Search

CD137-targeting immune therapy, which activates anti-tumor T effector cell responses, seems to be an attractive concept in clinical oncology. Recent evidence has demonstrated that tumor cells besides T cells and antigen-presenting cells are able to express CD137 and CD137L. Here we aimed to identify CD137/CD137L expression in established colon cancer cell lines and primary tumors (UICC stages I-IV) from patients with documented long-term follow-up. CD137/CD137L expression was highly upregulated in early to late-stage tumors while the inverse was observed in patient-derived peripheral blood mononuclear cells. High CD137L expression within primary tumors was mediated by tumor cells and significantly correlated with the occurrence of distant metastases and shortened survival in advanced stages of disease (UICC stage IV). Interestingly, induced tumor cell signaling via CD137L on its surface in vitro resulted in dual effects: (i) reduced tumor cell proliferation suggesting inhibitory signaling in all investigated cancers and (ii) increased epithelial-to-mesenchymal transition signaling events. Taken together CD137/CD137L expression was stage-dependently upregulated with shortened survival in patients with highly CD137L-expressing tumors. Our clinical and experimental data suggest that colon cancer cells predominantly express CD137L and thereby have negative impact on overall survival through a process of reverse signaling. Beside agonistic CD137 antibody therapy to foster T effector cell responses, CD137L-mediated intervention strategies may become instrumental to circumvent relapsed tumor growth through induced epithelial-to-mesenchymal transition and consecutive metastases formation.

Background Cardiovascular fibrosis is a major contributor to cardiovascular disease, the primary cause of death in patients with chronic kidney disease (CKD). We previously reported expression of endogenous Klotho in human arteries, and that CKD is a state of Klotho deficiency, resulting in vascular calcification, but myocardial expression of Klotho is poorly understood. This study aimed to further clarify endogenous Klotho's functional roles in cardiac fibrosis in patients with underlying CKD. Methods and results Human atrial appendage specimens were collected during cardiac surgery from individuals with or without CKD. Cardiac fibrosis was quantified using trichrome staining. For endogenous Klotho functional studies, primary human cardiomyocytes (HCMs) were treated with uremic serum from CKD patients or recombinant human TGF-β1. The effects of endogenous Klotho in HCMs were studied using Klotho-siRNA and Klotho-plasmid transfection. Both gene and protein expression of endogenous Klotho are found in human heart, but decreased Klotho expression is clearly associated with the degree of cardiac fibrosis in CKD patients. Moreover, we show that endogenous Klotho is expressed by HCMs and cardiac fibroblasts (HCFs) but that HCM expression is suppressed by uremic serum or TGF-β1. Klotho knockdown or overexpression aggravates or mitigates TGF-β1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-β1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs. Conclusions Our data from analysis of human hearts as well as functional in vitro studies strongly suggests that the loss of cardiac endogenous Klotho in CKD patients, specifically in cardiomyocytes, facilitates intensified TGF-β1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/β-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.

Background Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) play a crucial role in RA through producing inflammatory cytokines and proteases which could cause cartilage destruction. We showed previously that elevated expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in RA synovium correlated significantly with the severity of synovitis and the number of infiltrated inflammatory cells. The aims of this study are to detect the roles of TRAF6 in RA-FLSs. Methods Synovium were collected by closed needle biopsy from inflamed knees of active RA patients, and FLSs were isolated by modified tissue culture method. Expression of TRAF6 and CD55 in RA synivium was tested by double immunofluorescence (IF) staining. TRAF6 in RA-FLSs was inhibited using Lentiviral-TRAF6-shRNA transfection. Real-time PCR and ELISA were used to detect the mRNA expression and secretion of matrix metalloproteinase (MMP) and pro-inflammatory cytokines. Cell Counting Kit-8 was used to detect cell proliferation, flow cytometry was used to detect cell cycle, and Annexin V assay was used to detect cell apoptosis. Results We showed that in the intimal and subintimal area of RA synovium, TRAF6 was expressed obviously not only in CD55+ cells, but also in some other CD55− cells. TRAF6 expression in RA-FLSs was suppressed effectively by Lentiviral-TRAF6-shRNA transfection. Inhibition of TRAF6 in RA-FLSs mitigated the mRNA levels and secretion of pro-inflammatory cytokines and MMPs, such as IL-1β, IL-8, IL-6, TNF-α, MMP-13, and MMP-3. In addition, it decreased the proliferation of RA-FLSs, blocked RA-FLSs in G0/G1-phase, and inhibited the cells to go into S-phase and G2/M-phase, but not facilitated apoptosis of RA-FLSs. Conclusion TRAF6 plays direct roles in the pro-inflammatory effects and proliferation of RA-FLSs. TRAF6 may serve as a potential treatment target in RA.

