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.

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 inhibitory effect of methylprednisolone (MP) on the development of coronary arteritis in a murine model of Kawasaki disease (KD) induced with a candida albicaus watersoluble fraction (CAWS).Methods Forty-five C57BIL/6mice were evenly divided into three groups (the control group,the CAWS group and the MP group).Mice in the CAWS group were intraperitoneally injected phosphate buffer saline (PBS) for 5 days.MP or PBS was administered to the different group.The animals were sacrificed at day 3,day 10 and day 28,and the status of vasculitis in the coronary arteries and the aortic root was investigated histologically.One-way analysis of variance (ANOVA) was used to compare the differences among three groups,and t-test for two independent groups.Results The mice in CAWS group and MP group,which induced by CAWS,showed that the body weight and heart weight decreased significantly,and the spleen weight was increased at day 10 and day 28 (P＜0.05).Vasculitis was induced in the mice of those two groups,and the severity score was the highest at day 10 (12.7±0.5).In addition,the severity of the inflammation of the aortic root and the coronary arteries were reduced in MP group (t=6.35,5.55,2.8,P＜0.05).Elastic fiber staining showed that the layers of vascular walls were in disorder and elastic fibers were broken in the CAWS group.However,there was no disruption or breakage in the MP group.Conclusion MP can suppress the progression of coronary arteritis in this CAWS-induced murine vasculitis model,which indicates the efficacy of MP in KD patients with coronary artery lesions.

Objective To investigate the effects of 5-Aza-CdR( methylation transferase inhibitor) on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis (OA) group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients (amputation due to severe trauma) were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region ( - 280- + 79) was detected by epityper quantitative DNA methylation analysis. Results ( 1 ) After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. ( 2 ) The mRNA expression levels of leptin were significantly different among the four groups ( P ＜ 0. 05 ), and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. (3) Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.

Objective To explore the effect of different concentrations of iguratimod (IGU) on mouse model of bleomycin-induced pulmonary fibrosis.Methods A total of 108 female C57BL/6 mice were randomly divided into the control group,the model group,the low dose IGU group,the moderate dose IGU group,high dose with group and the methylprednisolone (MP) group (n=18 in each group).The mice in the control group were injected with 0.2 ml normal saline endotracheally,and others were injected with 0.2 ml bleomycin (5 mg/kg) from endotracheally to induce pulmonary fibrosis model.The next day,the mice in both control group and the model group were fed with 0.2 ml normal saline every day;The mice in the IGU groups and methylprednisolone group were fed with 0.2 ml iguratimod liquid the IGU (10,30,90 mg/kg) and 0.2 ml methylprednisolone (10 mg/kg) every dayrespectively.Finally the mice were sacrificed at day 7,day 14,day 28 respectively,and the lung tissue was examined by HE staining and Masson staining to evaluate the degree of alveolitis and fibrosis.Repeated measurement of variance analysis was used to compare the differences for time and group,and multi-factor analysis of variance LSD test was used for the comparison between groups.Results ① The body mass of mice in bleomycin-induced groups were decreased compared to the control group.② The pathological alveolitis scores in the high dose IGU group and methylprednisolone group were significantly decreased compared to those of the model IGU group at day 7 and day 14 (P＜0.05),and the pathological fibrosis scores were decreased dramatically compared with the model group at day 14 and day 28 (P＜0.05).Conclusion High concentration of IGU and methylprednisolone can reduce and inhibit lung inflammation and fibrosis of bleomycin-induced pulmonary fibrosis in mice.

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 5-Aza-CdR (methylation transferase inhibitor) on gene expression levels of interleukin-1β (IL-1β),transforming growth factor-β (TGF-β) and nitric oxide synthase (NOS) in chondrocytes.Methods Chondrocytes from patients with osteoarthritis (OA) (n=22),rheumatoid arthritis (RA) (n=3) or trauma without rheumatic diseases (n=10) were collected and cultured.The mRNA expression levels of IL-1β,TGF-β and NOS were detected by real-time polymerase chain reaction (RT-PCR).Chondrocytes in trauma group were treated with 5-Aza-CdR(10μmol/L) for 72 h,and the mRNA and protein expression levels of IL-1β,TGF-β and NOS were detected by RT-PCR and ELISA,respectively.Results The mRNA expression levels of IL-1β,TGF-β and NOS were increased in OA and RA group as compared with trauma group (P＜0.05),while they had no differences between OA and RA groups.After treated with 5-Aza-CdR,the mRNA and protein expression levels of IL-1β,TGF-β and NOS in chondrocytes rised in trauma group as compared with pretreatment (all P＜0.05).Conclusions The mRNA expression levels of IL-1β,TGF-β and NOS in chondroytes are higher in OA and RA patients.5-Aza-CdR could increase the mRNA and protein expression levels of IL-1β,TGF-β and NOS by inducing relative gene methylation,which suggests demethylation might play a role in OA pathogenesis by influncing the inflammatory signal pathway or cell apoptosis.

