Objective To explore the effect of IL-23-stimulated synovial fibroblasts (FLS) in rheumatoid arthritis (RA) on the formation of human osteoclast-like cell. Methods Fibroblasts from patients with RA and osteoarthritis (OA) were stimulated with various concentrations of IL-23 (1, 5 and 10 ng/ml). After incubation for 72 hours, the level of receptor activator of nuclear factor kappa B ligand (RANKL) mRNA were, measured by using real time PCR. Human monocytes (MN) were isolated and cocultured with IL-23-stimulated RA or OA FLS, and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. Osteoclast-like cell molecule markers were measured using real time PCR. Results Various concentrations of IL-23 upregulated RANKL expression in rheumatoid FLS, but failed in OA FLS. In the system of IL-23-stimulated RA FLS cocuhured with human MN, TRAP-positive osteoclast-like cell was enhanced and high level expressions of molecule markers were induced. Whereas in the system of non IL-23-stimulated RA FLS or IL-23-stimulated OA FLS cocuhured with human MN there was no TRAP-positive osteoclast-like cell formation. Conclusion The results suggest that IL-23 induced osteoclast-like cell formation is via RANKL expressions in rheumatoid FLS.

Objective: To evaluate the effect of multiple amino acid substitutions of CⅡ263-272 peptide on collagen-induced arthritis,and explore a therapeutic strategy of rheumatoid arthritis.Methods: A panel of altered CⅡ263-272 peptides was synthesized with substitutions of TCR-contact residues of CⅡ263-272 with alanine.The competitive inhibition of APL to wild type CⅡ263-272 was analyzed by ~3H incorporation assay.CIA model was induced by intradermal injection of bovine CⅡ.Altered CⅡ peptide was injected subcutaneously in different doses(1,10,100 ?g,respectively,twice per week) after onset of CIA.The arthritis index,radiologic and histologic scores were recorded to evaluate the severity change of arthritis.Serum levels of IFN-? were examined by ELISA.Results: Competitive inhibition to wild type CⅡ263-272 of the altered CⅡ263-272 peptides(APL1,APL2,APL3) was found in PBMC from RA patients.Among them,APL3 with substitution of residues 267(Q),270(K) and 271(G) to A showed the most significant effect and thus was used in the treatment of CIA rats.We observed significantly reduced arthritis scores in CIA rats treated with 100 ?g/dose of APL as compared with rats treated with PBS and peptide control.The mean radiographic and histologic scores was also markedly lower in APL-treated CIA rats than in PBS or peptide control-treated rats.On day 35 after immunization,the serum level of IFN-? in rats was examined and a significantly low level of serum IFN-? was found in APL-treated rats.Conclusion: CⅡ263-272 peptide with TCR-contact residues substitutions inhibited joint destruction in CIA rats and down-regulated IFN-? production,suggesting that altered CⅡ peptide might be potentially therapeutic in rheumatoid arthritis.

Objective:To analyze the clinical characteristics of interstitial pulmonary fibrosis (IPF) in patients with Rheumatoid arthritis (RA). Methods: We retrospectively analyzed 198 RA patients with or without IPF. Characteristics of RA-IPF in clinical and lab data were analyzed. Age, duration of disease,clinical and laboratory parameters, history of smoking and medicine were compared between the patients with and without IPF. Results: (1) Among the 198 RA patients, 15.2% (30/198) were found with IPF. 100% RA-IPF patients had HRCT findings. However, 63.3% (19/30) had positive findings in chest X-ray, and only 46. 7% ( 14/30) had the complaints of cough and short breath. Velcro rales were found in 50.0% (15/30) patients with IPF and no acropachy occurred. Only one patient suffered from hypoxemia. IPF presented after the joint symptoms in most patients. (2 ) RA-IPF patients were older than those without IPF [(65.50±9.71) vs (55.22±12.98) years, P＜0.01]; Higher positivity of anti-keratin antibodies ( AKA) were found in RA-IPF compared to patients without IPF (61. 5% vs 35.9% , P =0.014). Furthermore, the levels of anti-cyclic citrullinated peptide (CCP) antibody were significantly higher in RA-IPF [(4.38±2.08) vs (3.20±2. 12) , P =0.01]. No differentiation of duration of disease, history of smoking and medicine, IgM rheumatoid factor, IgG rheumatoid factor, anti-nuclear antibody, anti-SSA antibody and levels of immunoglobins and complements were found between the two groups of RA patients with and without IPF. Conclusion: The clinical symptoms of IPF in RA patients are mild and more common in older patients. AKA and anti-CCP antibody might be important antibodies associated with RA-IPF.