Objective.To determine serum glucose-6-phosphate isomerase (GPI) concentrations in patients with rheumatoid arthritis (RA), and to test whether they correlate with objective measures of disease activity.Methods.Sera from 116 patients with RA, 69 patients with non-RA rheumatic diseases, and 101 healthy controls were analyzed. Levels of soluble serum GPI were measured by ELISA. Histological disease activity was determined with the synovitis score in synovial needle biopsies from 58 of the 116 patients with RA. Thirty-one of the 58 synovium samples were stained for CD68, CD3, CD20, CD38, CD79a, and CD34 by immunohistochemistry. Demographic data were collected, as well as serological and clinical variables that indicate RA disease activity, for Spearman correlation analysis.Results.Serum GPI level correlated positively with the synovitis score (r = 0.278, p = 0.034). Significantly higher soluble GPI levels were detected in the RA sera compared with sera from healthy controls and the non-RA disease controls (2.25 ± 2.82 vs 0.03 ± 0.05 and 0.19 ± 0.57 μg/ml, respectively; p < 0.0001). The rate of serum GPI positivity was significantly higher in the RA patients than in the non-RA disease controls (64.7% vs 10.1%; p < 0.0001). Spearman analysis showed no significant correlation between serum GPI level and Disease Activity Score in 28 joints at baseline. After initiation of antirheumatic treatments, GPI levels decreased significantly (2.81 ± 3.12 vs 1.44 ± 2.09 μg/ml; p = 0.016), paralleling improvement of the disease activity indices.Conclusion.Elevated serum GPI may be involved in the synovitis of RA and may prove useful as a serum marker for disease activity of RA.

Immune-complex (IC) mediated glomerulonephritis (GN) is a common cause of chronic kidney disease associated with increased levels of tumor necrosis factor (TNF)-alpha in renal cells. TNF-alpha signaling pathways involve complicated interactions between multiple proteins including TNF-receptor-associated factor (TRAF)-2. We have previously found markedly up-regulated expression of TRAF-2 in renal tissues from IC mediated lupus nephritis patients. Here we investigated the effect of TRAF-2 on inflammatory response in rat mesangial cells (MCs). The results showed that treatment with soluble aggregated IgG (AIgG) resulted in a time- and dose-dependent increase in the expression of interleukin (IL)-1beta and IL-6. Significant cell proliferation was also observed after the treatment with soluble AIgG. Knockdown TRAF-2 by siRNA significantly suppressed soluble AIgG induced up-regulation of TRAF-2, IL-1beta, and IL-6. Meanwhile the cell proliferation was inhibited and apoptotic cells were increased. It was concluded that TRAF-2 could induce the proinflammatory and proliferative effects of soluble AIgG on rat MCs. Thus, TRAF-2 may represent a future target for therapy of IC mediated GN.

