bluuebunny/crossref_metadata_2025 huggingface dataset:
From Venus and Mars: Gender Specific Analysis of Blood Platelets
Authors: Schubert Peter , Coupland Danielle , Walsh Geraldine M. , Chen Cindy , Devine Dana V.
Published: October 2019

Cardiovascular disease epidemiological studies reveal compelling evidence that this disease is strongly influenced by gender. Other laboratory based studies have suggested that gender disparities can also be detected in platelet physiological measurements. These observations have led us to ask whether gender differences may play a role in platelet quality and integrity during storage. We have applied proteomic approaches to gain an understanding of the molecular mechanisms of gender-specific platelet signal transduction and its potential significance to transfusion medicine. Buffy coat platelet concentrates (N=4) pooled from four male or female donors, respectively, were analyzed by 2D gel electrophoresis and revealed 15 reproducible protein differences between their individual platelet proteomes. Validation by western blot analysis confirmed the proteomic data demonstrating differences in the expression profiling of proteins including 14-3-3, myosin, platelet factor 4 and SH3-domain binding protein glutamic acid-rich like protein as well as in posttranslational modification of proteins including pleckstrin and 14-3-3. These findings spurred a more detailed gender-specific analysis of single donors. In vitro measures (N=28 of each gender) for platelet function revealed that female platelets showed an averaged 15% higher level of activation as well as GPIIb/IIIa activation monitored by CD62P or Pac-1 binding, respectively. This point towards differences in signal transduction and western blot analyses of the platelet proteome of these individuals showed an averaged two to three-fold higher expression of signaling proteins including 14-3-3, Rap1, RhoA and RhoGDI. Furthermore, female platelets contain a two-fold higher level of activated Rap1 supporting the gender-influenced signaling hypothesis. To test whether these gender-specific results might have an impact on transfusion medicine, biochemical analyses of buffy coat platelet concentrates during storage were carried out. Preliminary data indicate that female platelets maintain the higher level of platelet activation during a 7-day storage period paralleled with elevated metabolic activity as monitored by glucose and lactate levels, respectively. These data can be used to assess other potential molecular mechanisms underlying the differences in the gender-specific signaling platelet proteome. Thus far, the clinical significance of these findings in transfusion medicine is unknown.
Gfi1 as a Regulator of Hematopoietic Differentiation.
Authors: Grimes H. Leighton , Velu Chinavenmeni S. , Horman Shane , Chaubey Aditya , Bourdeau Tristan , Xu Haiming , Zhu Jinfang , Paul William , Colmenares Clemencia , Williams David A. , Gebelein Brian
Published: October 2019

Growth Factor Independent-1 (Gfi1) is a transcriptional repressor originally identified as a target of Moloney leukemia virus insertion mutagenesis. Gfi1 potently collaborates with Myc and Pim oncogenes to mediate T cell transformation. Retroviral insertions which activate Gfi1 are among the most frequent events in MoMLV-induced T cell, but not myeloid, leukemias. Orthologous proteins in Drosophila (Senseless) and C.elegans (Pag-3) are key regulators of developmental decisions. Gfi1−/− mice display lymphopenia, neutropenia, and abnormally proliferative HSC and progenitors which eventually lead to bone marrow failure. Humans with bone marrow failure syndromes, such as Severe Congenital Neutropenia or Non-Immune Chronic Idiopathic Neutropenia of Adults, display mutations in GFI1, which produce dominant negative acting GFI1 proteins. The SCN-associated GFI1N382S mutant proteins derepress a subset of GFI1 target genes, including CSF1 and CSF1R, to block granulopoeisis. Normally, Gfi1 antagonizes monopoiesis mediated by Pu.1, Egr1, Egr2, and Nab2. Thus, Gfi1 loss of function may impair granulopoiesis by failing to successfully repress monopoeitic differentiation. GFI1 dysfunction may also underlie Specific Granule Deficiency. GFI1 is able to synergize with C/EBP transcription factors to activate genes, such as neutrophil collagenase. Gfi1 may, therefore, enhance granulopoietic differentiation through C/EBP factors. Though great progress has been made in understanding Gfi1 biological and biochemical functions, we are only beginning to understand the role of Gfi1 in integrating hematopoietic transcriptional programming. We find that Gfi1 signaling controls both the differentiation and transformation of myeloid progenitors through an evolutionarily conserved transcriptional and post-transcriptional network.
Immunological Memory: Remembering Our Pathogens.
Authors: Ahmed Rafi
Published: October 2019

