bluuebunny/crossref_metadata_2025_split huggingface dataset:
State-of-the-Art and Current Controversies in the Detection and Quantitation of Tissue Factor-Bearing Microparticles
Authors: Zwicker Jeffrey 
Published: October 2019

Abstract SCI-11
Microparticles are submicron vesicles that circulate in blood and are derived from a variety of vascular cells. Microparticles expressing tissue factor have been shown in animal thrombosis models to contribute to thrombus formation in vivo, which has led to the evaluation of tissue factor bearing microparticle populations in a variety of thrombotic disorders including malignancy, sepsis, and coronary thrombosis. Central to the evaluation of tissue factor-bearing microparticle populations in health and disease is the establishment of accurate means for qualitative or quantitative assessment of circulating microparticles. Whilst there is considerable interest in evaluating whether pathologic disturbances in tissue factor-bearing microparticles contribute to thrombosis, analytic techniques for microparticle measurement remain in the validation stage. The method most commonly employed for the detection of tissue factor-bearing microparticles is light-scatter flow cytometry, which relies on the sizing of particles based on light-scatter profile. Light scatter, however, is also influenced by variables independent of particle size, including the wavelength of incident light, particle shape, presence of surface absorptive material, and relative refractive indices of particles and suspension medium. A number of potentially more sensitive methodologies are currently under investigation such as impedance-based flow cytometry, quasi-elastic light scattering based on Brownian movement, and atomic force microscopy. Alternatively, assays have been developed that measure tissue factor antigen or activity following isolation of microparticles from plasma or whole blood but without determination of particle size. Absent a gold standard for the measurement of tissue factor-bearing microparticle populations by which these approaches can be validated, the development of accurate methodology will require inter-assay and intra-assay precision as well as validation of clinical relevance in human studies.

Disclosures:
No relevant conflicts of interest to declare.
Autophagy and Metabolism in Lymphoid Malignancies
Authors: Distelhorst Clark W. 
Published: October 2019

Abstract SCI-26
Cell stress, if left unchecked, leads to apoptosis and autophagy. Both are highly conserved, genetically encoded processes. Apoptosis is a programmed death mechanism, designed to eliminate cells that are defective or no longer needed as part of developmental programs. Autophagy, on the other hand, is a programmed survival mechanism designed to provide cells a temporary source of energy when nutritionally deficient. Since in autophagy (“self eating”) the cell generates energy at the expense of its own organelles, temporary survival leads to death if nutrition is not restored. Both apoptosis and autophagy are induced by glucocorticosteroid hormone (GH) treatment of lymphoid malignancies. Since the main effects of GH on lymphocytes are metabolic, it is likely that apoptosis and autophagy are responses to metabolic stress. Metabolic stress occurs because GH inhibits both glucose uptake and metabolism, as well as calcium signals required for optimal mitochondrial function. Gene expression profiling has provided insight into the complex pathways that mediate apoptosis and autophagy in GH-treated leukemia and lymphoma cells. Ultimately, the balance of pro-apoptotic and anti-apoptotic factors determines cell fate. Elevation of the pro-apoptotic protein Bim is a critical step in apoptosis induction by GH and is due to GH-mediated repression of a microRNA cluster (miR17-95) that normally represses Bim expression. In metabolically stressed cells, Bim elevation is counterbalanced by a proton-sensing G protein-coupled receptor, TDAG8, that signals an elevation of the anti-apoptotic protein Bcl-2, counterbalancing Bim elevation and inhibiting apoptosis. Gene expression profiling also provided insight into the mechanism of autophagy by detecting GH-mediated repression of the Src kinases Fyn and Lck and GH-mediated elevation of Dig2 (REDD1, RTP801). Fyn and Lck interact with the inositol 1,4,5-trisphosphate calcium channel and promote physiological calcium signals. By decreasing Fyn and Lck, GH inhibits calcium signals necessary for optimal mitochondrial metabolism, contributing to autophagy induction. Dig2 is a stress protein that inhibits mTOR signaling. The absence of the Dig2 stress response in Dig2 knockout mice significantly impairs autophagy induction, decreasing lymphocyte survival in GH-treated mice. In summary, both apoptosis and autophagy occur in lymphoid malignancies in response to metabolic stress, as illustrated by the effects of GH treatment. A goal for future studies is to tie these signaling components together into a logical signaling network and to target components of the signaling network for therapeutic purposes.

