Objective To study the effect of type Ⅱ collagen (C Ⅱ) from Zaoeys dummies (Cantor)on the immune functions of rats with collagen-induced arthritis and to understand the mechanisms of RA treated with Zaoeys dhumnades (Cantor).Methods The rats with collagen-induced arthritis were randomly divided into three group:C Ⅱ from Zaocys dhumnades,bovine C Ⅱ and arthritis control group,a normal control group was set up,too.Every group had 7 rats.The C Ⅱ from Zaocys dhumnades and the bovine C Ⅱ group were fed with C Ⅱ from Zaocys dhumnades (15 mg/kg) and bovine C Ⅱ (15 mg/kg) per day respectivelyfor 15 days.The CD4/CD8 subset ratio,serum levels of anti-C Ⅱ antibody,TNF-a,IL-10,IL-1 and IL-4 in.rats were measured.Results In the arthritis group,CD4/CD8 subset ratio (P＜0.05) and serum levels of type Ⅱ collagen antibody (P＜0.01) and TNF-a (P＜0.01) were significant increased and IL-10 (P＜0.01) was significantly decreased.In the C Ⅱ from Zaocys dhumnades and bovine C Ⅱ group,CD4/CD8 subset ratio (P＜0.05) and the level of TNF-a (P＜0.01) were significantly decreased compared with the arthritis group,and had no difference compared with the normal group.The level of anti-C Ⅱ antibody was declined significantly compared with the arthritis group (P＜0.05) and had statistical difference with the normal group (P＜0.01).The level of IL-10 was significantly increased(P＜0.01),but lower than the normal group(P＜0.05).There was no statistical difference in the level of IL-1 and IL--4 in.all four groups.Conclusion C Ⅱ from Zaoeys dhum-nades (Cantor) is as effective as bovine C Ⅱ in modifying the immune functions of collagen-induced arth-ritis in rats.They can decrease the level of anti-C Ⅱ antibody,the level of TNF-a,CD4/CD8 subset ratio and increase the level of IL-10 in the peripheral blood of rats with collagen-induced arthritis.C Ⅱ from Zaocys dhumnades may be one of the important pharmacological active components that have the potential in treating rheumatoid arthritis.

Objective To characterize and quantify the CD4 +CD25 + regulatory T (Treg) cell population in peripheral blood of patients with ankylosing spondylitis (AS) and to determine the influence of treatment with tumor necrosis factor (TNF)-a inhibitors on them.Methods Peripheral blood mononuclear cells (PBMC) were isolated from 25 patients with active AS,in which 10 patients were treated with 12 weeks of etanercept,and 21 healthy subjects.CD4+CD25high T cells were analyzed using flow cytometry,and mRNA expression of FOXP3 was determined by real-time polymerase chain reaction (PCR).Proliferation of T cells to PHA was measured by WST-1 assay using depleted CD25+ cells by immunomagnetic sorting.Results There was no significant difference in the percentage of CD4+CD25high cells in peripheral blood between patients with active AS and controls (P＞0.05).However,PBMC from patients with active AS expressed reduced levels of FOXP3 mRNA (P＜0.01) which were inversely correlated with C-reactive protein (CRP)(P＜0.01).CD4+CD25+ cells in peripheral blood of both active AS patients and controls exhibited suppressive capacity on the proliferation of effector T cells in vitro (both P＜0.01).Treatment with etanereept increased significantly CD4+CD25high cells and FOXP3 mRNA expression (both P＜0.01),with negative correlations between these increases and decrease in CRP levels (P＜0.05 and P＜0.01,respectively).Conclusion In AS patients,peripheral FOXP3-expressing CD4 +CD25 + Treg cells are abnormal,and are up-regulated by etanercept treatment.This suggests a possible pathogenesis of AS and a potential mechanism for clinical efficacy of TNF-α inhibitors.

