scholarly journals Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation

2019 ◽  
Vol 317 (4) ◽  
pp. F913-F921 ◽  
Author(s):  
Carl J. May ◽  
Gavin I. Welsh ◽  
Musleeha Chesor ◽  
Phillipa J. Lait ◽  
Lauren P. Schewitz-Bowers ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Soluble Mediator ◽  
Stimulation Of

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.

scholarly journals Overexpression of lncRNA TUG1 Alleviates NLRP3 Inflammasome-Mediated Cardiomyocyte Pyroptosis Through Targeting the miR-186-5p/XIAP Axis in Coronary Microembolization-Induced Myocardial Damage

2021 ◽  
Vol 12 ◽  
Author(s):  
You Zhou ◽  
Tao Li ◽  
Zhiqing Chen ◽  
Junwen Huang ◽  
Zhenbai Qin ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Myocardial Injury ◽  
Culture Supernatant ◽  
Myocardial Damage ◽  
Luciferase Reporter ◽  
Cell Culture Supernatant ◽  
Coronary Microembolization ◽  
Lncrna Tug1

Coronary microembolization (CME) is a complicated problem that commonly arises in the context of coronary angioplasty. The lncRNA taurine-up regulated gene 1 (TUG1), significantly contributes to cardiovascular diseases; however, its contribution to CME-induced myocardial damage remains elusive. Herein, we establish the rat CME model and investigate the role of TUG1 in CME. The cell viability was evaluated via CCK-8 assay. Serum and cell culture supernatant samples were evaluated via ELISA. The dual luciferase reporter (DLR) assay, RIP, and RNA-pull down were conducted to validate the associations between TUG1 and miR-186-5p as well as miR-186-5p and XIAP. The expression of TUG1, miR-186-5p, and XIAP mRNA were determined by RT-qPCR, and proteins were evaluated via immuneblotting. As a result, TUG1 and XIAP were significantly down-regulated, and the miR-186-5p level was found to be remarkably up-regulated in CME myocardial tissues. Overexpression of TUG1 alleviated CME-induced myocardial injury and pyroptosis, whereas TUG1 knockdown showed the opposite effects. The DLR assay, RIP, and RNA-pull down results reveal that TUG1 directly targets miR-186-5p and miR-186-5p directly targets XIAP. In vitro rescue experiments show that TUG1 overexpression alleviates LPS-caused cardiomyocyte injury and pyroptosis via sponging miR-186-5p and regulating XIAP, and depression of miR-186-5p reduces LPS-induced cardiomyocyte injury and pyroptosis by targeting XIAP. Concludingly, the overexpression of TUG1 alleviates NLRP3 inflammasome-mediated cardiomyocyte pyroptosis through targeting the miR-186-5p/XIAP axis in CME-induced myocardial injury.

scholarly journals Studies on the Anti-Inflammatory Effect and Its Mechanisms of Sophoridine

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Xiumei Huang ◽  
Bo Li ◽  
Lianzhong Shen
Keyword(s):  
Cell Culture ◽  
Inflammatory Response ◽  
Inflammatory Mediators ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
In Vitro Cell Culture ◽  
Anti Inflammatory ◽  
Inflammatory Effect

This work is to study the anti-inflammatory effect and its mechanisms of sophoridine in vitro and in vivo. For this aim, the influences of sophoridine on several inflammatory mediators were investigated. Excessive inflammatory response in vitro model was developed by using lipopolysaccharide (LPS) to stimulate the mouse peritoneal macrophages and HL-60 cells to produce IL-6 and IL-8. Carrageenin-induced mouse paw edema model was used as inflammatory response in vivo model. MTT method, ultraviolet spectrophotometric method, and radioimmunoassay were used to measure the changes of TNFα, IL-6, PGE2, and IL-8 in in vitro cell culture supernatant or in the local inflammatory exudates. The results showed that sophoridine inhibited the production of IL-8 in in vitro cell culture supernatant and inhibited the production of TNFα, PGE2, and IL-8 in the local inflammatory exudates but had no significant effects on the production of IL-6 in vitro and in vivo. It is demonstrated that sophoridine’s anti-inflammatory effect was due to its ability to inhibit the production of cytokine and inflammatory mediators.

