bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Double-Negative Regulatory T Cells Selectively Suppress mTOR Signaling and Metabolic Reprogramming of Conventional T Cells
Authors: Haug Tabea , Bruns Heiko , Aigner Michael , Mackensen Andreas , Voelkl Simon 
Published: November 2019

Abstract
Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). The subpopulation of TCRαβ+ CD4- CD8- (double-negative, DN) T cells has been described to suppress immune responses in both mice and humans. Of particular interest, infusion and/or activation of murine DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allogeneic hematopoietic stem cell transplantation (SCT). Moreover, clinical studies in patients after SCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD, suggesting a therapeutic potential of human DN T cells. However, the molecular mechanism of suppression still remains unclear. To gain a better understanding of DN T-cell functionality, we investigated whether human DN T cells modulate distinct TCR signaling processes in conventional T cells. We found that DN T cells selectively inhibit the mechanistic target of rapamycin (mTOR) signaling pathway but not activation of mitogen-activated protein kinases (MAPK). The crucial function of mTOR signaling was confirmed by treating effector T cells with a chemical activator of protein kinase Akt, which induces mTOR phosphorylation. Indeed, enforced activation of the mTOR pathway rendered conventional T cells unsusceptible to DN T cell-mediated suppression. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Intriguingly, DN T cells significantly diminished upregulation of the glycolytic machinery, expression of glucose transporters and glucose uptake in conventional T cells. These findings indicate that DN T cells inhibit metabolic reprogramming of conventional T cells by abrogating mTOR signaling, thereby inducing a quiescent phenotype. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune responses.

Disclosures
No relevant conflicts of interest to declare.
Regulation of Marginal Zone B Cell Differentiation By microRNA-146a Via the Numb-Notch Pathway
Authors: King Jennifer K , Ung Nolan , Paing May , Contreras Jorge R. , Alberti Michael O. , Fernando Thilini , Zhang Kelvin , Pellegrini Matteo , Rao Dinesh S. 
Published: November 2019

Abstract
B cell development in bone marrow is followed by specification into spleen subsets, including marginal zone (MZ) cells. MZ require elaboration of distinct gene expression programs for development. Given their role in gene regulation, its not surprising that microRNAs (miRNAs) influence cell development. Recent work demonstrated that deficiency of NF-κB feedback regulator, Mir146 (miR-146a), led to a range of hematopoietic phenotypes, but B cells have not been extensively characterized. Here, we found miR-146a deficient mice demonstrate a reduction in MZ B cells, likely from a T cell independent developmental block. Utilizing comparative analysis of developmental stage-specific transcriptomes, we show MZ cell differentiation was impaired due to decreases in Notch2 signaling. Further, we discovered that the cell-fate regulatory protein, Numb, is a direct target of miR-146a, and its derepression in miR-146a deficient B cells underlies the decreases in Notch2. Our studies reveal miR-146a-dependent B cell phenotypes regulated by the Numb-Notch2 pathway.

Disclosures
No relevant conflicts of interest to declare.
The Critical Roles of SIN1 in Platelet Activation and Myocardial Infarction
Authors: Xu Yanyan , Chen Xue , Liu Junling 
Published: November 2019

Abstract
Mammalian stress-activated protein kinase interacting protein 1 (SIN1) is an essential subunit of the mTORC2 complex, which regulates Akt activation by phosphorylation of Akt at Ser473 residue. Despite the function of Akt in platelet activation and thrombosis was well studied, the role of SIN1 in platelet activation and thrombosis remains unknown. In this study, we observed that megakaryocyte/platelet specific SIN1 deficiency caused 30% reduction of platelet counts in peripheral blood probably by blockage of megakaryocyte differentiation and enhancing platelet apoptosis, suggesting that SIN1 had an important role in thrombopoiesis. More importantly, SIN1 deficiency caused a defect in platelet aggregation in response to low level of thrombin, U46619, ADP and collagen. SIN1 deficiency also exhibited diminished ability of platelet to spread on immobilized fibrinogen and the decreased rate of clot retraction in platelet-rich plasma containing SIN1 deficient platelets. mTORC2 complex analysis revealed that the expression levels of Rictor, another mTORC2 component, were significantly diminished in SIN1 deficient platelet. And SIN1 deficiency attenuated agonist-induced phosphorylation of Akt at Ser473, Thr308 and Thr450, and Gsk3β at Ser9 in platelet. SIN1 could be phosphorylated at Thr86, which correlated with the phosphorylation of Akt at Ser473 in activated platelets. Further study demonstrated that the phosphorylation levels of SIN1 at Thr86 and Akt at Ser473 and Thr450, but not at Thr308 were enhanced in the platelets collected from ST-segment elevation myocardial infarction (STEMI) patients, indicating that SIN1 activation correlated with myocardial infarction process. A mouse model of chronic myocardial infarction (MI) was performed and the results demonstrated that platelet-specific SIN1 deficient mice had less platelet activation, reduced MI size, and improved post-MI heart function. In conclusion, SIN1 plays critical roles in platelet activation, MI and post-MI heart failure, therefore serves as a target for therapeutic intervention in the thrombosis and myocardial infarction.

