Objective To study the effect of Iguratimod on the collagen secretion of 3T6 cells. Method The median toxic concen- tration of Iguratimod was determination by MTT Assay. Iguratimod' s median toxic concentration to 3T6 cell was about 1142.6mg/L. Taking into the consideration of the cells performance after administration under spectacles, we chosen the administration concentration as 334.8 mg/L(DMS0:1?l/256?l) ,167.4mg/L,83.7mg/L and 41.85 mg/L. After administration of 12 hours, 24 hours, 36 hours and 48 hours, the supernatant was collected for assayed respectively. The collagens content was detected by the method of chloramine T. Results The collagen concentration of the drug concentration 334.375mg/L group increased (P

Objective To study the relationship between DNA methylation inhibitor and histone deacetylase inhibitor (HDACI) with HLA-B27 gene expression. Methods Hela-HLA-B27 promoter stable cell line was developed as the first step and followed by detecting the effect of DNA methylation inhibitor(5-Aza-CdR) and HDACI. The synergetic effect of HDACI and cytokines as well as the effect of anti-TNF-α antibody, anti-IFN-β antibody, anti-IFN-γ antibody and Infiximab on HLA-B27 promoter activity were tested by measuring luciferase value. Then HLA-B27 mRNA expression level in CCL6-B27 genomic DNA stable cell line was measured by real-time PCR. Results Sodium butyrate(NaB) and valproic acid(VPA)could significantly up-regulate HLA-B27 promoter activity at 24 h (24.0±1.7) times, ( 17.4±2.2 ) times respectively,(P＜0.05). The synergetic effect between VPA with TNF-α, IFN-α, IFN-β, IFN-γ and PMA on HLA-B27 promoter activity was found. None of the antibody could inhibit the effect of HDACI. It also showed that NaB,TSA, VPA and 5-Aza-CdR could significantly increase HLA-B27 mRNA expression in CCL6-B27 stable cell line. Conclusion DNA methylation and HDACI can up-regulate HLA-B27 gene expression level.

0 05) after treatment.At the same time,the clinical symptoms were improved significantly.Conclusion ① Testing expression level of HLA DR is helpful to diagnose and monitor the state of RA.② The change of expression level of HLA DR conforms to that of rheumatic factor.③ Examination of the change of expression level of HLA DR can help to judge the state and prognosis of RA.

Objective To study the gene expression profile of peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of patients with spondyloarthropathy (SpA) and try to identify genes which might be related to the pathogenesis of SpA.Methods Microarrays were carried out with 1 176 target gene filters using PBMC and SFMC of 5 patients with SpA,5 patients with rheumatoid arthritis (RA) and 6 healthy volunteers.More precise measurement was performed with semi quantitative PT PCR on 9 microarray positive genes.Results There was no significant difference in positive gene numbers between SpA and RA patients although the positive gene numbers were much higher in PBMC than in SFMC in both groups.It is noteworthy that in RA group there were 53 genes which expression in PBMC was much higher than in SFMC,while in SpA patients there were only 5 genes which expression in PBMC was much higher than in SFMC ( P

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.

AIM:To screen the effective chemicals,which can suppress the promoter activity of the HLA-B2705 gene as potential therapeutic agents.METHODS:The HeLa-HLA-B27,293T-HLA-B27 stable transfectants were used to monitor the effect of 12 264 chemicals through high throughput screening(HTS).Chemicals which regulates HLA-B*2705 promoter activity more than 150% or less than 60% were picked out for the further IC50/EC50 and cell viability detection.RESULTS:(1)The primary screening used by 293T-HLA-B27 stable transfectant yielded about 5.1% hits which either suppressed(556 chemicals)or enhanced(68 chemicals)the HLA-B*2705 promoter activity.(2)A reconfirmation screening was carried out with these 624 of the candidates using transfected HeLa-HLA-B27 cells.Seventy hits were confirmed.(3)Based on the bioinformatics of those positive hits,40 chemicals were selected for in-depth analysis by dose-response experiment and IC50/EC50 detection.Six suppressors showed potential pharmacological activities.Interestingly,two suppressors(celastrol and pristimerin)are derived from Leigongteng,a herbal medicine already used for several decades for treatment of immune regulatory and inflammatory diseases.Four active chemicals were computer designed with no relevance to the above structures.CONCLUSION:Chinese traditional herb Nansheteng and Leigongteng might be the potential drugs for HLA-B27 positive patients.These results provide new direction for research in both the therapeutics and the pathogenesis of spondyloarthritis.

