Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

2008 ◽  
Vol 1 ◽  
pp. CMBD.S507 ◽  
Author(s):  
Masato Mitsuhashi ◽  
Katsuya Endo ◽  
Kazuhiko Obara ◽  
Hiroshi Izutsu ◽  
Taishi Ishida ◽  
...  
Keyword(s):  
Whole Blood ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Drug Induced ◽  
Human Whole Blood ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Sensitivity Tests ◽  
Data Points ◽  
Mrna Markers

Apoptosis was induced in heparinized human whole blood by 3 different ways (radiation, bleomycin, or etoposide), and various mRNA were quantified using the method we reported (Clin. Chem. 2006; 52:634-642). We found that cyclin-dependent kinase inhibitor 1A (p21) and p53 upregulated modulator of apoptosis (PUMA) were the most sensitive and universal mRNA markers of apoptosis in leukocytes. In order to define positive and negative responses, a synthetic RNA was spiked into the lysis buffer and the fold increase was calculated. As a result, 837/880 (95.1%) of data points stayed between 0.75 and 1.5 fold increase, and 874/880 (99.3%) were within 0.5-2.0 fold increase. When blood samples from 40 healthy adults were stimulated with 22 different drugs, more than 75% of the samples responded to bleomycin (1 μM), idarubicin (2 μM), vincristine (1 μM), daunorubicin (2 μM), cytarabine (10 μM), to induce p21 and/or PUMA mRNA, and approximately 25% showed no induction. Significant correlation was found between p21 and PUMA mRNA responses, and between daunorubicin and cytarabine, idarubicin, and vincristine for both p21 and PUMA. The quantification of drug-induced mRNA in whole blood will be considered as ex vivo, and is a suitable platform for biomarker screening as well as a model system for drug sensitivity tests in future.

scholarly journals c-Myc inhibition is critical for HSC expansion and Rad51 expression

2018 ◽  
Author(s):  
Merve Aksoz ◽  
Esra Albayrak ◽  
Galip Servet Aslan ◽  
Raife Dilek Turan ◽  
Lamia Yazgi Alyazici ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Nos Inhibitor ◽  
Kinetics Of

c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclin-dependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose derived mesenchymal stem cells, however; it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

scholarly journals Surface modification of decellularized bovine carotid arteries with human vascular cells significantly reduces their thrombogenicity

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Eriselda Keshi ◽  
Peter Tang ◽  
Marie Weinhart ◽  
Hannah Everwien ◽  
Simon Moosburner ◽  
...  
Keyword(s):  
Surface Modification ◽  
Whole Blood ◽  
Carotid Arteries ◽  
Complete Blood Count ◽  
Ex Vivo ◽  
Vascular Grafts ◽  
Blood Perfusion ◽  
Vascular Cells ◽  
Perfusion System ◽  
Human Whole Blood

Abstract Background Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.

Identification of a Human Whole Blood-Based Endothelial Cell Impedance Assay for Assessing Clinical TRPV4 Target Engagement Ex Vivo

2020 ◽  
pp. JPET-AR-2020-000307
Author(s):  
Xiaoping Xu ◽  
Navin Goyal ◽  
Melissa H Costell ◽  
Theresa Roethke ◽  
Christian H James ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Whole Blood ◽  
Ex Vivo ◽  
Target Engagement ◽  
Human Whole Blood ◽  
Cell Impedance

Myc Targets Cks1 To Provoke Proliferation and Lymphomagenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2618-2618
Author(s):  
Ulrich Keller ◽  
Jennifer B. Old ◽  
Jonas Nilsson ◽  
Lisa Nilsson ◽  
Kirsteen Maclean ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Burkitt Lymphoma ◽  
Poor Prognosis ◽  
Transcriptional Activity ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Induced Apoptosis

Abstract Reduced levels of the cyclin dependent kinase inhibitor p27Kip1 connote poor prognosis in cancer. In human Burkitt lymphoma, and in pre-cancerous B cells and lymphomas arising in Eμ-Myc transgenic mice, p27Kip1 expression is markedly reduced. Furthermore, the Cks1 component of the SCFSkp2 complex that is necessary for p27Kip1 ubiquitylation and degradation, and to a lesser extent Skp2, are induced by Myc ex vivo and in Eμ-Myc B-cells and lymphomas, and up-regulation of CKS1 and SKP2 are hallmarks of Burkitt lymphoma. While loss of Skp2 has rather modest effects, the deletion of Cks1 in Eμ-Myc B-cells elevates p27Kip1 levels, reduces proliferation and delays lymphoma development. In contrast, Myc-induced apoptosis and transcriptional activity are not affected by Cks1 (or Skp2) loss. Therefore, Myc accelerates cell proliferation and promotes tumorigenesis through its ability to selectively induce Cks1.

Tigecycline has no effect on cytokine release in an ex vivo endotoxin model of human whole blood

2009 ◽  
Vol 33 (6) ◽  
pp. 583-586 ◽  
Author(s):  
Friederike Traunmüller ◽  
Christiane Thallinger ◽  
Johann Hausdorfer ◽  
Christopher Lambers ◽  
Stanislava Tzaneva ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Cytokine Release ◽  
Human Whole Blood

scholarly journals Ex Vivo Induction of mRNA in Human Whole Blood as a New Platform of Drug and Dietary Supplement Development

2008 ◽  
Vol 25 (5) ◽  
pp. 1116-1124 ◽  
Author(s):  
Masato Mitsuhashi ◽  
Mieko Ogura ◽  
Katsuya Endo ◽  
Kazuhiko Obara ◽  
Hiroshi Izutsu ◽  
...  
Keyword(s):  
Dietary Supplement ◽  
Whole Blood ◽  
Ex Vivo ◽  
Human Whole Blood

Naringenin has anti-inflammatory properties in macrophage and ex vivo human whole-blood models

2008 ◽  
Vol 43 (4) ◽  
pp. 400-407 ◽  
Author(s):  
C. Bodet ◽  
V. D. La ◽  
F. Epifano ◽  
D. Grenier
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Human Whole Blood ◽  
Anti Inflammatory
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