bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nox2 siRNA Transfection Down Regulates VEGF Induced-Angiogenesis on Endothelial Progenitor Cells Which Are Derived From HCB
Authors: Yoo Eun-Sun , Mun Yeung-Chul , Nam Eun Mi , Lee Kyoung Eun , Huh Jung Won , Lee Soon Nam , Cho Jung Yeon , Ahn Jee Young , Lee Solip , Seong Chu-Myong 
Published: October 2019

Abstract
Abstract 5188

Background:
Reactive oxygen species (ROS) such as superoxide and H2O2 have roles signaling for molecules on angiogenesis. NADPH oxidase Nox2 (gp91phox) is a major source of ROS. Previous, we had found that Nox2-based NADPH oxidase (gp91phox)-induced ROS may play important roles on EPCs migration and proliferation by VEGF (Blood. 2009;114:Abstract 1445). In the present study, we studied the impact of down-regulation of Nox2 on intracellular ROS level, proliferation, transmigration, and in vitro tube formation of HCB derived EPCs via Nox2 siRNA transfection.

Methods:
Outgrowing endothelial progenitor cells were established from mononuclear cells of human cord blood (Yoo et al, Stem cells. 2003;21:228-235) using EGM-2 media in a fibronectin-coated dish. EPCs were transfected with HiPerFection transfection reagent plus Nox2 siRNA or non-targeting control siRNA and cultured for 5 hours. 100ng/ml of VEGF was added to the transfected cells and cultured for overnight. Expression of Nox2 and pERK in the Nox2 siRNA transfected EPCs were detected by western blot analysis. Intracellular ROS level was analyzed by staining with 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and flow cytometry. Transmigration against VEGF was performed using transwell system (Costar) and in vitro tube formation was assayed using In vitro angiogenesis kit (Chemicon).

Results:
Intracellular ROS level was increased during endothelial progenitor cell culture which were derived from HCB by VEGF treatment. Proliferation, in vitro tube formation matrigel assay and migration assay on endothelial progenitor cells using VEGF were decreased with Nox2 siRNA transfection when compared with that of control group. In western blot data, Nox2-based NADPH oxidase (gp91phox) was increased by VEGF and decreased by Nox2 siRNA transfection. VEGF induced pERK expression was also decreased by Nox2 siRNA transfection as well.

Conclusions:
Based on our studies, Nox2-based NADPH oxidase (gp91phox)-induced ROS may have important roles on proliferation in HCB induced EPCs by VEGF stimulation. Furthermore, Nox2 siRNA transfection into HCB derived EPC down-regulated intracellular ROS production and pERK expression. Our data may be useful finding the new therapeutic targets for ischemic heart and ischemic limb diseases by manipulating the level of intracellular ROS via Nox2.

Disclosures:
No relevant conflicts of interest to declare.
Genome-Wide Array-Based Methylation Profiling Reveals Preferential Methylation of Homeobox Transcription Factor Genes In Mantle Cell Lymphoma and Pro-Apoptotic Genes In Chronic Lymphocytic Leukemia
Authors: Halldorsdottir Anna M , Kanduri Meena , Marincevic Millaray , Göransson Hanna , Isaksson Anders , Mansouri Mahmoud , Stamatopoulos Kostas , Sander Birgitta , Ehrencrona Hans , Rosenquist Richard 
Published: October 2019

Abstract
Abstract 536

Introduction:
Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies of different postulated origin, genetics, clinical presentation and prognosis. Several studies have reported that both MCL and CLL individually exhibit aberrant methylation in comparison to normal B-cells. However, a comprehensive comparison of the methylation profiles of these two B-cell disorders has not been performed yet. This strategy has the potential to identify cellular pathways and genes that are specifically targeted in each disease.

Methods:
We applied the genome-wide Illumina Infinium HumanMethylation27 BeadChip array (Illumina, San Diego, USA) which measures methylation levels at 27,578 CpG dinucleotides covering 14,495 genes, to compare the methylation profiles in: (i) 20 MCL cases; and, (ii) 30 CLL cases, 15 each with unmutated stereotyped subset #1 (IGHV1-5-7/IGKV1(D)-39) B cell receptors (BCRs) or mutated stereotyped subset #4 (IGHV4-34/IGKV2-30) BCRs, where these two subsets represent prototypes of unmutated and mutated CLL. The methylation status for each detected CpG site ranged between 0.1 (completely unmethylated) to 1 (completely methylated).