Acute viral infections induce long-term humoral and cellular immunity. However, the nature of T- and B-cell memory is different. Antiviral B-cell memory is usually manifested by continuous antibody production that lasts for many years after infection or vaccination. In contrast, the effector phase of the T cell response is short-lived (a few weeks), and “memory” in the T-cell compartment results from the presence of memory T cells, which are found at higher frequencies and can respond faster and develop into effector cells (i.e., CTL or cytokine producers) more efficiently than can naïve T cells. In this talk, I will discuss the following aspects of immunological memory:

Functional differences between naïve and memory T cells; Memory T cell differentiation and memory cell subsets; and Protective immunity by memory CD8 T cells.
Homeostasis of Mature T Cells.
Authors: Surh Charles D.
Published: October 2019

The overall size and composition of the mature T cell pool is regulated by homeostatic mechanisms. A hallmark of homeostatic regulation is the ability of the T cells to undergo spontaneous “homeostatic” proliferation in response to severe lymphopenia. By defining the factors that drive such homeostatic proliferation, we have determined that homeostasis of naïve and memory T cells is controlled by signals from contact with self-MHC/peptide ligands and/or two cytokines, namely IL-7 and IL-15. In addition, we have recently described a simple and highly effective way to administer IL-2, IL-7, IL-15, and other cytokines to strongly expand selective populations of T cell under normal physiological conditions. This new approach overcomes the current limitations that prevent therapeutic use of these essential T cell regulators in their native form. Identification of these homeostatic factors has already proven to be highly clinically relevant in devising novel therapeutic modalities for treatment of cancer and restoration of the immune deficiency from lympho-depleting regiments and advanced aging. The new method of delivering exogenous cytokines should further expand the clinical applicability of homeostatic cytokines.
Genome-Wide Epigenetics in Myeloid Leukemias.
Authors: Figueroa Maria Eugenia , Greally John , Delwel Ruud , Melnick Ari M.
Published: October 2019

While the role of genetic alterations in cancer is well-recognized, epigenetic deregulation has only recently been identified as a hallmark of malignant transformation. The term “epigenetic” refers to a heritable regulation of gene expression that is not dependent on changes in the DNA sequence. These epigenetic modifications – including but not limited to DNA methylation and covalent modifications of histone tails – play a crucial role in determining chromatin structure and gene expression. Abnormal epigenetic regulation can lead to aberrant chromatin structure and deregulation of transcriptional activity. Epigenetic lesions can affect cancer-related genes, such as CDKN2B, CDKN2A, RB, and BRCA1, and it is not rare for epigenetic lesions to accompany genetic mutations of these and other genes, suggesting that epigenetic deregulation can form a part of the multi-step process of oncogenesis. An alteration in the distribution of DNA methylation has been demonstrated in AML as well as in other malignancies. Generally, intergenic DNA methylation is reported to decrease and promoter methylation to increase. Hypomethylation of DNA can lead to genomic instability and further increase the number of genetic lesions, while promoter hypermethylation has been associated with aberrant silencing of tumor suppressor genes. Altered levels of acetylation at specific histone residues were also shown to be associated with aberrant chromatin structure and gene deregulation in AML. Several oncogenic transcription factors and fusion proteins, such as PML-RARalpha, and AML1-ETO, can introduce aberrant epigenetic programming in myeloid cells through recruitment of epigenetic modifying enzymes to their target genes. However, the emerging field of epigenomic profiling has yielded evidence that epigenetic deregulation in AML is more profound and cannot always be linked to the presence of a given fusion protein. The mechanisms leading to genome-wide epigenetic deregulation still remain largely unidentified, although environmental factors and aging can contribute to this process. Current epigenetic profiling studies have revealed that DNA methylation or histone modification patterns can identify biologically distinct forms of AML that may not be readily identified through other methods. New data suggest that specific DNA methylation profiles may be associated with response to therapeutic agents, including epigenetic-targeted drugs. Numerous epigenetic candidate biomarkers have been recently described in both myeloid and lymphoid malignancies. Integrative analysis of DNA methylation, histone modifications, and gene expression may synergize to identify in far greater depth than single platform studies differences in gene regulation among leukemias. Overall, the emerging field of epigenomics provide a new opportunity to more accurately identify biological variation and therapeutically target acute myeloid leukemias.
Imaging Asymmetric Division in Stem Cells and Cancer.
Authors: Reya Tannishtha
Published: October 2019

