikas , Gerloff Dennis , Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Mueller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard 
Published: October 2019

Abstract
Abstract 2368
The transcription factor CCAAT Enhancer Binding Protein alpha (C/EBPα) is crucial for normal granulopoiesis and frequently disrupted in acute myeloid leukemia (AML). Loss of expression or function of C/EBPα leads to a block of myeloid differentiation. MicroRNAs inhibiting translation of mRNA into protein were identified as critical players in stem cell development. We and others have already shown that C/EBPα exerts its effects by regulating microRNAs such as miR-223 and miR-34a. In a global microRNA-array screen we found miR-30c as a novel target of C/EBPα during granulocytic differentiation. Wild-type C/EBPα-p42 upregulates miR-30c expression, whereas the C/EBPα-p30 mutant, found in AML, does not. Furthermore, G-CSF upregulates miR-30c expression during granulocytic differentiation of primary human CD34-positive progenitor cells. C/EBPα induces miR-30c and downregulates Notch1, a putative target of miR-30c, on protein, but not mRNA level. A block of miR-30c by LNAs prevents C/EBPα–induced downregulation of Notch1 protein expression. miR-30c is a tumor suppressor and downregulated in various subtypes of AML. In mice, miR-30c shows a high expression in LSK (including hematopoietic stem cells), GMP (granulocytic monocytic precursors) and granulocytes. An induced knock-out of C/EBPα in mice leads to a significantly downregulation of miR-30c expression in bone marrow cells. Our data indicates that C/EBPα-induced miR-30c inactivates Notch1 during granulopoiesis and is downregulated in AML. These data reveal the importance of deregulated microRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Key Autophagy Genes in Primary AML Patients and Their Functional Role in Neutrophil Differentiation of APL Cells
Authors: Tschan Mario P. , Brigger Daniel , Schläfli Anna M. , Fey Martin F. 
Published: October 2019

Abstract
Abstract 2456
There is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. Autophagy may support the remodelling processes that are taking place during differentiation and maturation of different cell types. Generally, autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Furthermore, autophagy-inducing drugs support degradation of aggregated PML-RARA protein in acute promyelocytic leukemia (APL). We used customized 384-well micro fluidic cards to determine the expression of key autophagy genes (ATGs) in a large cohort of primary acute myeloid leukemia (AML) samples (n=122) and normal neutrophils (n=14). In general, we found significantly lower ATG mRNA levels in AML as compared to neutrophils for 21/38 autophagy genes investigated. Among the most significantly downregulated genes in AML were ATG1, ATG4D, ATG14, ATG16L2, WDFY3, BNIP3L, FIP200, GATE-16 and GABARAPL1. Given the low expression of these ATGs in AML, a disease mainly characterized by a block in differentiation, we hypothesized that these genes are implicated in myeloid differentiation. We therefore analyzed expression of selected ATGs in two APL cell line models for neutrophil differentiation. We found a significant 2- to 40-fold increase of ATG1, ATG7, WDFY3, GATE16 and GABARAPL1 in NB4 and HT93 APL cell lines upon all-trans retinoic (ATRA)-induced neutrophil differentiation. The expression of these genes did not change in ATRA-resistant NB4-R2 cells upon neutrophil differentiation indicating that their increased expression is functional in neutrophil differentiation. Next, we knocked down ATG1, WDFY3, GATE16 and GAPARAPL1 in NB4 cells using lentivirally delivered shRNAs. Inhibition of ATG1, WDFY3 and GATE16 but not GABARAPL1 significantly attenuated ATRA-induced neutrophil differentiation as measured by reduced levels of the myeloid markers CD11b, CEBPE and G-CSF-R. Analysis of the autophagic markers p62/SQSTM1 and LC3B revealed that inhibiting ATG1, WDFY3 and GATE16 not only diminished neutrophil differentiation of APL cells but also myeloid differentiation-associated autophagy (MDAA). In summary, low ATG expression is generally associated with an immature AML phenotype. Furthermore, knocking down key autophagy genes attenuates ATRA-induced neutrophil differentiation and MDAA of APL cells. Our studies indicate that autophagy is a putative tumor suppressor mechanism in AML/APL and already available autophagy-inducing drugs such as lithium chloride may be used in combination with differentiation-inducing drugs.