Disclosures:
No relevant conflicts of interest to declare.
Epigenetic Effects of IDH1/IDH2 Mutations
Authors: Melnick Ari M. , Levine Ross L , Figueroa Maria E , Thompson Craig B. , Abdel-Wahab Omar 
Published: October 2019

Abstract
Abstract SCI-33
Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ∼20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss-of-function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCpG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using α-ketoglutarate (αKG) as a substrate. 2HG can disrupt the activity of enzymes that use αKG as a cofactor, including TET2 and the jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibits TET2 hydroxymethylation and jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards increased histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is in abundance in hematopoietic stem cells and displays specific distribution patterns, yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning.

Disclosures:
Thompson: Agios Pharmaceuticals: Consultancy.
Platelets, Obesity, and the Metabolic Syndrome
Authors: Freedman Jane 
Published: October 2019

Abstract
Abstract SCI-34
Cardiovascular disease continues to be the major cause of morbidity and mortality in North America and a growing problem in many other parts of the world. The risk of premature atherosclerosis and cardiovascular events rises with increasing obesity and excess adiposity has been linked to increased all-cause mortality. Current estimates show that one in four ischemic heart disease events are attributable to excess weight. Inflammation of adipose tissue orchestrated by monocyte/macrophage infiltration and overproduction of proinflammatory and proatherogenic cytokines may mediate metabolic dysfunction and vascular disease in human obesity. Importantly, obesity has been associated with increased cardiovascular disease as well as enhanced platelet function and thrombosis. We recently measured expression of 48 genes by high-throughput quantitative reverse transcription PCR (qRT-PCR) in over 2250 participants of the Framingham Offspring and Omni minority cohorts (FHS) using RNA from isolated platelets and found that specific inflammatory transcripts including ICAM1, IL1R, IFNG, IL6, COX2, TNF, TLR2, and TLR4 were significantly and positively associated with higher body mass index (BMI) by regression analysis. With the additional measurement of 196 genes, the data has remained extraordinarily consistent. Other cardiovascular risk factors rarely associate with platelet gene expression and BMI remains specifically associated with inflammatory transcripts related to the NFkB-innate immune and inflammatory pathways. These surprising data led to the question: Why would the anucleate platelet have gene expression changes specifically in the setting of increased BMI and can this powerful clinical association be explored using both pre-clinical and clinical studies to find new targets for disease and define new mechanisms for platelet function beyond thrombosis?

Disclosures:
No relevant conflicts of interest to declare.
Oxygen Availability and Stem Cells: Impact On Development and Disease
Authors: Simon M. Celeste 
Published: October 2019

Abstract
Abstract SCI-37
Stem and progenitor cells reside in specialized microenvironments that regulate their function. While some stem/progenitor cells are perivascular, others clearly occupy hypoxic niches and may be regulated by O2 gradients. We are currently evaluating underlying mechanisms for the impact of O2 levels on stem and progenitor cells within distinct microenvironments. We have previously shown that neural stem cells within the adult hippocampus are closely associated with low O2 regions and that hypoxia-inducible factor 1α (HIF-1α), a principle mediator of hypoxic adaptations, modulates Wnt-β catenin signaling to maintain stem cell proliferation, differentiation, and neuronal maturation. We have extended these findings to other stem cell populations, such as those of adult muscle and bone marrow. Our findings will be presented at this meeting.

Disclosures:
No relevant conflicts of interest to declare.
Small Molecule Screens for Stem Cell Expansion
Authors: Cooke Michael P. , Boitano Anthony E. 
Published: October 2019

Abstract SCI-42
The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening >100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a >600-fold increase in the number of CD34+ cells, and a >2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use.

Disclosures:
Cooke: Novartis: Employment. Boitano:Novartis: Employment.
Hemophilia Gene Therapy
Authors: Arruda Valder R. 
Published: October 2019

