scholarly journals The Effect of miR-138 on the Function of Follicular Helper T Cells and the Differentiation of B Cells in Osteosarcoma

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Zhitao Wang ◽  
Jianxiao Liang ◽  
Shanyong Jiang ◽  
Gang Zhao ◽  
Jianshu Lu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Flow Cytometry ◽  
B Cells ◽  
Culture Medium ◽  
Membrane Surface ◽  
Surface Antigens ◽  
Cell Culture Medium ◽  
Tfh Cells ◽  
Follicular Helper ◽  
Cell Membrane Surface

Objective. To explore the effect of miR-138 on the function of follicular helper T (Tfh) cells and the differentiation of B cells in osteosarcoma. Methods. Clinically collect peripheral blood from osteosarcoma (OS) patients and healthy volunteers (HC), as well as OS tumor tissues (OS tumor) and adjacent tissues with normal histology (normal group). The CD4+CXCR5+Tfh cells of OS patients were screened and isolated by flow cytometry, and the expression of Tfh cell membrane surface antigens PD-1 and CTLA-4 was detected. In addition, qRT-PCR was used to detect the expression of miR-138 in tissues and Tfh cells, and the correlation relationship between miR-138 and PD-1 and CTLA-4 was analyzed. After interference or overexpression of miR-138 in Tfh cells, coculture with untreated B cells was done, and the levels of IL-10, IL-12, IL-21, and INF-γ in Tfh cell culture medium and the levels of IgM, IgG, and IgA in B cell culture medium after coculture were measured by ELISA. Flow cytometry was used to detect the expression of B cell membrane surface antigens CD27 and CD38 after coculture. Results. The rate of PD-1- and CTLA-4 positive cells in the peripheral blood and tissues of the OS group was significantly increased, the expression of miR-138 was significantly reduced, and the expression of miR-138 was negatively correlated with the expression of PD-1 and CTLA-4. In addition, upregulation of miR-138 can lead to a significant increase in the level of IL-10 in the supernatant of Tfh cells and a significant decrease in the levels of IL-12, IL-21, and INF-γ, which in turn leads to increased levels of IgM, IgG, and IgA released by B cells. At the same time, it significantly increases the rate of CD27- and CD38-positive cells and promotes the maturation of B cells. Downregulating miR-138 has the opposite effect. Conclusion. Downregulating the expression of miR-138 in osteosarcoma can improve the dysfunction of CD4+CXCR5+Tfh cells and promote the differentiation of B cells.

scholarly journals THU0231 IL-2 DRIVES THE CONVERSION OF T FOLLICULAR HELPER TO T FOLLICULAR REGULATORY CELLS THROUGH EPIGENETIC MODIFICATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 343.2-343
Author(s):  
H. Hao ◽  
S. Nakayamada ◽  
Y. Kaoru ◽  
N. Ohkubo ◽  
S. Iwata ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
B Cells ◽  
Histone Modifications ◽  
Lupus Erythematosus ◽  
Regulatory Cells ◽  
Systemic Lupus ◽  
Research Support ◽  
Tfh Cells ◽  
Follicular Helper

Background:Systemic lupus erythematosus (SLE) is a complex polygenic autoimmune disease characterized by immune-system aberrations. Among several types of immune cells, T follicular helper (Tfh) cells promote autoantibody production, whereas T follicular regulatory (Tfr) cells suppress Tfh-mediated antibody responses.(1)Objectives:To identify the characteristics of Tfr cells and to elucidate the mechanisms of conversion of Tfh cells to Tfr cells, we probed the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors.Methods:Peripheral blood mononuclear cells from SLE patients (n=44) and healthy donors (HD; n=26) were analyzed by flow cytometry. Memory Tfh cells were sorted and cultured under stimulation with T cell receptor and various cytokines. Expression of characteristic markers and phosphorylation of STATs (p-STATs) were analyzed by flow cytometry and quantitation PCR. Histone modifications were evaluated by chromatin immunoprecipitation.Results:The proportion of CXCR5+FoxP3+Tfr cells in CD4+T cells tended to increase (2.1% vs 1.7%, p=0.17); however, that of CD4+CD45RA-FoxP3hiactivated Tfr cells in Tfr cells was decreased (4.8% vs 7.1%, p<0.05), while CD4+CD45RA-FoxP3lownon-suppressive Tfr cells was increased (50.1% vs 38.2%, p<0.01) in SLE compared to HD. The percentage of PD-1hiactivated Tfh cells was significantly higher in SLE compared to HD (15.7% vs 5.9%, p<0.01). Furthermore, active patients had a higher ratio of activated Tfh/Tfr cells compared to inactive patients. In vitro study showed that IL-2, but not other cytokines such as TGF-β1, IL-12, IL-27, and IL-35, induced the conversion of memory Tfh cells to functional Tfr cells characterized by CXCR5+Bcl6+Foxp3hipSTAT3+pSTAT5+cells. The loci ofFOXP3at STAT binding sites were marked by bivalent histone modifications. After IL-2 stimulation, STAT5 directly bound on FOXP3 gene loci accompanied by suppressing H3K27me3. Finally, we found that serum level of IL-2 was decreased in SLE and that stimulation with IL-2 suppressed the generation of CD38+CD27+B cells by ex vivo coculture assay using Tfh cells and B cells isolated from human blood.Conclusion:Our findings indicated that the regulatory function of Tfr cells is impaired due to the low ability of IL-2 production and that IL-2 restores the function of Tfr cells through conversion of Tfh cells to Tfr cells in SLE. Thus, the reinstatement of the balance between Tfh and Tfr cells will provide important therapeutic approaches for SLE.References:[1]Deng J, Wei Y, Fonseca VR, et al. T follicular helper cells and T follicular regulatory cells in rheumatic diseases. Nat Rev Rheumatol. 2019; 15 (8): 475-90.Disclosure of Interests: :He Hao: None declared, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yamagata Kaoru: None declared, Naoaki Ohkubo: None declared, Shigeru Iwata: None declared, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin

