bluuebunny/crossref_metadata_2025_split huggingface dataset:
HS1 (HCLS1) Protein, a Binding Partner of HAX1, Induces G-CSF-Triggered Granulopoiesis Via LEF-1 Transcription Factor.
Authors: Skokowa Julia , Lan Dan , Uenalan Murat , Gupta Kshama , Welte Karl 
Published: October 2019

Abstract
Abstract 548
Mutations in HS1 (HCLS1) associated protein × 1 (HAX1) have been described in patients with severe congenital neutropenia (CN). HAX1 is a ubiquitously expressed and until now it was unclear how HAX1 mutations cause isolated neutropenia in CN patients. So we were interested in investigating G-CSF-dependent HS1 (HCLS1) protein expression and phosphorylation in CN patients compared to healthy individuals, since HS1 (HCLS1) is a hematopoietic-specific protein and may link HAX1 to the G-CSFR signaling via the tyrosine kinases lyn and syk. Indeed, we found that treatment of CD34+ hematopoietic cells with G-CSF leads to phosphorylation of HS1 (HCLS1) protein and recruitment of receptor thyrosine kinases lyn and syk. In CN patients harboring HAX1 mutations, G-CSF failed to upregulate expression and phosphorylation of HS1 (HCLS1), suggesting that for HS1 phosphorylation functional HAX1 protein is required. Indeed, inhibition of HAX1 in CD34+ hematopoietic cells from healthy individuals significantly abrogated G-CSF-triggered HS1 (HCLS1) phosphorylation. Previously, we found that LEF-1 is a master regulator of granulopoiesis and is severely diminished in myeloid cells of CN patients with HAX1 mutations. Moreover, these cells also exhibited severe defects in F-actin assembly. Using immunoprecipitation assay of endogenous proteins, we found that upon G-CSF stimulation of CD34+ hematopoietic cells, HS1 (HCLS1) protein additional to HAX1, also binds to LEF-1 protein modulating its expression, transcriptional activity and subsequently granulopoietic differentiation. Using CFU, qRT-PCR and reporter gene assays we demonstrated that inhibition of HS1 (HCLS1) severely disturbed G-CSF-triggered granulocytic differentiation in line with downregulation of LEF-1 expression and functions, as well as with pathological redistribution of F-actin.
Taken together, in this study we demonstrated for the first time the connection between G-CSF receptor, HAX1 mutations, downregulation of HS1 (HCLS1) and LEF-1, abnormal F-actin assembly and the defective granulocytic differentiation in CN patients. Moreover, we demonstrated for the first time an ultimate direct connection between Wnt signaling and G-CSF cytokine cascade in granulopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Lyl1 and Scl Regulate Bcl2 Expression and Control Apoptosis at Different Stages of Hematopoietic Development.
Authors: Souroullas George , Goodell Margaret 
Published: October 2019

Abstract
Abstract 569
The Lymphoblastic Leukemia gene 1 (Lyl1) encodes a basic-helix-loop-helix (bHLH) transcription factor that is involved in T cell acute lymphoblastic leukemia via chromosomal translocations. Lyl1 has an almost identical bHLH motif to Scl, a known master regulator of hematopoiesis. In our previous work, we used a double knock-out mouse model to demonstrate functional redundancy between Lyl1 and Scl in adult hematopoietic stem cells (HSCs), where in the absence of both genes in adult mice, HSCs and progenitors undergo rapid apoptosis. Despite their common and overlapping functions, the two genes appear to also function at different aspects of hematopoietic development. Lyl1 knock-out mice exhibit a unique defect in B-cell development. This phenotype is recapitulated in vitro, where Lyl1 progenitors grown on a layer of OP9 cells and allowed to differentiate with the addition of cytokines fail to differentiate to B cells as well as wild type progenitors. Rescue assays, where Lyl1 was transduced using a retrovirus into Lyl1 knock-out progenitors and transplanted into lethally irradiated recipients, showed complete rescue of the B cell lineage. Forced expression of Scl into Lyl1 knock-out progenitors failed to rescue, giving rise to only about 5% of peripheral blood B cells. This demonstrates the distinct functions of the two genes in differentiated cells types, despite their functional redundancy in the HSCs. We further sought to dissect the functions and differences between Lyl1 and Scl based on their target genes. Using bioinformatics analysis and a literature search, we identified a number of genes that were candidates for Lyl1 transcriptional regulation; these interactions with Lyl1 and Scl were confirmed by chromatin immunoprecipitation. We found that Bcl2, and genes belonging to this family, are direct target genes of both Lyl1 and Scl in progenitor cells, and that they are downregulated in Lyl1 and Scl single knock-out mouse models. Since Bcl2 is critical for B cell survival, we hypothesized that the B cell deficiency in Lyl1 knock-out mice could be partly due to a loss of Bcl2. We performed rescue assays where Bcl2 was transduced using a retrovirus into Lyl1 knock-out progenitors which were transplanted into lethally irradiated recipient mice. Bcl2 overexpression rescued the B-cell defect in Lyl1 knock-out bone marrow. Since Bcl2 was identified as a target gene for Scl as well, we tested whether Bcl2 could also rescue the rapid apoptosis phenotype of the Lyl1/Scl double knock-out progenitors. Forced expression of Bcl2 in these cells, followed by deletion of the floxed Scl allele, resulted in a delayed onset of apoptosis, but incomplete rescue of the progenitor cell death. These findings distinguish the activities of Lyl1 and Scl in B cells and demonstrate a possible mechanism for their functions, through in part by their activation of the antiapoptotic gene Bcl2.

