bluuebunny/crossref_metadata_2025_split huggingface dataset:
S1P1 Agonists for Use as Adjunct Mobilizing Agents
Authors: Harun Nadia , Thien Marilyn , Juarez Julius G , Bradstock Kenneth Francis , Bendall Linda J. 
Published: October 2019

Abstract
Abstract 826
Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB.
Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models.
Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF.
S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes.

Disclosures:
No relevant conflicts of interest to declare.
Ror1 Targeting Monoclonal Antibodies Induced Apoptosis of Chronic Lymphocytic Leukemia Cells– A Potential Novel Therapeutic Approach
Authors: Rabbani Hodjattallah , Daneshmanesh Amir Hossein , Khan Abdul Salam , Hojjat-Farsangi Mohammad , Jeddi-Tehrani Mahmood , Akhondi Mohammad Mehdi , Österborg Anders , Shokri Fazel , Mellstedt Håakan 
Published: October 2019

Abstract
Abstract 916

Methods:
A peptide-based mouse monoclonal antibody generation method was used for producing MAbs against the three extracellular domains of Ror1. Twenty CLL patients (10 with progressive and 10 with non-progressive disease) were enrolled in this study. Flow cytometry was used for surface staining of Ror1. Annexin V and propidium iodide (flow cytometry) as well as PARP cleavage (Western blot) was used for detection of apoptosis.

Results:
Six monoclonal antibodies of different isotypes (IgG, IgM) were produced against Ror1. The frequency of Ror1 positive cells were in the range of 24–89%. Patients with progressive disease had a significantly higher number of Ror1 positive cells as compared to those with non-progressive disease as well as in patients with unmutated compared to mutated IgVH genes. All six antibodies alone induced apoptosis (Annexin V and PARP cleavage) of CLL cells. A higher frequency of apoptotic CLL cells was induced by the antibodies against the CRD region (ligand binding site for Wnt proteins) as well as one antibody against the kringle domain (also a binding region for regulatory proteins) . Apoptosis induced by these three antibodies alone was significantly higher than that induced by Rituximab (p<0.001). Apoptosis induction by all antibodies was statistically significantly augmented by crosslinking of the Ror1 antibodies with anti-Fc F(ab’)2 fragment antibodies. None of the antibodies induced apoptosis of PBMC of healthy individuals and normal PBMC did not express Ror1.

Conclusion:
Monoclonal antibodies alone against the RTK, Ror1, were shown to selectively kill CLL cells. Development of MAb targeting Ror1 might be a novel therapeutic approach complementary to existing therapies.

Disclosures:
No relevant conflicts of interest to declare.
The Function of Thymic Innate TCRαß+ CD8+ T Cells Is Regulated by Constitutive Expression of B7-H1
Authors: Rafei Moutih , Hardy Marie-Pierre , Perreault Claude 
Published: October 2019

Abstract
Abstract 955
Innate T cells display features of Ag-experienced T cells (e.g., high levels of CD44) before any encounter with non-self antigens. They include NKT cells, CD8α/α T cells, as well as an undefined (and presumably very small) proportion of naïve CD4 and CD8 α/β T cells. We used the RAG2-GFP transgenic mouse model to differentiate naïve innate single-positive thymocytes (GFP+CD44high) from re-circulating effector/memory T cells (GFP-CD44high) T cells and naïve conventional (GFP+CD44low) thymocytes. We found that innate T cells represent about 10% of TCRαβ+ single-positive GFP+ thymocytes. To evaluate their proliferative ability under lymphopenic conditions, conventional and innate T cells were sorted and injected in TCRβKO mice. Four weeks later, expansion of conventional T cells was 2.3-fold greater than that of innate T cells. Comprehensive phenotypic analyses of GFP+CD8+CD44low vs GFP+CD8+CD44hi thymocytes revealed that B7-H1 was absent on conventional thymocytes but constitutively expressed on innate CD8+ thymocytes. Moreover, in vitro stimulation of innate T cells with anti-CD3 and anti-CD28 antibodies induced a strong proliferation and secretion of inflammatory mediators such as interferon (IFN)-gamma. The addition of anti-B7-H1 neutralizing antibody, however, inhibited proliferation and IFN-gamma production in a dose-dependent manner. To see whether B7-H1 ligation influenced the viability of CD8+ thymocytes, GFP+CD8+CD44low and GFP+CD8+CD44hi thymocytes were treated for 48 hrs with anti-B7-H1 antibody in vitro then stained for annexin-V and propidium iodine. Under these conditions, 70% of innate GFP+CD8+CD44hi T cells underwent apoptosis as opposed to 30% with conventional thymocytes. To test their bystander properties, we added conventional or innate T cells as third-party cells to an in vitro antigen stimulation assay. Briefly, B6 peritoneal macrophages pulsed with recombinant chicken ovalbumin (rOVA) presented OVA-derived peptides in the context of MHCI, and served as antigen-presenting cells (APCs). For responder cells, we added naive T cells from T cell receptor-transgenic OTI mice that recognize OVA. Interestingly, innate thymocytes were capable of promoting the activation of responding T cells; an effect that was lost upon treatment of innate CD8 T-cells with anti-B7-H1 neutralizing antibody. The sum of these data suggests a role for B7-H1 in the development, survival and proliferation of innate α/β T cells.

