bluuebunny/crossref_metadata_2025_split huggingface dataset:
Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.
Authors: Mao Yingying , Getz Todd M , Jin Jianguo , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 770
Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt.

Disclosures:
No relevant conflicts of interest to declare.
Rituximab Administration within 6 Months of Allogeneic SCT Carries Risk of Prolonged and Life Threatening Cytopenias.
Authors: McIver Zachariah A. , Stephens Nicole , Naguib Nicolas , Grim Andrew , Barrett A. John 
Published: October 2019

Abstract
Abstract 793
The use of the monoclonal anti-CD20 antibody Rituximab to treat chronic graft-versus-host disease (c-GVHD) occurring after stem cell transplantation (SCT) is generally effective and well tolerated. However this agent can cause neutropenia. Here we describe cytopenias following Rituximab treatment in patients developing c-GVHD after allogeneic SCT. Between 2006-2009, 50 patients received a myeloablative matched-sibling T-cell depleted or selectively-depleted SCT for a variety of hematological malignancies, mainly AML, ALL, CML, and MDS. Median age was 43 years (range, 13 – 68), and median CD34+ cell dose was 6.1 × 106/kg (range, 3.1-10.1). Thirty-one patients developed acute GvHD; 13 grade 1, 16 grade 2, and 1 patients with grade 3. Twenty-seven patients had chronic GvHD (17 limited, 10 extensive). Nineteen patients were treated with Rituxmab; 15 within 180 days, and 4 between 1- 7 years following SCT. Patients receiving Rituximab one year after SCT experienced only moderate neutropenia 3-5 months after treatment lasting 10-20 days while maintaining ANC > 1.0× 109/L. In contrast, neutropenia occurring within 4 weeks of treatment occurred in all 15 patients given Rituximab within the first 180 days after SCT. Twelve of these patients developed severe neutropenia (ANC < 0.5K/μL) lasting 2-8 months in duration, with 10 out of the 12 patients requiring hospitalization to treat neutropenic infections. Six of the 12 patients died of infection complicationg GVHD treatment. Six patients treated with Rituximab within 120 days of SCT experienced the worse complications. All 6 developed neutropenia within 4 weeks of treatment lasting 3- 7 months. Five required recurrent hospitalization for neutropenia associated infections; one died of invasive fungal pneumonia, one died of gram negative bacteremia and relapsed disease, and one required prolonged intubation for parainfluenza type 3 infection associated ARDS. Recovery of both neutrophil and lymphocytes was extremely delayed in this group, with a significantly lower ANC and ALC at 9 months and 12 months post SCT compared to patients with GvHD not treated with Rituximab (p < 0.02). Investigation of the etiology of neutropenia revealed evidence of T cell clonality in 4 patients; 2 patients were diagnosed with LGL with evidence of restricted rearrangement patterns detected by PCR, one patient had 95% NK cell dominated bone marrow infiltrate seen on flow cytometry, and one patient had idiopathic thrombocytopenia purpura and responded to appropriate immunosuppression. Although Rituximab resulted in rapid control of c-GVHD, we conclude that administration of Rituximab early after SCT is associated with prolonged, possibly autoimmune mediated profound cytopenias, and should be avoided.

Disclosures:
No relevant conflicts of interest to declare.
In Vivo Lentiviral Marking Demonstrates Long-Term Myeloid and Lymphoid Lineage Contribution of Individual Hematopoietic Stem Cell Clones to Murine Steady-State Hematopoiesis.
Authors: Zavidij Oksana , Ball Claudia R , Fessler Sylvia , Belle Daniela , Schmidt Manfred , von Kalle Christof , Glimm Hanno 
Published: October 2019