Disclosures:
No relevant conflicts of interest to declare.
Gabp Transcription Factor Is Required for Myeloid Differentiation, In Part, through Its Control of Gfi-1 Expression
Authors: Yang Zhong-Fa , Drumea Karen , Cormier James , Wang Junling , Rosmarin Alan G. 
Published: October 2019

Abstract
Abstract 740
GABP is an ets transcription factor which regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPβ, which contains the transcription activation domain. In order to examine the role of Gabp in myeloid differentiation, we created mice in which exons that encode the ets DNA-binding domain are flanked by LoxP recombination sites (Gabpa fl/fl or floxed mice), and bred them to mice that bear Cre recombinase under the control of the pIC-inducible promoter, Mx1. Injection of these mice with pIC induces conditional deletion of Gabpa in hematopoietic tissues, and these mice are referred to as Gabpa knock-out mice (KO). Control mice that carry floxed Gabpa but lack Mx1-Cre were also treated with pIC. KO mice rapidly lost myeloid cells in peripheral blood, bone marrow, and spleen. Bone marrow cells from control and KO mice were sorted for CD11b and Gr1 to distinguish mature granulocytes from immature granulocytes and monocytes. KO mice had few mature myeloid cells, and the residual cells exhibited a dysplastic phenotypic appearance that did not resemble either normal maturing monocytes or granulocytes; instead they exhibited morphologic characteristics of both lineages. These KO myeloid cells simultaneously expressed differentiation-associated monocyte-restricted and granulocyte-restricted genes and markers of immaturity (Ly6C and c-kit). Thus, the patterns of gene and antigen expression by these dysplastic KO myeloid cells did not correspond to either normal monocytes or granulocytes. We recognized that these KO myeloid cells resemble those seen in mice with disruption of the transcriptional repressor, Gfi-1. We isolated granulocyte-monocyte progenitor (GMP) cells from control and KO bone mice, and found that expression of Gfi-1 was significantly reduced in KO GMPs, compared to controls. Chromatin immunoprecipitation demonstrated that Gabp bound the Gfi-1 promoter in vivo and transfection analysis showed that Gabp activated the Gfi-1 promoter. To test the hypothesis that Gabp controls myeloid differentiation through its regulation of Gfi-1, we grew bone marrow cells from both control and KO mice in liquid culture with SCF, Il-3 and Il-6, or GM-CSF. We transduced these cells with retrovirus bearing Gfi-1, and analyzed myeloid colony formation, and expression of CD11b and Gr1. Transduction of KO bone marrow cells with Gfi-1 partially restored myeloid colony formation in semi-solid media, and the percentage of Gr1+CD11b+ cells in liquid culture. We conclude that GABP plays a non-redundant role in myeloid development, and it controls differentiation of myeloid cells, at least in part, through its regulation of the transcriptional repressor, Gfi-1.

Disclosures:
No relevant conflicts of interest to declare.
Genetic and Epigenetic Contributions to Longevity
Authors: Barzilai Nir 
Published: October 2019

Abstract
Abstract SCI-1
Aging is a natural process that involves a general decline in many physiological functions resulting in loss of function and eventually death, and is also the main risk of death from almost all diseases. Therefore we reason that if we could understand the biology of aging, we could design a therapy that will not affect a single disease at a time but rather will prevent many aging related diseases. Indeed, in the field of aging we have been successful in identifying several mechanism that may be involved in aging, but even more importantly, mechanisms that are protective against aging.
Here we will summarize the efforts to implicated protective pathways in relatively healthy subjects with exceptional longevity. These subjects are part of the LonGenity (or Longevity Genes Study) at Albert Einstein College of Medicine, which gathered the clinical phenotype and performed extensive genotyping on centenarians and their offspring. We will demonstrate that genetic factors that are associated with human longevity are also heritable and may contribute not only to increase life span, but also provide a protection from age dependent disease and exceptional good health.
We also will demonstrate that aging may be modified by epigenetics mechanism and will show results from high throughput methylation studies in aging rodents and humans.

