Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets.</p><p><b>METHODS</b>We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors.</p><p><b>RESULTS</b>Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013).</p><p><b>CONCLUSIONS</b>Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.</p>

Role of interleukin-10 and transforming growth factor beta 1 in otitis media with effusion / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Otitis media with effusion (OME) is a disease with complicated pathogeneses which are not clearly known. Increasing interest has been focused on immunological cells, cytokines and their roles in chronic inflammatory states. This study was designed to disclose the existence and roles of interleukin-10 (IL-10) and transforming growth factor beta1 (TGF-beta1) in the cause of OME in adults, and to investigate the probable role of Foxp3(+)CD4(+)CD25(+) T cells in OME.</p><p><b>METHODS</b>The concentrations of IL-10 and TGF-beta1 in the middle ear effusions (MEEs) and plasmas of 36 adults (45 ears) with OME were measured by means of enzyme linked immunosorbent assay (ELISA). As contrast, the concentrations of IL-10 and TGF-beta1 in the plasma of 30 normal volunteers were measured using the same method. Furthermore, the proportion of Foxp3(+)CD4(+)CD25(+) T cells in CD4(+) T cells of blood was tested by flow cytometry.</p><p><b>RESULTS</b>(1) The concentrations of IL-10 in all MEEs and plasmas of the chronic OME patients were higher than those in patients with acute OME (both P < 0.05), so was TGF-beta1 (both P < 0.01). The concentration of IL-10 in MEEs was significantly higher than that in plasmas, not only in acute OME (P < 0.01), but also in chronic OME (P < 0.01). In chronic OME, the concentration of TGF-beta1 in MEEs had no statistical difference with those in plasmas of the same patients. However, the concentration of TGF-beta1 in plasmas of patients with chronic OME was significantly higher than that in plasmas of normal volunteers (P < 0.01). (2) The concentrations of IL-10 and TGF-beta1 in MEEs of the patients who had been treated more than once were higher than those MEEs of the patients who were treated for the first time, respectively (P < 0.05, P < 0.01). The level of TGF-beta1 in plasmas of the patients who had been treated more than once was higher than in those of the patients who were treated firstly (P < 0.05), while the level of IL-10 in plasmas had no difference. The concentration of IL-10 in mucoid MEEs was higher than those in serous ones (P < 0.05), while TGF-beta1 had no statistical difference between mucoid and serous MEEs (P > 0.05). The concentration of IL-10 in MEEs had a strong correlation with the duration of the illness (r = 0.547, P < 0.01). The same correlation was also found between the concentration of TGF-beta1 in MEEs and the times patients being treated (r = 0.579, P < 0.01). (3) The proportion of Foxp3(+)CD4(+)CD25(+)T/CD4(+) T cells in the blood of chronic OME was not only significantly higher than that in the acute OME (P < 0.01), but also higher than that in normal volunteers (P < 0.01). In chronic OME, there was a correlation between the proportion of Foxp3(+)CD4(+)CD25(+) T/CD4(+) T cells in the blood and the concentration of IL-10 in the plasmas (r = 0.602, P < 0.05).</p><p><b>CONCLUSIONS</b>IL-10 and TGF-beta1, as two important immunoregulatory mediators, participate in middle ear inflammatory response, especially in chronic course of OME in adults. Foxp3(+)CD4(+)CD25(+) T cells may play some immunoregulatory roles in the course of this disease.</p>

Expression of a Novo Human Osteoprotegerin-Mycobacterial Heat Shock 70 Fusion Protein with Baculovirus-Insect Expression System / 中国生物工程杂志

Objective:Osteoprotegerin (OPG) is a key molecule negatively regulating osteoclast differentiation and activation; and the conserved mycobacterial heat shock 70 (HSP70) peptide p111-125 has also been found to inhibit inflammation reactions in chronic arthritis. BaculoDirectTM baculovirus expression system was selected to express recombinant OPG-HSP70 in insect cells.Methods:The human functional fragment (p22-194) of OPG and functional fragment (p111-125) of mycobacterial HSP70 gene were cloned into the transfer vector pENTRTM/SD/D-TOPO. The recombinant plasmid was performed an LR reaction with the BaculoDirectTM Linear DNA to generate recombinant baculovirus DNA. The cultured Sf9 insect cells were directly transferred with the recombinant baculovirus DNA,and the pure recombinant baculovirus was obtained. Then recombinant baculovirus was infected Sf9 insect cells again to express the OPG-HSP70 gene.Results:The target protein was detected at the time of 48h post infection,reached at highest yield at the time of 72h post-infection. A 28kDa protein immunostaining band was detected by Western blotting from lysate of those cells.And the purified protein was obtained by using Ni-NTA system. Functional stuies on the fusion protein showed it significantly reduce osteoclast cell number[(3.10?0.640) cells under each microscope field in treatment group by comparing to (10.70?0.817)cells in the control group] in the osteoclast inhibition test,and reduce the inflammation reaction in a delayed type hypersensitivity (DTH) mice model (P

Expression and Biological Characterization of Human Osteoprotegerin Fused with Mycobacteria Heat Shock Protein 65 / 中国生物工程杂志

A fused functional gene of human OPG and Mhsp65 was amplified by PCR,and cloned into the prokaryotic expression vector pET-28a.The BL21(DE3) strain of E.coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG.Result of SDS-PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body.The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis.The purified and refolded fusion protein could inhibit osteoclast proliferation and bone absorption in vitro.The results of mouse ear swelling assay and expressions of TNF-?,IFN-? and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity.The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.