Abstract SCI-48
Over the last decade, five clinical studies on gene therapy for hemophilia A and B using viral and nonviral vectors for liver-restricted or ectopic expression of clotting factor demonstrated safety with no inhibitor formation. Although efficacy in these early phase trials was not achieved because of sustained levels of clotting factor below 1%, these studies form the basis for the second generation of clinical trials. To date, two studies on adeno-associated viral (AAV) vector encoding a FIX transgene for liver-restricted expression are ongoing. In one study using a modified AAV genome pseudotyped with serotype 8, emerging data are encouraging and sustained therapeutic levels of FIX have been obtained in a dose-dependent manner. Novel approaches for hemophilia have been explored targeting hematopoietic stem cells (HSC) using lentiviral vectors for expressing FVIII or FIX genes. Transgene expression under the control of either a non-lineage specific promoter or a platelet-specific promoter showed biological activity and improvement of the disease phenotype. The platelet-restricted approaches did not increase circulating plasma clotting factor levels, but they resulted in enrichment of the factor at the injury site upon platelet activation. An advantage of targeting FVIII expression and storage in platelets is the protection of FVIII from neutralizing antibodies (inhibitors). In this model, platelet-FVIII provided superior hemostasis than elevated plasma FVIII levels upon hemostatic challenges in the microcirculation and macrocirculation. Recently, platelet-restricted expression of human FVIII gene using a lentiviral vector for ex vivo transduction of canine HSC resulted in improvement of the disease phenotype in severe hemophilia A dogs without unwanted immune responses to the transgene. Additional strategies to optimize gene- and/or cell-based approaches have also focused on use of clotting factors with enhanced biological activities will be discussed. Together with the generation of novel vectors, further enhancement of both the efficacy the safety of these approaches is envisioned.

Disclosures:
Arruda: Pfizer: Research Funding.
Ataxia Telangiectasia
Authors: Gatti Richard A. 
Published: October 2019

Abstract
Abstract SCI-7
Ataxia-telangiectasia (A-T) is the prototype for an expanded group of inherited radiation sensitive disorders that together define the XCIND syndrome: x-ray hypersensitivity, cancer, immunodeficiency, neurological dysfunction, and DNA repair deficiency. Although the clinical radiosensitivity of these disorders can be tested in the clinical laboratory, diagnostic methods remain limited and in need of further validation. Without exception, to date, sensitivity to ionizing radiation appears to be integrally associated with double strand break (DSB) repair defects and lymphoid cancer susceptibility, setting these disorders apart from single strand break repair disorders such as xeroderma pigmentosum. Responding within seconds to DSB damage are ATM kinase, the protein lacking in A-T, and the NMR complex (nibrin, Mre11, and Rad50). The latter three proteins are associated with three additional XCIND disorders (nibrin deficiency [aka nijmegen breakage syndrome], Mre11 deficiency [ATLD], and Rad50 deficiency). ATM kinase activates a myriad of other proteins that 1) halt DNA synthesis, replication, and the progression of the cell cycle; 2) form a complex protein “mesh” to physically stabilize the broken DNA strands; and 3) restore the integrity of the breaks before they unravel to create even larger chromosomal lesions and resulting malignancies. Another ATM-dependent cancer link involves the downregulation of ATM by microRNA-421. MicroRNA-421 is upregulated by the transcription factor N-myc. Despite this, neuroblastomas are not commonly observed in A-T or XCIND patients. Another subset of XCIND-associated disorders lacks proteins the drive the nonhomologous end joining pathway of DNA repair. Several of these diseases present in infancy as B−/T− severe combined immunodeficiency, or SCID, and are frequent candidates for stem cell transplantation. Attempts to ablate existing bone marrow prior to transplantation may further compromise such patients if they are inherently radiosensitive. Thus, attempts to preselect such patients and reduce radiation dosages may improve general post-transplantation survival. While most protein deficiencies can be diagnosed by immunoblots of appropriate cellular fractions, nonfunctional proteins are not detected by this platform. Colony survival assays (CSA) measure the ability of replicating cells (e.g., lymphoblasts or fibroblasts) to survive after exposure to radiation. Although causal proof that CSA can predict clinical radiosensitivity is lacking, the reduced percent survival fraction (i.e., radiosensitivity) of A-T, N-Bromosuccinimide, or Fanconi cell lines can be abrogated by introducing the mutated cognate gene. Other surrogate assays for radiosensitivity include kinetic studies, pre-irradiation and post-irradiation of γ-H2AX or SMC1 phosphorylation. Ultimately, DNA sequencing of a candidate gene can pinpoint the underlying pathogenesis of radiosensitivity in an XCIND disorder.

Disclosures:
No relevant conflicts of interest to declare.
Leukocyte Immunoglobulin-Like Receptor B Regulates Erythroblastic Island Size in Human in-Vitro Erythroblastic Islands
Authors: Perumbeti Ajay , Girnita Alin , Cancelas Jose A. 
Published: October 2019

