bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aberrant DNA Methylation and Transcriptional Silencing of DLC-1 in Waldenstrom's Macroglobulinemia.
Authors: Xu Lian , Ciccarelli Bryan , Hatjiharissi Evdoxia , Yang Guang , Zhou Yangsheng , Hunter Zachary , Sun Jenny , Gong Ping , Liu Xia , Tseng Hsiuyi , Ioakimidis Thea , Manning Robert , Hanzis Christina , Cao Yang , Lewicki Megan , Patterson Christopher J , Treon Steven 
Published: October 2019

Abstract
Abstract 2954
Poster Board II-930
The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with potential tumor-suppressor activity. Hypermethylation in the DLC-1 promoter is an aberrant epigenetic modification associated with transcriptional silencing of DLC-1 in various types of human cancers. To explore the epigenetic alteration of DLC-1 in Waldenström's macroglobulinemia (WM), we investigated the methylation status of the DLC-1 promoter and its correlation with DLC-1 mRNA expression in cell lines and primary WM patient samples. Initial analysis using methylation-specific PCR (MSP) showed that DLC-1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1. Using quantitative RT-PCR, DLC-1 mRNA expression was detectable in BCWM.1, but not WM-WSU. These results suggested a correlation between the DLC-1 methylation status and mRNA expression. Similarly, among multiple myeloma (MM) cell lines, we showed that RPMI and U266 exhibited complete methylation, whereas INA6 showed partial methylation, and no methylation was detectable in MM1S and MM1R cells. Similar in WM cells, DLC-1 methylation status was highly correlated with the mRNA expression in these MM cell lines. Of 37 WM patient samples examined, 24 (65%) exhibited methylation in the DLC-1 promoter. In contrast, no methylation of DLC-1 was observed in 4 healthy volunteers. The methylation status was further confirmed using bisulfite DNA sequencing in a subset of WM patients. Quantitative RT-PCR analysis showed that DLC-1 mRNA expression was significantly lower in WM patients compared to healthy volunteers (p=0.001). Treatment with demethylation agents azacytidine or 5-aza-deoxycytidine resulted in significant reactivation of DLC-1 transcription in the WM cell lines WM-WSU and BCWM.1. In addition, a synergistic induction of DLC-1 transcription was observed in the presence of azacytidine and the HDAC inhibitor Vorinostat in BCWM.1 and primary WM patient cells. Moreover, functional studies showed that overexpression of DLC-1 induced cell growth arrest and apoptosis in BCWM.1. DLC-1 methylation status was also correlated with serum sCD27 levels in WM patients (p=0.004), which is secreted by WM cells, serves as a marker of disease burden and facilitates CD40L directed paracrine stimulation by mast cells. Taken together, these results suggest that the down-regulation of DLC-1 via aberrant DNA methylation plays a role in the pathogenesis of WM, and represents a novel therapeutic target in the treatment of WM.

Disclosures:
No relevant conflicts of interest to declare.
Post-Thrombotic Syndrome (PTS) in Children: A Systematic Review of Prevalence, Validity of Outcome Measures, and Prognostic Factors.
Authors: Goldenberg Neil A , Kahn Susan , Crowther Mark A. , Kenet Gili , Nowak-Gottl Ulrike , Manco-Johnson Marilyn J. 
Published: October 2019

Abstract 2978
Poster Board II-949

BACKGROUND:
Post-thrombotic syndrome (PTS) is a condition of chronic venous insufficiency following deep venous thrombosis (DVT) that affects both adults and children. Nevertheless, investigation of PTS in children has lagged behind that in adults.

OBJECTIVE:
This systematic review was undertaken to summarize evidence from the pediatric literature on the prevalence of PTS, the validity of proposed measures of PTS, and prognostic factors for PTS in children.

METHODS:
A comprehensive Medline search was performed employing the following terms: “[pediatric OR children] AND [post-thrombotic syndrome OR post-phlebitic syndrome]”. Single case reports, narrative reviews, and commentaries were excluded.

