bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of RNA Interference on CD70 in Dendritic Cells of Patients with Immune Thrombocytopenia
Authors: Zhou Zeping , Wang Yunlong , Yang Renchi , Li Huiyuan 
Published: November 2019

Abstract
Objective: This study aims to assess the biological properties and functions of CD70 costimulatory molecule on dendritic cells (DCs) from patients with chronic immune thrombocytopenia (ITP).
Methods: DCs were generated from monocytes isolated from peripheral blood, the immunophenotype markers were detected by flow cytometry. Chemically synthesized siRNA was then transferred into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates. The mRNA expression and protein synthesis were analyzed by real-time RT-PCR and flow cytometry. The CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+ T cells and the production of IL-10 and IFN-γ by CD4+ T cells. The CD4+ T cells were co-cultured with DCs to assess the ability of DCs in the induction of Regulatory T cells (Tregs).
Results: Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h. The transfection efficiency was average 60%. After transfection (48 h), the results of real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected cells (p=0.046, siRNA1\2\3 Inhibition rates (74.5±6.1) %, (78.1±8.0) %, (90.1±2.5) % respectively, but not in negative control or in the absence of siRNA(p=0.157). The CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70. The expression of CD70 on the DCs with siRNA3 was also significantly lower(p=0.043), but showed no difference in negative control (p=0.157). DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients(p=0.008), but showed no difference in group of normal control (p=0.08). In chronic ITP patients and normal controls, the DCs from transfected group could inhibit the expression of IFN-γ compared with the group of negative control (p=0.018, p=0.043) and increase the expression of IL-10 (p=0.012, p=0.043). In chronic ITP patients and normal controls, the DCs from transfected group were defective in inducing the CD4+ T cell differentiating to CD4+CD25+CD127low Tregs compared to the negative control (p=0.012, p=0.043).
Conclusions: In our study, siRNA is capable of inducing RNAi in DCs, it can knock down the CD70 gene expression specifically and effectively. The DCs from transfected group could inhibit the CD4+ T cells and induce the CD4+ T cells differentiating to Tregs. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease.

Disclosures
No relevant conflicts of interest to declare.
Development of Platforms to Phenotype Variants of Uncertain Significance in VWF
Authors: Yee Andrew , Dai Manhong , Kretz Colin , Meng Fan , Ginsburg David 
Published: November 2019

Abstract
Platelet sequestration at sites of vascular injuries and factor VIII (FVIII) stabilization critically relies on von Willebrand factor (VWF). Specific domains within VWF coordinate these functions and may harbor mutations that result in von Willebrand disease (VWD). To survey all possible mutations that may lead to type 2 VWD, we developed prokaryotic and eukaryotic systems to display VWF fragments that are screened for a specific function and identified by DNA sequencing. We constructed an M13 filamentous phage display library consisting of ~2.8x106 independent clones that express random VWF fragments. Following a single round of selection for platelet binding, bound phage were eluted and analyzed by next generation DNA sequencing, revealing an optimal fragment spanning the A1 domain and a second region of weaker enrichment over the D4 domain. All A1 fragments encompassed C1272-C1458, indicating that intramolecular disulfide bridging of these cysteines optimizes platelet binding. Competitive binding assays between phage displaying a fragment of the D4 domain vs. a control phage resulted in enrichment of the former, suggesting a potential, novel role for the VWF D4 domain. Analogous to our previously reported method for analyzing VWF proteolysis (Kretz CA et al. Proc Natl Acad Sci USA. 2015), we mutagenized and displayed the VWF A1 domain to determine the spectrum of mutations that may alter the interaction between VWF and its platelet receptor, GPIbα. The mutant A1 library comprised ~5x106 independent clones, sufficient to represent all 3,933 single substitutions. Screening this library against platelets in the presence of excess control phage resulted in an enrichment of phage displaying an A1 fragment. DNA sequencing identified mutations located outside the VWF A1/platelet GPIbα interface, indicating allosteric control of this interaction. Screening phage displayed VWF fragments for FVIII binding, however, failed to identify critical residues; thus, we adapted our approach for eukaryotes. When displayed on mammalian cells, the VWF D'D3 domains retained their capacity to bind FVIII. Introduction of the VWD subtype 2N mutation, R816W, into the displayed VWF D'D3 domains abolished FVIII binding, demonstrating feasibility for mutational analyses. Together, these results demonstrate the utility of phage and mammalian display for structure-function analyses and that these platforms provide an efficient method for phenotyping variants of uncertain significance.