Disclosures:
No relevant conflicts of interest to declare.
MicroRNA in Platelet Production and Activation
Authors: Bray Paul F. 
Published: October 2019

Abstract
Abstract SCI-39
Alterations in gene expression are at the heart of both megakaryocytopoiesis and interindividual variation in platelet reactivity. Over the past decade there has been an increasing awareness of the important role played by microRNAs (miRNAs) in these diverse cell biologic and physiologic processes. MiRNAs are noncoding RNAs that target complementary sequences in mRNAs, leading to mRNA degradation or inhibition of translation. More than 1000 miRNAs have been identified, which are estimated to regulate 30%-90% of all coding genes. Expression of miRNAs is cell and developmental stage specific. MiRNAs regulate hematopoietic lineage commitment, as well as B lymphopoiesis, granulopoiesis, erythropoiesis and monocytopoiesis. Recent data also demonstrate a clear role for miRNAs in megakaryocytopoiesis (Meg-poiesis). The molecular genetic basis for the transition from one stage of Meg-poiesis to another requires fine-tuning of the various control elements, and miRNAs act as “rheostats” in this process. MiRNA-150 has a strong effect on Meg-erythroid progenitor cells, inducing commitment to the Meg lineage at the expense of erythropoiesis. MiRNA-150 directly down-regulates MYB, a transcription factor important in thrombopoiesis. Over-expressed miRNA-146a knocks down CXCR4 (receptor for SDF-1), reducing Meg proliferation and maturation. MiR-146a gene expression is negatively regulated by the promyelocytic zinc finger protein, PLZF. Thus, miRNAs act as intermediaries of transcription factors that control Meg-poiesis. An increasing number of genetic diseases are being described due to mutations in miRNA genes or their mRNA targets. Deletion of miR-145 and miR-146a on 5q is sufficient to cause the 5q- phenotype; miR-125b-2 up-regulates Meg-poiesis and may be involved in megakaryoblastic leukemia. Platelets appear to be a particularly rich source of miRNAs, many of which are expressed at very high levels. Notably, platelets contain Dicer, TRBP2 and Ago2, and are capable of synthesizing miRNAs from pre-miRNAs. MiRNAs regulate Bcl-xL and Bak, raising the possibility that platelet miRNAs affect platelet lifespan. Because miRNAs are very stable, they also represent a potential biomarker, and we have found sets of miRNAs that appear to classify platelet reactivity to epinephrine. Using bioinformatic approaches, we have identified miRNA-mRNA pairs differentially expressed in platelets with differing phenotypes. This approach permits functional characterization of novel platelet mRNAs, and elucidation of a potential genetic mechanism for adjusting megakaryocyte/platelet mRNA expression. Thus, information extracted from these RNA networks continue to provide insights into systems biology of higher organisms.

Disclosures:
No relevant conflicts of interest to declare.
The Cellular and Molecular Mechanisms of Neutropenia in Barth Syndrome
Authors: Aprikyan Andrew A , Makaryan Vahagn , Kulik Willem , Vaz Frederic , Allen Christopher , Dror Yigal , Dale David C. 
Published: October 2019

Abstract
Abstract 1105
Barth syndrome (BTHS) is a rare, X-linked recessive disease characterized by progressive cardiomyopathy and neutropenia with life-threatening bacterial infections. BTHS is attributable to loss-of-function mutations in the tafazzin gene, TAZ, a phospholipid acyltransferase localized to the mitochondrial membrane and involved in remodeling cardiolipin. BTHS children exhibit reduced blood levels of total cardiolipin; however, the molecular and cellular mechanisms of BTHS remain largely unknown.
We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrated a significant down-regulation in the TAZ gene expression, mimicking naturally-occurring truncation mutations. FACS analyses of cells with TAZ-specific but not scrambled shRNAs demonstrated nearly two-fold increase in proportion of annexin-V positive cells and significantly increased dissipation of mitochondrial membrane potential (MMP) as determined by DIOC6-staining. Similar pro-apoptotic effects were observed in U937 myeloid progenitors, but not in Jurkat or Ramos human lymphoid cell lines. Further studies revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knock-down. Treatment with caspase-specific inhibitor zVAD-fmk reduced apoptosis to near-normal levels. FACS analysis utilizing cryopreserved bone marrow cells from a BTHS patient showed evidence of accelerated apoptosis compared to corresponding cells from a healthy volunteer. Importantly, human myeloid progenitor cells and cardiomyoblasts/cardiomyocytes depend heavily on mitochondria, which may explain why the loss of mitochondrial protein tafazzin has more severe effects on these specific tissues in BTHS resulting in neutropenia and cardiomyopathy.
These data suggest that neutropenia in BTHS patients is attributable to increased dissipation of MMP, aberrant release of cytochrome c, activation of caspase-3 and accelerated apoptosis, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors. Further studies should evaluate the TAZ–regulated mitochondrial apoptosis pathway using primary cells from BTHS patients.

