Long Noncoding RNA HAFML Promotes Migration and Invasion of Rheumatoid Fibroblast-like Synoviocytes.

The aggressive phenotype exhibited by fibroblast-like synoviocytes (FLSs) is critical for the progression of joint destruction in rheumatoid arthritis (RA). Long noncoding RNAs (lncRNAs) have crucial roles in the pathogenesis of diverse disorders; however, few have been identified that might be able to control the joint damage in RA. In this study, we identified an lncRNA, ENST00000509194, which was expressed at abnormally high levels in FLSs and synovial tissues from patients with RA. ENST00000509194 positively modulates the migration and invasion of FLSs by interacting with human Ag R (HuR, also called ELAVL1), an RNA-binding protein that mainly stabilizes mRNAs. ENST00000509194 binds directly to HuR in the cytoplasm to form a complex that promotes the expression of the endocytic adaptor protein APPL2 by stabilizing APPL2 mRNA. Knockdown of HuR or APPL2 impaired the migration and invasion of RA FLSs. Given its close association with HuR and FLS migration, we named ENST00000509194 as HAFML (HuR-associated fibroblast migratory lncRNA). Our findings suggest that an increase in synovial HAFML might contribute to FLS-mediated rheumatoid synovial aggression and joint destruction, and that the lncRNA HAFML might be a potential therapeutic target for dysregulated fibroblasts in a wide range of diseases.

NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis.

OBJECTIVE: Recent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like synoviocytes (FLSs) functions and synovial immune cell infiltration in rheumatoid arthritis (RA). METHODS: FLSs were obtained from active established patients with RA. Protein expression was determined by western blotting or immunohistochemistry or multiplexed immunohistochemistry. Cell migration was measured using a Boyden chamber. ac4C-RIP-seq combined with RNA-seq was performed to identify potential targets of NAT10. RNA immunoprecipitation was used to validate the interaction between protein and mRNA. NAT10 haploinsufficiency, inhibitor remodelin or intra-articular Adv-NAT10 was used to suppress arthritis in mice with delayed-type hypersensitivity arthritis (DYHA) and collagen II-induced arthritis (CIA) and rats with CIA. RESULTS: We found elevated levels of NAT10 and ac4C in FLSs and synovium from patients with RA. NAT10 knockdown or specific inhibitor treatment reduced the migration and invasion of RA FLSs. Increased NAT10 level in the synovium was positively correlated with synovial infiltration of multiple types of immune cells. NAT10 inhibition in vivo attenuated the severity of arthritis in mice with CIA and DTHA, and rats with CIA. Mechanistically, we explored that NAT10 regulated RA FLS functions by promoting stability and translation efficiency of N4-acetylated PTX3 mRNA. PTX3 also regulated RA FLS aggression and is associated with synovial immune cell infiltration. CONCLUSION: Our findings uncover the important roles of NAT10-mediated ac4C modification in promoting rheumatoid synovial aggression and inflammation, indicating that NAT10 may be a potential target for the treatment of RA, even other dysregulated FLSs-associated disorders.

A novel long noncoding RNA ENST00000597482 serves as a potential biomarker for disease activity and diagnosis of systemic lupus erythematosus.