Objective:To evaluate the inhibitory effect of altered HA308-317 peptides on HLA-DR4 restricted CII specific T cell response.Methods:Three altered HA308-317 peptides and CII263-272 were synthesized using solid-phase techniques. The binding of altered HA308-317 peptides for HLA-DR4 molecules was assayed using flow cytometry. The suppressive effect of altered HA308-317 peptides on CII-mediated T cell proliferation was determined using 3H incorporation assay. The level of IL-2 in the supernatants was identified by ELISA. The expression of CD25 and CD69 on T cell surface were studied using flow cytometry.Results:The altered HA308-317 peptides were able to bind to HLA-DR4 molecules and competed with CII263-272. Altered HA308-317 peptides inhibited T cell proliferation and IL-2 production induced by CII263-272( P

Objective: To investigate the relationship between HLA-DR4/1 subtypes and T cell responses to type Ⅱ collagen or anti-CⅡ263-272 antibodies. Methods: Peripheral blood mononuclear cells and sera were obtained from 70 rheumatoid arthritis (RA) patients. T cell proliferative responses to CⅡ263-272 were evaluated by 3H thymidine incorporation assay. Antibodies specific to CⅡ263-272 were determined by ELISA. HLA-DR4/1 subtypes were analyzed by PCR-SSP. Results: In HLA-DR4/1 positive group, T cell proliferative responses to CⅡ263-272 were observed in 55.6% RA patients and anti- CⅡ263-272 antibodies were present in 66.7% RA patients. In HLA-DR4/1 negative group, T cell proliferative responses to CⅡ263-272 were observed in 30.3% RA patients and anti-CⅡ263-272 antibodies were present in 34.8% RA patients. The number of positive antibodies to CⅡ263-272 or T cell proliferative responses to CⅡ263-272 in HLA-DR4/1 positive group were higher than in negative group. Conclusion: There is a relationship between HLA-DR4/1 subtypes and T cell responses to CⅡ263-272 or anti-CⅡ263-272 antibodies.

Objective To identify interleukin (IL)-17-producing T cells and Foxp3 positive CD4 positive T ceils from patients with rheumatoid arthritis (RA), and investigate their cytokine levels as well as their correlations. Methods Flow cytometry was used to analyze the subsets of T cells in peripheral blood from 39 RA patients and 40 healthy donors. IL-17, IL-6, IL-23 and transforming growth factor (TGF)-beta levels in sera of these subjects were tested by ELISA. Results IL-17+CD4+ T cells increased significantly and IL-17, IL-6 and IL-23 elevated markedly in peripheral blood of RA patients compared to healthy donors (P< 0.01 ). However,Foxp3+CD4+T cells decreased evidently (P<0.01) and TGF-beta did not increase in RA pati-ents when compared to healthy donors (P>0.05). Conclusion This study suggests that Th17 cells predomin-ance coupled with relatively insufficient CD4+ regulatory T cells in RA is mainly caused by alterations of rela-ted cytokines.

Objective To explore the sensitivity of antibodies against RF,APF,AKA,anti-CCP in rheumatoid arthritis (RA) and analyze the relationship between these autoantibodies in RA.MethodsFive hundreds of RA patients were studied.Immunoturbidmetry was used to measure the RF-IgM.The enzyme-linked immunosorbent assay (ELISA) was used to measure the HRF-IgG,HRF-IgA and anti-CCP.Indirect immunofluorescence was used to measure the APF and AKA.ResultsThe seropositive rates of RF-IgM,anti-CCP,APF,HRF-IgG,AKA and HRF-IgA were 78.0%,70.8%,51.8%,48.4%,45.0% and 20.0% respectively.RF-IgM could not be detected in 110 patients while the seropositive rates of anti-CCP,APF,AKA,HRF-IgG and HRF-IgA were 41.8%,27.3%,22.7%,15.5% and 1.8% respectively.Anti-CCP could not be detected in 146 patients while the seropositive rates of RF-IgM,HRF-IgG,APF,AKA and HRF-IgA were 56.8%,24.7%,16.4%,8.9% and 7.5% respectively.The combined sensitivities of RF-IgM anti-CCP and APF were higher than others.The seropositive rates were 88.6%.ConclusionsRF-IgM and anti-CCP are sensitive antibodies for diagnosis of RA.Combinations RF-IgM with anti-CCP can significantly improve the sensitivity of diagnosis of RA.