Results:
As expected, major differences in methylation patterns between MCL and CLL were observed. When the methylation profiles of the two entities were compared, 51 genes were identified as differentially methylated in all comparisons (MCL versus both CLL subsets combined and each subset separately). Among the 19 genes highly methylated in MCL were six (32%) homeobox or homeodomain-containing transcription factors (e.g. POU4F1, PITX3), whereas genes enhancing cell proliferation and tumor progression such as MERTK and CAMP were hypomethylated in MCL. Of the 32 genes hypermethylated in CLL were six pro-apoptotic genes, including DYRK2 and CYFIP2, the tumor suppressor PRDM2 and the cell cycle regulator CCND1.

Conclusions:
We report for the first time disease-biased methylation profiles for different functional classes of genes in MCL or CLL. Homeobox genes were highly methylated in MCL, whereas CLL was characterized by methylation of apoptosis-related genes. The identified differences in global methylation profiles between MCL and CLL may assist in unfolding distinct epigenetic silencing mechanisms involved in the pathogenesis of these B-cell malignancies.

Disclosures:
No relevant conflicts of interest to declare.
A Two-Year Prospective Study on Bleeding Tendency In 105 Patients with Type 2 A and M Von Willebrand Disease
Authors: Castaman Giancarlo , Federici Augusto B. , Baronciani Luciano , Mannucci Pier M. , Rodeghiero Francesco 
Published: October 2019

Abstract
Abstract 543

Background:
Type 2 A and 2 M von Willebrand Disease (VWD) are both characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. The bleeding incidence and possible clinical differences between these two types have never been investigated prospectively in a large number of patients.

Aims and design of the study:
To investigate clinical history and determinants of bleeding in a cohort of patients (pts) with type 2 A and M VWD characterized by mutations over 24 months of follow-up.

Patients and Methods:
10 families with type 2 A VWD (47 patients) and 15 with type 2 M (58 patients) with type 2 M VWD were diagnosed according to the recommendations of the ISTH-SSC subcommittee on VWF and prospectively followed-up from April 2007 to March 2009. Bleeding score (BS) was calculated in all the patients at enrollment by the administration of the same questionnaire tested in type 1 VWD. In case of VWF:RCo levels < 10 U/dL, all patients were tested with a more sensitive ELISA assay to calculate ratios between VWF:RCo and VWF:Ag. VWF mutations were identified in all the patients.

Results:
The 105 patients were characterized by the following mutations: type 2 A R202W/R1583Q 1 pt, S1506L 11 pts, S1543F 1 pt, R1596W 3 pts, V1607D 8 pts, I1628T 1 pt, G1629R 1 pt, G1631D 9 pts, V1665E 8 pts, Q2520P 2 pts, multiple changes 1 pt. Type 2 M: L1278P 6 pts, R1315L 3 pts, R1315C 9 pts, Y1321C 9 pts, L1361W 4 pts, R1374H 23 pts, C1927/c.8155+6C>T 1 pt, c.3831del-3 3 pts. The table summarizes the main demographic and laboratory characteristics. All the patients had a VWF:RCo/VWF:Ag ratio <0.6. The mean BS and VWF:Ag were significantly higher in type 2 A (P < 0.01). No correlation between VWF:RCo levels and the severity of BS was observed.
During follow-up, the bleeding episodes in patients with type 2 M and R1374H or R1315C mutations were very few and mild while they were recurrent and severe in those with type 2 A and V1665E or G1631D mutations, the only laboratory difference being the lack of high molecular weight multimers in type 2 A VWD. Furthermore, 44 gastrointestinal bleeding episodes occurred in 15 patients with type 2 A (range 1–7) compared to 8 episodes in 2 patients with type 2 M (range 2–6). Older age was strongly related to the risk of recurrence of this type of bleeding.

