Molecular cloning of HLA-B27 gene promoter and transcriptional activity detection in HeLa cells / 中华风湿病学杂志

Objective To construct pGL4.14-1uc eukaryotic expression vector for HLA-B27 promoter gene and explore the activity regulation of this promoter in Hela cells.Methods The HLA-B27 gene promot- er(-419 bp～1 bp)was amplified by polymerase chain reaction and was cloned into pGL4.14-luc vector to construct eukaryotic expression vector pGL4.14/B27 pro-luc.The purified pGL4.14/B27 pro-luc was stablely transfected into HeLa cells and the activity of HLA-B27 gene promoter was detected by luminometer.Results About 432 bp gene fragment was amplified by PCR from genomie DNA and pGL4.14/B27 pro-luc vector was constructed successfully.The activity of HLA-B27 promoter was 1.67?0.20,1.79?0.71,2.94?0.68,1.98?0.45 in Hela stable cells after treated with TNF-?,IFN-?,IFN-?and IFN-?for 48 hours.Conclusion TNF-?. IFN-?,IFN-?and IFN-?can regulate the B27 promoter activity.The high specific activity of constructed HLA-B27 promoter eukaryotic expression vector may be a good method for further research.

The study of T cell clones of synovial fluids of reactive arthritis / 中华风湿病学杂志

Objective To compare the cytokine expression profile of 3 CD8+, 3 CD4+ and 3 gamma delta-positive T cell clones derived from the synovial fluids of reactive arthritis (ReA) patients and to explore the immunological pathogenesis of ReA. Methods Complementary DNA-based microarrays containing genes for 56 cytokines were used for screening the expression profile of 3 CD8+, 3 CD4+ and 3 gamma delta-positive T cell clones derived from the synovial fluids of 3 reactive arthritis (ReA) patients. Results Transcripts encoding for interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were expressed among all CD8+ and CD4+ T cell clones by microarray. Expression of these cytokines could be verified by RT-PCR in 14 out of 15 microarray experiments. Lymphotoxin (LT)-alpha and granulocyte macrophage colony-stimulating factor (GM-CSF) were also consistently expressed among CD4+ cells. However, ??+T cells revealed a different cytokine pattern, mainly expressing transforming growth factor (TGF)-beta 2 and GM-CSF. Conclusion CD8+ and CD4+ T cells mainly reveale a Th1-mediated profile, whereas ??+T cells expresse less pro-inflammatory cytokines resembling a Th3-driven pattern. T lymphocyte clones from the joint of ReA patients exhibit different cytokine expression profiles refelecting their different roles in pathogenesis.

Expression and significance of monocyte chemotactic protein in the pathogenesis of ankylosing spondylitis / 中华风湿病学杂志

Objective Previous study had shown that signal transduction was changed when Hela cells were transfected with HLA-B27gene.An increase of monocyte chemotactic protein-1(MCP-1)of Hela cells was observed.To determine the role of MCP in the pathogenesis of ankylosing spondylitis(AS),expression of MCP-1,2,3and4in peripheral blood mononuclear cells(PBMC),synovial fluid mononuclear cells(SFMC)and synovial tis-sues of AS patients were tested.Methods Gene expression profiles of PBMC from AS patients and healthy volun-teers were determined by cDNA microarray with1176target gene filter.The differentiated expressed gene MCP-1in PBMC,SFMC and synovial tissue of AS patients were confirmed by semi-quantitative RT-PCR.Results The gene expression profile of SFMC of AS patients was significantly different from those of PBMC from AS and PBMC from healthy volunteers.The MCP-1level was positively correlated with MCP-3(r=0.76,P=0.03).The expressions of MCP-1were higher in synovial tissues of AS than those of healthy volunteers(P=0.0035).MCP-1levels in monocytes of AS patients and control subjects were increased after LPS stimulation for4hours.Conclusions There is increased expression of MCP-1in SFMC and synovial tissue of AS patients.The results indicate that MCP-1may play a potential key role in the homing of cells migrating from blood to joint and in the pathogenesis of joint inflammation in AS patients.

ASAS20 at week 2 predicts subsequent improvement as well as the rate of relapse of ankylosing spondylitis infused with three pulses of infliximab / 中华风湿病学杂志

Objective To evaluate the clinical parameters that can predict whether a patient can get significant improvement at the 10th week,or whether a patient can have an extended length of remission after discontinuing the infusion in ankylosing spondylitis(AS)patients treated with three standard infusions of in- fliximab.Methods Sixty-three AS patients were given three infusions of 5 mg/kg of infliximab at week 0,2 and 6;and were evaluated serially before each infusion and week 10.Afterwards.patients were followed by telephone interview until their disease activity was≥60% of the baseline level.At that point,disease was con- sidered to relapse.Clinical parameters at baseline as well as at week 2 were used to identify factors which might predict an improvement at week 10,or predict a delayed relapse.A predictor was regarded as being use- ful if the area under the curve(AUC)more than 0.75 when analyzed by receiver operator calculations(ROC). Results No parameters at baseline have sufficient predictive value.However,ASAS20(Assessment in Anky- losing Spondylitis International working Group criteria)at week 2 predicts improvement at week 10.and also duration of remission after discontinuing the infliximab at week 6.Conclusion The response to one pulse of infliximab is the best predictor of subsequent response as well as rate of relapse after discontinuing the inflix- imab.

Effect of cytokines on transcriptional regulation of HLA-B27 promoter in HeLa cells / 中国病理生理杂志

AIM:To assess the effect of seven cytokines on transcriptional regulation of HLA-B27 promoter, NF-?B and ISRE cis-element activity in HeLa cells.METHODS: HeLa-HLAB27 promoter stable cell line was constructed. The HeLa-B27 promoter stable cell line, HeLa-NF-?B stable cell line and HeLa cells transfected with pISRE-luc were cultured with different cytokines (IL-1?, IL-1?, TNF-?, IFN-?, IFN-?, IFN-? and TGF-?).RESULTS: TNF-?, IFN-?, IFN-? and IFN-? increased the B27 promoter activity at 96 h. The strongest inducer was IFN-?, the luciferase activity increased by 5.4 times. TNF-?, IL-1? and IL-1? also induced the NF-?B activity at 8 h (increased around 30 times). IFN-? and IFN-? increased the interferon-stimulated response element(ISRE) activity 10 times at 6 h.CONCLUSION: TNF-? and interferons increase the B27 promoter activity. IFN-? might play an important role in the pathogenesis of B27 related diseases.