scholarly journals miR-138 Reduces the Dysfunction of T Follicular Helper Cells in Osteosarcoma via the PI3K/Akt/mTOR Pathway by Targeting PDK1

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Baoen Jiang ◽  
Xiuqin Kang ◽  
Gang Zhao ◽  
Jianshu Lu ◽  
Zhitao Wang
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Peripheral Blood ◽  
Mtor Pathway ◽  
Control Group ◽  
T Follicular Helper Cells ◽  
Tfh Cells ◽  
Helper Cells ◽  
Follicular Helper ◽  
Immature B Cells

Objective. To study the effect of miR-138 on the function of osteosarcoma (OS) T follicular helper cells (Tfh cells) and its mechanism. Methods. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with osteosarcoma (OS group) and healthy volunteers (control group). CD4+CXCR5+ Tfh cells and CD9+ B cells were sorted by flow cytometry. qRT-PCR was used to detect the expression of miR-138 and PDK1 in the peripheral blood and CD4+CXCR5+ Tfh cells. Flow cytometry was employed to detect the proportion of CD4+CXCR5+ Tfh cells in CD4+ T cells, the level of CD40L in CD4+CXCR5+ Tfh cells, and the expression of CD27 and CD38 in B cells. Western blot was used to determine the protein expression of PDK1, PI3K, p-Akt, Akt, p-mTOR, and mTOR. In addition, dual-luciferase reporter assay was performed to verify the relationship between miR-138 and PDK1. ELISA method was used to determine the levels of IgM, IgG, IL-10, and IL-21. Results. Compared with that of the control group, the expression of miR-138 in PBMC and CD4+CXCR5+ Tfh cells of the OS group was lower; overexpression of miR-138 could promote the maturation of Tfh cells and immature B cells. The results of the dual-luciferase report experiment showed that miR-138 can target and negatively regulate PDK1, and PDK1 can reverse the effect of miR-138 on the function of Tfh cells and immature B cells. Conclusion. miR-138 inhibits the PI3K/Akt/mTOR pathway by targeting and negatively regulating PDK1 to alleviate the dysfunction of T follicular helper cells in OS.

scholarly journals Effects of Environmental pH on the Growth of Gastric Cancer Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Wenjie Li ◽  
Ying Zhou ◽  
Chunyu Shang ◽  
Hui Sang ◽  
Hong Zhu
Keyword(s):  
Gastric Cancer ◽  
Cell Culture ◽  
Flow Cytometry ◽  
Cancer Cells ◽  
Culture Medium ◽  
Ph Value ◽  
Acid Suppression ◽  
Biological Behavior ◽  
Cell Culture Medium ◽  
Gastric Cancer Cells