Conclusions:
Bleeding tendency in type 2 A VWD is higher than that of type 2 M VWD and it is not related to the reduction of FVIII:C and VWF activity. The risk of gastrointestinal bleeding is greater in type 2 A and might be related to the lack of high molecular weight multimers and older age. Further analysis are required to evaluate the definite impact of some mutations on the different bleeding tendency.

Disclosures:
No relevant conflicts of interest to declare.
Antigen-Specific Transfer of Functional Programmed Death Ligand 1 From Antigen Presenting Cells Onto Human CD8+ T Cells Via Trogocytosis
Authors: Gary Regina , Voelkl Simon , Palmisano Ralf , Mackensen Andreas 
Published: October 2019

Abstract
Abstract 584
Specific T-cell responses are initiated by T-cell receptor (TCR) recognition of peptide-MHC-complexes on antigen presenting cells (APCs). Upon specific interaction of T cells with APCs T cells capture membrane fragments and surface molecules of APCs in a process termed trogocytosis. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. Here, we demonstrate that human antigen-specific CD8+ T cells do acquire the co-inhibitory molecule programmed death ligand 1 (PD-L1) from mature monocyte-derived dendritic cells (mDC) and tumor cells in an antigen-specific manner. The kinetics of PD-L1 transfer revealed a maximal PD-L1 expression on antigen-specific T cells within 3–4 hours after co-incubation with antigen-pulsed APCs, being detectable up to 72 hours. Antigen-pulsed immature DCs were less effective in transfering surface molecules such as PD-L1 onto CD8+ T cells after antigen-specific recognition. Using a transwell system we could show that the acquisition of PD-L1 requires cell-cell contact. Furthermore, PD-L1 cannot be acquired by T cells from a lysate of mDCs. The transfer process is impaired after pretreatment of T cells with concanamycin A, a specific inhibitor of vacuolar ATPases, playing an important role in membrane trafficking. Moreover, fixation of DCs with glutaraldehyde completely abrogated the acquisition of PD-L1 on T cells suggesting that an active interaction between APCs and T cells is required for trogocytosis. Of importance, CD8+ T cells which acquired PD-L1 complexes, were able to induce apoptosis of neighbouring PD-1 expressing CD8+ T cells, that could be completely blocked by an anti-PD-L1 antibody. In summary our data demonstrate for the first time that human antigen-specific CD8+ T cells take up functionally active PD-L1 from APCs in an antigen-specific fashion, leading to apoptosis of PD-1 expressing T cells. The transfer of functionally active co-inhibitory molecules from APCs onto human CD8+ T cells may serve to limit clonal expansion of antigen-specific T-cell responses but may also play a major role for T-cell exhaustion in chronic infection and tumor immunosurveillance.

Disclosures:
No relevant conflicts of interest to declare.
Chromatin Boundaries Require the Functional Cooperation Between hSET1 and NURF Complexes
Authors: Li Xingguo , Wang Shaohua , Li Ying , Qiu Yi , Huang Suming 
Published: October 2019

Abstract
Abstract 646
Chromatin modification and remodeling activities play a central role in organizing nuclear functions in eukaryotic genome. Chromatin domains with characteristic epigenetic marks are organized by chromatin insulator. The chicken b-globin insulator, 5′HS4, is an excellent model system to study how insulator maintains gene function and prevents the encroachment of repressive heterochromatin. We showed previously that USF1/2 bound 5′HS4 insulator mediates chromatin barrier activity by recruiting and organizing active histone modifications in the chicken b-globin locus. However, it is not clear how insulator establishes such a barrier. To further understand the physical function of USF1, we purified the USF1-associated protein complexes. We found that USF1 forms a multiprotein complex with hSET1 and NURF exhibiting histone H3K4 methyltransferase and ATP-dependent chromatin remodeling activities, respectively. Both hSET1 and NURF complexes are specifically recruited to the 5′HS4 insulator by USF1 to retain the active chromatin structure. Knock-down of BPTF, a component of NURF complex, resulted in a rapid loss of barrier activity that protects a transgene from chromatin silencing. The loss of barrier by the BPTF knock-down is accompanied by an alternation of nucleosome positioning that expands the nucleosome to the nucleosome free linker region in the HS4 insulator site and increases repressive H3K9 dimethylation. Furthermore, the suppression of hSET1, a H3K4 methyltransferase, leads to not only a loss of barrier activity, but also a loss of H3K4 dimethylation and H3K9/K14 acetylation, as well as the recruitment of BPTF at chromatin insulator sites. Thus, our data reveal a molecular mechanism in which histone modifying enzyme hSET1 and chromatin remodeling complex NURF collaborate together to maintain the 5′HS4 chromatin barrier activity and to prevent the encroachment of adjacent heterochromatin.