Objective To study the (-)-epigallocatechin-3-gallate (EGCG) function on the proliferation of T cells derived from the peripheral blood and synovial fluid of rheumatoid arthritis (RA) and RA-related cytokine levels and the role of EGCG on RA synovial fibroblasts (FLS) proliferation was investigated. Methods ① Mononuclear cells from RA peripheral blood (30 cases) and synovial fluid (23 cases)were isolated. Blank group, negative control group, positive control methotrexate (MTX) group and therapeutic group with three different concentrations of EGCG were set up. Incorporated isotope 3H was used to test T cell proliferation from RA-PBMC and SFMC. ELISA assay was used to test cytokine (TNF-α, IFN-γ, IL-1, IL-6 and IL-17A) levels. ② MTT assay was used to test FLS proliferation from RA synovial tissue (8 cases).Results ① The CPM value of the high-dose group of EGCG in the peripheral blood and synovial fluid of RA patients was [ ( 15 136±2910), ( 11 587±3135 ) ], which was declined significantly than the control group (42856±2127) (P＜0.01). The levels of TNF-α, IFN-γ, IL-1, IL-6 and IL-17A in the high-dose group of EGCG in the peripheral blood were [(321±13), (298±20), (132±12), (197±7), (59±8) pg/ml], which were decreased significantly than those of the control group [ (458±28), (505±26), (346±28), (405±25),(109±13) pg/ml ] (P＜0.05 or P＜0.01 ). The levels of TNF-o, IFN-γ, IL-1, IL-6 and IL-17A in the highdose group of EGCG in the synovial fluid were [(41.4±2.9), (182±16), (56.3±11.0), (34.2±1.9), (44±8)pg/ml ], which was decre-ased significantly than the control group [ ( 388.3± 19.3 ), (469±20), ( 104.2±17.8 ),( 114.5±4.8), ( 104±11 ) pg/ml] (P＜0.05 or P＜0.01 ). ② The level (A) of the high-dose group of EGCG in the FLS was (0.08±0.02), which was declined significantly than the blank group (0.27±0.04) (P＜0.05).Conclusion ① In vitro EGCG can inhibit T cell proliferation from peripheral blood and synovial fluid of RA patients and the TNF-α, IFN-γ, IL-1, IL-6 and IL-17A secretion are decreased. ② In vitro EGCG can inhibit the proliferation of RA FLS.

Objective To investigate the effects of simvastatin on the production of interleukin (IL)-17and B-cell activating transcription factor (BATF) in the peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients and healthy individuals.Methods PBMCs were isolated from heparinized blood of healthy donors or RA patients using Ficoll-Hypaque density gradient centrifugation.The cells were stimulated by PMA and ionomycin in the absence or presence of simvastatin or MVA at 37 ℃ 5％CO2.The mRAN level of IL-17,BATF and GAPDH was detected by RT-PCR; the protein level of IL-17 in supernatants was assayed by ELISA kit; and the protein level of BATF was detected by Western Blotting.The comparison between the two groups was carried out by paired-t test and Chi-square test was used for muhi-group comparison.Results PBMCs of healthy donors [(69.2±12.2) vs (8.1±2.2) pg/ml,P＜0.05; (76.6±14.7) vs (10.2±7.2) pg/ml,P＜0.05] and RA patients [(79.6±12.7) vs (15.8±5.8) pg/ml,P＜0.05; (90.3±9.7) vs (12.9±7.9) pg/ml,P＜0.05] were stimulated with PMA and ionomycin to produce high levels of IL-17.After treatment with simvastatin,the expression and secretion level of IL-17 in healthy controls and RA PBMCs were markedly decreased.The inhibition of simvastatin on the production of IL-17 was reversed by mevalonic acid (MVA),but no significant changes of BATF after treating with simvastatin.Conclusion Simvastatin inhibits the production of IL-17 in the PBMCs at gene and protein levels,which is not targeted at suppressing the expression of IL-17 transcription factor BATF.

