Comparative in vitro stimulation with lipopolysaccharide to study TNFα gene expression in fresh whole blood, fresh and frozen peripheral blood mononuclear cells

2010 ◽  
Vol 357 (1-2) ◽  
pp. 33-37 ◽  
Author(s):  
Jiwang Chen ◽  
Anke H. Bruns ◽  
Helen K. Donnelly ◽  
Richard G. Wunderink
2012 ◽  
Vol 44 (1) ◽  
pp. 59-75 ◽  
Author(s):  
Roby Joehanes ◽  
Andrew D. Johnson ◽  
Jennifer J. Barb ◽  
Nalini Raghavachari ◽  
Poching Liu ◽  
...  

Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL.


Author(s):  
Mariana Brayner Cavalcanti ◽  
Lídia Lúcia Bezerra Leite ◽  
Diogo Manoel de Queiroz ◽  
Thiago de Salazar e Fernandes ◽  
Wagner Luís Mendes de Oliveira ◽  
...  

2020 ◽  
Vol 10 (3) ◽  
pp. 125
Author(s):  
Simon Sioen ◽  
Karlien Cloet ◽  
Anne Vral ◽  
Ans Baeyens

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.


2016 ◽  
Vol 42 (04) ◽  
pp. 195-201
Author(s):  
Yafen Cheng ◽  
Chung-Hsi Chou ◽  
Hsiang-Jung Tsai

Mycobacterium bovis (M. bovis) is causative agent of tuberculosis in cattle and humans populations. To understand its effects on gene expression profiles, we conducted an in vitro time-course study to identify transcriptional changes in infected bovine peripheral blood mononuclear cells (PBMCs), using quantitative RT-PCR. We discovered a likely involvement of C-type lectin domain family 4, member E (CLEC4E) in triggering a series of negative intracellular signaling via Syk/CARD9 pathway for cytokines, as early as 24 h post-infection (hpi). This is the first report confirming induction of CLEC4E and the Syk/CARD9 pathway in PBMCs in response to M. bovis infection, and these findings support the view that M. bovis inhibits signaling pathways of antimycobacterial host defense in bovine cells. In addition, M. bovis infection in PBMCs may suppress apoptosis by interfering with TNF-[Formula: see text] signaling. This study, contributes to a better understanding of M. bovis-reduced signal transduction and microbial changes in PBMCs earlier than 24 hpi.


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