Objective To investigate the expressions and significance of interleukin 8 (IL 8) in patients with ankylosing spondylitis (AS). Methods Using microarray and RT PCR to detect the expressions of IL 8 in peripheral blood mononuclear cells (PBMC), synovial fluid mononuclear cells (SFMC) and synovial cells in patients with active AS. Comparing the results with those in PBMC of both healthy volunteers and patients with rheumatoid arthritis (RA), and also with those in synovial cells obtained from patients undergoing knee operations for trauma. Results The expressions of IL 8 in PBMC and SFMC of patients with active AS were significantly higher than those in PBMC of the healthy controls. The expression of IL 8 in PBMC of RA patients was also higher than that of healthy volunteers. Higher expression of IL 8 in the synovial cells were detected in AS than that of patients with knee joint trauma. Conclusions The expression of IL 8 is high in patients with AS. The result may indicate that IL 8 is possibly one of the important mediators of inflammation in AS, and it may mediate the induction and development of inflammation in synovium or other tissues involved in AS

Objective To consecutively understand the national clinical testing status and to reassure the quality-control of autoantibody detection. Methods Letter or telephone notification were conducted to the participating hospitals or departments. Autoantibodies for quality control survey included anti-nuclear anti-body (ANA), anti-double-stranded DNA (anti-dsDNA), anti-extractable nuclear antigens (A-ENA), anti-mitochondria antibody (AMA)/anti-smooth muscle antibody (ASMA), and anti-citrulline antibody (anti-CCP). Each had 3 control samples, and altogether 15 samples for testing. Sample distribution and data analysis were double-blinded. Results One hundred and two hospitals/departments participated in the national quality-control survey. The accurate rate for this survey was 70%, 88%, 93%, 85%, 79% respectively for ANA, anti-dsDNA, AMA, ASMA and anti-CCP. Anti-ENAs were further divided into anti-RNP, Sm, SSA, SSB and Scl-70 subgroups, and the accurate rate was 88%, 77%, 92%, 93% and 87% respectively. Conclu-sion Compared to the previous 4 national surveys, the accurate rates of ANA, anti-dsDNA, anti-ENAs, AMA in our country's autoantibody testing is improved, and the detection rate of ASMA and anti-CCP antibody is also increased. More hospitals and testing items can test these autoantibodies, which implies that autoantibody testing status has been improving in our country.

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.

Objective To investigate the effects of the leflunomide active metabolite (A771726) on the expression of phorbol-12-myristate-13-acetate (PMA) -induced CD147, matrix metallo-proteinase (MMP)-2 and MMP-9 on THP-1 cells. Methods THP-1 cells were cultured in RPMI-1640 medium supplemented with 10％ fetal bovine serum. For all experiments, THP-1 cells were cultured at an initial density of 5×105/ml. Before A771726 treatment, cells were cultured with serum-free RPMI-1640 medium for 12 h, THP-1cells were co-cultured with PMA at three different concentrations of A771726 (5, 15 , 45 μg/ml) for 24 h.The mRNA expression of CD147, MMP-2 and MMP-9 was measured by real-time PCR. CD147 expression on the cells were evaluated by flow cytometric analysis. The activity of MMP-2 and MMP-9 were evaluated by gelatin zymography. Statistical differences among the groups were tested by one-way ANOVA or KruskalWallis test. Results The expression of CD147, MMP-2 and MMP-9 were upgraded by the PMA. The expression of CD147 on THP-1 cells was inhibited significantly by A771726 in a dose-dependent pattern (P＜0.01). The mean fluorescence intensity (MFI) of CD147 in positive control group was 109.5±3.8, the MFI in A771726 (5, 15, 45 μg/ml) group were 73.3±2.5, 64.5±2.3, 40.9±2.7, respectively. The expression of MMP-2, MMP-9 mRNA and the activity of MMP-2, MMP-9 in the supernatant was inhibited significantly by A771726 (P＜0.01). The expression of CD147 mRNA was not inhibited significantly by A771726 (P＞0.05).Conclusion Leflunomide active metabolite (A771726) can inhibit the expression of PMA-induced CD147,MMP-2 and MMP-9 on THP-1 Cells.