Objective To compare the efficacy of the conventional PPD skin test and a new enzymelinked immunospot assay(TSPOT-TB)for diagnosing latent tuberculosis infection(LTBI)in patients with rheumatic diseases.Methods Two hundred and sixty rheumatic patients were enrolled,and all were screened for LTBI based on clinical history,chest X-ray,PPD skin test or TSPOT.Results The positive rate of TSPOT assay was 24.1%and that of PPD skin test was 39.4%.The overall concordance rate between the 2tests was 61.0%.Among PPD negative patients (n=149).29 were TSPOT(+)(19.5%).Among PPD(+)patients(n=98),69 were TSPOT(-)(70.0%).The patients who got BCG vaccination or had history of tuberculosis infection showed a significantly higher rate of positive result of PPD skin test than those who did not (P<0.05 or P<0.01).While in TSPOT assay,the BCG vaccination or history of tuberculosis infection did not show influence on TSPOT results(P>0.05).Of the 127 patients who received biological agents after screening for LTBI,9 patients were pretreated with isoniazide.Twenty-seven patients stopped biological agent treatment because of the positive results of PPD or TSPOT.Twenty three patients who had positive PPD but negative TSPOT results received biological agent treatment without isoniazide,and none of them developed active tubereulosis after 6 to 18 months of follow-up.Conclusion BCG vaccination affects the result of PPD test in rheumatic patients,but has no influence on TSPOT results.The infection rate of latent tuberculosis of rheumatic patients in our research is 23.8%detected by TSPOT.

Objective To evaluate a new enzyme-linked immunospot assay (TSPOT-TB) for the diagnosis of latent tuberculosis infection in patients with rheumatic diseases.Methods The rapid TSPOT-TB assay was applied to detect ESAT-6 and CFP-10 specific T cells in blood samples from 126 rheumatic disease patients.A PPD skin test was performed on all patients simultaneously.Results The positive rate of TSPOT assay was 23.8% and that of PPD skin test was 34.9%.The overall agreement between the 2 tests was 71.4%.Among PPD (-) patients (n=82),11 were TSPOT (+) ( 13.4% ).Among PPD (+) patients (n=44),25 were TSPOT(-) ( 56.8% ).The patients who got BCG vaccination showed a significantly higher rate of positive results of PPD skin test than those who did not(41% vs 19%,P＜0.05).While in TSPOT assay,the BCG vaccination did not show any influence on TSPOT results (22% vs 27%,P＞0.05).Conclusion BCG vaccina-tion affects the results of PPD test in patients with rheumatic diseases,but has no influence on TSPOT results.The infection rate of latent tuberculosis in patients with rheumatic diseases in our study is 23.8% detected by TSPOT.

Objective The therapeutic effect of epigallocatechin-3-gallate (EGCG) on the collageninduced arthritis model (CIA) was observed and its immunological mechanism was analyzed. Methods EGCG was administered to CIA mice and PBS was admitted as negative control. The severity of CIA was evaluated by clinical scores and histopathological assessment (H-E staining). Immunological mechanisms inv-suppressive effect on IL-17 secretion of CD4+T cells (EGCG group: 0.41%; PBS group: 4.05% ) and inhibitive activity of C Ⅱ -reactive splenocytes proliferation. There was statistical significant difference between IKB expression and down-regulate phosphorylated IKB expression in lymph node cells of CIA mice.Conclusion EGCG can significantly ameliorate the severity of CIA. The therapeutic mechanisms may be related to inhibition of C Ⅱ -reactive splenocyte proliferation and IL-17 secretion and via inhibiting the activity of NF-κB by inducing the expression of IKB and by suppressing the expression of phosphorylated IKB in CIA mice.

