bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prolylcarboxypeptidase Murine Hypomorphs Are Hypertensive and Prothrombotic
Authors: Adams Gregory N , Zhou Yihua , Larusch Gretchen , Nieman Marvin T , Hoit Brian , Jacobs Gretta , Yu Xin , Mahdi Fakhri , Shariat-Madar Zia , D’Alecy Louis , Okada Yoshio , Schmaier Alvin H. 
Published: October 2019

Abstract
Prolylcarboxypeptidase (PRCP) is a serine protease that degrades bradykinin and angiotensin II. It also activates prekallikrein (PK) (K =9 nM) to plasma kallikrein when bound to high molecular weight kininogen on cells and matrix independent of factor XIIa. PRCP polymorphisms are a risk factor for metabolic syndrome and pre-eclampsia. PRCP murine hypomorphs (PRCPgt/gt) were created from ES cells where the Prcp gene was interrupted by gene trap with a LacZ insertion in intron 4 (KST302gt/+). PRCPgt/+ mice were created and backcrossed 10 generations in a C57BL6 background. PRCPgt/gt mice contain <10% PRCP mRNA than littermate wild type (WT) mice. LacZ staining and immunostaining indicate PRCP is highly expressed in renal proximal tubules where it co-localizes with lotus lectin and arterial vessels (small, medium, carotid, aorta) where it co-localizes with alpha-SMA, a vascular smooth muscle cell marker. Studies show that PRCPgt/gt mice are hypertensive. Using carotid sensors and continuous telemetry BP monitoring for 7 days, the mean systolic and diastolic BP in PRCPgt/gt mice were ~138, 125, and 113 mm Hg, versus ~125, 113, and 111 mg Hg in WT mice. Transthoracic ultrasound studies show significantly reduced anterior and posterior cardiac muscle velocity and strain at 6 and 12 months in PRCPgt/gt mice versus WT. Murine micro-MRI show reduced ejection fraction (p=0.038) and an enlarged aortic root (p=0.048) in PRCPgt/gt mice. Additionally, PRCPgt/gt mice have shortened carotid artery closure times using the Rose Bengal (25±0.8 min PRCPgt/gt vs 51±4 min WT, p<0.0001) and ferric chloride (20.6±3.4 min PRCPgt/gt vs >60 min WT, p<0.0001) models of carotid artery thrombosis. Pharmacological inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with PSKI-571, SBTI, or Pro-Phe-Arg- CK in WT C57BL/6 or 129/SvJ mice resulted in shortened Rose Bengal carotid artery closure times. PRCPgt/gt murine hypomorphs also have reduced plasma factor XII (69±12% vs 114±15% WT, p<0.03), increased PK levels (110±4% vs 94±3% WT, p<0.006) with normal angiotensin II and bradykinin levels. PRCPgt/gt mice also have normal contact activation- or tissue factor-induced thrombin generation times, indicating that arterial thrombosis risk is independent of thrombin formation. These combined investigations indicate the PRCP is an important modulator of constitutive arterial blood pressure and thrombosis risk by mechanisms independent of thrombin generation. Further, reduced PK activation or pharmacologic inhibition of plasma kallikrein may increase arterial thrombotic risk.
Platelet Interaction with Bone Marrow Derived CD34+ Stem Cells: Potential Role of Chemoattractant Chemerin
Authors: Du Xiao-Yan , Leung Lawrence L. 
Published: October 2019

Abstract
Chemerin is a 16 kDa chemoattractant circulating in blood in an inactive form (prochemerin). Its chemotactic activity is released following proteolytic cleavage of its labile carboxyl-terminal amino acids by proteases of the coagulation, fibrinolytic and inflammatory cascades. Chemokine-like receptor 1 (CMKLR1) is a chemerin receptor expressed on plasmacytoid dendritic cells, natural killer cells and tissue macrophages. We identified the expression of CMKLR1 on mouse CD34 positive (CD34+) bone marrow-derived stem cells (BMSC) using anti-mouse CMKLR1 antibody by FACS. Fluorescein-labeled CD34+/CMKLR1+ BMSC adhered to immobilized chemerin dose-dependently. The EC50 of BMSC adhesion to human and mouse chemerin is 2.5 and 5 ug/mL respectively. BMSCs also adhered to SDF-1 (stromal cell-derived factor-1) with EC50 of 0.3 ug/mL, but did not bind to immobilized fibronectin. We have previously reported that platelets express chemerin and release it upon activation. CMKLR1 is also expressed on platelet surface as demonstrated by FACS. Immobilized chemerin could induce platelet adhesion and CD62P (p-selectin) expression as detected by fluorescein-labeled anti-CD62P antibody. BMSC formed complexes with both resting and activated platelets as demonstrated by fluorescein-labeled washed platelets incubated with unlabeled mouse CD34+/CMKLR1+ BMSCs using FACS analysis. Both anti-chemerin and anti-CD62P antibodies could partially block BMSCs binding to immobilized platelets at 50% and 35% respectively. In conclusion, chemerin directly mediates platelets interaction with BMSCs. Chemerin also facilitates platelet/BMSC complex formation by inducing platelet p-selectin (CD62P) expression. Platelet-derived chemerin, similar to SDF-1, may be involved in tissue regeneration by inducing progenitor cells mobilization and differentiation.
Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice
Authors: Cornelissen Ivo , De Candia Erica , Palmer Daniel , Coughlin Shaun R 
Published: October 2019

