bluuebunny/crossref_metadata_2025_split huggingface dataset:
PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,
Authors: Zaccariotto Tânia Regina , Ribeiro Daniela Maria , Machado-Neto Joao , Santos Magnun N N , Lanaro Carolina , Albuquerque Dulcinéia Martins , Saad Sara Teresinha , Costa Fernando Ferreira , Sonati Maria de Fátima 
Published: October 2019

Abstract
Abstract 3200

Background:
Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter.
This work was supported by FAPESP, CNPq and INCTS.

Disclosures:
No relevant conflicts of interest to declare.
Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,
Authors: Vazquez Carlos E , Prado Gregory N , Maldonado Enrique R , Saca Gabriela , Ortiz Iren M , Brito Giselle , Nieva Adriana , Rivera Alicia , Romero Jose R 
Published: October 2019

Abstract
Abstract 3206
Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,
Authors: Rafei Moutih , Rouette Alexandre , Ruiz Juan Vanegas , Perreault Claude 
Published: October 2019

Abstract
Abstract 3235
T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4+CD8+CD69−) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCRloCD5lo (DP1), TCRintCD5hi (DP2), and TCRhiCD5int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8+ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8int and CD8hi. When analyzed by flow-cytometry, ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells.
This work was supported by grant a from CIHR.

Disclosures:
No relevant conflicts of interest to declare.
A Glycan Wedge Between βTD and βI Domains Activates Integrin αIIbβ3,
Authors: Yamanouchi Jun , Hato Takaaki , Fujiwara Hiroshi , Yakushijin Yoshihiro , Yasukawa Masaki 
Published: October 2019

Abstract
Abstract 3259
Integrin αIIbβ3 undergoes allosteric conformational changes in its extracellular domains, resulting in integrin activation that allows high affinity binding with soluble ligands. The crystal structure of the integrin β subunit revealed an interaction of the β-tail domain (βTD) with the βI domain containing ligand-binding sites, suggesting that βTD may be involved in allosteric mechanism for integrin activation. However, previous studies have shown conflicting results on the functional role of βTD in integrin activation. In this study, we conducted site-directed mutagenesis in the βTD domain and tested ligand binding to αIIbβ3 mutants. We produced αIIbβ3 mutants in which the β3TD loop residues (DSSG) were substituted with the corresponding β1 (NGNN) or β2TD residues (DGMD). The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of PAC1, a ligand-mimetic anti-αIIbβ3 antibody. The NGNN, but not DGMD mutant bound significant PAC1 binding without any stimulation, indicating a constitutively active state. To identify the residue(s) responsible for αIIbβ3 activation in the βTD, we produced αIIbβ3 mutants in which the individual residues in the β3TD loop were substituted with the corresponding β1TD residues. Among them, only G675N bound significant PAC1 binding without any stimulation. Since G675N mutation creates a sequence known to be a consensus sequence for glycosylation (Asn-X-Ser/Thr), it is possible that the insertion of glycans into the βTD loop induces conformational changes in αIIbβ3 which allow ligand binding. To test this hypothesis, we added substitution of S677 with Thr, Ala or Asp to the G675N mutation. The resultant G675N/S677T double mutant, in which the N-glycosylation site was preserved, was constitutively active. In contrast, G675N/S677A and G675N/S677D, in which the N-glycosylation site was disrupted, were in an inactive state. These results suggest that an artificial glycan wedge between βTD and βI domains activates αIIbβ3. However, our study does not provide evidence that the βTD domain constrains wild type αIIbβ3 inactive although the separation of βTD and βI domains may be able to activate integrins.