Tumor necrosis factor (TNF)-alpha is not just a proinflammatory cytokine. It has also been proposed to be an immunoregulatory molecule that can alter the balance of T regulatory cells. Anti-TNF-alpha therapies have been provided clinical benefit to many patients and introduced for treating moderate to severe rheumatoid arthritis, Crohn's disease, and other chronic inflammatory disorders. However, their use also is accompanied by new or aggravated forms of autoimmunity, such as formation of autoantibodies, including antinuclear antibodies (ANAs), antidouble-stranded DNA (dsDNA) antibodies, and anticardiolipin antibodies (ACL). Systemic lupus erythematosus (SLE) is a disease with autoimmune disturbance and inflammatory damage. The role of TNF-alpha in human SLE is controversial. Here we review the role of TNF-alpha in the pathophysiological processes of SLE and the likely effects of blocking TNF-alpha in treatment of SLE.

The role of tumour necrosis factor (TNF-alpha) signalling adapters in lupus nephritis (LN) is poorly understood. This study investigated renal expression of TNF-alpha and TNF signalling adapter proteins, including TNF receptor-associated death domain protein (TRADD), receptor-interacting protein (RIP) and TNF receptor-associated factor-2 (TRAF-2) in patients with LN. The renal expression of proliferating cell nuclear antigen (PCNA) and CD68 was also measured. The study showed that glomerular and tubular expression of TNF-alpha, TRADD, RIP and TRAF-2 was significantly up-regulated in class III and IV LN in which the intense staining was observed on the crescents, proximal and distal tubules and interstitial mononuclear cells. The number of PCNA-positive cells and CD68-positive cells (macrophages) was increased obviously in class III and IV LN. There was a correlation between the expression levels of TNF-alpha, TRADD, RIP, TRAF-2 and the number of PCNA-positive or CD68-positive cells and active index of renal pathology. These findings suggest that TNF-alpha and TNF-alpha adapters in patients with LN play a role in immunopathogenic injury via transmitting abnormal cell proliferating and proinflammatory signals. The findings have provided further insights into the role of TNF-alpha and its adapter proteins in the pathogenesis of LN and have important therapeutic implications.

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine. Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease. However, until now, the expression and pathophysiological role of TNF adapters in SLE have been poorly understood. This study aims to investigate the expression of mRNA for the TNF adapter proteins including TNF receptor-associated death domain (TRADD) protein, Fas-associated death domain (FADD) protein, receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) from patients with SLE and to explore the relationship between the expression of these adapters and the SLE disease activity. PBMCs were isolated from the venous blood of 51 SLE patients and 17 healthy subjects. The expression of mRNA for TNF adapter molecules such as TRADD, FADD, RIP-1, and TRAF-2 in PBMCs were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT–PCR) and quantitative real-time RT–PCR. There were constitutive expressions of mRNA for TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from healthy subjects. The expression of mRNA for all the adapter molecules significantly decreased in PBMCs from patients with SLE, which were 0.38-, 0.69-, 0.59-, and 0.55-fold, respectively, compared to those of control subjects (P < 0.05). The expression of Caspase 3 was significantly increased in SLE patients (P < 0.01); however, the expression of IL-1beta was not significantly different between SLE and control subjects. The expression of TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from patients with SLE were negatively correlated with SLEDAI, the correlation coefficient of which was −0.285, −0.280, −0.307, and −0.298, respectively (P < 0.05). The expression of mRNA for TNF adapter molecules TRADD, FADD, RIP-1, and TRAF-2 decreased significantly in PBMCs from patients with SLE, and the expression of these adapters were negatively correlated with the SLE activity index. These abnormalities may be involved in the immunopathogenic injury mediated by the aberration TNF-alpha signaling pathway in SLE.