Abstract
Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y12 pathway by inducing irreversible alteration of the platelet receptor P2Y12, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.
In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y12 pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl3-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.
To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y12. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y12 double null mice, in both the tail bleeding assay and in a FeCl3-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y12 pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.
Use of Recombinant Activated Factor VII in Severe Postpartum Haemorrhage: Data from the South European Registry. A Multicentric Observational Retrospective Study
Authors: Barillari Giovanni ,   On behalf of the rFVIIa - PPH South European Registry Study Group
Published: October 2019

Abstract
Introduction: Major Post Partum Haemorrhage (PPH) is a life threatening complication of labor, which mainly occurs without warning, predictive signs and symptoms and often in absence of predisposing conditions. A retrospective survey of cases of severe primary PPH treated with Recombinant Activated Factor VII (rFVIIa) in Italy, Greece, Spain and Portugal was performed. The aim of our study was to report the south european real experience in clinical practice about the use rFVIIa in PPH treatment.
Methods: anamnestic, clinical and haemostatic data about fortyfive patients, treated between 2005 and 2007, were collected. Coagulative parameters and transfusion requirements before and after rFVIIa treatment were compared.
Results: INR significantly decreased, while levels of fibrinogen markedly increased after rFVIIa administration. The median need of packed red blood cells units, platelets units, fresh frozen plasma and crystalloids and colloids, before and after rFVIIa administration, dramatically reduced being respectively 6 and 2 units (p<1.2exp-6), 1.5 and 0 units (p = 0.001), 1250 and 0 mL (p<4.4exp-5), 3000 and 1250mL (p<0,0042). No cases of adverse effects or thromboembolic complications were observed.
Conclusions: Our clinical and haemostatic data suggest that recombinant activated factor VII may be a safe and helpful adjunctive therapy in the management of postpartum haemorrhage.
Sickle Cell Disease Is Associated with Reduced Adenosine Deaminase Catalytic Activity, Resulting in Altered Adenosine Metabolism
Authors: Eiymo Mwa Mpollo Marthe-Sandrine , Malik Punam 
Published: October 2019

Abstract
Abstract 1078
Sickle cell disease (SCD), a chronic hemolytic anemia due to a mutant beta-globin, is associated with prominent inflammatory features with elevated levels of inflammation mediating molecules and an oxidative state, with elevated levels of reactive oxygen species (ROS). Additionally, elevated levels of adenosine (Ado) have recently been shown to play a role in exacerbating sickle cell pathophysiology. However, the etiology of increased Ado is unclear; and presumed to occur secondary to increased ATP release from cellular damage (Xu et al, Nature Medicine, 2010). Herein, we show that increased levels of Ado in the plasma of patients with SCD and the mouse model of SCD [Berkeley sickle (SS) mice] were accompanied by significantly reduced catalytic activity of the Ado degrading enzyme, adenosine deaminase (ADA) in sickle red blood cell (RBCs). ADA activity in Berkeley sickle (SS) RBCs was 246 ± 50 U/L as compared to 570 ± 36 U/L in normal mouse RBCs (derived from Berkeley AA/WT mice), as measured by a color-based enzymatic assay. Notably, despite lower enzyme activity of ADA, immunoblotting assays showed that the levels of ADA protein in sickle RBC were similar, or even slightly higher compared to that in wild type (WT) RBC, suggesting that ADA catalytic activity was impaired in sickle RBCs. In vitro plasma swap experiments, where WT RBC are incubated with sickle plasma and vice versa for an hour, revealed that sickle plasma induced a significant decrease in ADA activity in WT RBC, suggesting that plasma from sickle mice contains a soluble factor that is capable of impairing ADA enzyme activity. Interestingly enough, the decrease in ADA activity correlated inversely with the levels of intracellular RBC ROS in sickle mice. Next we directly assess in vivo the effect of induced oxidative stress on RBC ADA activity in sickle mice by injecting sickle mice with 35umoles/Kg body weight of hemin and saline respectively. Preliminary data suggests a decrease in RBC ADA activity in hemin–injected mice (294 ± 197 U/L) compared to the saline-injected group (346 ± 185.8 U/L), and this data was consistent with a concomitant increase in intracellular RBC and white blood cells ROS levels. We conclude that increase in oxidative stress levels mediates a post translational alteration of ADA catalytic activity and alters Ado metabolism in SCD.