Abstract
Abstract 813
Most of the knowledge to date on the in vivo blood forming activity of individual hematopoietic stem and progenitor cells was gained in transplantation experiments of defined cell populations into syngeneic or xenogeneic murine hosts. Consequently, stem and progenitor cells are solely defined by their role in post-transplant reconstitution and very little is known on their clonal activity in steady-state hematopoiesis. To gain new insights into the clonal activity of stem and progenitor cells under steady-state conditions we used a genetic in vivo lentiviral marking strategy and subsequently monitored the clonal activity of marked hematopoietic cells for up to one year by highly sensitive integration site amplification using LAM-PCR. Highly concentrated GFP-expressing lentiviral vectors (LV) were injected intravenously (IV, n=10) or intrafemorally (IF, n=15) into GFP-tolerant B6.Cg-Tg (Krt1-15-EGFP) 2Cot/J (Krt15) mice to directly mark hematopoietic stem and progenitor cells. 5 mice from each of the two cohorts were treated with 5-Fluorouracil (5-FU, 150 mg/kg) to mobilize hematopoietic stem cells prior to LV-marking. The clonality of the transduced myelopoiesis and lymphopoiesis was analyzed by LAM-PCR. A small proportion of all peripheral blood cells in LV-injected mice consistently expressed GFP for up to one year (5-100 GFP+ cells per 20000 PB cells analyzed). Pre-treatment with 5-FU did not affect the percentage or lineage distribution of marked blood cells even when the vector was injected intravenously. Even though the initial percentage of marked cells was similar after IV and IF vector injection (p>0.05) the marking kinetics were different. Whereas the percentage of GFP expressing cells in PB of IF-marked mice remained stable over the whole observation period for up to 1 year, a 2-fold decline of the percentage of marked cells was detected two weeks after IV-marking indicating that predominantly short-lived more mature cells were transduced after IV vector injection. LAM-PCR analyses of sorted cell lineages showed that multiple clones contributed to the marked myeloid and lymphoid long-term hematopoiesis after IF-injection. In summary, our data demonstrate stable marking of steady-state hematopoiesis for up to one year. Our results demonstrate that remarkably stable stem cell clones with myeloid and lymphoid differentiation potential contribute to murine steady-state long-term hematopoiesis. In vivo marking will further allow to directly address the response of individual stem cell clones to hematopoietic stress including chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Increased Immune Suppressive CD14+ hla-DRneg Circulating Monocytes Are Found in Aggressive Non-Hodgkin's Lymphoma and Correlated with Increased Arginase I Level.
Authors: Lin Yi , Gustafson Michael P. , Bulur Peggy A , Maas Mary , Mohr Amy , Witzig Thomas E , Dietz Allan 
Published: October 2019

Abstract
Abstract 970
Despite advances in treatments for patients with non-Hodgkin's lymphoma (NHL), the relapse rates remain high and 40% of diffuse large B-cell NHL (DLBCL) patients die of disease. These patients have variable levels of immunodeficiency that may impact their prognosis. Previously we reported that monocytes in relapsed NHL patients suppress both autologous and allogeneic T cell proliferation and that these monocytes were unable to mature into dendritic cells in vitro. These suppressive functions were associated with increased frequency of CD14+HLA-DRneg monocytes in NHL patients (NHL: 29.4 ± 3.2%, n = 36; healthy donors 7.54 ± 0.97%, n = 27; p < 0.0001). This phenotype is distinct from other myeloid suppressors (Lin−CD33+HLA-DR−) or non-classical monocytes (CD16+), neither of which was different in numbers between NHL and healthy donors. We have now expanded our study cohort and found that the percentage of CD14+HLA-DRneg cells among circulating monocytes was increased in patients with more aggressive disease: DLBCL 37.5 ± 5.11%, n = 16 vs SLL and MALT lymphoma 15.8 ± 4.02%, n = 7; p = 0.01. Two newly diagnosed patients and three patients with localized disease amenable to resection all had frequencies of CD14+HLA-DRneg monocytes closer to that of healthy donors whereas 5 of the 6 patients with low grade NHL that transformed to DLBCL had significantly elevated percentage of these suppressive monocytes (49.1 ± 4.70%). To better understand the mechanism of immune suppression by CD14+HLA-DRneg monocytes, we investigated production of IFN-alpha induced by CpG oligodeoxynucleotide. Monocytes from NHL patients were less effective at IFN-alpha production (0.54 ± 0.22%, n = 4) compared to healthy donors (1.37 ± 0.25%, n = 4; p = 0.04). The corresponding culture supernatants also had a 4-fold reduction in IFN-alpha level by ELISA (p = 0.01). Reduced IFN-alpha production and subsequent decreased stimulatory response may contribute to diminished treatment response to CpG in NHL. Finally, we also found that the frequency of CD14+HLA-DRneg monocytes correlated with increasing plasma levels of arginase I, a known mediator of T cell suppression (n = 16, p = 0.06). Such correlation was not observed for circulating VEGF, IL-6 or IL-10. Taken together we have described a population of immunosuppressive CD14+HLA-DRneg monocytes in NHL patients that is more prevalent in aggressive disease and likely suppresses T cell function through multiple mechanisms including arginine metabolism. This work was supported through the University of Iowa and Mayo Clinic Lymphoma SPORE CA097274 and the Gerstner Family Foundation.