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Objective.The efficacy of B cell depletion in the treatment of patients with rheumatoid arthritis (RA) has revitalized interest in the pathogenic role(s) of B cells in RA. We evaluated the distribution of synovial B lineage cells and their correlation with histologic disease activity and joint destruction in RA.Methods.Synovial tissue samples were obtained by closed-needle biopsy from 69 Chinese patients with active RA, from 14 patients with osteoarthritis (OA), and from 15 with orthopedic arthropathies (OrthA) as disease controls. Serial tissue sections were stained immunohistochemically for CD79a (pro-B cell to plasma cell), CD20 (B cells), CD38 (plasma cells), CD21 (follicular dendritic cells), CD68 (macrophages), CD3 (T cells), and CD34 (endothelial cells). Densities of positive-staining cells were determined and correlated with histologic disease activity (Krenn 3-component synovitis score) and radiographic joint destruction (Sharp score).Results.Mean sublining CD79a-positive cell density was significantly higher in RA than in OA (p Conclusion.Synovial CD79a-positive B cells may be a helpful biomarker for histologic disease activity in RA and may be involved in the pathogenesis of joint destruction in RA.

Objective.To investigate the expression of tumor necrosis factor receptors (TNFR1 and TNFR2) and adapter proteins (TRADD, RIP, and TRAF2) in peripheral blood mononuclear cell (PBMC) subsets from patients with systemic lupus erythematosus (SLE).Methods.PBMC were isolated from 45 SLE patients and 25 controls, and stained with labeled antibodies that enabled identification of various T cell, B cell, and monocyte subpopulations. Expression of TNF-related signaling molecules was measured by staining with labeled antibodies either directly or following fixation and permeabilization. Apoptosis was quantified using an anti-active caspase 3 antibody. RNA expression of TNF-related signaling molecules was assessed by quantitative RT-PCR and serum levels of TNF-α by ELISA.Results.SLE patients had increased levels of TNFR1, TNFR2, and TRAF2, together with decreased levels of RIP, on various B, CD4+ T, and CD8+ T cell subsets as compared to controls. This altered expression was seen in both naive and memory subpopulations, and reflected altered staining of the whole population rather than a subset of cells that were activated. The levels of these molecules were not significantly correlated with serum TNF-α levels or their RNA expression in whole peripheral blood. TNFR1 and TNFR2 expression was negatively correlated with disease activity. There was no association between the proportion of apoptotic cells in any of the subpopulations and serum TNF-α levels or expression of TNF-related signaling molecules.Conclusion.Patients with SLE had altered expression of TNF-related signaling molecules, suggesting that there may be an imbalance in TNF-α signaling favoring cellular activation as opposed to proapoptotic pathways.

Tumor necrosis factor (TNF)-α is not just a proinflammatory cytokine. It has also been proposed to be an immunoregulatory molecule that can alter the balance of T regulatory cells. Anti-TNF-α therapies have been provided clinical benefit to many patients and introduced for treating moderate to severe rheumatoid arthritis, Crohn's disease, and other chronic inflammatory disorders. However, their use also is accompanied by new or aggravated forms of autoimmunity, such as formation of autoantibodies, including antinuclear antibodies (ANAs), antidouble-stranded DNA (dsDNA) antibodies, and anticardiolipin antibodies (ACL). Systemic lupus erythematosus (SLE) is a disease with autoimmune disturbance and inflammatory damage. The role of TNF-α in human SLE is controversial. Here we review the role of TNF-α in the pathophysiological processes of SLE and the likely effects of blocking TNF-α in treatment of SLE.