Disclosures:
No relevant conflicts of interest to declare.
Batf3-Dependent CD11blow/− Peripheral Dendritic Cells Are GM-CSF-Independent and Are Not Required for Th Cell Priming After Subcutaneous Immunization
Authors: Edelson Brian T. , Murphy Kenneth M. 
Published: October 2019

Abstract
Abstract 1113
Granulocyte/macrophage colony stimulating factor (GM-CSF) regulates the development and activity of several myeloid cell types, including dendritic cells (DCs), thereby influencing the initiation and maintenance of adaptive immune responses. Mice lacking GM-CSF or its receptor show decreased antigen specific T cell priming against the encephalitogenic peptide of myelin oligodendrocyte glycoprotein (MOG35–55), and are resistant to experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. Beyond this role in priming adaptive immune responses, GM-CSF acts to sustain ongoing T cell effector responses, both in EAE initiated by MOG35–55 peptide and in other models of autoimmunity.
A recent report has argued that part of the role for GM-CSF in EAE is based on its requirement for the development of a subset of dermal DCs, termed CD11blow/−Langerin+CD103+ DCs (J Exp Med. 207:953–61, 2010). These dermal DCs represent one anatomic subtype of peripheral tissue CD11blow/−CD103+ DCs, and share the properties of efficient antigen cross-presentation and IL-12 production with lymphoid tissue-resident CD8α+ DCs. Peripheral tissue CD11blow/−CD103+ DCs and lymphoid tissue-resident CD8α+ DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. Mice deficient in any one of these transcription factors selectively lack these DC subsets, with Batf3−/− mice representing the best model for studying the role of these DCs in vivo, as Irf8−/− and Id2−/− mice display more widespread immune defects.
Here, we directly compared the development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb−/−) and Batf3−/− mice. We confirmed that Csf2rb−/− mice displayed poor priming of MOG35-55 peptide-specific T cell responses and resisted induction of EAE. Importantly, however, we found that Batf3-dependent dermal CD11blow/−Langerin+ DCs did, in fact, develop in Csf2rb−/− mice, but that these DCs expressed reduced, but not absent, levels of the surface marker CD103. In contrast, Batf3−/− mice, lacking all peripheral CD11blow/− DCs and lymphoid tissue-resident CD8α+ DCs, showed robust Th cell priming after subcutaneous immunization, and were fully susceptible to EAE. These results exclude the hypothesis that defective T effector cell priming and resistance to EAE exhibited by Csf2rb−/− mice result from the absence of dermal CD11blow/−Langerin+CD103+ DCs, and instead suggest that GM-CSF is acting via different mechanisms to promote autoimmunity.

Disclosures:
No relevant conflicts of interest to declare.
Ewing Sarcoma Cells Are Sensitized to NK Cell Mediated Killing by SNDX-275, An HDAC Inhibitor
Authors: Ray Anish K. , Lee Dean A. 
Published: October 2019

Abstract 1126
Natural Killer (NK) cells are an important a part of the innate immune system response to infection and malignancy. Tumors may escape NK cell surveillance by upregulating expression of inhibitory ligands (HLA), or downregulating expression of activating ligands (MICA/B, ULBP). Histone Deacetylase (HDAC) inhibitors promote cell cycle arrest, differentiation, and apoptosis in tumor cells by decompacting chromatin to a more transcriptionally active state. In previous studies, we and others have shown that HDACi can sensitize tumor cells to NK cell mediated cytotoxicity by modulating expression of activating or inhibitory receptors. We investigated whether HDACi could modulate sensitivity of Ewing Sarcoma (EWS) cell lines to lysis by NK cells. EWS cells were cultured in vitro in the presence of SNDX-275, a Class I HDAC inhibitor, for 24, 48 and 72 hours, and then assessed for viability, cell proliferation, expression of activating and inhibitory receptors, and susceptibility to NK cell mediated cytolysis. We found a dose and time dependent inhibition of EWS cell proliferation mediated by SNDX-275. We also found a dose and time dependent decrease in the expression of HLA on tumor cell surface, which correlated with drug treated cells being more susceptible to NK cell mediated killing. SNDX-275 not only has a direct toxicity on Ewing sarcoma cells but it also appears to improve NK cell mediated cytolysis of these cells by down regulation of HLA, an inhibitory receptor of NK cells.