Disclosures:
No relevant conflicts of interest to declare.
ITI with a VWF-Stabilised FVIII Concentrate in Haemophilia A Patients with Inhibitors and a Poor Prognosis for ITI Success: Progress Report on octanate® in the Observational ObsITI Study,
Authors: Escuriola-Ettingshausen Carmen , Vdovin Vladimir , Zozulya Nadezhda , Plyushch Olga , Kreuz Wolfhart , Schwartz Bruce A. 
Published: October 2019

Abstract
Abstract 3310
FVIII inhibitors that neutralise FVIII efficacy are the most serious complication of FVIII replacement therapy in patients with haemophilia A (HA) and can be overcome with high-dose FVIII (Bonn protocol) during immune tolerance induction (ITI) therapy.
The ongoing investigator-initiated Observational Immune Tolerance Induction (ObsITI) study systematically documents the tolerisation process, retrospectively and prospectively, in patients with HA and inhibitors, including those with a poor prognosis for ITI success. Here we report interim data on Octanate®, a human plasma-derived, von Willebrand factor-stabilised FVIII product.
Data were analysed for a subgroup of 18 prospective patients who received octanate® as the sole FVIII replacement therapy for a treatment/follow-up period of 36 months. ITI success was determined by fulfilment of three stringent criteria: (I) inhibitor titre < 0.6 Bethesda Units, (II) incremental in vivo recovery ≥ 80% of 1.5% per IU/kg body weight reference and (III) FVIII half-life ≥ 7 hours. According to the criteria fulfilled, patients achieved the following: partial response (I), partial success (I, II), complete success (I, II, III) or failure. Following complete success, patients received prophylaxis with octanate® every second day.
Of 18 patients, with at least one risk factor for a poor ITI outcome, 14 patients (77.8%) achieved complete success; 2 patients (11.1%) had partial response and 2 patients (11.1%) failed the ITI. Cox regression analysis of time to achievement of treatment outcome, suggested that both commencing ITI at ≤ 7 years of age and having had no prior ITI attempt appear to be positive prognostic factors for earlier ITI success in this population.
Despite the stringent success criteria, complete or partial ITI success was achieved in 77.8 % of the patients. Treatment with octanate® administered according to the Bonn protocol is safe and effective in the induction of immune tolerance, even in patients with a poor-prognosis for ITI success.

Disclosures:
Schwartz: Octapharma usa: Employment.
The Biological Efficacy Profile of a Chemically Modified Recombinant Factor VIII Molecule,
Authors: Valentino Leonard A. , Hakobyan Narine , Enockson Candace , Cong Lin , Scheiflinger Friedrich , Muchitsch Eva M , Turecek Peter L 
Published: October 2019

Abstract
Abstract 3324
Prophylactic administration of anti-hemophilic factor concentrates can prevent bleeding and limit the complications of hemophilia. The current regimes for prophylaxis require factor concentrates to be administered frequently. This need for frequent venipuncture poses one of the major barriers to more widespread use of prophylaxis. Development of drugs with a longer circulating half-life and duration of action would be beneficial in reducing this requirement and result in less demanding treatment regimes.
In this study the duration of the effect of a chemically modified recombinant factor VIII based on the ADVATE™ manufacturing process (CMrFVIII) in comparison to unmodified ADVATE™ (rFVIII) was determined in a murine model of hemophilic joint bleeding.
CMrFVIII and rFVIII were infused at a concentration of 280 IU/Kg at specified time points prior to joint injury to induce hemarthrosis. Animals were sacrificed three days following injury and assessed for the presence of blood in the joint.
CMrFVIII and rFVIII both prevented bleeding, assessed by the total bleeding score (TBS). Pretreatment with CMrFVIII protected against bleeding more effectively compared to rFVIII at all time points assessed after 24 hours (figure). 17% of mice pretreated with CMrFVIII had bleeding at 72 hours compared to 100% of those pretreated with rFVIII.
Our data indicate that the effective duration of action in protecting against joint bleeding was longer after prophylaxis with CMrFVIII compared to rFVIII. This mouse model is an inexpensive, rapid and reliable tool to assess new factor concentrates in clinical development for hemophilia.