To investigate the mRNA expressions of the TNF adapter proteins, including TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) of lupus nephritis (LN) patients of various TCM asthenia syndromes. Methods Fifty-one inpatients with LN were differentiated according to TCM syndrome differentiation, 13 cases of yin-deficiency with inner heat syndrome (A); 26 cases of both qi-yin deficiency syndrome (B), 12 cases of Pi-Shen yang-deficiency syndrome (C). Peripheral venous blood samples from the 51 LN patients and 17 healthy subjects were collected to separate PBMCs. The mRNA expressions of TNF adapter molecules (TRADD, FADD, RIP-1 and TRAF-2), as well as Caspase-3 and interleukin-1beta (IL-1beta) were analyzed by quantitative real-time PCR and the differences among them were compared.(1) As compared with the healthy subjects, expression of TRADD mRNA in patients of syndrome A, B and C was lowered to 0.54, 0.32, and 0.38-fold, respectively (P0.05, P0.01), showing insignificant difference among the three syndromes; (2) FADD mRNA lowered to 0.79, 0.62, and 0.72-fold respectively, only with significance shown in syndrome B (P0.05); (3) RIP-1 mRNA lowered to 0.79, 0.50, and 0.60-fold respectively with significance shown in syndrome B and C (P0.01, P0.05), and insignificant difference was shown among the three syndromes; (4) TRAF-2 lowered to 0.70, 0.52, and 0.50-fold respectively (P0.01, P0.01, P = 0.07), significance shown in syndrome B and C (P0.01), but with insignificant difference among the three; (5) Caspase-3 elevated in all patients of the three syndromes (all P0.01); (6) IL-1beta in syndrome A was apparently lower ed to the normal range and also lower than that in the other two syndromes (both P0.05).Expressions of TRADD, FADD, RIP-1 and TRAF-2 mRNA decreased in all the patients of various TCM asthenia syndromes, the decrement in patients of syndrome B and C was lesser than that in syndrome A. These abnormal low expressions of signal proteins might be the substantial bases for asthenia syndromes of LN patients, and the apoptotic signal mediated by them may involve in the formation of asthenia syndrome in LN.

To investigate the mRNA expression of the tumor necrosis factor (TNF)-adapter proteins, TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), and to explore the relationship between the expression of these adaptors and the SLE disease activity.PBMC were isolated from venous blood of 51 SLE patients and 17 healthy subjects. The mRNA expression of TRADD, FADD, RIP-1, TRAF-2, Caspase 3, and interleukin (IL)-1beta were analyzed by quantitative real time RT-PCR. Disease activity was measured using the SLE Disease Activity Index (SLEDAI).All healthy subjects showed mRNA expressions of TRADD, FADD, RIP-1, and TRAF-2. The mRNA expression levels of TRADD, FADD, RIP-1, and TRAF- in the PBMC from the patients were 0.38, 0.69, 0.59, and 0.55 tomes that from the control subjects (all P0.05). The expression levels of these 4 adapters of the SLE patients with the SLEDAI/= 10 were significantly lower than those of the SLE patients with the SLEDAI10 (all P0.05). The Caspase3 mRNA expression of the SLE patients was significantly higher than that of the healthy controls (P0.01); however, the IL-1beta mRNA expression was not significantly different between the SLE and control subjects. The mRNA expression levels of TRADD, FADD, RIP-1 and TRAF-2 in the PBMC from the SLE patients were all negatively correlated with the SLE activity index with the coefficient correlation of -0.285, -0.280, -0.307, and -0.298 respectively (all P0.05).The mRNA expression levels of the TNF adapter molecules, such as TRADD, FADD, RIP-1, and TRAF-2, decrease significantly in the PBMC from the SLE patients, and are negatively correlated with the SLE activity index. These abnormalities may participate in the immunopathogenic injury mediated by the aberration TNFalpha signaling pathway in SLE.

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis which leads to bone destruction. Matrix metalloproteinase (MMP) 3 can activate other MMPs, degenerate extracellular components and aggravate inflammation. Previous studies have reported correlation of serum MMP-3 and clinical disease activity in RA. However, little was known about the relationship between MMP-3 and histological synovitis. Objectives To explore the correlation of serum and synovial MMP-3 and histological synovitis in RA. Methods Sixty-two patients with active RA were recruited and their serum MMP-3 was detected by ELISA. Synovial tissues were obtained from RA patients as well as 12 OA and 8 orthopedic arthropathies (Orth.A) patients. Serial tissue sections were stained with H&E and immunohistochemically for MMP-3, CD3, CD20, CD38, CD68, CD15 and CD34. Krenn9s synovitis score was assessed semi-quantitatively and the density of positive-staining cells were quantitatively determined. Results (1) In RA synovium, MMP-3 expressed strongly in the endochylema of lining cells (both macrophage-like and fibroblast-like synoviocytes) with absence in the sublining area and the percentage of MMP-3+ lining cells (median 47%, IQR 39–52%) was significantly higher than that in OA (median 20%, IQR 17–24%, P 4, n=27) and low grade (≤4, n=35) synovitis. The percentage of MMP-3+ lining cells in patients with high grade synovitis was significantly higher than that in patients with low grade synovitis (median 51%, IQR 47–56% vs median 42%, IQR 36–49%, P Conclusions Our results implied that MMP-3 is involved in synovitis and serum MMP-3 may be a helpful biomarker of histological synovitis. Acknowledgements This work was supported by Chinese National Natural Science Research Grant (grant no. 81373183 and 81001334), Specialized Research Fund for the Doctoral Program of Higher Education (grant no.20130171110075) and Guangdong Natural Science Foundation (grant no.S2013010014396). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3622

