bluuebunny/crossref_metadata_2025_split huggingface dataset:
Imaging Asymmetric Division in Stem Cells and Cancer.
Authors: Reya Tannishtha 
Published: October 2019

Abstract
While it has long been speculated that mammalian stem cells undergo asymmetric and symmetric division, whether this actually occurs has not been clearly demonstrated. We have used a transgenic Notch reporter mouse, in which the GFP status of a cell acts as a sensor for the differentiated state, to image how mammalian hematopoietic stem cells divide and respond to signals during growth, differentiation, and oncogenic transformation. We show that mammalian hematopoietic stem cells have the ability to undergo both symmetric and asymmetric divisions and that the balance between these divisions is not hardwired in precursors but, instead, responsive to extrinsic and intrinsic cues. In addition, we show that oncoproteins can also influence a cell’s choice between symmetric and asymmetric division. Cumulatively, this work not only establishes a novel system in which division of early hematopoietic precursor cells can not only be tracked in real time, but also indicates that the distribution of symmetric and asymmetric division can be modulated in response to the microenvironment and subverted by oncogenes.
The Ontogeny of Definitive Hematopoiesis in the Zebrafish.
Authors: Bertrand Julien , Kim Albert , Cisson Jennifer , Stachura David , Traver David 
Published: October 2019

Abstract
Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed “primitive” and “definitive.” Primitive hematopoiesis rapidly generates erythrocytes and macrophages through monopotent precursors for immediate use in the developing embryo. Definitive hematopoiesis arises later and generates multipotent precursors, including hematopoietic stem cells (HSCs). We have recently performed the first prospective isolation and functional characterization of hematopoietic stem and progenitor cells in the zebrafish and shown that definitive hematopoiesis generates two distinct precursor types during embryogenesis. First to arise are erythromyeloid progenitors (EMPs) in the posterior blood island (PBI), cells that possess robust but transient proliferation potential but lack self-renewal and lymphoid differentiation capacities. Next to develop are HSCs in the aorta/gonad/mesonephros (AGM) region. Unlike EMPs, HSCs colonize the developing thymus to initiate T-lymphopoiesis and seed the pronephros, the site of adult hematopoiesis. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. By contrast, fate mapping of CD41:eGFP+ cells residing in the AGM region demonstrate robust colonization of the pronephros and thymus. Using time-lapse microscopy, we have observed directly the behaviors of the first HSCs to arise in the embryo. AGM HSCs, marked by a CD41:eGFP or c-myb:eGFP transgene, enter circulation to seed the thymic anlage and migrate along the pronephric tubules to seed the pronephros. We are currently performing retrospective time-lapse analyses to determine where in the early embryo HSC and EMP precursors are born. These data will be informative in analyzing where instructive ligands are expressed, including the Delta genes that provide paracrine signals to cells expressing the Notch receptor. We have demonstrated that Notch signaling is necessary for the generation of HSCs but dispensable for EMP formation. We have utilized new and existing Notch reporter lines to determine more precisely where and when HSC precursors receive Notch signals. Together, these studies highlight the power of the zebrafish system in combining genetic approaches with the direct imaging of hematopoietic stem and progenitor cells in living embryos.
Novel Mechanisms of Translational Control Linking Inflammation and Thrombosis.
Authors: Weyrich Andrew , Schwertz Hansjörg 
Published: October 2019