Disclosures
No relevant conflicts of interest to declare.
Dabigatran Is Associated with a Significantly Lower Risk of Abnormal Uterine Bleeding Than Warfarin in Female Patients of Childbearing Age with Venous Thromboembolism
Authors: Ferreira Melanie , Klok Frederikus A , Feuring Martin , Fraessdorf Mandy , Kreuzer Jörg , Huisman Menno V 
Published: November 2019

Abstract

Introduction: While Direct Oral Anticoagulants (DOACs) have been shown to have an overall equal efficacy as and better safety profile than warfarin in patients with acute venous thromboembolism (VTE), Xa-inhibitors have been associated with a higher risk of abnormal uterine bleeding (AUB). No data regarding AUB with the direct thrombin inhibitor dabigatran etexilate (dabigatran) have been reported to date.
Aim: To determine the risk of AUB during anticoagulation with dabigatran compared to warfarin in female VTE patients aged 18-50 years.
Methods: We performed a post hoc analysis of the pooled RE-COVER studies, two double-blind randomized trials evaluating dabigatran administered at a dose of 150mg twice daily versus warfarin after an initial treatment with parenteral anticoagulants, and the RE-MEDY trial, a double-blind randomized trial that studied the extended use of dabigatran at the same dose versus warfarin, all in patients with VTE. We compared the incidence of AUB, based on a defined preferred terms search in adverse events, in female patients aged 18-50 years who were treated with dabigatran versus who were treated with warfarin. Patients who rolled-over from RE-COVER into RE-MEDY and stayed on the same treatment were accounted for as "only one patient" taking the whole treatment period into consideration, whereas patients who switched treatment were analyzed as two different study subjects.
Results: Of the 2964 women included in the above mentioned trials, 1280 women were in the relevant age category and included in the current analysis. A total of 643 patients were randomized to treatment with dabigatran and 637 to treatment with warfarin. The overall rate of AUB was 5.9% for the women treated with dabigatran and 9.6% in those treated with warfarin, for an Odds Ratio of 0.59 (95% confidence interval 0.39-0.90).
Conclusion: In female VTE patients of 18-50 years old, dabigatran was associated with a significantly 41% lower risk of AUB than warfarin.

Disclosures
Ferreira: BMS-Pfizer: Consultancy, Other: Grant support; Bayer: Other: Grant support. Klok:Boehringer Ingelheim Pharma GmbH & Co.KG: Research Funding; Bristol-Myers Squibb: Research Funding; Bayer HealthCare: Research Funding; Daiichi-Sankyo: Research Funding. Feuring:Boehringer Ingelheim Pharma GmbH & Co.KG: Employment. Fraessdorf:Boehringer Ingelheim Pharma GmbH & Co.KG: Employment. Kreuzer:Boehringer Ingelheim: Employment. Huisman:GlaxoSmithKline: Other: Grant support; Boehringer Ingelheim Pharma GmbH & Co.KG: Other: Grant support; Bayer HealthCare: Other: Grant support; Pfizer: Other: Grant support; Actelion: Other: Grant support.
So Difficult to Understand : Readability Index Analysis of Online Patient Information on Lymphoma from NCI- Designated Cancer Center.
Authors: Kaur Supreet , Kumar Abhishek , Mehta Dhruv , Maroules Michael 
Published: November 2019

