Antigen presentation and T cell activation by dendritic cells in radiation damage / 中国辐射卫生

@#<b>Objective</b> To explore dendritic cells (DCs)-mediated antigen presentation for radiation-injured cells by using the <i>in vitro</i> cell co-culture technology to simulate the <i>in vivo </i>microenvironment of the lung tissue. <b>Methods</b> ⁶⁰Co γ-irradiated mouse lung epithelial cells (MLE-12) were cultured with bone marrow-derived DCs and/or splenic T lymphocytes for 48 hours. Flow cytometry was used to measure the expression levels of costimulatory molecules (CD80/86) and antigenic peptide recognition complexes (the major histocompatibility complex [MHC] class Ⅰ/Ⅱ) on DCs and T cell activation markers (CD69/28/152) as well as the numbers of CD4⁺ and CD8⁺ T cells. <b>Results</b> ⁶⁰Co γ irradiation significantly increased the apoptosis rate of MLE-12 cells in a dose-dependent manner, and significantly stimulated the expression of CD80/86 and MHC Ⅱ on DCs, without direct activation of T cells. After γ (6 Gy)-irradiated MLE-12 cells were co-cultured with DCs and T lymphocytes for 48 h, there were significant increases in the expression of CD69 and CD28 on T cells, the numbers of CD4⁺ and CD8⁺ T cells, and the expression of CD86 and MHC I on DCs, as compared with the control groups. <b>Conclusion</b> Radiation-injured cells can stimulate antigen presentation by DCs and activate T cells.

An LC-MS/MS method for simultaneous analysis of the cystic fibrosis therapeutic drugs colistin,ivacaftor and ciprofloxacin / 药物分析学报

Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung in-fections in cystic fibrosis patients.In this study,we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin,colistin and ivacaftor in rat plasma,human epithelial cell lysate,cell culture medium,and drug transport media.An aliquot of 200 μL drug-containing rat plasma or cell culture medium was treated with 600 μL of extraction solution(acetonitrile containing 0.1％ formic acid and 0.2％ trifluoroacetic acid (TFA)).The addition of 0.2％ TFA helped to break the drug-protein bonds.Moreover,the addition of 0.1％ formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility.After vortexing and centrifuging,the sample components were analyzed by HPLC-MS/MS.The multiple re-action monitoring mode was used to detect the following transitions:585.5-101.1 (colistin A),578.5-101.1 (colistin B),393.2-337.2 (ivacaftor),332.2-314.2 (ciprofloxacin),602.3-101.1 (polymyxin 81 as internal standard (IS)) and 595.4-101.1 (polymyxin B2 as IS).The running time of a single sample was only 6 min,making this a time-efficient method.Linear correlations were found for colistin A at 0.029-5.82 μg/mL.colistin B at 0.016-3.14 μg/mL.ivacaftor at 0.05-10.0 μg/mL,and ciprofloxacin at 0.043-8.58 μg/mL.Accuracy,precision,and stability of the method were within the acceptable range.This method would be highly useful for research on cytotoxicity,animal pharmacokinetics,and in vitro drug delivery.

Effect of miR-20a on pulmonary surfactant synthesis of alveolar epithelial cells A549 and its mechanism / 中华实用儿科临床杂志

Objective To explore the role of miR-20a on pulmonary surfactant synthesis of alveolar epithelial cells A549 and its potential mechanism.Methods Lentivirus miR-20a overexpression vector(miR-20a group) or lentivirus no-load vector(no-load group) was transfected into A549 cells,and the expression of green fluorescent protein(GFP) was observed to determinate the transfection effficiency;cell proliferation was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT);the bioinformatics software and database were applied to predict and analyze the target genes of miR-20a about lung development;expressions of miR-20a,pulmonary surfactant-associated protein A(SP-A),pulmonary surfactant-associated protein B(SP-B),pulmonary surfactant-associated protein C(SP-C) and pulmonary surfactant-associated protein D(SP-D) mRNA were detected by using quantitative real-time PCR(qPCR);the expressions of SP-A protein,SP-B protein,SP-C protein,SP-D protein and protein signal transducers and activators of transcription 3 (STAT3) were detected by using Western blot.Results Observation of GFP expression under a fluorescent microscope indicated similar transfection efficiency,and real time-PCR showed that the expression of miR-20a increased after being transfected with lentivirus miR-20a overexpression vector(3.85 ± 0.18)compared with the normal group (0.99 ± 0.04)and the no-load group (1.21 ± 0.12),and the differences were significant(t =10.85,9.64,all P ＜0.001).As a result,lentivirus miR-20a overexpression vector was constructed successfully.Online software predicted that STAT3 gene was likely to be the target gene of miR-20a.Compared with the normal group (24 h,48 h,72 h:0.23 ± 0.01,0.39 ± 0.01,0.56 ± 0.03) and the no-load group (24 h,48 h,72 h:0.25 ± 0.01,0.44 ± 0.05,0.59 ± 0.01),miR-20a did not change the cell proliferation at different time points(24 h,48 h,72 h:0.26 ± 0.01,0.41 ± 0.02,0.58 ± 0.02) (all P ＞ 0.05).Compared with the normal group (1.00 ± 0.05,1.24 ± 0.20,1.31 ± 0.09,0.89 ± 0.12) and the no-load group (0.76 ± 0.10,1.31 ± 0.13,1.50 ± 0.11,1.01 ± 0.11),miR-20a up-regulated the mRNA expressions of SP-A,SP-B,SP-C and SP-D (2.05 ± 0.17,2.14 ± 0.10,2.84 ± 0.09,1.66 ± 0.08),and the differences were significant (all P ＜ 0.05).Compared with the normal group (0.46 ± 0.01,0.27 ± 0.03,0.69 ± 0.01,0.43 ± 0.01) and no-load group (0.43 ± 0.01,0.21 ± 0.01,0.79 ± 0.02,0.44 ± 0.02),miR-20a also increased the protein expressions of SP-A,SP-B,SP-C and SP-D (0.55 ±0.01,0.47 ±0.05,0.96 ±0.02,0.59 ±0.03),the diffe-rences were statistically significant (all P ＜0.05).The expression of STAT3 in miR-20a group(0.37 ±0.05) was significantly lower than that in the normal group(0.60 ±0.04) and the no-load group (0.68 ±0.06),and the differences were statstically significant (all P ＜ 0.05) in A549.Conclusions STAT3 is a downstream target gene of miR-20a.miR-20a can promote pulmonary surfactant synthesis of alveolar epithelial cells A549 by inhibiting STAT3.