Abstract
Abstract 3190
Erythropoiesis occurs in a unique bone marrow (BM) microenvironment known as erythroblastic islands. Erythroblastic islands are composed of erythroid precursors in close contact with specialized stromal macrophages, also referred to as central or island macrophages. Human erythroblastic island macrophages have been difficult to discern from other macrophage subpopulations by cell surface markers, although they have reduced expression of C3b receptor (CD35) (Lee et al., 1988). The unique non-antigen presenting nature of erythroblastic island macrophages led us to hypothesize that it may share surface receptors of other tolerogenic macrophages subpopulations, including leukocyte immunoglobulin-like transcript B receptors (LILRB).
We first identified a distinct subset of human bone marrow macrophages with flow cytometry, which express high amounts of LILRB1 and LILRB2 surface receptors, have low expression of C3b surface receptor (p<0.05), and have increased expression of VCAM-1 surface receptor. This macrophage subpopulation consisted of 1 in 500 (0.02%) of bone marrow mononuclear cells. Functionally, when we FACS sorted human BM enriched erythroid precursors (CD45−/CD71+) and combined them with FACS sorted LILRB1+/LILRB2+ (LILRB+) or LILRB1−/LILRB2- (LILRB−) BM macrophages, there were no significant differences in erythroblastic island reconstitution, suggesting that the adhesion of primary BM erythroblasts to island macrophages is not related to macrophage LILRB expression. Since our ex-vivo erythroid population was enriched for late precursors, we tested island reconstitution with CD34+ cell-derived cultured erythroblasts and FACS sorted LILRB+ or LILRB- BM macrophages, which resulted in a significantly higher number of large proliferative erythroblastic islands (>25 erythroblasts/island macrophage) in LILRB+ compared with LILRB- macrophages (p<0.05) (Figure 1). Incubation with neutralizing anti-LILRB1/LILRB2 antibodies did not prevent primary erythroblastic island reconstitution independent of expression of LILRB by macrophages. However, anti-LILRB1/LILRB2 did prevent the formation of large erythroblastic islands indicating a specific role of LILRB in optimal island reconstitution and erythropoiesis (p<0.05) (Figure 1). In conclusion, we demonstrate that LILRB+ BM macrophages increase the formation of large in-vitro erythroblastic islands and LILRB receptors are important candidate receptors for functional regulation of erythropoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Immunosuppressive Drugs up-Regulate the Immunoinhibitory Receptor Osteoactivin On Human Monocyte-Derived Dendritic Cells
Authors: Braun Sabine , Gutknecht Michael , Schwarzbich Mark-Alexander , Kanz Lothar , Salih Helmut R , Grünebach Frank , Rittig Susanne M 
Published: October 2019

Abstract
Abstract 3284

Introduction:
Dendritic cells (DC) abundantly express the type I transmembrane glycoprotein Osteoactivin (OA) - also known as transmembrane glycoprotein NMB and DC-HIL - compared to low expression levels on monocytes. Antigen-presenting cells interact via OA with the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells which inhibits T cell activation. We previously reported on increased expression of OA upon exposure of monocyte-derived DC (moDC) to immunosuppressive drugs (e.g., Gutknecht et al ASH annual meeting 2011). Here we extended these analyses and comparatively analyzed the impact of various immunsuppressive drugs (ID) on moDC phenotype and function.

Methods:
The moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Clinically relevant concentrations of ID were added to the culture medium every second day starting with the first day of culture (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml, mycophenolat-mofetil 0.1μM, methotrexat 230ng/ml). Cells were harvested for immunophenotyping by flow cytometry, western-blotting and real-time PCR. Cytokine release by moDC was determined on day 7 by ELISA. Functional properties were determined by mixed lymphocyte reactions (MLR) on day 7 of culture.

Results:
Exposure of moDC to therapeutic concentrations of prednisolone resulted in significantly reduced expression of the costimulatory molecules CD83 and CD86 and increased levels of the monocyte marker CD14, indicative of impaired differentiation. Tacrolimus significantly increased CD14 expression and reduced CD83 expression, while the other ID did not cause significant alterations. All ID altered the release of the immunomodulatory cytokines IL-10, IL-6 and TGF-ß. Notably, all ID except cyclosporine A caused a substantial upregulation of the immunoinhibitory receptor OA in moDC. The extent of OA expression increased over time of exposure to ID during differentiation and resulted in reduced capacity of the moDC to stimulate allogenic T cells which could be restored by disruption of OA/SD-4 interaction using a blocking OA antibody.

Conclusion:
Increased expression of OA on moDC upon exposure to ID contributes to inhibition of T-cell activation. The mechanisms underlying the differential effect of cyclosporine A are presently under study. Our results indicate that targeting OA/SD-4 interaction may hold promise for modulation of T cell responses in various pathophysiological conditions and immunotherapeutic strategies.

Disclosures:
No relevant conflicts of interest to declare.