Acknowledgments
Moutih Rafei holds a Fellowship from the CIHR. Claude Perreault holds a Canada Research Chair in Immunobiology. This work was supported by grant # 42384 from the CIHR.

Disclosures:
No relevant conflicts of interest to declare.
JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS
Authors: Olnes Matthew J. , Poon Andrea , Tucker Zachary , Young Neal S. , Sloand Elaine M 
Published: October 2019

Abstract
Abstract 973
The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML.

Disclosures:
No relevant conflicts of interest to declare.
Genomic Variants Associated with Childhood Acute Lymphoblastic Leukemia
Authors: Relling Mary V. 
Published: October 2019

Abstract
Abstract SCI-11
Genome-wide interrogations have a role in addressing both the etiology and the responsiveness of childhood acute lymphoblastic leukemia (ALL). Recent work by our own1 and other groups identified common polymorphisms in ARID5B, and to a lesser extent in IKZF1, as predisposing to the development of childhood ALL. Polymorphisms in these two genes can account for ∼ 40% of the population attributable risk of ALL, and differences in the frequency of the minor allele among different ancestral groups can account for a large portion of the differences in the incidence of ALL among different race groups. The association of these inherited polymorphisms with specific ALL subtypes (e.g. ARID5B with risk of hyperdiploid ALL) indicates that germline polymorphisms affect not only risk of ALL but may also affect or confound association analyses between germline variants and the probability of cure of ALL. The biological basis for the prognostic differences that exist among ALL subtypes remain largely unknown, and it is plausible that inherited polymorphisms may affect both susceptibility to subtypes of ALL as well as to drug responsiveness. Genome-wide analyses have also identified polymorphisms associated with eradication of MRD across multiple treatment protocols.2 Polymorphisms in IL15 indicate variants that likely affect the inherent pharmacodynamic responsiveness of ALL to drug-induced apoptosis. Approximately 20% of the polymorphisms associated with MRD were also associated with rapid drug clearance in the host, even though pharmacokinetic data were only available for 2 of the 4–8 medications used during induction. These findings lead us to suggest that perhaps half of the polymorphisms associated with eradication of ALL are related to effects on host drug clearance, and about half have penetrant effects on the inherent responsiveness of the ALL cells themselves. A genome-wide analysis for the determinants of clearance of one such drug, methotrexate, identified a strong effect of polymorphisms in the SLCO1B1 transporter.3 Genome-wide approaches have identified the importance of genes that decades of candidate gene approaches did not reveal, illustrating the utility of an agnostic approach to genotype-phenotype association studies in childhood ALL.
1. Treviño LR, Yang W, French D, et al. Germline genomic variations associated with childhood acute lymphoblastic leukemia. Nat Genet 41:1001–5, 2009.
2. Yang J, Cheng C, Yang W, et al. Genome-wide interrogation of germline genetic variation associated with treatment response in childhood acute lymphoblastic leukemia. JAMA 301:393–403, 2009.
3. Treviño LR, Shimasaki N, Yang W, et al. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J Clin Oncol 27:5972–8, 2009.

Disclosures:
Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.
Heme, Iron, and the Kidney
Authors: Nath Karl A. 
Published: October 2019

