Ex vivo [11C]-(+)-PHNO binding is unchanged in animal models displaying increased high-affinity states of the D2receptor in vitro

Synapse ◽  
2009 ◽  
Vol 63 (11) ◽  
pp. 998-1009 ◽  
Author(s):  
Patrick N. McCormick ◽  
Shitij Kapur ◽  
Greg Reckless ◽  
Alan A. Wilson
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
High Affinity

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography

2004 ◽  
Vol 9 (4) ◽  
pp. 719 ◽  
Author(s):  
Kostadinka Bizheva ◽  
Angelika Unterhuber ◽  
Boris Hermann ◽  
Boris Považay ◽  
Harald Sattmann ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Animal Models ◽  
Ex Vivo ◽  
Brain Morphology ◽  
Optical Coherence ◽  
Ultrahigh Resolution

scholarly journals An Ex Vivo Vessel Injury Model to Study Remodeling

2018 ◽  
Vol 27 (9) ◽  
pp. 1375-1389 ◽  
Author(s):  
Mehmet H. Kural ◽  
Guohao Dai ◽  
Laura E. Niklason ◽  
Liqiong Gui
Keyword(s):  
Shear Stress ◽  
Animal Models ◽  
Intimal Hyperplasia ◽  
Vascular Remodeling ◽  
Ex Vivo ◽  
Vessel Injury ◽  
Injury Model ◽  
Human Arteries

Objective: Invasive coronary interventions can fail due to intimal hyperplasia and restenosis. Endothelial cell (EC) seeding to the vessel lumen, accelerating re-endothelialization, or local release of mTOR pathway inhibitors have helped reduce intimal hyperplasia after vessel injury. While animal models are powerful tools, they are complex and expensive, and not always reflective of human physiology. Therefore, we developed an in vitro 3D vascular model validating previous in vivo animal models and utilizing isolated human arteries to study vascular remodeling after injury. Approach: We utilized a bioreactor that enables the control of intramural pressure and shear stress in vessel conduits to investigate the vascular response in both rat and human arteries to intraluminal injury. Results: Culturing rat aorta segments in vitro, we show that vigorous removal of luminal ECs results in vessel injury, causing medial proliferation by Day-4 and neointima formation, with the observation of SCA1+ cells (stem cell antigen-1) in the intima by Day-7, in the absence of flow. Conversely, when endothelial-denuded rat aortae and human umbilical arteries were subjected to arterial shear stress, pre-seeding with human umbilical ECs decreased the number and proliferation of smooth muscle cell (SMC) significantly in the media of both rat and human vessels. Conclusion: Our bioreactor system provides a novel platform for correlating ex vivo findings with vascular outcomes in vivo. The present in vitro human arterial injury model can be helpful in the study of EC-SMC interactions and vascular remodeling, by allowing for the separation of mechanical, cellular, and soluble factors.

scholarly journals In vitroandex vivomeasurement of the biophysical properties of blood using microfluidic platforms and animal models

The Analyst ◽  
2018 ◽  
Vol 143 (12) ◽  
pp. 2723-2749 ◽  
Author(s):  
Yang Jun Kang ◽  
Sang-Joon Lee
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
Biophysical Properties ◽  
Microfluidic Platforms

Several techniques for thein vitroandex vivomeasurement of hemorheological properties using microfluidic platforms and animal models were reviewed.

CIB1 Is Required for FAK Activation during Outside-in Signaling through Platelet Integrin αIIbβ3

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2873-2873 ◽  
Author(s):  
Ulhas Pandurang Naik ◽  
Meghna Ulhas Naik
Keyword(s):  
Resting State ◽  
Ex Vivo ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Platelet Spreading ◽  
Inside Out ◽  
Dependent Interaction

Abstract Platelets play an important role in the processes of hemostasis and thrombosis. Platelet integrin αIIbβ3 mediates bi-directional signaling during these processes. Agonist-dependent activation of integrin αIIbβ3 through inside-out signaling results in high-affinity binding of soluble ligands, such as fibrinogen. Fibrinogen binding induces a cascade of signaling through the integrin, termed outside-in signaling that results in platelet aggregation and clot retraction. Previously, we have characterized CIB1, a calcium- and integrin-binding protein that specifically interacts with the cytoplasmic domain of αIIb. Previous reports using in vitro and ex vivo studies implicated that CIB1 is involved in maintaining αIIbβ3 in its resting state, agonist-induced activation of the integrin, and outside-in signaling resulting in platelet spreading. Here, we show that platelet filopodia formation induced by fibrinogen binding to integrin αIIbβ3 needs Ca2+, but is independent of the Ca2+-dependent interaction of CIB1 with αIIb. Additionally, dynamic rearrangement of the cytoskeleton is required for the recruitment of FAK to the CIB1-αIIb complex at the filopodia and FAK activation. Moreover, disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or CIB1 antibody inhibited FAK activation. Furthermore, Cib1 null platelets acquired a spiky morphology and failed to fully spread on immobilized fibrinogen. Interestingly, FAK activation was significantly reduced in Cib1 null platelets exposed to immobilized fibrinogen. Our results suggest that during outside-in signaling, a rise in the intracellular Ca2+ level and filopodia formation occurs prior to the interaction of CIB1 with αIIb. Additionally, Ca2+ bound CIB1 recruits FAK to the αIIbβ3 complex at the filopodia, where FAK is activated, resulting in platelet spreading. Thus, our results have provided a mechanism through which CIB1 regulates outside-in signaling through integrin αIIbβ3.