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

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

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.
Assembly, Storage, and Secretion of Von Willebrand Factor.
Authors: Sadler J. Evan
Published: October 2019

Von Willebrand factor (VWF) plays a central role in hemostasis, and dysregulation of VWF can cause either bleeding or thrombosis. Endothelial cells assemble VWF multimers in two stages that occur in distinct intracellular compartments: proVWF subunits dimerize in the endoplasmic reticulum through “tail-to-tail” disulfide bonds between C-terminal CK domains, and the proVWF dimers form enormous multimers in the Golgi through “head-to-head” disulfide bonds between N-terminal D3 domains. The finished multimers are packaged into ordered tubules within storage organelles called Weibel-Palade bodies (WPB), and tubular packing is necessary for the secretion of VWF filaments that have normal hemostatic function. We have recreated VWF tubule assembly in vitro, starting with pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D’D3 domains. No other cellular proteins or components of WPB are needed. Assembly requires low pH and calcium ions, similar to conditions in the Golgi. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D’D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Secretion into the blood exposes VWF tubules to neutral pH conditions, releasing the constraints that maintain tubular packing and permitting the orderly unfurling of large VWF multimers. Some VWF multimers bind platelets and initiate the growth of platelet-rich thrombi. Under normal circumstances, these thrombi are limited in size by ADAMTS13, a metalloprotease that cleaves VWF multimers and releases the platelets. The absence of large VWF multimers causes bleeding that is typical of von Willebrand disease. Conversely, congenital or acquired deficiency of ADAMTS13 prevents the dissolution of VWF-platelet aggregates, which can cause the widespread microvascular thrombosis that characterizes thrombotic thrombocytopenic purpura (TTP). Interestingly, ADAMTS13 deficiency alone need not trigger TTP. Some patients persist for months or years without active disease but become ill whenever they suffer additional inflammatory stress associated with infection, surgery, or pregnancy. Thus, interactions between inflammatory mediators and endothelial cells can determine the course of VWF-dependent thrombosis. The mechanisms responsible for these phenomena remain poorly understood.
On Our Way to a Better Understanding of CD8 T Cell Differentiation and Memory T Cell Development.
Authors: Kaech Susan
Published: October 2019

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

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 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 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 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 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 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.
Disruption of Bis Leads to Deterioration of Mesenchymal Stromal Cells Predominantly Affecting Vascular Compartment of Hematopoietic Stem Cell Niche.
Authors: Oh Il-Hoan , Kwon Kyong-Rim , Ahn Ji-Yeon , Kim Myungshin , Lee Jeong-Hwa
Published: October 2019