Disclosures:
Valentino: Baxter Bioscience, Bayer Healthcare, GTC Biotherapeutics, NovoNordisk, Pfizer, CSL Behring, Inspiration Bioscience, and Biogen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheiflinger:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
Hypofibrinolysis and the Risk of Recurrent Venous Thromboembolism: A Prospective Cohort Study,
Authors: Eichinger Sabine , Traby Ludwig , Heinze Georg , Kollars Marietta , Eischer Lisbeth , Kyrle Paul Alexander 
Published: October 2019

Abstract
Abstract 3331
Hypercoagulability reflected by enhanced activity of clotting factors or a natural inhibitor deficiency is a risk factor of venous thromboembolism (VTE). Whether defects in the fibrinolytic system confer an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic system activity (reflected by clot lysis time [CLT]) and the risk of recurrent VTE.
We followed 704 patients (378 women; mean age 48 years) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with a natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, female hormone use, homozygosity or double heterozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. The fibrinolytic potential was assessed using a plasma based clot-lysis assay according to Lisman et al. (1). A tissue factor-induced clot was lysed by adding tissue-type plasminogen activator and the optical density was measured at 405 nm in an absorbance microplate reader every 30 seconds in order to get a clot-lysis turbidity profile. Study end point was symptomatic recurrent VTE.
135 (19%) patients had recurrent VTE. Recurrence was unprovoked in 117 patients and was more frequent in men (97/326, 29.8%) than in women (38/378, 10.1%). When CLT was entered as continuous variable in a Cox proportional hazards model, hazard ratio (HR) for recurrence was 1.13 (95% CI 1.02–1.25; p=0.02) for each 10 minutes prolongation. The HR for recurrence was 1.11 (95% CI 1.11–1.24; p=0.046) after adjustment for age and 1.08 (95% CI 0.98–1.20; p=0.13) after additional adjustment for sex. After 5 years, the likelihood of recurrence was 23.8% (95% CI 19.3%-28.3%) in patients with a CLT longer than 63.5 minutes (25th percentile) as compared with 14.1% (95% CI 7.4%-20.8%; p=0.04) among those with a shorter CLT. Men and women differed with regard to CLT (76.1±16.2 vs. 71.5±13.6 sec, p<0.001). After adjustment for age, the HR was 1.04 (95% CI 0.92–1.18; p=0.54) among men and 1.14 (95% CI 0.93–1.14; p=0.22) among women.
Hypofibrinolysis confers an increased risk of recurrent VTE. The effect was more pronounced in women than in men.

Disclosures:
No relevant conflicts of interest to declare.
The Point of Care Monitor Coagucheck-XS Displays the Lowest Coefficient of Variation for Determination of Rivaroxaban Compared to Prothrombin Time and Chromogenic Assays,
Authors: Harenberg Job , Giese Christina , Marx Svetlana 
Published: October 2019

Abstract
Abstract 3353
The new oral direct coagulation inhibitor rivaroxaban prolongs coagulation times of various coagulation assays in a concentration-dependent manner. A point of care determination of the anticoagulant effect of rivaroxaban would improve patient care and patient's safety in case of overdose of rivaroxaban. We have analysed intraindividual coefficients of variations (CV) of the Coagucheck XS compared to the STA Neoplastin Plus and STA Rotachrom heparin assays.
Rivaroxaban was spiked to whole blood and plasma from 10 healthy donors (50ng/ml, 200 ng/ml, and 600 ng/ml). Coagulation times and ratios were measured by means of the Coagucheck XS (recombinant human thromboplastin) from whole blood and plasma samples and STA Neoplastin Plus (rabbit brain thromboplastin). The concentration of rivaroxaban was determined using the factor Xa specific chromogenic substrate STA Rotachrom heparin assay. The intraindividual CVs were calculated from 10 determinations at 10 days for the methods at all concentrations of rivaroxaban.
Rivaroxaban prolonged coagulation times of all coagulation assays. The ratios increased from 0 ng/ml to 600 ng/ml rivaroxaban from 1.0 (50 ng/ml) to 1.20 (200 ng/ml) and 1.63 (600 ng/ml) using the whole blood Coagucheck XS method, from 1.0 to 1.33 to 1.85 using plasma on Coagucheck XS, and from 1.0 to 1.51 to 1.80 using the STA Neoplastin Plus assay, respectively. Values of the STA Rotachrom assay were used for comparison of the CVs of the methods. CVs were calculated from the original data (seconds or optical density). CVs were calculated from the intraindividual variation over 10 days for every volunteer. The median, minimum and maximum values are displayed as well as the p-value for the difference of the CVs between methods for every concentration of rivaroxaban (Kruskal Wallis test).MethodRivaroxaban: 0ng/ml50ng/ml200ng/ml600ng/mlCogucheck XSCV median1.040.991.040.87whole bloodmin-max0.38–1.700.65–1.430.37–1.680.40–1.40Coagucheck XSCV median1.081.011.041.80plasmamin-max0.83–2.380.46–2.800.61–2.360.52–3.17Neoplastin PlusCV median1.831.743.974.30min-max1.06–2.171.33–2.671.75–4.832.02–6.09RotachromCV median1.481.843.972.99min-max0.96–2.390.97–2.392.46–6.492.08–6.15p-value0.01150.0011<.0001<.0001
The point of care monitor Coagucheck XS using whole blood determines with the highest accuracy and precision the amount of rivaroxaban. The method may be used to screen for overdose in patients on treatment with rivaroxaban.