Abstract
The anatomy of platelets is unique and deceptively simple with a key feature being that platelets circulate without a nucleus. This has led to a common misconception that platelets lack the machinery to produce new proteins. However, over 40 studies from 1966 to 1997 collectively demonstrated and/or inferred that platelets synthesize protein. Recent investigations have not only confirmed these earlier reports but show that platelets possess a sophisticated translational apparatus consisting of template mRNAs, ribosomes, and translation initiation factors, such as eukaryotic initiation factor(eIF)-4E and eIF-2a. Current estimates indicate that platelets retain 4,000–6,000 transcripts and platelet-derived mRNAs are capped and polyadenylated at the 5′ - and 3′-untranslated regions, respectively. Data from our group also demonstrates that platelets contain a pool of precursor mRNAs (premRNAs), uridine-rich small nuclear RNAs, and essential splicing machinery. In response to activation, platelets splice specific pre-mRNAs into mature mRNAs. Published results from our group and others demonstrate that platelets use their splicing and translational machinery to synthesize new proteins. B-cell lymphoma 3 (Bcl-3), cyclooxygenase-1 (COX-1), integrin αIIbβ3, interleukin-1β (IL-1β), Na+-dependent transporter SVCT2, plasminogen activator inhibitor-1 (PAI-1), and tissue factor (TF) are among the most recently characterized proteins reported to be synthesized by platelets. The synthesis of these proteins is highly regulated and involves numerous checkpoints of control. Synthesis of Bcl-3, for example, is controlled by the mammalian Target of Rapamycin (mTOR), one of the most abundantly expressed proteins in platelets. In contrast, platelets use premRNA splicing pathways to regulate the production of pro-IL-1β protein. Processing of pro-IL-1β into its mature form is dependent on an innate cytosolic molecular complex referred to as the inflammasome. The inflammasome has been described in nucleated cells and consists of nucleotide-binding oligomerization domain (NOD)-like receptors, such as NALP-3, as well as caspase-1. Although not previously-described, recent studies from our group demonstrate that platelets possess a functional inflammasome that regulates pro- IL-1β maturation. Identifying and characterizing gene expression pathways in platelets will be an exciting area of investigation throughout the next decade. Our group is actively searching for new targets and, in addition to the inflammasome, we found that platelets harbor reverse transcriptase (RT) activity. Inhibition of RT activity modulates protein synthesis and differentiation responses in platelets. Gene expression pathways including the inflammasome and RT provide platelets with previously-unrecognized mechanisms for controlling thrombosis and inflammation and may be targets for future therapeutic interventions.
On Our Way to a Better Understanding of CD8 T Cell Differentiation and Memory T Cell Development.
Authors: Kaech Susan 
Published: October 2019

Abstract
As certain acute viral or bacterial infections resolve, two types of effector CD8 T cells are formed that differ in their longevity and memory T-cell potential. These cell types can be distinguished from one another by surface markers, namely IL-7R and KLRG1. Memory precursor effector cells are enriched in the IL-7Rhi KLRG1lo cell population, and short-lived effector cells are enriched in the KLRGhi IL-7Rlo population. These two populations become discernible during the peak of clonal expansion. Upon expression of KLRG1, the effector cells are committed to becoming short-lived. Our recent progress toward identifying the signals and genetic pathways that control the formation of these different types of effector T cells will be discussed.
Biology, Therapy, and Resistance of Myeloid Malignancies Initiated by Hyperactive Ras.
Authors: Shannon Kevin 
Published: October 2019

Abstract
Ras proteins regulate cell fates by cycling between active GTP-bound and inactive GDP-bound states (Ras·GTP and Ras·GDP). Ras·GTP modulates cell fates by activating effector pathways that include the Raf/MEK/ERK, phosphoinositol 3’-kinase (PI3K)/Akt, and Ral·GDS cascades. Signaling terminates when Ras·GTP is hydrolyzed to Ras·GDP. GTPase activating proteins (GAPs) are negative regulators of Ras output that increase the rate of GTP hydrolysis. Mammalian cells express two major GAPs – p120GAP and neurofibromin. The latter is encoded by the NF1 tumor suppressor gene, which is mutated in persons with neurofibromatosis type 1 (NF1). Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and other cancers. Somatic RAS mutations are also common in myeloid malignancies, and other leukemia-associated mutations, such as FLT3 internal tandem duplications, PTPN11 point mutations, and the BCR-ABL fusion protein deregulate Ras signaling. Together, the prevalence of oncogenic RAS mutations and the existence of these alternative genetic mechanisms establish hyperactive Ras as a major therapeutic target. However, developing inhibitors of oncogenic Ras proteins is extremely challenging due to structural considerations and because an effective drug must restore normal biochemical activity (i.e., repair a broken enzyme). Strains of mice carrying conditional mutant alleles of Nf1, oncogenic Kras, and oncogenic Nras are novel reagents for understanding how cells remodel signaling networks in response to hyperactive Ras and for performing pre-clinical trials. Use of the Mx1-Cre transgene to ablate Nf1 or to activate oncogenic KrasG12D or NrasG12D expression in hematopoietic cells causes myeloproliferative disorders (MPDs) that model JMML and chronic myelomonocytic leukemia (CMML). We are using retroviral insertional mutagenesis (RIM) to identify cooperating mutations that might induce progression from MPD to acute myeloid leukemia (AML). CI-1040, a potent inhibitor of MEK, unexpectedly had no beneficial effects in Nf1 mutant mice with MPD. By contrast, MEK inhibition induced regression of Nf1-deficient AMLs. These AMLs uniformly developed resistance in vivo, despite equivalent biochemical inhibition of the target in paired sensitive and resistant clones. Analysis of retroviral insertions in resistant AMLs revealed outgrowth of a pre-existing clone during CI-1040 administration, and we have implicated RasGRP1 and p38α as modulating resistance in vivo. These data emphasize the importance of cell context in the response to targeted agents and establish a tractable in vivo system for identifying genes that modulate therapeutic efficacy and for probing mechanisms of acquired resistance.
Transient Inhibition of Transforming Growth Factor-β1 in Human Blood-Derived CD34+ Cells Enhances Survival and Proliferation in Vitro and Vascular Reparative functions In Vivo.
Authors: Bartelmez Stephen , Bhatwadekar Ashay , Iversen Patrick , Ruscetti Francis W , Grant Maria 
Published: October 2019