Abstract 3627

Poster Board III-563

The stem cell niche plays an important role in the microenvironmental regulation of hematopoietic stem cells (HSCs), but the integration of niche activity remains poorly understood. In this study explored the hematopoietic defect of mice disrupted with Bis/BAG-3/CAIR-1, a protein related to apoptosis and response to cellular stress and show that functional loss of bis leads to series of hematopoietic derangements due to perturbation of vascular stem cell niche. First, mice with targeted disruption of bis (bis−/−) exhibited severe hypocellularity in the bone marrows and spleen starting from 16 days after birth. Affected mice exhibited loss of primitive neonatal HSCs (CD34+Lin-Sca-1+c-kit+) and defect in early stage B-lymphopoiesis including common lymphoid progenitors (IL-17R+LSK), pre-B and pro-B cell populations, but not for mature recycling B-lymphocytes (IgD+B220+). However, this hematological defect of bis−/− mice was not reproduced when bis−/− bone marrow cells were transplanted into wild-type (WT) recipients, pointing to the microenvironmental origin of the phenotypes. Subsequent analysis of bis−/− mice bone marrow revealed a characteristic defect in the mesenchymal stromal component that included a quantitative loss of stromal cells (CD45-CD31-TER119-CD105+) in the bone marrows and rapid sensescence of stromal cells comprising colony forming unit-fibroblast (CFU-F) when re-plated in the ex-vivo culture. Moreover, mesenchymal stromal cells expressing CXCL-12 or IL-7 was lost in the affected bone marrows with lowered density of vascular development, together indicating a perturbation of peri-vascular stem cell niche in the bone marrow. In contrast, no abnormalities were observed in the growth and hematopoietic supporting activities of osteoblasts obtained from bis−/− mice. Collectively, these results indicate that Bis functions to mediate cellular regulation of the stem cell niche activities selectively on the vascular compartment without affecting osteoblastic niche, and suggest that Bis may serve as a molecule that can bridge the microenvironment niche and cellular stress/apoptotic signals during the in-vivo orchestration of hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
MT1-MMP Is Required for Hematopoietic Maturation in the BM Niche.
Authors: Nishida Chiemi , Beate Heissig , Seiki Motoharu , Nakauchi Hiromitsu , Hattori Koichi
Published: October 2019

Abstract 3634

Poster Board III-570

Specialized niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. Activation of the fibrinolytic pathway via matrix metalloproteinase-9 (MMP-9) resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate e.g. MMP-9. To investigate the role of MT1-MMP in hematopoiesis, we used MT1-MMP deficient mice. MT1-MMP−/− mice examined 12 days after birth showed pancytopenia and reduced numbers of bone marrow mononuclear cells (BMMCs) and splenocytes. BM cytospins from MT1-MMP−/− mice showed mild perturbations in erythropoiesis and a more severe impairment of myelopoiesis. Although all lineages were present, the ratio of erythroid to myeloid precursors increased from 0.36 in wildtype to 0.60 in MT1-MMP−/− mice. Myeloid and erythroid cell differentiation was impaired in MT1-MMP−/− BMMCs. The numbers of colony forming unit cells (CFU-C) was reduced in MT1-MMP−/− BMMCs. In contrary, the number of immature hematopoietic cells (CFU-S8, KSL cells) was augmented in MT1-MMP−/− BMMCs. FACS analysis of BM cells showed a decrease in the percentage of mature B cells with an increase number of Pro-B and immature B cells in MT1-MMP−/− BMMCs relative to controls. MT1-MMP−/− BM cells showed lower expression of CXCL12 and KitL, typical niche growth factors important for myelopoiesis and lymphopoisis. Thus, MT1-MMP is required for normal hematopoietic differentiation of lymphoid and myeloid lineage cells, most likely due to growth factor defective niche cells.

Disclosures:

No relevant conflicts of interest to declare.
Modified International Prognostic Score (IPS) for Predicting the Need for Bone Marrow Biopsy (BMB) in Hodgkin's Lymphoma (HL).
Authors: Muslimani Alaa A , Spiro Timothy , Chaudhry Asif A. , Taylor Harris C , Jaiyesimi Ishmael , Vashist Praveen , Daw Hamed A
Published: October 2019

Abstract 3661

Poster Board III-597

Introduction

BMB is frequently performed during the staging process of patients (pts) with HL. Previous data suggested routine use of this procedure may be unnecessary. The Ann Arbor classification is currently used to detect pts requiring BMB, despite sensitivity (sen) of 100%, specificity (spec) is only 40%. We sought to determine whether a modified IPS [Hasenclever and Diehl (1998)] would yield greater spec while maintaining good sen.

Methods

We retrospectively reviewed charts of 1215 histologically proven HL pts from Jan 2000-Dec 2008 at Cleveland Clinic Taussig and Moll Cancer Centers. Of 1215 pts, 1089 had BMB (90%). 876 pts were included in our study. 213 were excluded due tor un-interpretable BMB or missing data. Each component of IPS was equally weighted such that one point is assigned to each positive variable [male sex, age ≥45, albumin (alb) <4 g/dL, hemoglobin (hem) <10.5 g/dL, stage IV, white blood cell (WBC) ≥15,000/mm3, lymphocyte (lymph) <600/mm3 and/or <8% of total WBC], Multivariable logistic regression was initially used to assess the association between bone marrow involvement (BMI) and each factor in the IPS. All 7 factors were significant (p<.001 for sex, age, albu, hem, stage and lymph; .07 for WBC); and therefore recursive partioning algorithm was used to identify a cutoff for determining BMI.