Disclosures:
No relevant conflicts of interest to declare.
Bypassing Agents in Children with Inhibitors: How Much Do We Use and How Much Is Too Much?
Authors: Gordon Deana , Wang Michael , Ruegg Katherine , Villalobos-Menuey Elizabeth , Manco-Johnson Marilyn J. 
Published: October 2019

Abstract
Abstract 1217

Background:
Children with hemophilia A and high-titer inhibitors suffer frequent severe bleeding events and often respond to bypassing agents more poorly than adults resulting in increased dosing and cost. Aims: This study was conducted to determine the amount and variability of bypass agent therapy currently given to children with inhibitors at a large combined adult and pediatric hemophilia treatment center in comparison to treatment of adult inhibitor patients. Methods: All subjects were enrolled onto a single institution prospective inceptional cohort study of bleeding disorders with appropriate IRB-approved consent and assent. All children who developed high-titer inhibitors were treated with immune tolerance induction, and children treated with bypassing agents primarily represent difficult inhibitors. Data regarding treatment agents, dose and duration were extracted for 24 bleeding events in 6 children in comparison to comparable data on 26 bleeding events in 5 adults with high-titer inhibitors. Treatment decisions were made clinically by academic hematologists in consultation with hemophilia nurses, patients and parents based on perceived severity of bleeding events and therapeutic response. Treatment was given as recombinant factor VIIa (NovoSeven, N, n=28), prothrombin complex concentrates (FEIBA, F, n=10) or alternating doses of both (A, n=12). Median results for individual children and aggregated adult data are shown on Table 1. Children received seven times the treatment doses and duration compared with adults. In addition, children received both N and F for half of the bleeding events, while this was used for only one adult event. Treatment duration in children was not affected by initial dose or dose frequency of N (p > 0.05 for both). Bleeding events were severe and difficult to manage; significant long-term morbidity in these 6 children include ankle arthropathy in 3, elbow contracture following compartment syndrome in 2 and quadrucep pseudotumor in 1.

Discussion:
Children with hemophilia A and inhibitors experience bleeding episodes that are poorly responsive to both N and F, resulting in higher dosing, longer duration and enormous costs. Children require better inhibitor therapies. Clinical trials of new bypassing agents designed specifically for children are urgently needed.

Disclosures:
No relevant conflicts of interest to declare.
SAR1 Is Critical to the HbF-Inducing Effect of HU and Other Therapeutic Cytotoxic Agents
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai Wulin , Kumkhaek Chutima , Liu Wenli , Rodgers Griffin P. 
Published: October 2019

Abstract
Abstract 1306
Hydroxyurea (HU) is a drug that is effectively used in the management of the β-hemoglobinopathies sickle cell disease and β-thalassemia by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear, as does the reason why some proportion of the patients failed to show a beneficial therapeutic response. A better understanding of how hydroxyurea acts at the molecule levels and induces fetal hemoglobin production would offer targeted molecular therapy with higher specificity, and less toxicity and may provide alternative effective therapies to current non-responders. Secretion-associated and ras-related protein (SAR1) is a small guanosine triphosphate (GTP)-binding protein that is induced by HU and closely mimics the known effects of HU on erythroid cells, including gamma-globin induction and cell-cycle regulation. Here we report of our further investigation on whether SAR1 is an obligatory mediator to the HbF-inducing effect of HU and other therapeutic cytotoxic agents. Our results indicate that blocking SAR1 expression not only significantly lowered the basal and HU-elicited HbF production in CD34+ and K562 cells (see table), it also abolished HU-mediated S-phase cell-cycle arrest (see table) and cell apoptosis (P<0.001).Transcriptional factor BCL11A, which controls globin switching and suppresses HbF, is involved in SAR1 and HU regulated gamma-globin expression, and SAR1 is required for HU, 5-AZA and Ara-C mediated BCL11A reduction. We also found HU acts through the interaction of NFκB with its conjugated binding site on SAR1 promoter to regulate its transcriptional expression. Our data show that SAR1 is not only sufficient, but indeed necessary and central, to HU's beneficial effects. Our data suggests that SAR1 is not only sufficient, but indeed necessary and central, to HU's erythroid effects. Further exploration of the mechanistic details underlying SAR1 effects may reveal novel therapeutic targets for the beta-globin disorders.MockControl shRNA or microRNASAR1 shRNA or microRNAHU−HU+HU−HU+HU−†pHU+‡pHbF positive CD34 + cells27.00 ± 1.01%50.70 ± 1.88%20.59 ± 0.76%0.00331.77 ± 1.17%0.00001HbF positive K562 cells18.21 ± 0.21%41.00 ± 1.41%19.72 ± 2.11%43.37 ± 0.2%14.29 ± 1.2%0.00426.63 ± 1.23%0.00002Fraction of S-phase cells24.94 ± 0.84%32.38 ± 0.59%24.08 ± 0.59%34.26 ± 0.54%24.88 ± 0.4%0.09727.64 ± 0.25%0.00192†p compared with control HU-‡p compared with control HU+

Disclosures:
No relevant conflicts of interest to declare.