Abstract
Abstract 3546
Poster Board III-483
CD34+ cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation as well as diminished migration prowess which is largely due to lower levels of bioavailable nitric oxide (NO). We asked whether a transient TGF-β1 blockade in CD34+ cells from diabetics would improve their reparative ability given that TGF-β is a key factor modulating stem cell quiescence. Peripheral blood lin-CD34+ cells or lin-CD34+CD38+/− cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1 -PMO), demonstrated to inhibit TGF-β1 protein expression in stem cells. Cells were then analyzed for cell surface TGF-β Receptor 2 (TGF-β R2) and CXCR4 expression, their ability to generate NO, their ability to migrate toward SDF-1, their ability to survive in the absence of added growth factors, and tested in vivo for their vascular reparative ability. After TGF-β1-PMO treatment, healthy and diabetic CD34+CD38+ and - cells downregulated TGF-βR2, upregulated CXCR4 expression, survived in the absence of added growth factors ex vivo and migrated more efficiently to SDF-1 compared to controls. TGF-β1-PMO treated diabetic CD34+ cells restored NO production to non-diabetic levels. In contrast, TGF-β1-PMO did not enhance NO generation in CD34+ cells from healthy subjects. Using an in vivo retinal ischemia reperfusion model, we observed that TGF-β1-PMO treatment increased the ability of both healthy and diabetic CD34+ cells to home to injured capillaries compared to control PMO treated cells. As also observed in our current study, a reduction of TGF-β1 levels in murine hematopoietic stem cells correlates with a reduction in TGF-βR2 expression which may induce proliferation in vivo. We also show that both diabetic and healthy lin-CD34+CD38+ cells express TGF-βR2 by FACS. In contrast, only healthy lin-CD34+CD38- cells expressTGF-βR2 while diabetic lin-CD34+CD38 - cells express essentially no cell surface TGF-βR2 (<5 % of cells are TGF-βR2+). Our results suggest that a transient blockade of TGF-β1 may represent a promising therapeutic strategy in restoring vascular reparative function in diabetic CD34+ cells.

Disclosures:
Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Head, SRB, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iversen:AVI-Biopharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Genetically-Engineered Endothelial Cells Implanted Into the Omentum of Hemophilia A Dogs Provides Long-Term Circulating FVIII Resulting From Sustained FVIII Expression and Persistent Cell Viability.
Authors: Ozelo Margareth C , Matsui Hideto , Hegadorn Carol A. , Harpell Lori , Powell Sandra , Hulvat James F , Winterborn Andrew , Handforth Janine , Hough Christine , Lillicrap David 
Published: October 2019