Disclosures:
Witzig: Novartis: Research Funding.
Mitochondrial Physiology in Health and Disease: Changes with Aging.
Authors: Wallace Douglas C. 
Published: October 2019

Abstract
Abstract SCI-1
The incidence and cost of the age-related metabolic and degenerative diseases is rising in the face of ever increasing investment. Therefore, something must be missing in our current hypotheses about these diseases. Until now, the prevailing paradigms in Western medicine have been the anatomical paradigm of disease which posits that tissue-specific symptoms are due to tissue-specific structural defects and the Mendelian paradigm of genetics which posits that if a disease is inherited according to the Laws of Mendel it is genetic and if not it is environmental. The epigenome is invoked to explain the environmental modulation of the nuclear gene expression. However, life requires both structure and energy and the over 1500 mitochondrial energy genes are dispersed across the chromosomes plus the maternally inherited mitochondrial DNA. Moreover, the cells and tissues most affected by aging and the age-related disease are those most reliant on mitochondrial energy and the mitochondrial lies at the interface between environmental calories and human physiology, diabetes and obesity. The epigenome and the signal transduction pathways are regulated by protein phosphorylation by ATP, acetylation via acetyl-CoA, and methylation by S-adenosylmethionine, all driven by mitochondrial high energy substrates modulated by available calories. Furthermore, mitochondrial redox chemistry regulates reactive oxygen production and thiol/disulfide chemistry and these also regulate cellular signaling and function. Therefore, bioenergetics and mitochondrial genetics are the missing factors which have inhibited our capacity of address the biology and genetics of the age-related metabolic and degenerative diseases, cancer and aging.

Disclosures
No relevant conflicts of interest to declare.
Telomere Dysfunction in Bone Marrow Failure.
Authors: Lansdorp Peter M. 
Published: October 2019

Abstract
Abstract SCI-12
In order to distinguish a normal telomere from a double strand break, a minimum number of telomere repeats must “cap” each chromosome end. The length of each repeat array will reflect a unique history of addition and losses. Telomere losses are known to occur sporadic as well as with every replication cycle. Losses of telomeric DNA are countered by the telomerase enzyme containing telomerase RNA (encoded by the TERC gene) and a reverse transcriptase protein (encoded by TERT gene) as minimal components. Telomerase levels are high in cells of the germline and immortal cellines and the telomere length is typically maintained in such cells. In contrast, telomerase activity is limiting in most human somatic (stem) cells and as a result the average length of telomere repeats in most somatic cells shows a highly significant decline with age. The hypothesis that loss of telomere repeats acts as a “mitotic clock” and a tumor suppressor mechanism in stem cells is strongly supported by recent studies of patients with mild telomerase deficiency resulting from haplo-insufficiency for either the TERC or TERT gene. Such genetic defects can give rise to various disorders including autosomal dominant Dyskeratosis Congenita (DKC), aplastic anemia, liver fibrosis and pulmonary fibrosis. Other recent studies have revealed that amplification of the hTERT gene is one of the most common genetic abnormalities in various cancers. Paradoxically, it is becoming clear that SNPs within the TERT locus are among the most reproducible risk factors for the development of different types of cancer including lung cancer, acute myeloid leukemia and chronic lymphocytic leukemia. The links between hypo- and hyperproliferative consequences of inborn telomerase deficiencies and SNP's in the TERT gene are poorly understood. It seems plausible that the increased risk of leukemia development in aplastic anemia, myelodysplastic syndrome and Dyskeratosis Congenita, results from stem cell failure. Could reduced stem cell numbers by itself provide a risk factor for tumor development? More direct measures of stem cell numbers in vivo are needed to examine this possibility and the relationship between stem cell numbers and tumor development in patients with defective telomere maintenance as well as in normal individuals as a function of age. Measurements of the average telomere length as well as the length of telomere repeats at individual chromosome ends in specific cells and tissues will further calrify the involvement of telomeres in bone marrow failure, normal aging and tumor biology.