scholarly journals Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Tumor Biology ◽  
2019 ◽  
Vol 41 (8) ◽  
pp. 101042831986636 ◽  
Author(s):  
Abel Jacobus Bronkhorst ◽  
Vida Ungerer ◽  
Stefan Holdenrieder
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Cell Types ◽  
Cell Culture Supernatant ◽  
Cell Free Dna ◽  
Free Dna

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA–based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

Obtaining and Characterization of Alhagi persarum Boiss. et Buhse Callus Cell Cultures, Isoflavonoid Producers

2020 ◽  
Vol 36 (6) ◽  
pp. 35-48
Author(s):  
D.V. Коchkin ◽  
G.I. Sobolkovа ◽  
А.А. Fоmеnkov ◽  
R.А. Sidorov ◽  
А.М. Nоsоv
Keyword(s):  
Fatty Acids ◽  
Cell Culture ◽  
Liquid Chromatography ◽  
Secondary Metabolites ◽  
Cell Cultures ◽  
Mass Spectrometric ◽  
Mass Spectrometric Detection ◽  
Gas Liquid Chromatography ◽  
Callus Cell

The physiological characteristics of the callus cell cultures of Alhagi persarum Boiss et Buhse, a member of the legume family, widely used in folk medicine, have been studied. It was shown that the source of the explant was an important factor in the initiation of callusogenesis: more intense callusogenesis (almost 100%) was observed for explants from various organs of sterile seedlings, rather than intact plants (less than 30%). As a result, more than 20 lines of morphologically different callus cell cultures were obtained, and the growth parameters for the 5 most intensively growing lines were determined. The composition of fatty acids (FA) of total lipids and secondary metabolites in the most physiologically stable callus line Aр-207 was analyzed. Using capillary gas-liquid chromatography with mass spectrometric detection (GLC-MS), 19 individual C12--C24 FAs were identified, the main fraction of which were palmitic (~ 23%), stearic (~ 22%), linoleic (~ 14%) and α-linolenic (~ 33%) acids. The established atypical ratio of FAs (a simultaneous high content of both saturated FAs and polyunsaturated α-linolenic acid) is possibly due to the adaptation of cells to in vitro growth conditions. Phytochemical analysis of the secondary metabolites was carried out using ultra-performance liquid chromatography with electrospray ionization mass spectrometric detection (UPLC MS). Compounds belonging to different structural groups of isoflavones were found. Aglycones (calycosin, formononetin and afrormosin isomer), glucosides (formononetin glucoside), as well as esters of glucosides (malonylglycosides of calicosin, formononetin, afrormosin isomers, glycitein and genistein) were detected. These secondary metabolites are widespread in plants of the Fabaceae family; however, isoflavones are rare in representatives of the Alhagi genus. The presence of malonylated isoflavone glycosides in Alhagi spp. was shown for the first time. endemic plant species, Alhagi, in vitro cell culture, callus cell culture, isoflavones, fatty acids All studies were carried out using the equipment of the "Experimental Biotechnological Facility" and the "All-Russian Collection of Cell Cultures of Higher Plants" of IРР RAS. This work was supported by the Russian Foundation for Basic Research (RFBR), contract no.18-54-06021 (Az_a), and the Government of the Russian Federation, Megagrant Project no. 075-15-2019-1882.