Abstract
Abstract SCI-26
The kidney is often the target of injury by heme proteins and states of iron overload. Exogenous heme proteins, such as hemoglobin delivered to the kidney in hemolytic states, or myoglobin imposed upon the kidney following rhabdomyolysis, can injure the kidney via pathways that include vasoconstriction, direct cytotoxicity, and tubular cast formation. The former two pathways critically involve the heme prosthetic group: heme can bind vasodilator gases such as nitric oxide and carbon monoxide, while free heme can be cytotoxic because it is lipophilic, lipid membrane-transmissible, lipid membrane-destabilizing, prooxidant, and proinflammatory. Renal injury can also arise from endogenous heme proteins, such as cytochrome P450 enzymes, which can be destabilized by ischemic and nephrotoxic insults; such destabilization leads to the release of heme and incurs heme-dependent injury. The toxicity of heme may also reflect, at least in part, the release of iron and the attendant increase in labile cellular iron, the latter representing a potent catalyst for oxidative stress; increased levels of cellular iron may also originate from intracellular nonheme sources. Heme-iron can not only induce acute kidney injury, but also can provoke chronic kidney disease by virtue of its proinflammatory and profibrotic effects. Mechanisms that protect against heme-dependent and iron-dependent toxicity include heme oxygenase (HO), the rate-limiting enzyme in heme degradation, and increased synthesis of ferritin. Induction of HO-1, the inducible HO isoform, is protective against renal injury because of the following mechanisms: 1) the prevention of acute elevation in cellular heme concentrations otherwise incurred by cell injury; 2) the safe sequestration of iron in iron-binding proteins such as ferritin, or the cellular extrusion of iron by iron-exporting proteins; 3) the generation of antioxidant, anti-inflammatory metabolites such as bile pigments; and 4) the generation of carbon monoxide which is an antiapoptotic, anti-inflammatory, and vasorelaxant gas. Carbon monoxide can also be cytoprotective by binding cytochrome P450 enzymes, and thereby preventing their destabilization and the release of heme that subsequently occurs. In addition to these areas, this presentation discusses the pathobiologic and clinical significance of the siderophore-binding protein, NGAL (Neutrophil gelatinase-associated lipocalin): NGAL protects against renal ischemic injury through mechanisms that require the induction of HO-1; NGAL is increasingly utilized as a biomarker of acute kidney injury. The presentation concludes by discussing the use of iron supplementation in the treatment of anemia of chronic kidney disease and therapeutic strategies that may be designed from understanding endogenous and adaptive mechanisms that protect against the toxicity of heme-iron.

Disclosures:
No relevant conflicts of interest to declare.
Functional Genomics Approaches to Platelet Signaling
Authors: Ouwehand Willem Hendrik , Soranzo Nicole 
Published: October 2019

Abstract
Abstract SCI-37
The count, volume and function of platelets varies in the population and there is ample evidence that all 3 quantitative traits (QT) are highly heritable. The control of platelet function is complex with a large number of forward and reverse regulatory pathways that promote and inhibit thrombus formation, respectively. We used 4 parallel genomics approaches with the aim to identify novel regulators of platelet function. First we established a genome-wide expression (GWE) HaemAtlas of all 8 haematological elements, including erythroblasts and megakaryocytes (MKs)(1). Mining of this data identified 279 transcripts that were relatively over-expressed in MKs if compared with the results obtained with the 7 remaining blood cell types. Of these, 75 transcripts encode membrane proteins, many with known and several with a hitherto unknown function (2). Second we determined the platelet response to ADP and a collagen mimetic in 500 healthy subjects of the Platelet Function Cohort (PFC)(3). WGE studies of platelet RNA samples from 37 PFC subjects, selected to be representative of the observed variation, identified 63 transcripts that were correlated with function. Third the 500 PFC samples were typed for 1536 SNPs tagging 108 candidate genes for sequence variation and this identified 19 associations (p-value ≤ 0.005) (3). All 19 QT loci (QTLs), but the GP6 gene, were novel. Based on the above 9 genes were selected for functional studies with platelets from genotyped healthy subjects and by morpholino-based gene knockdown in a model of laser-induced thrombus formation in Danio rerio (2,3). This identified BAMBI, COMMD7, LRRC32 and LRRFIP1 playing a role in the promotion and DCBLD2, ESAM, G6B and GTF2A2 in the inhibition of thrombus formation. Finally 2 of the 15 QTLs for platelet volume and count identified in about 15,000 healthy subjects with genome-wide typing information showed an effect on platelet function demonstrating that sequence variants that modify platelet volume may also exert an effect on function (4). (1) Macaulay, I. C. et al., Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 109 (8), 3260 (2007); Watkins, N. A. et al., A HaemAtlas: characterizing gene expression in differentiated human blood cells. Blood 113 (19), e1 (2009); (2) O'Connor, M. N. et al., Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood 113 (19), 4754 (2009).; (3) Jones, C. I. et al., A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways. Blood 114 (7), 1405 (2009); (4) Soranzo, N. et al., A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet 41 (11), 1182 (2009).

Disclosures:
No relevant conflicts of interest to declare.
Angioblast Behavior in Arterial-Venous Segregation
Authors: Herbert Shane P. , Stainier Didier 
Published: October 2019

Abstract
Abstract SCI-43
Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from pre-existing vessels (angiogenesis). Using high resolution imaging of zebrafish vascular development we discovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated in part by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial/venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Disclosures:
No relevant conflicts of interest to declare.
Complementary Costimulation of Human T Cell Subpopulations by CD28 and CD81
Authors: Levy Shoshana , Sagi Yael 
Published: October 2019

Abstract
Abstract 1124
CD81 is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells - primary human naïve T cells are better costimulated by CD81, while the memory T cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared to that by CD28. We found that IL-6 played a role in the activation of the naïve T cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.

Disclosures:
No relevant conflicts of interest to declare.