Abstract
Objectives - In current era of information technology, there is abundance of medical information for the patients and families. It is recommended that the online patient information (OPI) should be written no greater than sixth grade as per The National Institute of Health (NIH), American Medical Association, Department of Health & Human Services. We aim is to assess whether OPI on lymphoma from NCI-Designated Cancer Center (NCIDCC) and various cancer associations websites meet the current recommendations on a panel of readability indexes.
Methods - OPI from patient only section of NCIDCC and cancer associations websites were collected. This text was analyzed by 7 commonly used readability tests - Flesch Reading Ease score(FRE), Gunning Fog(GF), Flesch-Kincaid Grade Level(FKGL), The Coleman-Liau Index(CLI), The Simple Measure of Gobbledygook (SMOG) Index, Automated Readability Index(ARI) and Linsear Write Formula(LWF). Text from each article was pasted into Microsoft Word and analyzed using the online software Readability formulas.
Results - The mean FRE score is 55.4 (range 37.4-67.9) which corresponded to difficult level grade. The mean GF score is 12.9 (range 10.8-15.2) that comes between difficult and hard. The FKGL score is 9.9 (range 7.2-11.9) that corresponds to above the level of ninth grader.The mean CLI score is 11.09 (range 9-12) which represented text of twelfth grade. The mean SMOG index is 9.6 (range 8.1-11.1) which corresponded to greater than seventh grade level. While the mean ARI score was 10.5 (range 7.7-12.5) which represents readability suitable for people more than tenth grade. The mean LWF was 11.29 (range 7.8-14.4) that corresponds to tenth grade level of text.
Conclusion - The currently available OPI on lymphoma did not met the set national recommendations level on seven different validated readability indexes. Currently, available literature is difficult to understand and comprehend for average patient and their kins. There is a dire need to revise the currently available material for easy comprehension and understanding by the general patient population.

Disclosures
No relevant conflicts of interest to declare.
the Use of Tailored Feedback to Improve Hematologist Competence and Clinical Decision Making for the Treatment of Patients with Multiple Myeloma: a Novel Web-Based Educational Solution
Authors: Repetto Patricia , Raje Noopur S. , Fagerlie Sara R 
Published: November 2019

Abstract
Introduction/Background:
Recent advances in the understanding and treatment of multiple myeloma (MM) have led to improvements in patient management, including stratifying patients according to disease- and patient-specific risk factors, identifying appropriate patients for autologous stem cell transplantation, selecting treatment and incorporating new therapies into practice, and managing adverse effects.
Materials and Methods: An online educational program using clinical problem-based instruction methodology was developed for hematologists and posted November 30, 2015 (http://www.medscape.org/viewarticle/853712). Each patient case included interactivity in the form of clinical decision questions and knowledge assessmentquestions. Tailored feedback and potential consequences in response to clinical decision questions was provided to each learner. Learners who answered a question incorrectly on the first attempt were provided feedback without revealing the correct answer and given the opportunity to answer the question again (second attempt) [Figure 1]. To determine measurable improvements in competence and clinical decision making, first and second attempt answer choices were evaluated for the clinical decision questions. Overall effect size was calculated using Cohen's d to show the magnitude and strength of the consequence-based feedback learning method. Data were collected through February 17, 2016.
Results: A total of 404 hematologists participated in the activity during the study period, and responses from 129 (those who completed all clinical decision questions) were assessed. Responses to the 6 clinical decision questions show that a range of 28% to 67% of learners answered a question correctly on the first attempt. After receiving feedback specific to each incorrect answer, there was an overall 71% relative improvement in hematologists/oncologists who answered a question correctly on the second attempt [Figure 2]. The consequence-based feedback had a large impact on hematologists' ability to make clinical decisions correctly as demonstrated by the overall effect size of d=.94.
Conclusions: This study demonstrated the success of a web-based CME activity with tailored feedback to learners' responses. Participation in this activity led to large improvements in clinician knowledge and the ability to select appropriate evidence- based practice choices, which may ultimately improve outcomes of patients being treated for multiple myeloma.

Disclosures
No relevant conflicts of interest to declare.
Outcome of Myelodysplastic Syndromes over Time in the US: A National Cancer Data Base Study from 2004-2013
Authors: Al-Kali Aref , Zblewski Darci , Bartley Adam C , Patnaik Mrinal M , Gangat Naseema , Begna Kebede H. , Tefferi Ayalew , Elliott Michelle , Hogan William J , Litzow Mark R. , Go Ronald S. 
Published: November 2019