Abstract
Abstract 3578
Poster Board III-515
Ex vivo delivery of therapeutic transgenes provides an additional level of safety as it avoids systemic administration of viral vectors. Our group has shown that autologous blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector delivery system containing the FVIII transgene is a promising gene therapy strategy for hemophilia A. We have shown that subcutaneous implantation of factor (F) VIII-expressing BOECs in a murine model of hemophilia A can produce therapeutic levels of FVIII that are sustained for more than 6 months. However, to improve the levels of FVIII expression and cell viability we wanted to evaluate the omentum as an alternative site for BOEC implantation. Initially this strategy was evaluated in two normal dogs. One and three months after delivery of the cells, immunostaining of biopsies from the injection sites showed the presence of viable cells expressing FVIII and evidence of neovascularization. To evaluate the efficacy of this strategy, a hemophilia A dog received 5×108 transduced autologous BOECs that expressed high levels of FVIII in vitro (1.5 IU/106 cells/24hrs). We used autologous fibrinogen as a vehicle for the cells along with canine endothelial growth factors (VEGF and bFGF). For the implantation procedure the dog received prophylaxis with canine cryoprecipitate transfusions. FVIII antigen levels (FVIII:Ag) of between 20 and 50 ng/mL continue to be detected in the plasma 8 months post-implantation, indicating that these cells remain viable and express persistent high levels of FVIII over an extended period of time. However, two weeks after the procedure, the dog developed an anti-FVIII immune response comprising both inhibitory and non-inhibitory antibodies, and therefore no FVIII coagulant activity (FVIII:C) was detected. With a view to preventing the development of the anti-FVIII immune response, we used immunosuppression with cyclophosphamide in two additional hemophilia A dogs. Each of these dogs received 5 × 108 transduced autologous BOECs. In place of fibrinogen, these cells were delivered in a gel comprised of synthetic, heparin-binding peptide-amphiphiles (HBPA) and heparan sulfate, along with canine VEGF and bFGF. The peptide gel prolongs the activity of these growth factors and protects them from proteolysis, enhancing their angiogenic activity. HBPA gel has been shown to increase vascularization of cell transplant sites, which should improve BOEC survival in the omentum. The procedure of implanting the genetically modified BOECs was completed without complications in all hemophilia A dogs and we are continuing to evaluate the efficacy of this strategy.

Disclosures:
Hulvat: Nanotope Inc.: Employment.
Prochemerin Is a New Substrate for Thrombin.
Authors: Du Xiaoyan , Myles Timothy , Morser John , Leung Lawrence L. 
Published: October 2019

Abstract
Abstract 3591
Poster Board III-528

Introduction
Prochemerin is a 163 amino acid precursor protein with a C-terminal domain highly susceptible to proteolysis. Its chemotactic activity is unmasked upon C-terminal cleavage by proteases of the coagulation, fibrinolytic and inflammatory systems. Here, we studied whether thrombin is able to cleave prochemerin to generate active forms of chemerin.

Methods
The 15mer peptide prochemerin C-terminal sequence (YFPGQFAFSKALPRS) or prochemerin was incubated with thrombin at different concentrations and times. The reactions were stopped by addition of PPACK before determining their chemotactic activity in a transwell assay using CMKLR1-transfected cells and their mass by mass-spectrometry.

Results
Thrombin (0-100 nM) dose-dependently cleaved 15mer to 14mer (YFPGQFAFSKALPR). Over a longer reaction time, the 10mer (YFPGQFAFSK) was also detected. The 15mer was almost inert in the chemotaxis assay but thrombin-cleaved 15mer caused migration of CMKLR1 transfectants. The 14mer and 10mer at 1 μM induced CMKLR1 cell migration at a rate of 3200 and 2800 cells/ml, but 1 μM 15mer did not induce any chemotaxis. Thrombin can also cleave the 14mer to a 10mer as determined by mass spectrometry. Using selected thrombin mutants for the Na+ binding site (E229K) and the active site YPPW-insertion loop (W50A), we found that thrombin hydrolysis of the 15mer was dependent on the Na+ bound “fast” form of thrombin and active site topology. The 10mer could be further activated by carboxypeptidase N (CPN) by removing the C-terminal lysine, whereas the C-terminal arginine of 14mer could not be cleaved by CPN, which did not affect its activity. Full-length prochemerin was also activated by thrombin (100nM) and the chemotactic activity further increased by CPN (50nM) about 6 fold.

Conclusions
Prochemerin is a new substrate for thrombin. Thrombin-cleaved chemerins are active chemoattractants in chemotaxis. This extends the molecular link between blood coagulation and CMKLR1-mediated immune responses.