Disclosures:
Harenberg: Roche Diagnostics: Research Funding.
Can Heparin-Based Therapy in Children Be Monitored on Samples Drawn Through Ports? An Analysis From Clinical Trials Data,
Authors: Manco-Johnson Marilyn J. , Goldenberg Neil , Mitchell Lesley G , Jacobson Linda J. 
Published: October 2019

Abstract
Abstract 3365

Background:
Based on preliminary data, children clear heparins more rapidly than adults, and thus most pediatric hematologists monitor heparin therapy. Many pediatric deep vein thrombosis patients have indwelling central venous lines (CVLs) due to co-morbid conditions and patients and clinicians desire to use these CVLs for blood sampling. Standard protocols are used to flush and clear CVLs prior to sample collection, but validated approaches to exclude contamination of heparin in dwell-volume (i.e., hep-lock) from systemic anticoagulant have not been established. Aims: This study was conducted to determine the accuracy of heparin monitoring in samples collected from CVLs. Methods: Anticoagulant effects of unfractionated heparin (UH) and dalteparin low molecular weight heparin (D) were determined by assay of anti-IIa activity, anti-Xa activity and the ratio of the two using chromogenic assays (Biophen). Normal pooled plasma was spiked with 2 U/mL of either drug and dilutions were made to 1.5, 1, 0.75, 0.5, 0.25 and 0.1 U/mL in normal pooled plasma. The assays and ratios were applied to plasma samples obtained by standard protocol from CVLs or peripheral venipuncture in two separate clinical trials. Results: Intra-assay CV for anti-IIa was 14.2 and 13.3% for UH and D, respectively; intra-assay CV for anti-Xa was 8.3 and 2.8% for UH and D, respectively. The ratio of anti-IIa to anti-Xa activity for UH was > 0.75 at all concentrations, while the ratio of anti-IIa to anti-Xa for D was < 0.5 when anti-Xa was within the therapeutic range of 0.5–1.0 U/mL, but overlapped with UH at concentrations ≥ 1.5 U/mL and ≤ 0.25 U/mL. In trial 1, DAVINCI, 4/26 (15.4% of) samples drawn from CVLs with therapeutic anti-Xa activity had ratios > 0.5 suggestive of UH contamination, while 0/8 peripherally drawn samples had ratios in that range. In trial 2, 5/26 (19.2% of) patient samples drawn from CVLs had ratios > 0.5. Conclusions: An anti-IIa/anti-Xa ratio < 0.5 is specific for uncontaminated D effect when anti-Xa is within the therapeutic range. However, without measuring ratios 15–20% of CVL samples used to monitor D will overestimate therapeutic response. Therefore, we favor peripherally drawn samples in order to reliably monitor anticoagulation in children.

Disclosures:
Manco-Johnson: Easai: Research Funding. Goldenberg:Easai: Research Funding.