RESULTS:
Seven case series and cross-sectional studies, nine registries and cohort studies, and one uncontrolled clinical trial were identified that met inclusion criteria; these studies reported on a total of 1316 children with DVT (Table 1). The prevalence of PTS differed substantially between observational and non-observational studies: 15% of 1042 children versus 46% of 274 children, respectively. No pediatric studies have evaluated quality of life (QOL) associated with PTS. Two reports from a single-institutional cohort have identified elevated levels of factor VIII and D-dimer and non-use of thrombolytic therapies as potentially prognostic of PTS in children, particularly among those with veno-occlusive thrombi; these are the only reports employing a PTS outcome measure that has been validated in children (the Manco-Johnson instrument).

CONCLUSIONS:
Overall, high-quality evidence on pediatric PTS is lacking. Collaborative prospective cohort studies and trials that use validated measures of pediatric PTS are needed to assess the incidence of, prognostic factors for, and QOL impact of PTS in children.

Disclosures:
No relevant conflicts of interest to declare.
The Epidemiology of Venous Thromboembolism in the Elderly: a Population-Based Study.
Authors: Tagalakis Vicky , Kahn Susan 
Published: October 2019

Abstract 2980
Poster Board II-954

Background.
There are few studies that examine incidence trends of venous thromboembolism (VTE) among the elderly, and moreover data on changes in the prevalence over time of VTE risk factors such as hospitalization are limited.

Objectives.
Using the Province of Québec's administrative health claims (“RAMQ”) and hospitalization (“MED ECHO”) databases, we determined among individuals 65 years of age and older the trend in annual VTE incidence over a ten-year period, and examined the prevalence of hospital vs. community acquired VTE over time.

Methods.
Using RAMQ medical service and prescription claims data, we identified a cohort of elderly persons with incident VTE between 1983 and 1994. All individuals 65 years of age and older between January 1, 1994 and December 31, 2004 with at least one medical service (i.e. physician) claim linked to a VTE ICD-9 code in conjunction with a prescription claim for an anticoagulant in the subsequent 60 days, and who had no prior VTE-coded claim between 1983 and 1994 were included in the cohort. The first (incident) VTE-coded claim (index claim) during the period between January 1, 1994 and December 31, 2004 defined entry into the cohort. Using Québec population census data, we determined annual VTE incidence estimates, adjusting for the population's age and sex distribution as per 1999 census data. An index VTE event that occurred during a hospitalization and up to 90 days following hospital discharge was defined as hospital-acquired VTE, otherwise it was considered to be community-acquired.

Results.
A total of 27 758 persons were included in our cohort. The age and sex adjusted annual VTE incidence among individuals 65 years of age and older was 2.1, 2.7, and 2.8 per 1000 population in 1994, 1999 and 2004, respectively (p trend < 0.001 (across years)). The incidence rates increased with age and were slightly higher in men than women. Overall, 35% of VTE events were hospital-acquired (n=9598) and 65% occurred in the community (n=18160). There was a significant trend over time of an increase in the proportion of VTE associated with hospitalization with 32% of VTE being hospital acquired in 1994 and 39% in 2004.

Conclusion.
In a general population of individuals 65 years of age and older, VTE is a common problem, and its overall annual incidence has increased over time. Our results suggest that hospital admission and recent hospitalization are increasingly important risk factors for VTE occurrence in this population, and that efforts are needed to improve and optimize VTE preventative strategies in the hospitalized elderly.

Disclosures:
No relevant conflicts of interest to declare.
Risk Stratification of Recurrent Venous Thromboembolism.
Authors: Zotz Rainer B. , Gerhardt Andrea 
Published: October 2019

Abstract
Abstract 2981
Poster Board II-956

Background:
A better risk stratification for recurrent venous thromboembolism (VTE) in patients with a first episode of idiopathic venous thromboembolism (VTE) is urgently needed.

Methods:
Retrospective study covering more than 20 years after a first venous thromboembolic event in a group of 1,440 patients with VTE.