Evidence for Associations Between Th1/Th17 “Hybrid” Phenotype and Altered Lipometabolism in Very Severe Graves Orbitopathy

2020 ◽  
Vol 105 (6) ◽  
pp. 1851-1867 ◽  
Author(s):  
Sijie Fang ◽  
Shuo Zhang ◽  
Yazhuo Huang ◽  
Yu Wu ◽  
Yi Lu ◽  
...  
Keyword(s):  
T Cell ◽  
Th17 Cell ◽  
Th17 Cells ◽  
T Cell Subsets ◽  
Th1 Cell ◽  
Effector T Cell ◽  
Cell Lineages ◽  
Graves Orbitopathy ◽  
Ifn Γ

Abstract Purpose The purpose of this article is to investigate the characteristics of Th1-cell and Th17-cell lineages for very severe Graves orbitopathy (GO) development. Methods Flow cytometry was performed with blood samples from GO and Graves disease (GD) patients and healthy controls, to explore effector T-cell phenotypes. Lipidomics was conducted with serum from very severe GO patients before and after glucocorticoid (GC) therapy. Immunohistochemistry and Western blotting were used to examine orbital-infiltrating Th17 cells or in vitro models of Th17 polarization. Results In GD, Th1 cells predominated in peripheral effector T-cell subsets, whereas in GO, Th17-cell lineage predominated. In moderate-to-severe GO, Th17.1 cells expressed retinoic acid receptor-related orphan receptor-γt (RORγt) independently and produced interleukin-17A (IL-17A), whereas in very severe GO, Th17.1 cells co-expressed RORγt and Tbet and produced interferon-γ (IFN-γ). Increased IFN-γ–producing Th17.1 cells positively correlated with GO activity and were associated with the development of very severe GO. Additionally, GC therapy inhibited both Th1-cell and Th17-cell lineages and modulated a lipid panel consisting of 79 serum metabolites. However, in GC-resistant, very severe GO, IFN-γ–producing Th17.1 cells remained at a high level, correlating with increased serum triglycerides. Further, retro-orbital tissues from GC-resistant, very severe GO were shown to be infiltrated by CXCR3+ Th17 cells expressing Tbet and STAT4 and rich in triglycerides that promoted Th1 phenotype in Th17 cells in vitro. Conclusions Our findings address the importance of Th17.1 cells in GO pathogenesis, possibly promoting our understanding of the association between Th17-cell plasticity and disease severity of GO.

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

2013 ◽  
Vol 8 (3) ◽  
pp. 352-362 ◽  
Author(s):  
Paula A. J. Rosa ◽  
Ana M. Azevedo ◽  
S. Sommerfeld ◽  
Martina Mutter ◽  
Werner Bäcker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

scholarly journals The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+Smokers

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Igor Z. Barjaktarevic ◽  
Ronald G. Crystal ◽  
Robert J. Kaner
Keyword(s):  
Tissue Destruction ◽  
Epithelial Lining Fluid ◽  
Protein Levels ◽  
Knowing That ◽  
Lung Epithelial ◽  
Interleukin 23 ◽  
Metalloproteinase 9 ◽  
Stimulation Of

Rationale.Matrix metalloproteinase-9 (MMP-9) expression is upregulated in alveolar macrophages (AM) of HIV1+smokers who develop emphysema. Knowing that lung epithelial lining fluid (ELF) of HIV1+smokers contains increased levels of inflammatory cytokines compared to HIV1−smokers, we hypothesized that upregulation of lung cytokines in HIV1+smokers may be functionally related to increased MMP-9 expression.Methods.Cytokine arrays evaluated cytokine protein levels in ELF obtained from 5 groups of individuals: HIV1−healthy nonsmokers, HIV1−healthy smokers, HIV1−smokers with low diffusing capacity (DLCO), HIV1+nonsmokers, and HIV1+smokers with lowDLCO.Results. Increased levels of the Th17 related cytokine IL-23 were found in HIV1−smokers with lowDLCOand HIV1+smokers and nonsmokers. Relative IL-23 gene expression was increased in AM of HIV1+individuals, with greater expression in AM of HIV1+smokers with lowDLCO. Infection with HIV1in vitroinduced IL-23 expression in normal AM. IL-23 stimulation of AM/lymphocyte coculturesin vitroinduced upregulation of MMP-9. Lung T lymphocytes express receptor IL-23R and interact with AM in order to upregulate MMP-9.Conclusion. This mechanism may contribute to the increased tissue destruction in the lungs of HIV1+smokers and suggests that Th17 related inflammation may play a role.

Sign in / Sign up

Export Citation Format

Share Document