AB0026 A Simple Method of Removing Low Density Granulocytes from Peripheral Blood Mononuclear Cells in Autoimmune Diseases

2016 ◽  
Vol 75 (Suppl 2) ◽  
pp. 905.3-905
Author(s):  
S. Zhang ◽  
H. Shen
Keyword(s):  
Autoimmune Diseases ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Low Density ◽  
Peripheral Blood Mononuclear ◽  
Simple Method ◽  
Blood Mononuclear Cells

scholarly journals Low-Density Granulocyte Contamination From Peripheral Blood Mononuclear Cells of Patients With Sepsis and How to Remove It – A Technical Report

2021 ◽  
Vol 12 ◽  
Author(s):  
Judith Schenz ◽  
Manuel Obermaier ◽  
Sandra Uhle ◽  
Markus Alexander Weigand ◽  
Florian Uhle
Keyword(s):  
Healthy Volunteers ◽  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15+ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15+ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

scholarly journals Specific TLR4 Blocking Effect of a Novel 3,4-Dihydropyrimidinone Derivative

2021 ◽  
Vol 11 ◽  
Author(s):  
Mingqian Zhou ◽  
Yiqi Wang ◽  
Xiaoying Lin ◽  
Jieping Wan ◽  
Chengping Wen
Keyword(s):  
Molecular Docking ◽  
Autoimmune Diseases ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Blood Mononuclear Cells

Background: Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. Dihydropyrimidinone derivatives are medicinally important molecules with diverse pharmacological activities, including anti-inflammatory activity. The present study focused on novel synthesized 3,4-dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4.Methods: A series of 3,4-dihydropyrimidinone derivatives were recently synthesized and evaluated for their TLR4 inhibition activities and cytotoxic on HEK-BlueTM hTLR4 cells with the help of QUANTI-Blue assay and MTS assay. Selected compound 3 was analyzed for its molecular docking with TLR4 by using Autodock vina 1.1.2. Its effect on the TLR4 pathway related cytokines was also evaluated in THP-1 cells and human peripheral blood mononuclear cells by using real-time PCR, ELISA and western blot.Results: Five compounds were synthesized and characterized for effectiveness based on 3,4-dihydropyrimidinone. Compound 3 was found to be the potent hybrid among the synthesized compounds, with high TLR4 inhibition activities and low cytotoxic activities against HEK-BlueTM hTLR4 cells. Molecular docking analysis showed that two hydrogen bonds between compound 3 and residues Asp209(TLR4) and Asp99(MD-2) mainly contribute to the TLR4 inhibition. In addition, compound 3 suppressed LPS-induced of the mRNA expression of TLR4, IP-10, TNF-α, IL-6, IL-12A, and IL-12B, the protein expression of pIRF3 and pNFκB and the secretion of IP-10, TNF-α in THP-1 cell line. Compound 3 also inhibited LPS-induced expression of TNF-α, IL-6, and IL-1β but increased IP-10 at mRNA levels in human peripheral blood mononuclear cells.Conclusion: Our study reveals compound 3, a novel 3,4-dihydropyrimidinone derivative, is a potential TLR4 antagonist, which opens up new research avenues for the development of promising therapeutic agents for inflammatory and autoimmune diseases.

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