Disclosures:
No relevant conflicts of interest to declare.
Activated K-RAS Down-Regulated Small GTPases and Cell Motility of Myeloid Sarcoma-Inducing Leukemia Cells Through Erk Signaling Cascade
Authors: Fu Jen-Fen , Shih Lee-Yung 
Published: October 2019

Abstract
Abstract 2459
The molecular mechanism of myeloid sarcoma (MS) formation in the patients with acute myeloid leukemia (AML) is not clear. By analyzing the genetic mutations, we found that AML patients with combined MLL/AF10 and activated N-/K-RAS mutation had a high occurrence of association with MS. We had previously performed a retroviral transduction/transplantation assay and demonstrated that the activated KRAS mutation could not immortalize murine bone marrow (BM) cells, whereas MLL/AF10 alone induced myeloproliferative disease-like myeloid leukemia (MPD) in the transplanted mice. Combination ofMLL/AF10 and activated K-RAS mutation induced MPD in a shorter latency, in addition, multiple MS were detected in the abdominal-, subcutaneous-, or intramuscular fat of the transplanted mice. To understand the role of activated KRAS mutation in cooperation with MLL/AF10 in the MS formation, we performed an in vivo imaging analysis to compare cell migration between cells carrying single and combined mutations. Our results showed that the cells carrying combined mutations were slowly migrating to the omental and gonadal adipose tissues of the transplanted mice after intra-peritoneal injection. On the contrary, the cells carrying only MLL/AF10 were rapidly condensed and entered into blood stream. An in vitro transwell migration assay confirmed that the cells carrying combined mutations exhibited a reduced cell motility in response to SDF-1 treatment. Analysis of the Cxcr4 gene expression did not show significant difference between cells carrying single and combined mutations, suggesting that the slow migration was not attributed to lacking receptor for SDF-1. Further determination of the cell migration-related small GTPases revealed that the amount of total and GTP–bound Rac, Cdc42 and Rho was reduced in the cells carrying combined mutations. Moreover, treatment of SDF-1 further down-regulated protein expression and activity of these small GTPases. To understand which signaling pathway(s) linked the KRAS mutation to the down-regulated small GTPases, we analyzed the amount of total and phosphorylated signaling molecules and found that Erk, but not Jnk1 and p38 Mapk, had a similar protein expression and activation pattern as those of the GTPases, suggesting that the reduction in cell motility of the MS-inducing leukemia cells might be mediated via KRAS-Mek-Erk axis.

Disclosures:
No relevant conflicts of interest to declare.
Source and Purity of Factor VIII Products As Risk Factors for Inhibitor Development In Previously Untreated Patients with Severe Hemophilia A
Authors: Mancuso Maria Elisa , Mannucci Pier Mannuccio , Rocino Angiola , Garagiola Isabella , Tagliaferri Annarita , Santagostino Elena 
Published: October 2019