Disclosures:
No relevant conflicts of interest to declare.
Crosstalk Between Inflammation and Thrombosis: The Surprising Role of Extracellular Histones
Authors: Xu Jun , Lupu Florea , Esmon Charles T. 
Published: October 2019

Abstract
Abstract MRG-1
Activated protein C (APC) is effective in treating septic patients who have already experienced multiorgan failure. The mechanisms responsible for the efficacy remain unclear. Macrophages were activated with endotoxin and gamma interferon, the supernatant harvested and subjected to incubation with activated protein C. The toxicity of the supernatant toward endothelium was blocked by APC incubation. APC incubation generated 3 new peptides which sequence determination demonstrated were derived from histones including H3 and H4. Histones incubated with endothelium were cytotoxic. APC cleaved and eliminated this cytotoxicity. Histones were identified in the circulation of mice challenged with endotoxin, baboons challenged with E. coli and some septic human patients. In mice, blocking the histone toxicity with a monoclonal antibody effectively reduced endotoxin-mediated death. Histone injection resulted in death with pathologies resembling those of sepsis including fibrin deposition and neutrophil infiltration. APC could also prevent death in a histone-mediated model. The histones elicit an inflammatory response, suggesting some recognition by the immune system. Using 293 cells expressing individual toll receptor reporter constructs, we found that the histones activated toll 2 and toll 4. Mice with either of these tolls deleted were resistant to histone-mediated death. This data, coupled with earlier studies demonstrating that histones can kill bacteria, suggest that histones play a role in innate immunity but are also toxic. The results suggest that the inflammatory response in trauma may be triggered by the cellular destruction that releases histones and further suggest that histone antagonists might be an effective mechanism to block progressive organ failure.

Disclosures:
No relevant conflicts of interest to declare.
A Dual Proto-Oncogenic and Tumor Suppressive Role of LRF/POKEMON In Hemopoietic Malignancies through Control of Cell Fate Decision
Authors: Pandolfi Pier Paolo 
Published: October 2019

Abstract
Abstract SCI-14
LRF (Leukemia/lymphoma-related factor, also known as POKEMON) is a member of the POZ and Kruppel (POK) family of transcription factors. LRF has been shown to play an essential role in embryonic development and to act as a master regulator of cellular differentiation in virtually any tissue where it is found expressed, including the hemopoietic compartment. As we will discuss, LRF inactivation in the mouse blocks cellular differentiation in both myeloid/erythroid and lymphoid compartments. On the other hand, LRF has been shown to possess a potent proto-oncogenic activity both in vitro and in vivo. In fact, LRF itself can transform primary cells in combination with known oncogenes and is also essential for cellular transformation of mouse embryonic fibroblasts. In addition, overexpression of LRF in immature B and T progenitor cells in vivo in the mouse lead to lethal precursor T-cell lymphoblastic lymphoma/leukemia. In agreement with this notion, LRF is aberrantly expressed in a variety of human cancers, including diffuse large B cell and follicular lymphomas, but also ovarian and breast cancers. Further, the LRF gene is found amplified in a subset of non-small cell lung cancers (NSCLCs), illustrating a direct role in human cancer. However, we speculated that due to the key role of LRF in cell fate decisions, LRF/POKEMON loss could also contribute to tumorigenesis by blocking cellular differentiation. We will discuss provocative in vivo data in support of the notion that LRF/POKEMON can indeed act as a bona fide tumor suppressor representing a compelling example of two-faced cancer genes.