OBJECTIVES: Emerging evidence indicate that long noncoding RNAs (lncRNAs) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE) however, the contribution of lncRNAs to SLE remains largely unclear. Our study aimed to explore the lncRNA expression profiles in peripheral blood mononuclear cells (PBMCs) from SLE patients. METHODS: LncRNA sequencing was used to detect differentially expressed genes in PBMCs from 5 SLE-MIX samples and 3 healthy controls (HC)-MIX samples, and the expression of selected lncRNAs was further verified by real-time quantitative polymerase chain reaction (RT‒qPCR). The correlation of lncRNA expression with laboratory indicators as well the SLE disease activity index 2000 (SLEDAI‒2K) score from 72 SLE patients was assessed by Spearman's test. The association between lncRNA ENST00000597482 and organ involvement in SLE patients was determined by the Mann‒Whitney U test. Moreover, lymphocyte subsets in peripheral blood from SLE patients were measured by flow cytometry. In addition, the diagnostic value of lncRNAs in predicting SLE was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: The lncRNA expression profiles demonstrated 218 differentially expressed lncRNAs, including 121 upregulated genes and 97 downregulated genes, in PBMCs from SLE patients compared to HCs. Among the 10 candidate genes selected, only lncRNA ENST00000597482, which was lower in SLE PBMCs than in HCs, was consistent with the sequencing results. LncRNA ENST00000597482 expression was negatively correlated with SLEDAI-2K score and the titres of ANA antibodies and anti-double-stranded DNA (anti-dsDNA) antibodies. Of note, SLE patients with lower expression of lncRNA ENST00000597482 were prone to develop organ involvement. Furthermore, lncRNA ENST00000597482 exhibited potential diagnostic value in differentiating SLE patients from HCs. CONCLUSIONS: LncRNA ENST00000597482 expression was lower in PBMCs from SLE patients than HCs and was negatively correlated with the SLEDAI-2K score and autoantibody titres. In addition, lncRNA ENST00000597482 could act as a novel biomarker for disease activity and diagnosis of SLE.

Impaired left ventricular systolic synchrony in patients with lupus Nephritis: A speckle tracking echocardiography study.

OBJECTIVES: We aimed to explore the value of two-dimensional speckle tracking echocardiography measurements of the global longitudinal strain (GLS) and left ventricular mechanical dispersion (LVMD) in the assessment of early stage left ventricular systolic dysfunction and heterogeneity of myocardial contraction in patients with lupus nephritis (LN). METHODS: Patients with LN and extra-renal systemic lupus erythematosus (SLE) and healthy participants in the control group underwent echocardiography for the traditional measurement of the left ventricular systolic and diastolic function and speckle tracking measurements of the GLS and LVMD. GLS was defined as the average value of the peak strain during systole of the left ventricular 17 segments, and LVMD was defined as the standard deviation. The demographic characteristics including age, sex, and body mass index (BMI) of all the participants were collected. The clinical and laboratory characteristics of the patients with LN were collected. RESULTS: We included 41 healthy control, 37 patients with extra-renal SLE, and 73 patients with LN. There were statistically significant differences in the GLS and LVMD between the extra-renal SLE and LN groups (GLS -19.36% vs. -17.61%, p < 0.001; LVMD 35.62 ms vs 42.96 ms, p<0.001). There was a statistically significant difference in the LVMD between the extral-renal SLE and control groups (35.62ms vs 25.51ms, p<0.001), but not in GLS (-19.36% vs -19.52%, p > 0.05). Multiple regression analyses were conducted in a subset of patients, and 24-hour proteinuria was independently associated with LVMD (ß [SE], 0.793 [0.302], p < .05). CONCLUSIONS: Patients with LN have more severe myocardial involvement than patients with extra-renal SLE. The asynchrony in myocardial contraction represented by the LVMD can be recognized earlier than that of the overall contractile functional impairment represented by GLS. In patients with LN, the 24-hour proteinuria was associated with LVMD. This indicates that the heterogeneity in the contractile function may be associated with the severity of renal damage.

Postsystolic shortening and early systolic lengthening for early detection of myocardial involvement in patients with systemic lupus erythematosus.

BACKGROUND: Cardiac involvement predicts a poor prognosis in patients with systemic lupus erythematosus (SLE). Two-dimensional speckle-tracking echocardiography (2D-STE) are used to identify subclinical myocardial involvement in various diseases. This study objected to evaluate postsystolic shortening (PSS) and early systolic lengthening (ESL) by 2D-STE for early detection of myocardial involvement in patients with SLE. METHODS: A total of 121 patients with preserved left ventricular ejection fraction (LVEF) in SLE and 30 healthy controls underwent standard 2D-STE in our study. According to SLE disease activity index (SLEDAI), we divided SLE patients into two groups: the group of inactive disease (SLEDAI ≤ 4) and active disease (SLEDAI ≥ 5). The maximum of postsystolic strain index (PSImax ) and early systolic strain index (ESImax ) were acquired from 17 segments of left ventricular (LV). We also compared the PSImax and ESImax of basal, medial, and apical segments between SLE patients and controls. RESULTS: Compared with healthy controls and the group of SLEDAI ≤ 4, the group of SLEDAI ≥ 5 had higher PSImax and ESImax value of global LV and basal segments. The absolute value of global longitudinal strain (GLS) had no difference between the group of active disease and inactive disease. Multivariate analysis demonstrated that PSS was independently associated with SLEDAI and diabetes mellitus. CONCLUSIONS: Detection of PSS and ESL enable to identify LV systolic impairment in SLE patients at an early stage.