Abstract
Abstract 25

Background:
Inhibitor development is influenced by several genetic and environmental factors and the type of factor VIII (FVIII) products may play a role. Methods: We designed a cohort study whose novelty resides in the classification of products not only according to the plasmatic (pdFVIII) or recombinant (rFVIII) source of FVIII but also to the degree of purity expressed as FVIII specific activity per mg of protein. The role of FVIII product as risk factor for inhibitor development was evaluated in a multivariate model adjusting for potential confounders (i.e. age at first FVIII exposure, intensive treatment and prophylaxis). Cumulative incidences of all and high-responding inhibitors were calculated for the whole cohort of 721 patients with severe and moderate hemophilia A followed-up in 3 Italian Hemophilia Centers. Detailed treatment data from the first FVIII infusion up to inhibitor development or 150 exposure days were available for 377 patients and in this group of patients risk factors for inhibitor development including the type of FVIII product and its degree of purity (i.e. low/intermediate-, high-purity pdFVIII and rFVIII) were analysed. Results: The overall cumulative incidence of inhibitors was 22% (n=160; 130 high-responders, 18%) and it was lower in patients first treated with pdFVIII (107/586, 18%) than in those treated with rFVIII (53/135, 39%). Similar results were obtained by evaluating only high-responding inhibitors and patients with severe hemophilia. The adjusted hazard ratio of inhibitor development was 4.9 with rFVIII and 2.0 with high-purity pdFVIII (95%CI: 2.9–8.3 and 1.1–4.0), taking as reference low/intermediate-purity pdFVIII. There was no difference in the frequency of inhibitor testing between treatment groups. Sensitivity analyses - in patients who never switched product type, previously untreated patients, those treated on-demand and those with high-risk F8 mutations - confirmed an increased inhibitor risk in patients first treated with rFVIII or high-purity pdFVIII than in those treated with low/intermediate-purity pdFVIII. In fact, in all the aforementioned subgroups by multivariate analysis the risk of inhibitor development was invariably 3- to 6-fold higher in patients first treated with rFVIII than in those first treated with pdFVIII, and similar results were obtained for both all inhibitors and high-responding inhibitors. Conclusions: This study shows that the degree of purity of FVIII products influences inhibitor development independently from other risk factors, and emphasizes that differences exist also within pdFVIII products.

Disclosures:
No relevant conflicts of interest to declare.
The TOR Kinase Inhibitor INK128 Is Effective in Pre-B Acute Lymphoblastic Leukemia Models
Authors: Fruman David A , Janes Matthew Robert , Mallya Sharmila , Vu Collin C , Shieh Marie P. , Lilly Michael B. , Sender Leonard S , Martin Michael B , Ren Pingda , Liu Yi , Rommel Christian 
Published: October 2019

Abstract
Abstract 2585
Pre-B Acute Lymphoblastic Leukemia (B-ALL) remains a significant cause of morbidity and mortality in both adults and children. Targeting signaling pathways that sustain B-ALL cell survival is a promising strategy to improve treatment outcomes. The target of rapamycin (TOR) is a serine/threonine kinase that integrates signals from oncoproteins and the microenvironment to support ALL cell proliferation and survival. A single TOR enzyme in cells forms two multiprotein complexes, TORC1 and TORC2, with distinct regulation and substrates. The classical TOR inhibitor rapamycin inhibits only some functions of TORC1 and does not acutely inhibit TORC2. A new generation of TOR inhibitors has been developed that inhibit all outputs of both TORC1 and TORC2. These ATP-competitive, TOR kinase inhibitors (TOR-KIs) have improved anti-cancer activity in models of both solid tumors and hematological malignancies. Previously we reported that the TOR-KI compound PP242 is effective in mouse models of Philadelphia Chromosome-positive (Ph+) B-ALL, with minimal toxicity to normal hematopoietic cells or immune function (MR Janes et al., Nat. Med. 2010; 16:205). Here we present promising results on the efficacy of a second generation TOR-KI, INK128, in both Ph+ and non-Ph B-ALL. INK128 is an orally active, highly selective TOR-KI that is currently in phase I clinical trials for solid tumors, multiple myeloma and Waldenstrom's Macroglobulinemia. We find that INK128 has similar biological effects as PP242 on Ph+ B-ALL cells, but these effects are apparent at 5-fold lower concentrations in vitro and 80-fold lower doses in mice. In methylcellulose assays using Ph+ and non-Ph B-ALL specimens, INK128 greatly reduces colony numbers and enhances the efficacy of standard-of-care drugs. Both adult and pediatric non-Ph B-ALL specimens are sensitive to growth suppression by INK128. INK128 also causes apoptosis in B-ALL cells cultured on stromal cells. Importantly, INK128 suppresses human Ph+ B-ALL xenograft expansion in mice at doses that do not impair normal hematopoietic cell proliferation. INK128 is also less immunosuppressive than rapamycin. Together these results provide rationale for testing INK128 and other TOR-KIs in clinical trials of B-ALL therapy.

Disclosures:
Fruman: Intellikine: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janes:Intellikine: Employment. Martin:Intellikine: Employment. Ren:Intellikine: Employment. Liu:Intellikine: Employment. Rommel:Intellikine: Employment.