Disclosures
No relevant conflicts of interest to declare.
Aberrant Phosphorylation of STAT3 Protein of the CD4+ T Cells in Patients with Primary Immune Thrombocytopenia
Authors: Li Feng , Ke Yang , Cheng Yunfeng , Zhan Yanxia , Wu Boting 
Published: November 2019

Abstract
Background: Primary immune thrombocytopenia (ITP) is an autoimmune mediated bleeding disorder characterized by decreased platelet count. Dysfunctional cellular immunity is considered important in the pathogenesis of ITP. Signal transducer and activators of transcription 3 (STAT3) is critical for the differentiation of T cells, abnormal activation of STAT3 pathway has been confirmed in a variety of autoimmune diseases. However little is known in its role in ITP. The present studiesaimed to analyze the STAT3 protein phosphorylation of CD4+ T cells before / after therapy, response group versus no response group, in newly diagnosed ITP patients.
Methods: Twenty-nine adult patients with newly diagnosed ITP and sixteen healthy controls enrolled in this study. The IL-6 level was measured by ELISA; the phosphorylation STAT3 protein and the percentage of Th17 cells of the CD4+ T cells were analyzed by flow cytometry; the STAT3 mRNA expression was analyzed by Real-time PCR.
Results: The levels of IL-6 in the response group were higher than that in the controls. The basal level of pSTAT3 in the response group was higher than that in the no response group. The basal level of pSTAT3 correlated with the response. There was no statistical difference between the three groups both in the STAT3 mRNA expression and the percentage of Th17 cells of the CD4+ T cells.
Conclusion: The higher basal level of pSTAT3 might have good response with the corticosteroids therapy. This suggested the level of pSTAT3 might has prognostic role in ITP patients.

Disclosures
No relevant conflicts of interest to declare.
Evaluation of Recombinant Fixfc-Xten Bleeding Efficacy in Hemophilia-B Mouse Models
Authors: Liu Zhan , van der Flier Arjan , Ismail Ayman , Simpson Allison , Kurt Lutfiye , Schellenberger Volker , Loh Christine , Peters Robert 
Published: November 2019

Abstract
Introduction: Prophylactic treatment for hemophilia has demonstrated clear benefit over on demand treatment in hemophilic patients. Alprolix (rFIXFc) is the first of a new generation of long acting clotting factors we made to reduce the frequency of intravenous infusions (Powell, NEJM, 2013). Nevertheless, intravenous injections can be particularly challenging for young patients and patients with limited venous access and are time consuming. We are developing a novel rFIXFc fusion protein, using XTEN recombinant technology, which is suitable for prophylactic subcutaneous dosing in hemophilia B. In the current studies we explore the in vivo bleeding efficacy of subcutaneously dosed rFIXFc-XTEN in hemophilia B mouse model.
Methods: recombinant rFIXFc-XTEN was produced in HEK293 cells and affinity purified. The subcutaneous PK profile of rFIXFc-XTEN was compared to that of intravenously dosed rFIXFc in hemophilia B mice. The prolonged and acute bleeding efficacies were evaluated in HemB mouse tail-vein transection (TVT) and tail-clip bleeding models.
Results and conclusions: Subcutaneous dosing of rFIXFc-XTEN shows a tmax around 20 hours post dosing in mice, and improved plasma activity levels compared to similar (IU/kg) intravenously dosed rFIX or rFIXFc. Using the TVT bleeding model in HemB mice we show that at 72 hours post dosing, subcutaneously dosed rFIXFc-XTEN has improved in vivo efficacy compared to intravenously dosed rFIXFc at all tested doses. Similarly, acute efficacy testing in the HemB mouse tail-clip bleeding model showed improved efficacy of intravenously dosed rFIXFc-XTEN compared to rFIXFc. These data support the potential of once weekly or less frequent subcutaneous prophylactic dosing of rFIXFc-XTEN in humans.