Objective To detect v raf murine sarcoma viral oncogene homologue B1 (BRAF) in the synovial fluid of rheumatoid arthritis (RA) and to investigate its clinical significance in RA.Methods Synovial fluid samples were obtained from patients with RA and osteoarthritis (OA).Serum samples were obtained from patients with RA,OA and heathy controls.The presence of BRAF in the synovial fluid and sera were examined by enzyme-linked immunosorbent assay (ELISA).Western blotting was used to detect the expression of BRAF protein in the synovial tissue of RA and OA.The associations between the BRAF and the clinical features and laboratory parameters of RA were evaluated.Data analysis were performed using t test and Spearman's association analysis.Results ① The level of BRAF in the synovial fluid of RA [(84±59) ng/ml] was significantly higher than OA [(38±41) ng/ml] (t=3.290,P=0.002).② The level of BRAF in the sera of RA patients [(22.0±12.5) ng/ml] was also higher than OA [(6.8±7.5) ng/ml,t=3.882,P＜0.01] and healthy controls [(4.8±2.2) ng/ml,t=6.766,P＜0.01].③ In RA patients,the BRAF protein level in the synovial fluid [(102±52) ng/ml] was significantly higher than that in the serum [(21±12) ng/ml] (t=-4.316,P=0.003).④The expression level of BRAF in the synovial tissue of RA (0.284±0.045) was higher than that in OA patients (0.191±0.013,t=3.169,P=0.034).⑤ The level of BRAF in the synovial fluid had a negative correlation with disease duration (r=-0.40,P=0.019) and a positive correlation with rheumatoid factor (RF) levels (r=0.37,P=0.03).Conclusion The presence of BRAF in the synovial fluid and synovium of RA indicates that BRAF may play a role in the pathogenesis of RA,especially in the early stage.

To explore the action target of praziquantel (PZQ) and its underlying mechanism of action, adult male worms of Schistosoma japonicum were collected from the hepatic vein of Kunming mice infected at least 6 or more weeks previously with single-sex cercariae of S.japonicum by perfusion method. These worms were subjected to the action of calcium channel blockers (CCBs) or actin depolymerizing/stabilizing agents interfering with function of the calcium channel. The adult male worms in DMEM culture medium were co-cultivated with near-lethal dose of PZQ(14 μmol/L) overnight(16 hours).Then, the parasites were washed 3 times with sterile physiologic saline next morning after cultivation, re-suspended in new and drug-free medium and then observed under stereo-microscopy during the following 5 days. The experimental results showed that majority of adult male worms of S.japonicum were killed by the action of PZQ in a dose of 14 μmol/L in vitro under normal condition; while the worms pre-incubated with the actin depolymerizing agent cyto chalasin D (CyD) were able to survive in the condition containing 14 μmol/L of PZQ with a survival rate of 100%, and the worms pre-incubted with CCBs, such as nitrendipine and nifedipineu showed a survival rate of about 50% under the same condition. The results of this study suggest that the calcium channel of Schistosomes may be involved in the action target of PZQ and its underlying mechanism.

Objective To explore the early diagnosis of Alport′s syndrome(AS).Methods Renal and skin biopsy was carried out in 5 patients who manifested with isolated hematuria and nephritic syndrome(NS).By using indirect immunofluorescence method,the expression of type Ⅳ collagen ? chains was detected on epidermal basement membrane(EBM) and glomerular basement membrane(GBM).Results ?_1 chains on EBM and GBM were expression in all patients,but ?_5 chains on EBM and ?_3,?_5 chains on GBM form 2 female patients were segmental expression.Thus the goal for early diagnosis was achieved.Conclusions ? chains for EBM type Ⅳ collagen and GBM type Ⅳ collegan should be investigated if condition permits for those patients with isolated hematuria,NS(steroid-resistant) and thinned GBM in electron microscopy.It can be useful for diagnosis and differenial diagnosis of AS.