Role of small ubiquitin-like modifier proteins-1 (SUMO-1) in regulating migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is featured by erosive cartilage and bone destruction. The enhancing aggressive property of fibroblast-like synoviocytes (FLSs) plays a critical role in this process. Small ubiquitin-like modifier (SUMO) proteins, including SUMO-1, SUMO-2, SUMO-3 and SUMO-4, participate in regulating many cellular events such as survival, migration and signal transduction in some cell lines. However, their roles in the pathogenesis of RA are not well established. Therefore, we evaluated the role of SUMO proteins in RA FLSs migration and invasion. We found that expression of both SUMO-1 and SUMO-2 was elevated in FLSs and synovial tissues (STs) from patients with RA. SUMO-1 suppression by small interference RNA (siRNA) reduced migration and invasion as well as MMP-1 and MMP-3 expression in RA FLSs. We also demonstrated that SUMO-1 regulated lamellipodium formation during cell migration. To explore further into molecular mechanisms, we evaluated the effect of SUMO-1 knockdown on the activation of Rac1/PAK1, a critical signaling pathway that controls cell motility. Our results indicated that SUMO-1-mediated SUMOylation controlled Rac1 activation and modulated downstream PAK1 activity. Inhibition of Rac1 or PAK1 also decreased migration and invasion of RA FLSs. Our findings suggest that SUMO-1 suppression could be protective against joint destruction in RA by inhibiting aggressive behavior of RA FLSs.

Piperlongumine inhibits the proliferation, migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

OBJECTIVES: Recent studies have indicated that piperlongumine (PLM) may exert anti-inflammatory effects. In the present study, we determined the effect of PLM on the proliferation, apoptosis, migration and invasion of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) (referred to herein as RA FLS). We further explored the mechanisms by which the studied compound inhibits the functions of RA FLS. METHODS: RA FLS viability and apoptosis were tested using MTT and Annexin V/PI assays, respectively. We performed an EDU assay to examine the proliferation of RA FLS. The migration and invasion of these cells were measured using a transwell chamber method and wound closure assay. The MMP-1, MMP-3, and MMP-13 levels in the culture supernatants of RA FLS were detected using a Luminex Assay kit. The intracellular ROS levels were detected using DCFH-DA. The expression levels of signal transduction proteins were measured using western blot. RESULTS: We found that PLM induced apoptosis in RA FLS at concentrations of 15 and 20 µM. The proliferation of RA FLS was downregulated by PLM at concentrations of 1, 5 and 10 µM. Migration and invasion of RA FLS were reduced by PLM at concentrations of 1, 5 and 10 µM. PLM also inhibited cytoskeletal reorganization in migrating RA FLS and decreased TNF-α-induced intracellular ROS production. Moreover, we demonstrated the inhibitory effect of PLM on activation of the p38, JNK, NF-κB and STAT3 pathways. CONCLUSIONS: Our findings suggest that PLM can inhibit proliferation, migration and invasion of RA FLS. Moreover, these data suggests that PLM might have therapeutic potential for the treatment of RA.

Efficiency of dose reduction strategy of etanercept in patients with axial spondyloarthritis.