Abstract
Background: Over 10,000 new cases of myelodysplastic syndrome (MDS) are diagnosed in the US annually (MA X, Cancer 2007). Hypomethylating agents (azacitidine and decitabine), immunomodulating drugs (lenalidomide) and erythropoietin stimulating agents (epoetin α and darbepoetin) have been approved for more than a decade for the treatment of MDS. However, changes in overall outcome after the approval of these treatments are unknown. We used the National Cancer Data Base (NCDB), a nationwide oncology registry covering 70% of the US cancer population to answer these questions.
Methods: We identified patients with MDS diagnosed from 2004-2013 using ICD-O-3 codes 9980-9983 and 9985-99912. Overall survival (OS) was estimated using the Kaplan-Meier method. We compared OS using the log-rank test according to demographic subgroups (sex, age [<40, 41-65, and ≥65 years], race [White, Black, Native American, Asian/Pacific Islander, and other], and ethnicity) across years of diagnosis (Period 1: 2004-2006, Period 2: 2007-2009, and Period 3: 2010-2013).
Results:
i) Characteristics: There were 54,953 MDS patients in our study. The median age at diagnosis was 76 years (78.7% older than 65) and 44% were females. White race (88.9%) was the most common, followed by black (7%). Four percent were Hispanics. A third (30.8%) of all patients were treated in academic centers. Three quarters (75.8%) had either Medicaid or Medicare insurance.
ii) MDS Subtypes: 5q Syndrome was seen in 2008 cases (3.7%) while the most common subtype was MDS-Unclassifiable (U) (55.6%). Therapy related MDS (t-MDS) was seen in 1125 cases (2%), however coding for t-MDS was discontinued in 2009. Refractory anemia (RA), refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), and refractory anemia with excess blasts were seen in 9%, 7.6%, 6%, and15.6%, respectively.
iii) OS: Compared to their counterparts, patients who were male, older, had a diagnosis of RAEB, covered by Medicare insurance, and treated at non-academic centers had the worse OS (all P <0.001). Across Periods 1-3, we did not see improvement in OS among demographic subgroups except for those who were <40 years old (P=0.03).
Conclusions: In the past decade, the overall outcome of MDS did not improve despite the advent of new therapies. More than half of the cases were classified as MDS-U reflecting the difficulty of subtyping MDS based on World Health Organization classification. We found disparity in outcomes by demographics and type of treatment facility. This is the largest study of MDS outcome to-date and should help in understanding the gaps and areas that need future research.
Figure Figure.

Disclosures
Al-Kali: Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding.
Male Gender Increases the Risk of Liver Fibrosis in Patients with Thalassemia Major Independent of Iron Overload
Authors: Al-Khabori Murtadha K. , Al Busaifi Said , Al Omairi Al Ghaliya , Hassan Moez , Al Dhuhli Humoud , Al Ajmi Umaima , Al Rahbi Sarah , Daar Shahina 
Published: November 2019

Abstract
Introduction and Objectives: Iron overload in patients with Thalassemia Major (TM) leads to various complications including liver fibrosis. The independent impact of gender on this risk has been previously investigated but not yet confirmed. We, therefore, planned to assess the independent impact of gender in patients with TM on the risk of liver fibrosis.
Methods: We included 96 patients with TM followed and transfused in one academic tertiary hospital. Patients underwent assessment of liver fibrosis using ultrasound elastography (FibroScan device) with a cut off value of 7.8 kPa. The mean ferritin in the 5 years prior to elastography assessment was used to represent iron overload. Association was tested using Chi-squared and the independent impact of gender was confirmed in the multivariable logistic regression with a model that included mean ferritin and gender.
Results: The median age of the 96 included patients was 26 years (Interquartile range [IQR]: 22-30). Males constituted 45% of patients and 33% of patients were splenectomised. The median alanine transaminase, aspartate transaminase, albumin and total bilirubin were 30 U/L (IQR: 18-64), 30 U/L (IQR: 18-46), 46 g/L (IQR: 44-48) and 21 µmol/L (IQR: 14-32) respectively. The median ferritin and liver iron concentration assessed by MRI T2* were 1293 µg/L (IQR: 753-2715) and 6.7 mg/gdw (IQR: 3.5-16.1) respectively. Thirty seven percent of patients had positive serology for HCV while 1% of patients had positive serology for HBV. The proportion of patients with fibrosis as assessed by elastography was 59%. The proportion of male patients with fibrosis was 70% compared to 51% in female patients with a trend towards statistical significance (odds ratio [OR] of 2.2 with a p value of 0.094). In the multivariable logistic regression model, both gender (OR of 3.0, P value of 0.0188) and ferritin (OR of 1.0004, p value of 0.0036) were statistically significant independent predictors of liver fibrosis.
Conclusion: Male gender increases the risk of liver fibrosis independent from iron overload. Our study confirms the previously suspected but unproven association. Follow up and therapy may be tailored to include gender as a decision factor. Larger studies are needed to further confirm these results.