Disclosures
Lansdorp: Repeat Diagnostics Inc.: Equity Ownership.
Nuclear Transcription Factories.
Authors: Fraser Peter 
Published: October 2019

Abstract
Abstract SCI-16
The discovery of long-range intrachromosomal and interchromosomal interactions in higher eukaryotes (1, 2) points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of such interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of interactions between transcriptionally active genes at transcription factories using the mouse globin genes in erythroid tissues. Our results show that the transcripitonally active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the erythroid-specific transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, suggesting that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hotspots optimized for efficient and coordinated transcriptional control. 1. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P. Long-range chromatin regulatory interactions in vivo. Nat. Genet. 2002;32:623-626. 2. Osborne CS, Chakalova L, Brown KE, et al. Active genes dynamically co-localize to shared sites of ongoing transcription. Nat. Genet. 2004;36:1065-1071.

Disclosures
No relevant conflicts of interest to declare.
Mast Cells and Tumor Progression.
Authors: Clapp D. Wade , Yang Fengchun , Ingram David A. , Robertson Kent A. , Hutchins Gary D. , Parada Luis F. 
Published: October 2019

Abstract
Abstract SCI-33
Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Neurofibromas form in association with peripheral nerves and are composed of Schwann cells, blood vessels, fibroblasts and degranulating mast cells. Genetic studies in engineered mice indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation in a genetically engineered murine model and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Recent studies in our group have focused on evaluating the role of bone marrow-derived cells, and particularly mast cells, in the tumor microenvironment. Previous work by our group established that human and murine Nf1 deficient Schwann cells secrete high concentrations of kit-ligand. Kit-ligand has a central role in multiple mast cell functions including chemotaxis, proliferation and degranulation. In a series of bone marrow transplantation studies, we established that Nf1 haploinsufficiency in bone marrow is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling diminishes neurofibroma initiation and progression. Collectively, the studies implicate mast cells as active participants in tumor formation and identify therapeutic targets for human phase 1-2 clinical trials.

Disclosures
Off Label Use: The drug imatinib mesylate was used to treat plexiform neurofibromas in genetically engineered mice, in a child with a plexiform neurofibroma, and it is currently being tested in a phase 2 clinical trial.
Ontology of Megakaryopoesis.
Authors: Geddis Amy E. 
Published: October 2019

Abstract
Abstract SCI-8
Compared to red cells, whose passage from embryonic to adult stages is marked by the expression of distinct forms of hemoglobin, the development of megakaryocytes during embryogenesis is less well understood. However, certain shared characteristics between megakaryocytes, endothelial cells, hematopoietic stem cells and erythrocytes infer developmental relationships between these lineages. Recent data support the model that hematopoietic stem cells derive from the hemangioblast, and that megakaryocytes and erythrocytes develop from a common precursor both in primitive and adult hematopoiesis. Evidence of these common origins can be found in the genetic programs that are activated during hematopoiesis, in that many of the cell surface markers and transcriptions factors that are characteristic of megakaryocytes can also be found in endothelial cells, stem cells and erythrocytes. In this session I will review current views on developmental thrombopoiesis, key megakaryocytic transcription factors and the experimental and clinical phenotypes associated with their disruption, and current controversies in lineage choice during megakaryocyte differentiation.

Disclosures
Geddis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.