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Zhang J.,CAS Institut Pasteur of Shanghai | Chen C.,CAS Institut Pasteur of Shanghai | Hou X.,Shanghai JiaoTong University | Gao Y.,CAS Institut Pasteur of Shanghai | And 10 more authors.
Journal of Biological Chemistry | Year: 2013

The expression of the transcription factorGATA3inFOXP3+ regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the downregulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the posttranslational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Shi H.,Fudan University | Lu L.,Shanghai Second Peoples Hospital | Zhang N.-P.,Fudan University | Zhang S.-C.,Fudan University | Shen X.-Z.,Fudan University
World Journal of Gastroenterology | Year: 2012

AIM: To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells. METHODS: HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 105 HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS: Compared to lamivudine alone (22.63% ± 0.12%), both sequential (51.50% ± 0.17%, P = 0.034) and cytokine treatment (49.66% ± 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% ± 0.02% vs 82.48% ± 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 ± 0.04 (control), 3.09 ± 0.08 (lamivudine), 2.55 ± 0.13 (cytokine), 2.32 ± 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 ± 0.01 (control), 3.08 ± 0.08 (lamivudine), 2.57 ± 0.15 (cytokine), 2.34 ± 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells (58.03% ± 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002]. CONCLUSION: Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load. © 2012 Baishideng.

Liu T.,Shanghai JiaoTong University | Liu T.,Donghua University | Liu T.,Shanghai University of Traditional Chinese Medicine | Huang Y.,Sino America United Stem Cell Research Center | And 9 more authors.
Stem Cells and Development | Year: 2013

Sperm abnormalities are one of the main factors responsible for male infertility; however, their pathogenesis remains unclear. The role of microRNAs in the development of sperm abnormalities in infertile men has not yet been investigated. Here, we used human induced pluripotent stem cells to investigate the influence of miR-122 expression on the differentiation of these cells into spermatozoa-like cells in vitro. After induction, mutant miR-122-transfected cells formed spermatozoa-like cells. Flow cytometry of DNA content revealed a significant increase in the haploid cell population in spermatozoa-like cells derived from mutant miR-122-transfected cells as compared to those derived from miR-122-transfected cells. During induction, TNP2 and protamine mRNA and protein levels were significantly higher in mutant miR-122-transfected cells than in miR-122-transfected cells. High-throughput isobaric tags for relative and absolute quantification were used to identify and quantify the different protein expression levels in miR-122- and mutant miR-122-transfected cells. Among all the proteins analyzed, the expression of lipoproteins, for example, APOB and APOA1, showed the most significant difference between the two groups. This study illustrates that miR-122 expression is associated with abnormal sperm development. MiR-122 may influence spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated with sperm development. © 2013 Mary Ann Liebert, Inc.

An Z.-M.,Shanghai Second Peoples Hospital | Dong X.-G.,Shanghai Second Peoples Hospital | Guo Y.,Shanghai Second Peoples Hospital | Zhou J.-L.,Fudan University | Qin T.,Fudan University
Journal of Huazhong University of Science and Technology - Medical Science | Year: 2015

Diabetic nephropathy (DN) is a common and serious clinical complication of diabetes and presently there are no effective ways to prevent its occurrence and progression. Recent studies show that pentoxifylline (PTX) can improve renal hemodynamics, reduce urinary protein excretion, and alleviate or delay renal failure in DN patients. In this study, we focused on the anti-oxidative stress effect of PTX on alleviating renal damages of DN using rat models. DN rats were established with injection of streptozotocin. Blood glucose, urinary protein excretion, serum cystatin C, renal biopsy, superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and renal homogenate and renal nitrotyrosine levels were analyzed before and 12 weeks after the treatment of PTX. Before treatment, all the DN rats had elevated blood glucose, increased urinary protein excretion and elevated serum cystatin C. Morphologically, DN rats exhibited renal tissue damages, including swelling and fusions of foot processes of podocytes under electron microscope. Masson staining revealed blue collagen deposition in glomeruli and renal interstitium. With treatment of PTX, symptoms and renal pathological changes of DN rats were alleviated. Furthermore, the MDA levels were increased and the SOD levels were decreased in the serum and kidneys of DN rats, and these changes were reversed by PTX. The expression of nitrotyrosine was up-regulated in DN rat model and down-regulated by PTX, indicating that PTX was able to inhibit oxidative reactions in DN rats. PTX could alleviate renal damage in DN, which may be attributable to its anti-oxidative stress activity. © 2015, Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg.

Wen C.J.,Shanghai Second Peoples Hospital
Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery | Year: 2011

To study the clinical efficacy of sublingual immunotherapy using standardized Dermatophagoides farinae extract for children with combined allergic rhinitis and asthma syndrome. Fifty-two children, from 4 to 14 years of age, with mite-sensitive combined allergic rhinitis and asthma syndrome were treated sublingually with standardized Dermatophagoides farinae extract. The clinical efficacy was evaluated by monthly follow-up visits. After treatment for 1 or 2 years using the standardized Dermatophagoides farinae extract, the asthma and rhinitis symptom scores, medication scores and adverse reactions before and after treatment were evaluated. SPSS 17.0 software was used to analyze the data. The allergic asthma symptom scores before treatment during the day were 3.22 ± 0.66 and at night 2.05 ± 0.57. After 1 year of treatment, the day and night scores (1.68 ± 0.61, 0.94 ± 0.32) respectively, were decreased significantly (q values were 15.25 and 13.78 respectively, all P < 0.01). After 2 years of treatment, the scores (0.61 ± 0.28, 0.43 ± 0.13) were also decreased significantly (q values were 10.29 and 6.07 respectively, all P < 0.01). The allergic rhinitis symptom scores and medication scores were 2.34 ± 0.59 and 3.09 ± 1.01 respectively before treatment and 1.21 ± 0.46 and 1.89 ± 0.64 after 1 year of treatment. The differences were significant (q values were 15.48 and 18.61 respectively, all P < 0.01). The allergic rhinitis symptom scores and medication scores were 1.02 ± 0.37 and 1.49 ± 0.38 after 2 years of treatment. There was no significant difference between 2 years of treatment and 1 year of treatment (q values were 2.53 and 2.78 respectively, all P > 0.05). There were no severe adverse events during the treatment, except for mild mouth cavity discomfort. Sublingual immunotherapy using standardized Dermatophagoides farinae extract is safe and effective in the treatment of children with combined allergic rhinitis and asthma syndrome.

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