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis, resulting in joint destruction and disability. Matrix metalloproteinase (MMP) 3 plays essential role in the degradation of extracellular components and cartilage. Several studies reported that serum MMP-3 could predict progression of joint damage in RA. However, there were seldom reports about dynamic change of serum MMP-3 and its relationship with radiographic progression in RA. Objectives To monitor serum MMP-3 and explore its correlation with radiographic progression in RA. Methods Sixty-eight patients with active RA who fulfilled the 1987 revised criteria of ACR were recruited and were divided into erosive and non-erosive group according to 2013 EULAR definition of erosive disease in hand radiographs. All patients were followed up at regular interval (1, 3, 6, and 12 months). Serum MMP-3 was detected by ELISA and clinical data was collected simultaneously at baseline and each visit. X-ray assessment of hand/wrist was repeated at 12 month visit and radiographic progression was defined as the change of total Sharp score more than 0.5 from baseline to one year. Results (1) Baseline serum MMP-3 in erosive RA patients (n=31, median 384 ng/ml, IQR 218–589 ng/ml) was significantly higher than that in non-erosive RA patients (n=37, median 204 ng/ml, IQR 91–338 ng/ml, P=0.004). Spearman9s rank order correlation showed significant correlations between baseline serum MMP-3 with space narrow score (r=0.349), erosion score (r=0.397) or total Sharp score (r=0.371, all P Conclusions Serum MMP-3 may be a helpful diagnostic biomarker of erosive disease and continually elevated serum MMP-3 may be a significant predictor for radiographic progression in RA. Acknowledgements This work was supported by Chinese National Natural Science Research Grant (grant no. 81373183 and 81001334), Specialized Research Fund for the Doctoral Program of Higher Education (grant no.20130171110075) and Guangdong Natural Science Foundation (grant no.S2013010014396). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2434