Disclosures:
Aprikyan: Barth Syndrome Foundation: Research Funding. Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies
Authors: Nalivaiko Kristina , Hofmann Martin , Grosse-Hovest Ludger , Krammer Peter H , Rammensee Hans-Georg , Salih Helmut R. , Jung Gundram 
Published: October 2019

Abstract
Abstract 1114
Antibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells.

Disclosures:
No relevant conflicts of interest to declare.
A Humanin Analog Attenuates Platelet Responses to Vascular Injury
Authors: Ren Lijie , Li Qiang , Zeng Zhao , Mou Peipei , Liu Xiaohui , Tang Xiaorong , Lu Qiongyu , Xu Xingshun , Zhu Li 
Published: October 2019

Abstract
Abstract 1131
Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P<0.05) and 71.9% (P<0.001), respectively. Similarly, HNG significantly reduced ATP release stimulated by collagen or convulxin. Convulxin-induced P-selectin expression and fibrinogen binding on single platelet was inhibited by HNG, as measured by flow cytometry. Moreover, HNG reduced platelet spreading on the fibrinogen coated surface by 62.9 % (P <0.05). Western blot revealed a reduction of platelet AKT phosphorylation by HNG upon collagen stimulation, implying the involvement of PI3K pathway. In addition, MAPK P38 phosphorylation by collagen and convulxin was also reduced by HNG. HNG effects on thrombus formation were tested in vivo in a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of HNG (25μg/kg) to male C57BL6/J mice significantly extended the first occlusion time (7.3±0.4 min, N=10), when compared to the saline injected littermates (5.4±0.7 min, N=12) (P <0.05). Furthermore, the number of mice that formed stable thrombus was less in the HNG–treated group (3/13) than the control group (6/13), while the non-occlusion mouse number was more in the HNG-treated group (3/13) than the control group (1/13). Together, these data show that HNG inhibits platelet activation and arterial thrombus formation. This might suggest that the protective effects of HNG against ischemia reperfusion injury could be, in part, via attenuating platelet activation. Therefore, HNG could be a potential therapeutic agent in thrombotic and cardiovascular disorders.

Disclosures:
No relevant conflicts of interest to declare.
Hypoxia-Induced Pulmonary Arterial Hypertension: The Role of TLR4
Authors: Ma Liping , Chang Jianxing , Wu Hui , Chen Yabing 
Published: October 2019

Abstract
Abstract 1146
Pulmonary arterial hypertension (PAH) is often as a result of COPD and contributes to morbidity and mortality of chronic cor pulmonale. Hypoxemia is its main clinical features. The toll-like receptors are a group of type I transmembrane proteins that play a central role in specific recognition of pathagen-associated molecular patterns and are critical for the induction of innate immunity and inflammation. Expression of these innate immune receptors, especially TLR4 in healthy and pathological arteries has implicated its role in the homeostasis of vasculature. Chronic hypoxia-induced PAH is mediated by increased production of reactive oxygen species (ROS). ROS have been linked to activation of toll-like receptor 4 (TLR4) signaling. The present studies investigated the role of TLR4 in the pathogenesis PAH and the underlying mechanisms.
In vitro, pulmonary arterial smooth muscle cells (PASMC) derived from TLR4−/− mice exhibited increased production of intracellular reactive oxygen species (ROS). Furthermore, under hypoxia, the expression of TLR4 in PASMC from WT mice was decreased, ROS production in PASMC from WT mice were increased, suggesting down-regulation of TLR4 may contribute to hypoxia-induced PAH.
Using a hypoxia-induced PAH model, we found that hypoxia induced PAH and increased pulmonary arterial wall thickness in wild type (WT) mice. In contrast, mice deficient in TLR4 spontaneously developed PAH and increased pulmonary arterial wall thickness, which were not further enhanced by hypoxia. Consistent with the development of PAH in TLR4−/− mice, echocardiography confirmed right ventricular hypertrophy and decreased pulmonary arterial acceleration time compared to WT mice. Above results suggest that TLR4 plays an important role in maintaining normal pulmonary vasculature, and that hypoxia induces PAH via TLR4.
In short, these studies demonstrate an important role of TLR4 in maintaining normal pulmonary vasculature and in the development of hypoxia-induced PAH. Genetic ablation of TLR4 and inhibition of TLR4 expression by hypoxia induces PASMC proliferation and vascular remodeling, in association with redox signaling in the pathogenesis of PAH.

Disclosures:
No relevant conflicts of interest to declare.