Disclosures:
No relevant conflicts of interest to declare.
TLR7/8 Differentially Regulates Fcγ Receptor Expression and Function.
Authors: Butchar Jonathan P , Mehta Payal , Justiniano Steven E , Guenterberg Kristan D , Kanneganti Thirumala-Devi , Amer Amal , Muthusamy Natarajan , Jarjoura David , Marsh Clay B , Carson William E , Byrd John C , Tridandapani Susheela 
Published: October 2019

Abstract
Abstract 3594
Poster Board III-531
Activation of Toll-like Receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors, and such compounds are in clinical use. We examined the effect of one agonist, R848, on monocyte Fcγ receptor (FcγR) expression. Results showed that overnight treatment with R848 increased expression of FcγRI, FcγRIIa and the common γ-subunit. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. We then treated bone marrow-derived macrophages from Wild-type, TLR7−/−, MyD88−/−, TRIF−/− and Cryopyrin−/− mice with R848 to test which pathway influenced FcγR expression. FcγR expression remained unchanged in TLR7−/− and MyD88−/− mice, suggesting that the TLR7 / MyD88 pathway was required. In order to test the functional effect of R848 treatment on monocyte FcγR, we treated monocytes with R848 and then co-incubated them with Rituximab-coated Raji B-cell lymphoma cells. Flow cytometric analysis showed that the treated monocytes led to a significantly greater decrease in intact antibody-coated Raji cells. Further, R848 treatment synergistically increased monocyte FcγR-mediated cytokine production. In conclusion, there are both regulatory and functional links between the TLR7/8 and FcγR pathways. This has implications for both antitumor immunotherapy and autoimmune diseases.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of a Novel Anti-P-Selectin Aptamer ARC5690 On Venous Inflammation Following Acute Venous Thrombosis.
Authors: Diaz Jose Antonio , Lester Patrick A , Kurz Jeffrey C , Wrobleski Shirley K , Hawley Angela E , Sigler Robert E , Wakefield Thomas W , Schaub Robert G , Myers Daniel 
Published: October 2019

Abstract
Abstract 3599
Poster Board III-536

Introduction
P-selectin is a pro-inflammatory molecule that increases neutrophil recruitment to the vein wall at the site of thrombosis. Aptamers are short single-stranded oligonucleotides with the ability to bind small molecules and diverse proteins such as P-selectin. P-selectin could be targeted by a specific inhibitor or novel aptamer to limit inflammation following deep vein thrombosis (DVT). In this study, the effect of a novel anti-P-selectin aptamer on reducing DVT associated inflammation was evaluated.

Methods
Male C57BL/6J mice (20-25g) were placed into groups: anti-P-selectin aptamer ARC5690 (APSA, 2mg/kg, 5mg/kg, and 10mg/kg intraperitoneal [IP]), anti-P-selectin control aptamer “scrambled sequence” (PSCA 1mg/kg and 10 mg/kg IP), anti-P-selectin antibody (APSB 0.2mg/kg IP), ligation only (LO), or true control (TC). Inferior vena cava (IVC) ligation, below the renal vein, was performed on all groups except TC. Groups PSCA and APSA received daily injections starting two days pre-IVC ligation and continued up to 3 days post-IVC ligation. Groups TC and LO did not receive test compounds. At 3 days post-IVC ligation, mice were euthanized. The IVC was harvested and weighed or submitted for vein wall morphometric inflammatory cell histological analysis. Blood was collected via cardiac puncture for plasma soluble P-selectin (sP-sel) protein analysis. Data was analyzed using a student t-test and Kruskal-Wallace test with a Dunn's multiple comparison test.

Results
The total leukocyte counts in APSA groups 5 mg/kg and 10 mg/kg were significantly reduced compared to the PSCA groups and APSA group 2 mg/kg (p<0.05). The inflammatory cell count in APSA groups 5mg/kg and 10mg/kg confirmed this same trend for neutrophils, monocytes, lymphocytes versus the PSCA and APSA 2 mg/kg groups. Increased neutrophil counts were observed in the PSCA group compared to the TC group Figure 1). The thrombus weight (TW) was significantly decreased in the APSA 5mg/kg group compared to the LO group and the PSCA 1mg/kg group (p<0.05). The TW in the APSB group was significantly decreased compared to the LO group, the PSCA group (1mg/kg and 10mg/kg). There was a positive correlation between TW and sP-sel (Spearman correlation r=0.55).

Conclusions
ARC5690 showed a significant anti-inflammatory effect at the 5 mg/kg and 10 mg/kg doses. A reduction in TW was observed using an anti P-selectin aptamer with a positive correlation between TW and sP-sel levels, providing evidence that sP-sel could be a clinical biomarker of venous thrombosis. The reduction of venous inflammation by p-selectin blockade could have benefit in reducing the sequelae of DVT including venous fibrosis.

Disclosures:
Kurz: Archemix: Research Funding. Schaub:Archemix: Research Funding.