OBJECTIVES: To evaluate the efficacy of different tapering or discontinuation strategies of etanercept in a cohort of axial spondyloarthritis from South China. METHODS: We performed a retrospective cohort study. Axial SpA patients who achieved clinical remission for at least 6 months after receiving a standard dose of etanercept therapy were enrolled. Different tapering or discontinuation strategies were compared. RESULTS: Altogether, 258 cases were enrolled. No differences were found in baseline characteristics among the three groups. Significantly more patients on discontinuation group (19%) than tapering group (5.4%, p<0.001) relapsed as early as 6 months. Almost all of the patients (103/107, 96.3%) in taper 25% group and more than 80% (71/88, 80.7%) of the patients in taper 50% group maintained low disease activity (LDA) or clinical remission during the first year. At the end of the 2-year follow-up, the percentage of patients maintaining LDA or remission were 28.6% (discontinuation), 55.7% (taper 50%), 84.1% (taper 25%), respectively. Activity indexes were significantly lower in taper 25% group compared to the other two groups. Patients in discontinuation group and tapering 50% group, with longer SpA duration were more likely to relapse, and remission>12 months before discontinuation/tapering helped to reduce relapse. CONCLUSIONS: It is feasible to slowly increase the dosing interval and transit to the lowest effective dosing interval for some patients in remission/LDA. Prolonging the time under remission before tapering help to improve the outcome. Tapering 25% of the etanercept dose every 3 months may be a pragmatic approach for more cost-effective use of the drug.

Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis.

Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis.

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

Prophylaxis of acute flares when initiating febuxostat for chronic gouty arthritis in a real-world clinical setting.

OBJECTIVE: Flare prophylaxis is recommended during urate-lowering therapy (ULT) despite lack of proven benefit especially when initiating febuxostat. We investigated if colchicine or steroids administration during initiation of febuxostat for chronic gouty arthritis reduces the frequency and/or severity of acute gout flares. METHODS: Patients with confirmed diagnosis of gout starting febuxostat were retrospectively studied. Frequency, severity, and length of flares were analyzed. Assessment of severity based on a visual analog scale (VAS). RESULTS: Two hundred and seventy-three patients were studied. The mean dose of colchicine and steroids was 0.53 ± 0.15 mg PO QD and 7.55 ± 1.30 mg prednisone equivalent PO QD; while the duration was 6.13 ± 1.14 and 6.20 ± 1.36 months, respectively. Subjects treated with colchicine and steroids suffered fewer total flares (0.30, 0.96 vs 2.47, p = .000), fewer flares from 0 to 3 months (0.26, 0.71 vs 1.72, p = .000), less severe flares assessed by VAS than those without prophylactic therapy (3.65, 3.49 vs 5.54, p = .000). Both total flares (p = .003) and flares from 0 to 3 months (p = .008) of the colchicine group were fewer than the steroids group. There were no significant differences in length of flares among groups (p = .815). Both colchicine and steroids were well tolerated. CONCLUSION: The use of colchicine or steroids prophylaxis reduces the frequency and severity of acute gout flares during initiation of febuxostat for chronic gouty arthritis. Colchicine is superior to steroids in flares prophylaxis. Prophylactic therapy with colchicine 0.5 mg PO QD or steroids 7.5 mg prednisone equivalent PO QD for 6 months is suggested.

Role of protein arginine methyltransferase 5 in inflammation and migration of fibroblast-like synoviocytes in rheumatoid arthritis.

To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up-regulated by stimulation with IL-1ß and TNF-α. Inhibition of PRMT5 by EPZ015666 and siRNA-mediated knockdown reduced IL-6 and IL-8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseß and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF-κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.

Indirubin inhibits the migration, invasion, and activation of fibroblast-like synoviocytes from rheumatoid arthritis patients.

OBJECTIVES: To evaluate the inhibition of indirubin in FLSs migration, invasion, activation, and proliferation in RA FLSs. METHODS: The levels of IL-6 and IL-8 in cultural supernatants were measured by ELISA. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction protein expression was measured by western blot. FLS proliferation was detected by Edu incorporation. F-actin was measured by immunofluorescence staining. RESULTS: We found that indirubin reduced migration, invasion, inflammation, and proliferation in RA FLSs. In addition, we demonstrated that indirubin inhibited lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of indirubin on PAK1 and MAPK activation. Our results indicated that indirubin inhibited the activity of PAK1 and MAPK. CONCLUSIONS: Our observations suggest that indirubin may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, activation, and proliferation.

Bromodomain and extra-terminal domain bromodomain inhibition prevents synovial inflammation via blocking IκB kinase-dependent NF-κB activation in rheumatoid fibroblast-like synoviocytes.