Aim To observe mesenchymal stem cell differentiation into osteoblast induced by TSPG, and explore how TSPG enhances the promotion of hemato-poiesis of osteoblast differentiation from mesenchymal stem cell. Methods MSCs were cultured by TSPG combined with osteoinductive medium. The cellular vi-ability of proliferation was detected with MTT assay. The content of alkaline phosphatase in the cultural su-pernatant was tested with pNPP assay. The ability of MSCs to form calcium nodes was also observed after a-lizarin red stain. The protein expression of RUNX2 was analyzed with Western blot. The content of cytokines associated with hematopoiesis was tested with Elisa as-say. The ability of promoting hematopoiesis was detec-ted with hematopoietic colony forming assay. Results Both MTT and pNPP assay showed that optical den-sity ( OD) values were increased in response to TSPG treatment in a dose-dependent manner. The mineraliza-tion formation ability was enhanced with TSPG-treat-ment. Meanwhile, the expression of RUNX2 protein was up-regulated in TSPG-treated cell. Moreover, the content of cytokines associated with hematopoiesis and the number of hematopoietic progenitor colony were in-creased by TSPG-treatment compared with the control group. Conclusion TSPG could induce MSCs differ-entiation in to osteoblast and enhance the effect of oste-oblast differentiation from MSCs on promoting hemato-poiesis.

BACKGROUND: Human leukocyte antigen (HLA)-B27 is closely connected to the occurrence of some rheumatic diseases such as ankylosing spondylitis and can be used as an important factor for evaluating the diagnosis of ankylosing spondylitis. Immunomagnetic separation and enzymelinked immunosorbent assay (IMS-ELISA) has been applied to the detection of HLA-B27.OBJECTIVE: To explore the accuracy, sensitivity and specificity of IMSELISA for detecting HLA-B27 and its value in the auxiliary diagnosis of ankylosing spondylitis.DESIGN: A clinical trial in comparison with the gold standard.SETTING: Departments of Rheumatology and Clinical Laboratory, Third Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: Eighty-six patients suffering from low back pain and/or arthritis who were treated for the first time in Department of Rheumatology from December 2002 to April 2003. Inclusion criteria: ① Presence of manifestations of low back pain and/or arthritis; ② Thorough documentation of clinical and other examinations; ③ Informed consent to HLA-B27 examination; ④ Treatment for the first time in the Third Affiliated Hospital of Sun Yet-sen University. Those with other serious diseases or with incomplete record of clinical and/or accessory examinations were excluded. The 86 patients included 56 male and 30 female patients aged from 12 to 65 years.METHODS: Blood sample was detected for HLA-B27 by both IMS-ELISA and microlymphocytotoxicity test, and the latter was selected as the gold standard. The coincidence rate of the results detected by the two methods as well as the sensitivity, specificity, positive and negative predictive values of IMS-ELISA were calculated.MAIN OUTCOME MEASURES: ① The coincidence rate of the results of the two methods. ② The sensitivity and specificity of IMS-ELISA for detecting HLA-B27.RESULTS: None of the patients was lost. For the 33 patients with ankylosing spondylitis, the positivity rate of IMS-ELISA (90.9％) was higher than that of microlymphocytotoxicity test (87.9％), but the difference was not statistically significant (P＞0.05). The total coincidence rate of the two methods was 93.0％ in all the 86 patients. The sensitivity, specificity, positive and negative predictive values of IMS-ELISA were 90.0％, 95.7％,94.7％ and 91.7％ respectively.CONCLUSION: Both IMS-ELISA and microlymphocytotoxicity test are capable of reliable examination of HLA-B27 with high sensitivity and specificity.