Results:
In the subgroup of patients (n=515) with a first spontaneous VTE, the yearly incidence of a recurrent spontaneous VTE was 8% for the time period 0-2 years and 4-6% in the following 8 years, after a first VTE, triggered by a transient risk factor (oral contraceptives, surgery or immobilization, pregnancy), the yearly incidence of a recurrent spontaneous VTE was 2% (first 2 years) and 1.3-3% (following 8 years). The hazard ratio for recurrent spontaneous VTE in patients with a first spontaneous VTE for specific predictors were as follows: prothrombin mutation heterozygous 1.2 (95% CI 0.9-1.7), FVL heterozygous 1.3 (95% CI 0.92-1.8), male sex 1.9 (95% CI 1.4-2.7), D-Dimer 2.3 (0.9-6.4), protein C (<60% activity) 2.6 (95% CI 1.2-5.7), FVL homozygous 3.0 (1.3-7.7), AT (<60% activity) 3.0 (95% CI 0.96-9.6). In Conclusion, in patients with a first spontaneous VTE the yearly recurrence rate of 5% is more than doubled in the presence of relevant thrombophilic risk factors supporting the need of long-term oral anticoagulant therapy after a first idiopathic VTE. In contrast to current ACCP recommendations, thrombophilic risk factors are of clinical relevance.

Disclosures:
No relevant conflicts of interest to declare.
LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.
Authors: Ma Liping , Wei Jing , Chang Jian-Xing , Zhang Cheng , Pei Zhi-Xin , Yang Qiu-Hong 
Published: October 2019

Abstract
Abstract 3001
Poster Board II-978
Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The present experiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets.

Disclosures:
No relevant conflicts of interest to declare.
SHIP-1 Associates with Tyrosine Phosphorylated Protein Kinase C-Delta at 155 Residue.
Authors: Chari Ramya , Bhavanasi Dheeraj , Daniel James , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 3004
Poster Board II-981
Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1.
This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate.

Disclosures:
No relevant conflicts of interest to declare.
Preliminary Identification of ATP Compartment Associated Proteins On Erythrocyte Membrane.
Authors: Chu Haiyan , Hoffman Joseph F. , Low Philip S. 
Published: October 2019

Abstract
Abstract 3019
Poster Board II-995
A compartment/pool of ATP that is generated by glycolytic enzymes has been reported to exist on the erythrocyte membrane where it channels ATP directly to the Na+/K+-ATPase (Proverbio F and Hoffman JF. J. Gen. Physiol. 1977, 69:605-632. Mercer RW and Dunham PB. J. Gen. Physiol. 1981, 78:547-567). In order to identify the protein components enclosing this compartment of ATP, a photoactivatable probe, 8-azido-[αa-32P]ATP, was loaded into porous erythrocytes under conditions that fill the compartment with ATP, and the radiolabeled ATP was induced to label proximal proteins by illumination with UV light. Analysis of radiolabeled bands reveals that spectrin, adducin, protein 4.1, and actin constitute major components of the compartment. To further verify the involvement of these proteins in the ATP compartment, antibodies against the aforementioned proteins were pre-incubated with porous erythrocytes before attempting to load the compartment with ATP. Analysis of the efficiency of ATP loading in the presence of these blocking antibodies reveals that antibody binding prevents normal filling of the ATP pool. These findings confirm Hoffman's earlier hypothesis that a membrane-bound compartment of ATP exists. The data also suggest that the location of the compartment might reside at the junctional complex, and that the complex of enzymes that fill the ATP pool is different from the complex organized around band 3.

Disclosures
No relevant conflicts of interest to declare.
Role of Spleen Tyrosine Kinase Signaling in Early B Cell Acute Lymphoblastic Leukemia.
Authors: Perova Tatiana , Nutter Lauryl , Matei Irina , Grandal Ildiko , Pine Polly , Hitoshi Yasumichi , Hitzler Johann K. , Guidos Cynthia J , Danska Jayne S 
Published: October 2019

Abstract
Abstract 3092
Poster Board III-29

Introduction
Early B cell acute lymphoblastic leukemia (B-ALL) is the most common type of childhood malignancy, characterized by abnormal accumulation and proliferation of progenitor-B or precursor-B (pre-B) cells. Current challenges associated with B-ALL treatment include fatal relapses, treatment-related toxicities and long-term morbidities underscoring a need to develop new targeted therapies aimed at eradicating leukemia cells and their stem cells. To achieve this, a better understanding of molecular mechanisms involved in leukemia initiation and progression is required. Our laboratory developed p53-/- PrkdcSCID/SCID double mutant (DM) strain as a mouse model of early B-ALL. We showed that DM leukemias progress through discrete developmental stages of leukemogenesis despite the absence of a pre-B cell receptor (pre-BCR), a crucial checkpoint in B cell development. Spleen tyrosine kinase (SYK), a key proximal component of pre-BCR signaling, was activated in the DM leukemias despite the absence of pre-BCR and was required for their survival. Approximately 70% of pediatric pre-B-ALLs also do not express pre-BCR, which lead us to investigate SYK signaling in human pre-B-ALL and to test potential therapeutic application of SYK inhibition in these leukemias.