Disclosures
No relevant conflicts of interest to declare.
Quantitation of Suicidal Netosis Using Convolutional Neural Networks in Human Neutrophils
Authors: Elsherif Laila , Metts Carrington A. , Sciaky Noah , Cooper Joshua N. , Holly Stephen P. , Parise Leslie V. 
Published: November 2019

Abstract
Suicidal NETosis (Neutrophil Extracellular Traps) is a mode of cell death involving de-condensation of chromatin and fusion with cytoplasmic granule proteins to create a net-like structure for entrapping pathogenic elements. Despite its name, NETosis does not occur exclusively in neutrophils but rather in all myeloid cells of the immune system except dendritic cells. A growing list of diseases and conditions implicate NETs in their etiology and the progression of symptoms. These conditions include sepsis, cystic fibrosis, lupus nephritis, acute respiratory distress syndrome (ARDS) and deep vein thrombosis. There are several different quantitative methods to assess NETosis making comparative analysis across the field of NETosis research extremely challenging. To further define NETosis and facilitate new discoveries in this field, we have developed a novel assay that combines high-content microscopy with Convolutional Neural Networks (CNN). CNN is the revolutionary image recognition technology used in popular social media facial features recognition systems to 1) quantify the percentage of NETotic and non-NETotic nuclei in a cell population, 2) classify NETotic, necrotic and normal nuclei in a mixed population of neutrophils and 3) differentiate between two different NETotic nuclei generated by two of the most commonly used agonists in NETosis research, the PKC activator PMA and A23187, a Ca+2 ionophore. Furthermore we have optimized neutrophil isolation and culture conditions to yield reproducible results with low inter-and intra-assay variability. Image acquisition was performed on adherent neutrophils using the automated BD Pathway Bio-imaging Systems with autofocusing function that allows imaging of approximately 10% of a well in a 96-well plate. An algorithm was created for image segmentation and recognition of NETotic nuclei based on size, DNA staining intensity and pattern. Data generated by this semi-automated analysis system were used to train a CNN to count NETotic and non-NETotic nuclei. These methods allowed us to calculate the EC50 for PMA (2.2 nM) and A23187 (2.5 uM) and to differentiate between their distinct mechanisms of NETosis. CNNs are able to classify NETotic phenotypes, without the use of expensive antibodies or reagents. Our assay represents a crucial first step for standardizing and quantitating NETosis, which will allow better data interpretation across different labs and provide a potential screening tool for known and as yet undiscovered NETosis agonists.

Disclosures
No relevant conflicts of interest to declare.
Ibrutinib Abrogates TREM-1 Mediated Neutrophil Activation
Authors: Stadler Nicole , Hasibeder Astrid , Aranda Lopez Pamela , Desuki Alexander , Kriege Oliver , Weber Alexander N.R. , Michel Christian , Teschner Daniel , Hess Georg , Theobald Matthias , Radsak Markus 
Published: November 2019

Abstract
Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor on neutrophils (PMN) and important in the innate host defence against microbial pathogens. Here we examined the influence of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib on TREM-1 dependent activation of human PMNs. Firstly, ibrutinib specifically inhibited TREM-1 mediated PMN activation of the oxidative burst and CD62L shedding, whereas TLR mediated activation remained unaffected. Correspondingly, ibrutinib suppressed ERK phosphorylation after TREM-1, but not after TLR ligation. To clarify whether this TREM-1 specific effect of ibrutinib was also relevant in vivo, we treated mice with ibrutinib, and analyzed fungal clearance in vivo upon pulmonary challenge with A. fumigatus and PMN activation ex vivo upon TREM-1 or TLR ligation, again finding that TREM-1, but not TLR induced activation was suppressed. Equivalent results were obtained in PMNs of six lymphoma patients under treatment with ibrutinib. In summary, BTK plays an important role in TREM-1 mediated PMN activation, providing a potential mechanism for the frequently observed infectious complications in patients treated with ibrutinib, and fosters studies using this drug as a novel tool to modulate TREM-1 activity for therapeutic purposes, i. e. in sepsis or autoimmune diseases.

Disclosures
Hess: Pfizer: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding; Roche: Honoraria; Novartis: Honoraria.