Disclosures
Liu: biogen: Employment, Equity Ownership. van der Flier:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment, Equity Ownership. Simpson:Biogen: Employment, Equity Ownership. Kurt:Biogen: Employment, Equity Ownership. Schellenberger:Amunix: Employment, Equity Ownership. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder.
Bleeding in Factor XI Deficient Mice
Authors: Ay Cihan , Hisada Yohei , Cooley Brian C. , Mackman Nigel 
Published: November 2019

Abstract
Introduction: Factor XI (FXI) is a key component of the intrinsic or contact pathway of coagulation. It can be activated by either FXIIa or thrombin and amplifies thrombin generation during clot formation. Humans with congenital FXI deficiency, a rare bleeding disorder also referred to as hemophilia C, have a mild hemostatic defect with rare spontaneous bleeding but bleeding after hemostatic challenge, as occurs with some instances of surgery or trauma (e.g. dental surgery, tonsillectomy, prostate surgery or in the postpartum period). In previous studies, FXI deficient mice did not have increased bleeding in a tail transection model. In contrast, we observed increased surgical wound bleeding in FXI deficient mice compared to wild-type mice, which promted us to further investigate the effect of an absence of FXI in mice on bleeding in different models.
Methods: We assessed the hemostatic capacity of FXI-/- mice using two quantitative bleeding models (tail transection and saphenous vein injury). In the saphenous vein bleeding model, the saphenous vein was partially transected, clots that formed were removed and the total number of clots formed within a 30 minutes period (i.e. number of hemostatic events) were recorded under the microscope. In this model, a larger number of reforming clots corresponds to a higher hemostatic capacity.
Results: We found that tail bleeding times of FXI-/- mice were similar to wild-type mice (median [range]: 63 [42 - 90] vs. 70 [60 - 114] seconds, p=0.119), which is consistent with previous studies. In contrast, we observed an impaired hemostatic capacity in FXI-/- mice compared with wild-type controls in the saphenous vein bleeding model (median number of hemostatic events [range]: (19 [9 - 26] vs. 25 [21 - 30], p=0.003). However, this phenotype was milder than the bleeding observed in FIX-/- mice (median number of hemostatic events [range]: 2 [0 - 4], p<0.001).
Conclusions: Our results indicate that FXI-/- mice have a mild hemostatic defect after laparotomy and injury to the saphenous vein but not after tail transection. The mild increase in bleeding in FXI deficient mice after hemostatic challenge is similar to the increased bleeding observed in patients with FXI deficiency. Therefore, these mice may be useful in studying bleeding associated with FXI deficiency and in the presence of FXIa inhibitors.

Disclosures
No relevant conflicts of interest to declare.
Escalating Single Doses of PF-05230907 (Recombinant Factor Xa Variant FXaI16L) Are Safe and Demonstrate Hemostatic Pharmacology in Healthy Volunteers
Authors: Arkin Steven , Hua Fei , Kantaridis Constantino , Li Gang , Parsons-Rich Dana , Pittman Debra D 
Published: November 2019