Patients and Methods
We examined 22 viably frozen primary pediatric pre-B-ALL bone marrow samples to test their responses to SYK inhibition in vitro and in vivo and have investigated the molecular basis for aberrant SYK-mediated signaling in B-ALL.

Results
Western blot analyses revealed that SYK and BLNK, a dominant target of SYK, were expressed in pre-B-ALL patient samples. The majority of human pre-B ALL samples tested (14/22) displayed significantly attenuated proliferation in the presence of SYK inhibitors suggesting that SYK is necessary for their survival and/or proliferation. Treatment with SYK inhibitor R406 prevented phosphorylation of downstream SYK targets including BLNK and PLC-γ2. We are continuing to study the effects of SYK inhibition using phospho-flow cytometry and genome wide expression arrays. Preliminary data will also be presented on therapeutic efficacy of an orally bioavailable form of R406-mediated SYK inhibition in vivo by xenotransplantation of human leukemias into immuno-deficient mice.

Conclusions
Understanding the molecular mechanisms of pre-BCR-independent SYK activation involved in proliferation and survival of leukemic blasts may provide a rational basis for development of effective treatment for ALL. Specifically, targeted therapeutic inhibition of SYK signaling may be effective B-ALL treatment that may improve outcomes of current treatment regiments with minimal additional treatment-related toxicity.

Disclosures
Pine: Rigel Pharmaceuticals: Employment, Equity Ownership. Hitoshi:Rigel Pharmaceuticals: Employment, Equity Ownership.
Warfarin Use May Be Associated with Increase Prevalence of Valvular Calcification.
Authors: Sekhri Arunabh , Lerner Robert G. , Aronow Wilbert S , Palaniswamy Chandrasekar , Ahn Chul , Singh Tarunjit , Sandhu Rasham , McClung John A 
Published: October 2019

Abstract
Abstract 3137
Poster Board III-74

Background
Warfarin affects the synthesis and function of the matrix Gla protein, a vitamin K–dependent protein, which is a potent inhibitor of tissue calcification. We aimed to study the association of use of warfarin with calcification of the mitral valve, mitral annulus, and aortic valve.

Methods
We reviewed 1155 patient's charts with a diagnosis of nonvalvular atrial fibrillation. Based on the chart review, patients were grouped into the warfarin group and the no warfarin group. The following parameters were recorded in both groups: age, ethnicity, glomerular filtration rate, calcium-phosphate product, lipid profile, coronary artery disease, diabetes, hypertension and intake of medications including aspirin, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, statins, and beta blockers. All the echocardiograms were analyzed by two cardiologists blinded to the clinical details of subjects but alerted to evaluate for the presence of mitral valve calcium (MVC), mitral annular calcium (MAC), aortic valve calcium (AVC), or any of the three. Stepwise logistic regression analysis was used to identify significant independent risk factors associated with any calcification using twenty five variables.

Results
Out of 1155 patient records reviewed, 725(63%) were in the warfarin group and 430(37%) were in the no warfarin group. MVC, MAC, or AVC was present in 473 of 725 patients (65%) on warfarin versus 225 of 430 patients (52%) not on warfarin (p<0.0001). Stepwise logistic regression analysis showed significant independent risk factors for any valve calcification were use of warfarin (Odds ratio 1.972, p-value< 0.001), age (Odds ratio 1.078 per one year increase, p-value <0.001), dyslipidemia (Odds ratio 0.235,p-value <0.001), calcium-phosphate product (Odds ratio 1.015, p-value 0.024), and coronary artery disease ( Odds ratio 13.563, p-value <0.0001).

Conclusion
Warfarin intake is associated with a significantly higher prevalence of MVC, MAC or AVC. Further studies are needed to confirm this association and the duration of warfarin intake for development of calcification. A pilot study has been started to study the effect of oral vitamin K on valvular calcification.

Disclosures
Off Label Use: Oral Vitamin K in preventing calcification.