Objective To observe the clinical efficacy of electroacupuncture plus oral administration ofZeling Guanjie Xiaozhong Heji in treating rheumatoid arthritis (RA) due to liver-kidney yin deficiency.Method Totally 126 patients with active RA due to liver-kidney yin deficiency were randomized into a treatment group and a control group, 63 cases in each group. The control group was prescribed with orally taking Methotrexate tablets and Leflunomide tablets, while the treatment group was intervened by electroacupuncture plus oral administration ofZeling Guanjie Xiaozhong Heji in addition to the medications given to the control group. The Disease Activity Score 28 (DAS28) was evaluated before and after intervention, and the therapeutic efficacies were compared based on the criteria of the American College of Rheumatoid (ACR) and syndrome of traditional Chinese medicine (TCM).Result The ACR total effective rate was 87.3% in the treatment group versus 65.1% in the control group, and the difference was statistically significant (P<0.01). The total effective rate based on TCM syndrome was 87.3% in the treatment group versus 73.0% in the control group, and the difference was statistically significant (P<0.05). There was a significant difference in comparing the DAS28 score between the two groups after intervention (P<0.01).Conclusion Electroacupuncture plus oral administration of Chinese medication and western medication is an effective approach in treating RA due to liver-kidney yin deficiency, and it can significantly enhance the therapeutic efficacy based on ACR20 and TCM syndrome.

Objective Rheumatoid arthritis (RA) is a systemic autoimmune disease, which mainly involves joints across the body, resulting in joint stiffness and loss of daily activity. Recent evidence suggests that numerous self-reacting T cells, including Th1 and Th17, infiltrate the synovium in RA patients, accompanied by functionally-compromised Treg. Iguratimod, a new small molecule with anti-inflammatory and immunomodulatory effects, has shown curative effects in animal models of arthritis. In this study, we aimed to test the clinical effects of Iguratimodˊs on RA patients and its role in immunoregulation. Methods We examined the clinical effects of iguratimod on RA patients in a random controlled clinical trials and analyzed its effects on Th1, Th17 and Treg as well as their associated cytokines and transcription factors by flow cytometry and real-time polymerase chain reaction (PCR). Then t-test, chi-square test and rank sum test were used to conduct statistical analysis. Results Our results revealed that iguratimod therapy provided significantly greater clinical benefit [ACR20, ACR50, ACR70 reached 50%, 20%, 15% respectively in iguratimod treatment group, Z=-2.216,P=0.027] than placebo group with the reduction of Th1 and Th17 but increment of Treg after iguratimod treatment [Th1: week 0 (26.5 ±8.0)%, week 24 (14.2 ±7.3)%, P<0.01; Th17:week 0 (1.7±0.7)%, week 24 (1.3±0.4)%, P<0.05;Treg:week 0 (6.8±1.6)%, week 24 (8.9±2.9)%, P<0.05], which was statistically significant. Conclusion Our results provide theoretical and clinical based evidence for the impact of iguratimod on immunomodulation of RA.

Objective Infliximab is a kind of recombinant human mouse chimeric anti-tumor necrosis factor monoclonal antibody. Here we aimed to examine the impact of infliximab therapy on RANK/RANKL/OPG system in the peripheral blood of rheumatoid arthritis (RA) patients. Methods Fifty patients with RA were rigorously screened and randomly divided into 2 groups. One group was treated with infliximab (3 mg/kg)and methotrexate (MTX). As control, the other group was treated with MTX alone. Infliximab was administered at weeks 0, 2, 6 and 14. The expression of RANK, RANKL mRNA in the peripheral blood, serum OPG and clinical indicators changes at week 0 and 18 were compared.x2-test or t-test were used for statistical analysis.Results After treated with infliximab, bone damage of joints were slowed down when examined by radiography in RA patients compared with the control group. And the ratio of OPG/RANKL was also decreased in RA peripheral blood (w0: 80.25;w 18: 63.2); (control group w0: 83.37; w18: 30.87)(P＞0.05). Although after the treatment with either MTX alone [w0: (238±15) pg/ml; w18: (118±10) pg/ml] or infliximab combined with MTX [(w0: (223.1±6.2) pg/ml; w18:(162.4±5.5) pg/ml], the serum levels of OPG were all decreased (P＞0.05), the level of OPG in infliximab treatment group was declined slower than those in the control group. Conclusion RA bone destruction can be inhibited by the combination therapy of infliximab and MTX. The mechanism may be partly through the RANK/RANKL/OPG system.