OBJECTIVE: To explore the roles of the bromodomain (Brd) and extra-terminal domain (BET) of chromatin adaptors in regulating synovial inflammation in RA. METHODS: Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissue from RA patients. A specific BET inhibitor, JQ1, and short hairpin RNA (shRNA) for Brd2 or Brd4 were used to evaluate the role of the BET Brd in inflammatory responses. Protein expression was measured by western blot or immunofluorescence staining. Nuclear factor kappa B (NF-κB) gene activity was detected by luciferase assay. The secretion and gene expression of cytokines and MMPs were evaluated by ELISA and real-time PCR, respectively. FLS proliferation was detected by BrdU incorporation. RESULTS: Four Brd proteins, including Brd2, Brd3, Brd4 and Brdt, were expressed in FLSs from patients with RA and OA; however, the expression of Brd2 and Brd4 was increased in RA compared with that in OA. Treatment with JQ1, Brd2 shRNA or Brd4 shRNA decreased the production of pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-8), MMPs expression (MMP-1, MMP-3 and MMP-13) and proliferation by RA FLSs. BET inhibition downregulated TNFα-induced NF-κB-dependent transcription and expression of the NF-κB target genes. JQ1 suppressed the phosphorylation of IκB kinaseß and IκBα, and nuclear translocation of p65. Intraperitoneal injection of JQ1 in mice with collagen-induced arthritis reduced synovial inflammation, joint destruction and serum levels of the anti-CII antibodies TNFα and IL-6. CONCLUSION: This study implicates BET Brds as important regulators of IκB kinase/NF-κB-mediated synovial inflammation of RA and identifies BET proteins as novel therapeutic targets in inflammatory arthritis.

Genetic markers in CYP2C19 and CYP2B6 for prediction of cyclophosphamide's 4-hydroxylation, efficacy and side effects in Chinese patients with systemic lupus erythematosus.

AIMS: The aim of the study was to investigate the combined impact of genetic polymorphisms in key pharmacokinetic genes on plasma concentrations and clinical outcomes of cyclophosphamide (CPA) in Chinese patients with systemic lupus erythematosus (SLE). METHODS: One hundred and eighty nine Chinese SLE patients treated with CPA induction therapy (200 mg, every other day) were recruited and adverse reactions were recorded. After 4 weeks induction therapy, 128 lupus nephritis (LN) patients continued to CPA maintenance therapy (200-600 mg week(-1)) for 6 months, and their clinical outcomes were recorded. Blood samples were collected for CYP2C19, CYP2B6, GST and PXR polymorphism analysis, as well as CPA and its active metabolite (4-hydroxycyclophosphamide (4-OH-CPA)) plasma concentration determination. RESULTS: Multiple linear regression analysis revealed that CYP2B6 -750 T > C (P < 0.001), -2320 T > C (P < 0.001), 15582C > T (P = 0.017), CYP2C19*2 (P < 0.001) and PXR 66034 T > C (P = 0.028) accounted for 47% of the variation in 4-OH-CPA plasma concentration. Among these variants, CYP2B6 -750 T > C and CYP2C19*2 were selected as the combination genetic marker because these two SNPs contributed the most to the inter-individual variability in 4-OH-CPA concentration, accounting for 23.6% and 21.5% of the variation, respectively. Extensive metabolizers (EMs) (CYP2B6 -750TT, CYP2C19*1*1) had significantly higher median 4-OH-CPA plasma concentrations (34.8, 11.0 and 6.6 ng ml(-1) for EMs, intermediate metabolizers (IMs) and poor metabolizers (PMs), P < 0.0001), higher risks of leukocytopenia (OR = 7.538, 95% CI 2.951, 19.256, P < 0.0001) and gastrointestinal toxicity (OR = 7.579, 95% CI 2.934, 19.578, P < 0.0001), as well as shorter median time to achieve complete remission (13.2, 18.3 and 23.3 weeks for EMs, IMs and PMs, respectively, P = 0.026) in LN patients than PMs (CYP2B6 -750CC, CYP2C19*2*2) and IMs. CONCLUSIONS: Our findings have indicated that genetic markers of drug metabolizing enzymes could predict the 4-hydroxylation, adverse reactions and clinical efficacy of CPA. This is a necessary first step towards building clinical tools that will help assess clinical benefit and risk before undergoing CPA treatment in Chinese SLE patients.