Result

88 pts (10%) had BMI, we used a modified IPS formally by calculating a score that uses “weights” based on the results of a multivariable model including all 7 factors and the relative magnitude of the associated regression coefficients (RC), [modified IPS= WBC+2x(sex+age+hem)+3x(stage+alb)+4xlymph].

This model suggests lymphopenia has a stronger impact on the presence of BMI, while leukocytosis has the least impact. The percentage of BMI was 0.1,17,48, and 87 % for pts with a score of 0-8, 9-10, 11-12, and >12 respectively. Using a score of >8 as a rule for BMI, sensitivity was 99% and specificity was 87%. The prevalence of BMI by histology was 4% lymphocyte-rich, 5% nodular sclerosis, 20% mixed-cellularity and 21% lymphocyte-depleted, further analysis showed using modified IPS, sensitivity and specificity were similar across all types of histologies.

Conclusions

Using a modified IPS of > 8 for predicting BMI in HL doubled the spec associated with Ann Arbor classification. The implementation of modified IPS is a practical and reliable tool that will allow physicians to predict BMI in HL pts. Therefore eliminate unnecessary painful procedure in many pts.

Disclosures:

No relevant conflicts of interest to declare.
Serum TARC Levels Correspond with Tumor Load and Response to Chemotherapy in Classical Hodgkin Lymphoma Patients.
Authors: Diepstra Arjan , van der Graaf Anne Marijn , Vos Hans JTWM , Visser Lydia , Van Den Berg Anke , van Imhoff Gustaaf
Published: October 2019

Abstract 3663

Poster Board III-599

Introduction

Thymus and Activation Related Chemokine (TARC) is produced in high amounts by Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). It attracts CCR4 positive Th2 and Treg cells, which are typically present in large numbers in the reactive infiltrate in cHL. TARC can be measured in the serum of cHL patients and is usually elevated in the presence of active disease, as compared to healthy controls. Higher levels are observed in advanced stage disease, compared to stage I disease. The goal of the current study was to correlate TARC serum levels to metabolic response to therapy as measured by FDG-PET scanning.

Patients and methods

Newly diagnosed cHL patients treated in our hospital were asked to participate. Serum samples were taken before the start of treatment, at the time of the mid treatment PET scan (after 2 cycles of ABVD for stage I and II, or mid BEACOPP or ABVD treatment for stage III and IV disease) and at the time of the PET scan after completion of chemotherapy. TARC serum levels were measured by ELISA and TARC immunohistochemistry was done on primary involved lymph node tissue.

Results

A total of 59 cHL patients, median age 40 (range 16-80) and 49% male participated. Eight patients had stage I, 26 stage II, 14 stage III and 10 stage IV disease. The known association of high pretreatment TARC levels with advanced stage was also significantly present in our data, supporting the hypothesis that serum TARC levels reflect tumor burden. Moreover, in stage I and II patients, bulky disease (27%) was associated with a significant higher TARC level, lending further support to this hypothesis. Compared to pretreatment values, TARC levels had decreased significantly at mid treatment and treatment completion to the range of healthy controls. Only in stage I there was no significant difference between pretreatment and mid or after treatment samples (see figure), probably due to the relatively large number of patients without elevated TARC before the start of therapy. In patients with a positive PET scan at mid treatment (26%), there was a trend for a higher TARC level (P=0.08) compared to PET negative patients. After completion of chemotherapy PET positivity (19%) was significantly associated with a higher TARC level. Three patients had a relapse and at that time TARC levels had clearly increased in comparison to the levels after treatment.

Conclusions

serum TARC levels are associated with tumor burden and metabolic response to therapy as measured by FDG-PET scan. Measurement of serum TARC levels is a potential tool to evaluate the response to therapy and to be used in follow up after completion of chemotherapy. Inclusion of more patients is necessary to further assess the clinical value of TARC serum levels and is ongoing.

Disclosures:

No relevant conflicts of interest to declare.