Characterization of Cigarette Smoke Extract (CSE)-induced Cell Death in Lung Epithelial Cells / 결핵및호흡기질환

Emphysema is characterized by air space enlarge?ment and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was per?formed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cy?tochrome c release was confirmed by using immuno?fluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near com?pletely blocked by N-acetylcystein and bcl-2 over?expression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the patho?genesis of emphysema.

Characterization of Nitric Oxide (NO)-Induced Cell Death in Lung Epithelial Cells / 결핵

BACKGROUND: Nitric Oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in other cell types. This study was performed to characterize NO-induced cell death in lung epithelial cells and to investigate the roles of cell death regulators including iron, bcl-2 and p53. METHODS: A549 cells were used for lung epithelial cells. SNP (sodium nitroprusside) and SNAP (S-nitroso-N-acetyl-penicillamine) were used for NO donor. Cytoxicity assay was done by MTT assay and crystal violet assay. Apoptotic assay was done by fluorescent microscopy after double staining with propidium iodide and hoecst 33342. Iron inhibition study was done with RBCs and FeSO4. For bcl-2 study, bcl-2 overexpressing cells (A549-bcl-2) were used and for p53 study, Western blot analysis and p53 functionally knock-out cells (A549-E6) were used. RESULTS: SNP and SNAP induced dose-dependent cell death in A549 cells and fluorescent microscopy revealed that SNAP induced apoptosis in low doses but necrosis in high doses while SNP induced exclusively necrotic cell death. Iron inhibition study using RBCs and FeSO4 significantly blocked SNAP-induced cell death. And also SNAP-induced cell death was blocked by bcl-2 overexpression. Finally, we found that SNAP activate p53 by Western blot analysis and that SNAP-induced cell death was decreased in the abscence of p53. CONCLUSION: In lung epithelial cells, NO can induce cell death, more precisely apoptosis in low doses and necrosis in high doses. And iron, bcl-2, and p53 play important roles in NO-induced cell death.

Pro-inflammatory cytokine expression through NF-kappaB/IkappaB pathway in lung epithelial cells / 결핵

BACKGROUND: The importance of pro-inflammatory cytokines, especially tumor necrosis factor α(TNF-α) and interleukin-1β(IL-1β), have been extensively documented in the generation of inflammatory lung disease. Lung epithelial cells are also actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. Understanding the mechanism of pro-inflammatory cytokine expression in lung epithelial cells is crucial to the development of new therapeutic modalities for inflammatory lung disease. Transcription of most pro-inflammatory cytokines is dependent on the actiation of NF-κB. However, the relationship between pro-inflammatory cytokine expression and NF-κB/IκB pathway in lung epithelial cells is not clear. METHODS: BEAS-2B, A549, NCI-H719 cells were stimulated with IL-1β or TNF-α at various times, and then IL-8 and TNF-αmRNA expressions were assayed by Northern blot analysis. IL-1β or TNF-α-induced NF-κB activation was assessed by the nuclear translocation of p65 NF-κB subunit. The degradation of IκBα and IκBβ by IL-1βor TNF-α stimulation was assayed by Western blot analysis. The phosphorylation of IκBαwas evaluated by Western blot analysis after pre-treating cells with proteasome inhibitor followed by IL-1β or TNF-α stimulation. The basal level of IKKα expression was evaluated by Western blot analysis. RESULTS: IκBαand IκBβ was repidly degraded after 5 minutes of incubation with IL-1β or TNF-α in BEAS-2B, A549, and NCI-H157 cells. The activation of NF-κB and the induction of IL-8 and TNF-α mRNA expressions were observed by IL-1β or TNF-α stimulation in these cells. In contrast, neither the changes in NF-κB/IκB pathway nor IL-8 and TNF-α mRNA expression was induces by IL-1β or TNF-α stimulation in NCI-H719 cell. IL-1β and TNF-α-induced IκB phoshorylation was observed in BEAS-2B, A549, and NCI-H157 cells, but not in NCI-H719 cells. The basal level of IKKα expression was not different between cells. CONCLUSION: NF-κB/IκB pathway plays an important role in the ixpression of pro-inflammatory cytokine in most lung epithelial cells. The absence of the effect on NF-κB/IκB pathway in NCI-H719 cells seems to be due to the defect in the intracellular signal transduction pathway upstream to IKK.