Abstract
PF-05230907 (FXaI16L) is a zymogen-like variant of recombinant activated coagulation factor X (FXa), which is more resistant to inactivation by plasma protease inhibitors (compared to endogenous FXa). Previous studies showed that its cofactor, Factor Va (FVa), facilitates its transition to an active conformation, promoting hemostatic activity. The properties of PF-05230907 are consistent with its use as a short acting hemostatic agent to control bleeding and it currently is in clinical development for treatment of individuals with intracerebral hemorrhage (ICH).
We conducted a Phase 1 First-in-Human study to determine if single escalating doses of PF-05230907 were safe and tolerable and to characterize pharmacokinetics (PK) and pharmacodynamics (PD). The study was conducted in healthy adult volunteers aged 18-35 years with no risk factors for thrombosis and was approved by the local ethics committee. Subjects were assigned to one of six ascending single dose cohorts, each with 2 placebo treated subjects and 6 subjects treated with PF-05230907 (0.1, 0.3, 1, 2, 3, or 5 µg/kg). Assessments included safety monitoring, PD, PK, and immunogenicity testing. All placebo treated subjects were to be included in the safety and PD analyses. All PF-05230907 treated subjects were to be included in the safety, PK and PD analyses.
Forty-nine male subjects were enrolled after informed consent. One subject from the 2 µg/kg cohort received less than 80% of the planned PF-05230907 dose and was replaced. This subject was included in the safety analysis, but excluded in PK and PD analysis. PF-05230907 exhibited a fast clearance rate with a terminalhalf-life of approximately 4 minutes. Exposure of PF-05230907 appeared to increase proportionally with dose. Treatment-related PD changes were observed for activated partial thromboplastin time (aPTT), thrombin-anti-thrombin III complexes, prothrombin fragment 1+2, D-dimer and thrombin generation assay (lag time and peak thrombin). PF-05230907 was safe and well tolerated at all dose levels tested with no dose limiting toxicity. There were no serious adverse events. One subject treated with PF-05230907 had a weak and transient non-neutralizing anti-drug antibody response, which did not cross react with native FX or native FXa.
PF-05230907 was safe and tolerated and exhibited pharmacologic effect in healthy adults when administered at doses up to 5 µg/kg. The safety profile, PK, and PD observed in this study support further development of PF-05230907 for hemostatic treatment in individuals with acute hemorrhagic conditions.

Disclosures
Arkin: Pfizer Inc: Employment. Hua:Pfizer Inc: Employment. Kantaridis:Pfizer Inc: Employment. Li:Pfizer Inc: Employment. Parsons-Rich:Pfizer Inc: Employment. Pittman:Pfizer: Employment.
Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation
Authors: Lollar Pete , Parker Ernest T. , Healey John F. , Doering Christopher B. 
Published: November 2019

Abstract
Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.
Exome Sequencing in Venous Thromboembolic Disease Identifies Excess Mutation Burden in PROS1, PROC, SERPINC1 and STAB2
Authors: Desch Karl C. , Ozel Ayse Bilge , Halvorsen Matt , Jacobi Paula M. , Germain Marine , Tregouet David Alexandre , Reitsma Pieter H. , Goldstein David , Ginsburg David 
Published: November 2019

Abstract
Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, and seven other loci are associated with VTE. Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 have been linked to VTE in family studies. In order to identify new genetic variants altering the risk for VTE, we performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with unprovoked VTE and compared results to a previously sequenced control cohort of 5784 unrelated Europeans. To avoid variant calling bias, only SNVs from exons with >10X coverage and less than 5% difference in coverage between cases and controls were included, removing 11,813 of 188,689 intervals. We used an emerging framework for a "collapsing" analysis on genes, defining qualifying variants on the basis of annotation and minor allele frequency <0.05%, and assumed a dominant model of inheritance. Tests were performed via a Fisher's exact test for a total of 11,585 CCDS genes. Strikingly, ranked by p-value, the top 4 genes were PROS1 (P= 2.01E-09, OR 11.8), STAB2 (P=2.70E-7, OR 3.37), PROC (P=3.24E-05, OR 11.0) and SERPINC1 (P=1.10E-04, OR 8.5). We detected 29 qualifying variants in 373 cases and 106 qualifying variants in 5784 controls in STAB2. This gene encodes Stabilin-2, which is a transmembrane glycoprotein scavenger receptor. Common variants at ABO are associated with VTE and are also known to regulate von Willebrand Factor (VWF) and coagulation Factor VIII (F8) plasma levels. Common variants in STAB2 are also associated with VWF/F8 levels in a large GWAS and with VTE risk in a smaller candidate gene study, suggesting that haploinsufficiency for Stabilin-2 may increase VTE risk through elevated levels of VWF/F8. Although replication and functional testing of these findings is warranted, this study demonstrates the utility of collapsing analyses using WES data to identify multiple loci harboring an excess of rare variants in individuals with a common complex disease trait.

Disclosures
Ginsburg: Shire: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: recombinant VWF and recombinant ADAMTS13; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.