Background. Proton pump inhibitor (PPI) and other acid-suppressing drugs are widely used in the treatment of gastrointestinal ulcer, upper gastrointestinal bleeding, gastritis, and gastric cancer (GC). About 80% of GC patients receive acid suppression treatment. PPI suppresses the production of gastric acid by inhibiting the function of H+/K+-ATPase in gastric parietal cells and raises the pH value to achieve therapeutic purposes. Some studies have found that PPI had a certain antitumor effect in the proliferation and apoptosis of tumor cells. But the effects of environmental pH on the growth of GC cells and its mechanism are unknown. Therefore, we hoped to find the effects of culture medium pH on the biological behavior of GC cells by in vitro experiments and provide guidance for the use of acid-suppressing drugs in GC patients. Aims. We aimed to observe the effects of pH changes in GC cell culture medium on the cell biological behavior of cancer cells and to analyze the potential mechanisms. We hoped to find out the effect of acid suppression on the growth of GC cells. Methods. The GC cell lines (SGC-7901 and MKN45) were used as the research object. We adjusted the pH value in the cell culture medium to observe the changes in cell viability (MTT), apoptosis (flow cytometry), and invasion (Transwell) at pH 6, pH 7, and pH 8. qRT-PCR and western blot (WB) assays were used to determine the expression changes of genes and proteins (mTOR, AKT, Wnt, Glut, and HIF-1α) at pH 6, pH 7, and pH 8. Results. The results of MTT showed that the viability of SGC-7901 and MKN45 in the pH 8.0 group was significantly weaker than that in the pH 6.0 or pH 7.0 group (P<0.001). Flow cytometry results showed that the apoptosis of SGC-7901 and MKN45 in the pH 8.0 group was more obvious than that in the pH 6.0 or pH 7.0 group (P<0.001). The results of Transwell showed that the invasion ability of SGC-7901 and MKN45 in the pH 8.0 group was significantly weaker than that in the pH 6.0 or pH 7.0 group (P<0.001). As shown by PCR and WB results, with the increase of pH, the expression of mTOR, AKT, Wnt, Glut, and HIF-1α in SGC-7901 and MKN45 was downregulated (P<0.05). Conclusions. Compared with the microacid environment, the microalkaline environment inhibited the viability, invasion, and expression of genes and proteins (mTOR, AKT, Wnt, Glut, and HIF-1α) but promoted the apoptosis of GC cells and thus inhibited the growth of GC.

3-iodothyronamine (T1AM) is taken up and rapidly metabolized in cell culture medium only at low concentration

2020 ◽  
Author(s):  
Federica Saponaro ◽  
Marco Borsò ◽  
Sara Verlotta ◽  
Lavinia Bandini ◽  
Alessandro Saba ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Cell Culture Medium ◽  
Low Concentration

Effect of Atmospheric-Pressure Helium Plasma Jet on Cell Culture Medium

2013 ◽  
Vol 133 (5) ◽  
pp. 278-285
Author(s):  
Norimitsu Takamura ◽  
Douyan Wang ◽  
Takao Satoh ◽  
Takao Namihira ◽  
Hisato Saitoh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Atmospheric Pressure ◽  
Culture Medium ◽  
Plasma Jet ◽  
Helium Plasma ◽  
Cell Culture Medium

scholarly journals Defined MSC exosome with high yield and purity to improve regenerative activity

2021 ◽  
Vol 12 ◽  
pp. 204173142110086
Author(s):  
Jun Yong Kim ◽  
Won-Kyu Rhim ◽  
Yong-In Yoo ◽  
Da-Seul Kim ◽  
Kyoung-Won Ko ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Density Lipoprotein ◽  
High Yield ◽  
Human Umbilical Cord ◽  
Cell Culture Medium ◽  
Human Coronary Artery ◽  
Isolation Methods ◽  
Regenerative Activity ◽  
Yield And Purity

Exosomes derived from mesenchymal stem cells (MSCs) have been studied as vital components of regenerative medicine. Typically, various isolation methods of exosomes from cell culture medium have been developed to increase the isolation yield of exosomes. Moreover, the exosome-depletion process of serum has been considered to result in clinically active and highly purified exosomes from the cell culture medium. Our aim was to compare isolation methods, ultracentrifuge (UC)-based conventional method, and tangential flow filtration (TFF) system-based method for separation with high yield, and the bioactivity of the exosome according to the purity of MSC-derived exosome was determined by the ratio of Fetal bovine serum (FBS)-derived exosome to MSC-derived exosome depending on exosome depletion processes of FBS. The TFF-based isolation yield of exosome derived from human umbilical cord MSC (UCMSC) increased two orders (92.5 times) compared to UC-based isolation method. Moreover, by optimizing the process of depleting FBS-derived exosome, the purity of UCMSC-derived exosome, evaluated using the expression level of MSC exosome surface marker (CD73), was about 15.6 times enhanced and the concentration of low-density lipoprotein-cholesterol (LDL-c), known as impurities resulting from FBS, proved to be negligibly detected. The wound healing and angiogenic effects of highly purified UCMSC-derived exosomes were improved about 23.1% and 71.4%, respectively, with human coronary artery endothelial cells (HCAEC). It suggests that the defined MSC exosome with high yield and purity could increase regenerative activity.

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