MEN1112, a Novel Humanized De-Fucosylated Monoclonal Antibody with High Affinity and Specificity for Bst1/CD157 Antigen and Enhanced CD16 Binding

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3607-3607
Author(s):  
Dee Aud ◽  
Rachel Dusek ◽  
Arnima Bisht ◽  
Sudha Swaminithan ◽  
Rahel Awdew ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Phage Display Library ◽  
Primary Diagnosis ◽  
Fc Receptor ◽  
Effector Cells ◽  
High Affinity ◽  
Adp Ribosyl Cyclase

Abstract Bst1/CD157 is a GPI-anchored transmembrane protein belonging to the ADP-ribosyl-cyclase family and it is expressed on some blood cells such as monocytes and neutrophils. Importantly, we demonstrated that this antigen is expressed in blast cells from almost 100% of Acute Myeloid Leukemia patients (either at primary diagnosis or at relapse). MEN1112 is a humanized, de-fucosylated antibody targeting the Bst1/CD157 antigen with high affinity and inducing potent in vitro and ex vivo Antibody Dependent Cell-mediated Cytotoxicity (ADCC) responses against AML cell lines and blasts, respectively. MEN1112 was discovered as a mouse Fab antibody from a phage display library raised against recombinant Bst1/CD157 protein. The Fab was selected base upon its ability to recognize recombinant Bst1/CD157 protein by ELISA and for its ability to recognize Bst1/CD157 positive endogenous cancer cell lines by FACS (fluorescence activated cell sorting). The Fab was also chosen based upon its ability to recognize cynomolgus Bst1/CD157 antigen on recombinant and endogenous cells. The selected Fab sequence was humanized and incorporated into the Biowa Potelligent cell line to produce a de-fucosylated full IgG1 version (MEN1112). The binding affinity of MEN1112 for the Bst1/CD157 antigen showed an EC50 of 1nM as assessed by flow cytometry (FACS) analysis using multiple cell lines. No binding was observed on antigen-negative cell lines demonstrating specificity of MEN1112. High affinity of MEN1112 for the recombinant antigen was confirmed in an ELISA assay (EC50 0.4 nM). Beyond the affinity for the antigen, it is of relevance for its ADCC activity that MEN1112 is characterized by an improved affinity for Fc receptors on effector cells over the parental antibody. In particular, MEN1112, lacking fucose residues in its Fc domain, showed an impressive high affinity binding to the Fc receptor CD16A both high affinity and low affinity alleles. Internalization studies demonstrate the slow internalization of the Bst1/CD157 antigen following binding to MEN1112 which favors the ADCC efficacy of the antibody. Overall our results indicate that MEN1112 has the potential to exert anti-leukemia actions through a powerful ADCC in view of: (i) superior affinity for blast cell membrane antigen Bst1/CD157, (ii) enhanced affinity for all variants of CD16A Fc receptor expressed on effector cells and (iii) minimal internalization following antigen binding. Disclosures Aud: Oxford BioTherapeutics: Employment. Dusek:Oxford BioTherapeutics: Employment. Bisht:Oxford BioTherapeutics: Employment. Swaminithan:Oxford BioTherapeutics: Employment. Awdew:Oxford BioTherapeutics: Employment. Trang:Oxford BioTherapeutics: Employment. Lin Lou:Oxford BioTherapeutics: Employment. Manzini:Menarini Ricerche SpA: Employment. Martinez Mogarra:Menarini Biotech srl: Employment. Bressan:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment.

scholarly journals Atherosclerosis and Thrombosis: Insights from Large Animal Models

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Gemma Vilahur ◽  
Teresa Padro ◽  
Lina Badimon
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
Large Animal ◽  
Isolated Cells ◽  
Large Animal Models ◽  
Thrombosis Research ◽  
Human Pathology ◽  
Thrombotic Complications

Atherosclerosis and its thrombotic complications are responsible for remarkably high numbers of deaths. The combination ofin vitro, ex vivo, andin vivoexperimental approaches has largely contributed to a better understanding of the mechanisms underlying the atherothrombotic process. Indeed, different animal models have been implemented in atherosclerosis and thrombosis research in order to provide new insights into the mechanisms that have already been outlined in isolated cells and protein studies. Yet, although no model completely mimics the human pathology, large animal models have demonstrated better suitability for translation to humans. Indeed, direct translation from mice to humans should be taken with caution because of the well-reported species-related differences. This paper provides an overview of the availableatherothrombotic-likeanimal models, with a particular focus on large animal models of thrombosis and atherosclerosis, and examines their applicability for translational research purposes as well as highlights species-related differences with humans.

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