Factors Associated with Erosive Arthritis in Rheumatoid Arthritis and Other Connective Tissue Diseases: A Retrospective Study From a Southern Chinese Population.

OBJECTIVE: To investigate the clinical predictors of erosive arthritis (EA) in patients with rheumatoid arthritis (RA) and other connective tissue diseases. METHODS: Four hundred and one consecutive patients with newly diagnosed RA between January 2010 and January 2013 were enrolled in the study. During the study period, 729 consecutive patients with non-RA connective tissue diseases were also included, and a cross-sectional study was performed. Medical records were reviewed. Only those patients with data for 2 years were considered in the analysis (338). RESULTS: Erosive arthritis was noted in 60.4% (204 /338) of patients with RA and occurred early in RA. The multivariate logistic regression analysis indicated that rheumatoid nodules, anemia, and positive anticyclic citrullinated peptide antibody (ACPA) were strongly associated factors for the occurrence of EA in RA patients. Erosive arthritis was also noted in 1.5% of patients with SLE, 5.8% of patients with primary Sjögren syndrome, and 9.1% (3/33) of patients with systemic sclerosis. When compared with patients without EA, high level and prominently higher positive rate of ACPA was found in these patients with EA. On receiver operating characteristic curve analysis, ACPA exhibited a maximum sensitivity with a cutoff value of 1.6 U/mL and 0.6 U/mL for RA and SLE patients, respectively. CONCLUSION: Erosive arthritis had a high prevalence in Chinese RA patients and occurred early. Anemia, rheumatoid nodules, and ACPA were associated with EA in RA. Erosive arthritis also could be detected in SLE, primary Sjögren syndrome, and systemic sclerosis. Anticyclic citrullinated peptide antibodies were also associated with EA in these diseases. Intensive monitoring for erosions was recommended for RA patients with a cutoff of ACPA greater than 1.6 U/mL and greater than 0.6 U/mL for SLE patients.

Early-stage hip involvement in patients with ankylosing spondylitis: A Chinese study based on magnetic resonance imaging.

OBJECTIVES: To evaluate the clinical characteristics and identify potential factors of the early-stage hip involvement in patients with ankylosing spondylitis (AS) based on the magnetic resonance imaging (MRI). METHODS: A cross-sectional retrospective study of 655 consecutive patients was performed. Patients with hip pain or limited hip function but lacking definitive evidence of hip involvement on radiography underwent hip MRI. Based on the results of the imaging tests, the patients were classified into three categories: (1) no hip involvement; (2) early-stage hip involvement according to MRI but not radiography; (3) advanced-stage hip involvement according to a Bath Ankylosing Spondylitis Radiology Index-hip score ≥2. RESULTS: One hundred and sixty-eight patients had early-stage hip involvement and 103 patients had advanced-stage hip involvement. Multivariate logistic regression analysis indicated that younger age at onset, worse BASMI score, and more active inflammation in the sacroiliac joints were associated with the occurrence of early-stage hip involvement. CONCLUSION: Negative plain radiography results cannot be used to rule out hip involvement. MRI was superior to radiography for the detection of early-stage hip involvement. Susceptible AS patients with symptoms or risk factors for hip involvement should undergo hip MRI to test for lesions in the early stage.