Disclosures:
No relevant conflicts of interest to declare.
Does Declining Mitochondrial NAD+ and Unscheduled Opening of Mitochondrial Transition Pore Promote Mammalian Aging?
Authors: Sinclair David , Hafner Angela V. , Gomes Ana , Liang Jing , Cen Yana , Lanza Ian , Nair Sree , Sauve Anthony , Rozenzweig Anthony 
Published: October 2019

Abstract
Abstract SCI-3
The processes that lead to the susceptibility of post-mitotic tissues to diseases of aging are poorly understood, but mitochondria are considered a key regulator. SIRT3 is mitochondrial sirtuin, a member a family of NAD+-dependent deacetylases. We have previously shown that increased levels of NAD+ in mitochondria promote cell survival in a SIRT3-dependent manner, but how this protection is mediated is unknown. Here we show that mitochondrial NAD+ levels decline dramatically with age and that SIRT3 is a critical component and regulator of the mitochondrial transition pore (MTP). Mice lacking SIRT3 exhibit hyperacetylation of the MTP with age, and as a result, develop multiple defects in post-mitotic tissues as they age, including mitochondrial defects, susceptibility to cardiac failure, and learning and memory deficits. In a mouse model of Alzheimer's disease (AD), SIRT3 knockout mice show striking phenotypes including small size, kyphosis, and a markedly reduced lifespan. Similar to mice, human brain samples show that acetylation of the MTP increases with age and with AD. These findings indicate that declining NAD levels with age lead to memory deficits and accelerate age-related diseases in post-mitotic tissues due to a decline in SIRT3 activity and unscheduled opening of the MTP.

Disclosures:
Sinclair: GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Expansion of Platelets for Transfusion
Authors: Fuentes Rudy , Wang Yuhuan , Poncz Mortimer 
Published: October 2019

Abstract
Abstract SCI-47
The clinical need for platelets transfusion continues to grow. Platelet units have all the usual concerns with other donor-derived blood products and in addition have concerns over the need to store the platelets at room temperature and of the resulting increased risk of bacterial contamination. Several groups have been able to develop platelets from embryonic stem cells, but achieving significant numbers of pro-platelets and/or platelets from in vitro grown megakaryocytes (Megs) remains elusive. Three sites of thrombopoiesis in vivo have been described: 1) intramedullary, 2) at the medullary/vascular junction, or 3) intrapulmonary from circulating Megs. We investigated whether infusing murine Megs into mice can release a large number of functional platelets both to better understand thrombopoiesis and also to see if such an approach may be an alternative to generating platelets for clinical usage. We show that infused fetal-liver-derived Megs results in 100–200 platelets/Meg. Levels as high as 12% of the total platelets in the recipient mouse were from the infused Megs. These platelets had a delayed presentation, peaking at 90 mins. In spite of these being new platelets, their half-life was only ∼75% of infused platelets. The shortened half-life was not due to the fact that these platelets were fetal in nature in that infused adult-liver-derived Megs had a similar temporal profile. These derived platelets had normal-size distribution, were not activated, and had not been affected by ADAM17 during in vitro culture as determined by surface glycoprotein 1b alpha. Platelet functionality was shown by their incorporation into clots in arterioles and venules in a cremaster laser injury model and in a ferric chloride carotid artery injury model. Using BrdU-labeled Megs we show that most of the Megs are trapped in the lungs with a few also appearing in the splenic red pulp. None were seen in other tissues. Staining with species-specific CD41 antibodies showed that detectable Meg cytoplasm peaked in the pulmonary vasculature at 30 min, and like the nuclei, was mostly gone by 5 hrs. We estimate that only ∼1% of the available pulmonary capillary bed is affected by the infused Megs, and most studied animals survived the infusion of the Megs. These studies support the lungs as a potential site for thrombopoiesis and that in vitro differentiated Megs can be infused directly avoiding the challenge of developing ex vivo technology to release large number of non-activated, but functional platelets.

Disclosures:
No relevant conflicts of interest to declare.