Inhibitory effects of niclosamide on inflammation and migration of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

OBJECTIVES: This study evaluated the anti-inflammatory effect of niclosamide in tumor necrosis factor (TNF)-α-stimulated human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and inhibitory effects on migration and invasion in RA FLS and investigated the signal mechanism, and further explored the treatment activity of niclosamide on collagen-induced arthritis (CIA). METHODS: The levels of interleukin (IL)-1ß, IL-6, IL-8, IL-10,IL-17A and interferon (IFN)-γ in cultural supernatants were measured by multiplex cytokine assay kits. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction proteins expression was measured by western blot. The in vivo suppressive effects of niclosamide were elucidated on CIA in a mouse model. RESULTS: Niclosamide reduced the secretion of IL-1ß, IL-6, IL-8, IL-17A and IFN-γ from TNF-α-induced RA FLS in a dose-dependent manner. Niclosamide inhibits FBS-induced migration and invasion and exhibits F-actin alterations in RA FLS. Niclosamide decreased the phosphorylation of c-Jun N-terminal kinase and ERK in TNF-α-stimulated RA FLS and blocked TNF-α-induced IKK, IκBα phosphorylation and translocation of p65. Niclosamide treatments reduced the severity of CIA model. CONCLUSIONS: Our data suggest for the first time that niclosamide posses the anti-inflammatory effect in RA both in vitro and in vivo.

Niclosamide inhibits the inflammatory and angiogenic activation of human umbilical vein endothelial cells.

OBJECTIVE: Niclosamide is known to have anti-cancer and anti-inflammatory activities; however, its therapeutic mechanism has not been defined. In this study, to explain the therapeutic mechanism of niclosamide, we examined the effect of niclosamide on endothelial cell activation,leukocyte integration, proliferation, migration and angiogenesis in vitro. METHODS: Endothelia-leukocyte adhesion assays were used to assess primary cultures of human umbilical vein endothelial cells' (HUVECs) activation following TNF-α treatment. Each step of angiogenesis was evaluatedin vitro, including endothelial cell proliferation, migration and tube formation. Proliferation was examined using EdU assays, while wound migration assays and transwell assays were used to evaluate cell migration; cord like structure formation assays on Matrigel were used to assess tube formation. In vivo matrigel plug assay was used to assess angiogenesis. The protein expression was measured using western blot. RESULTS: Niclosamide reduced the adhesion of human monocyte cells to HUVECs. Niclosamide also reduced protein expression of VCAM-1 and ICAM1 in HUVECs.Niclosamide significantly inhibited HUVEC proliferation,migration and cord-like structure formation. Niclosamide also suppresses VEGF-induced angiogenesis in vivo.Niclosamide attenuated IKK-mediated activation of NF-κB pathway in TNFα-induced endothelial cells. Niclosamide also suppresses VEGF-induced endothelial VEGFR2 activation and downstream P-AKT, P-mTOR and P-p70S6K. CONCLUSIONS: Niclosamide exerted a potent effect on HUVECs activation, suggesting that it might function via an endothelia-based mechanism in the treatment of various diseases, including rheumatoid arthritis and cancer.

Increased phosphorylation of ezrin is associated with the migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

OBJECTIVE: Increasing evidence indicates that the cytoskeletal protein ezrin may play a critical role in cell motility. This study aims to investigate the role of ezrin in regulating the migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with RA. METHODS: Synovial tissues were obtained from 12 patients with RA and 6 with OA, and then FLSs were separated from synovial tissues. The expression of ezrin and phosphorylated ezrin (p-ezrin) was examined by Western blotting or IF staining. A specific inhibitor of ezrin phosphorylation and small interference RNA-mediated ezrin knockdown were used to inhibit the phosphorylation of ezrin. Migration and invasion of FLSs in vitro were measured by the Boyden chamber assay. RESULTS: Increased expression of p-ezrin protein was found in synovial tissue and FLSs in patients with RA compared with patients with OA. Stimulation with TNF-α and IL-1ß increased ezrin phosphorylation in RA FLSs. Inhibition of p-ezrin protein by a specific inhibitor of phosphorylation of ezrin and small interfering RNA-mediated knockdown reduced in vitro migration and invasion, as well as actin stress fibre formation in RA FLS. Furthermore, rho kinase and p38 mitogen-activated protein kinase (MAPK) signal pathways were involved in the phosphorylation of ezrin and invasion of RA FLSs. CONCLUSION: Increased expression of p-ezrin may contribute to aberrant aggressive behaviours of RA FLSs, which are mediated by rho kinase and the p38 MAPK pathway. This suggests a novel strategy targeting phosphorylation of ezrin to prevent synovial invasiveness and joint destruction in RA.