Background Approximate 7.2% Chinese have been suffered from chronic hepatitis B virus (HBV) infection manifesting positive HBsAg. Our previous study showed the positive rate of HBsAg in Chinese rheumatoid arthritis (RA) patients was 11.3% (2013, Clin Rheumatol). Screening of HBV infection prior to immunosuppressive therapy, monitoring and antiviral prophylaxis during DMARDs treatment are recommended for HBsAg(+) RA patients by ACR 2008/2012 recommendations. However, little is known about the real-world clinical practice of Chinese physicians. Objectives To investigate Chinese physicians9 awareness of HBV infection in RA patients and to identify the important targets of education. Methods A cross-section survey of 100 physicians who treated RA patients in daily clinic was conducted with a modified ACR questionnaire (2010, Arthritis Care & Research). The questionnaire was composed of demographic data and 10 questions concerning essential areas of screening HBV infection, monitoring and antiviral prophylaxis (or therapy) during immunosuppressive therapy for RA patients. Each question has 4-7 options for choice. The questionnaires with ≥8 answered items were regarded as valid for statistical analysis. Results 1. 100 physicians were recruited for the survey and 92 questionnaires with ≥8 answered items were valid for statistical analysis. Among them, 73% were rheumatologist, 27% were non-rheumatologists including internal medicine physicians (14%), orthopedists (8%), traditional Chinese medical physicians (2%) and other specialists (3%). The mean working seniority was 11.3 years (range, 1-32) and 73% physicians were consulted more than 5 RA patients per month. 2. Twenty-three percentage of physicians reported no routine screening for HBV infection before immunosuppressive therapy for outpatients with RA, comparing 92% for hospitalized RA patients. During immunosuppressive therapy for HBsAg(+) RA patients, 36% physicians reported no regular monitoring of HBV DNA and 12% reported no regular monitoring of aminotransferases. Twenty-four percentage of physicians considered antiviral prophylaxis for HBsAg(+) RA patients and regarding to the detailed indications of antiviral prophylaxis, 62% (57/92) physicians considered high HBV load, 39% (36/92) considered elevated aminotransferases, and 32% (29/92) considered both HBsAg(+) and HBeAg(+). 3. Eighty-eight percentage of physicians had clinical experience of antiviral prophylaxis or therapy for RA patients. Among them, 62% prescribed antiviral drug before or at the initiation of immunosuppressive therapy and 38% prescribed antiviral drug after HBV reactivation. Conclusions Our results showed that Chinese physicians have poor awareness of HBV infection for RA patients. The further CME should emphasize the routine screening for HBV infection especially in RA outpatients, regular monitoring of HBV DNA for HBsAg(+) RA patients, and antiviral prophylaxis considered to all HBsAg(+) RA patients with immunosuppressive treatment. Acknowledgements This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education (no.20130171110075) and Natural Science Foundation of Guangdong Province, China (no.S2013010014396) to L. Dai; Medical Scientific Research Foundation of Guangdong Province, China (no.A2013201) to Y-Q. Mo; Chinese National Natural Science Research Grant (Grant no. 81373183 and 81001334) to L-J. Zhu. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4022

Background Rheumatoid arthritis (RA) is a heterogeneous disease and subtyping of RA may help optimal therapeutic strategies and outcome prediction. However, there has been no available satisfying classification method till now. Synovium is the target tissue involved in RA and it is potential to classify RA patients according to synovitis type. Recently, normal synovium was reported to be well-defined into areolar, fibrous and fatty types (Kelley’s Textbook of Rheumatology, 8 th edition). Here we set up an innovative histological classification of rheumatoid synovitis. Objectives To explore the relationship of histological classification of rheumatoid synovitis with clinical disease activity and joint destruction. Methods Synovial tissue was obtained by needle biopsy from inflamed knees of 152 consecutive patients with active RA and stained with HE (2) Fibrous (Fi) synovitis: mainly composed of dense connective tissue or fibrous tissue, with poorly vascularized structure; (3) Fatty (Fa) synovitis: abundant mature adipose tissue with little collagen deposition. Kruskal-Wallis ranks test was used to compare the difference of clinical characteristics among three groups. Results (1) Among 152 RA patients, 128 (84%) were female, median age was 52 years (range 23~80) and median disease duration was 42 months (range 3~480). (2) As shown in Figure 1, 27 (18%) RA patients had A type synovitis, 98 (64%) had Fi type and 18 (12%) had Fa type; while 9 (6%) patients had more than one types (7 patients had A+Fa type; 2 patients had Fi+Fa type). (3) 143 patients with unique synovitis type were divided into three groups. There was significant difference of disease duration, CRP, ESR, DAS28-crp, DAS28-esr, SDAI, RF among three groups (all p p 2 =7.619, p =0.006). Disease duration in Fa group was significantly shorter than that in Fi group (χ 2 =9.729, p =0.002) or A group (χ 2 =11.862, p =0.001). (4) Sharp narrowing score, erosive score and total score showed significant difference among three groups (all p p p Image/graph Conclusions Our study proposed an innovative histological classification of rheumatoid synovitis based on the major components of synovial subintima and suggested higher clinical disease activity in RA patients with Fi type syovitis, and severer joint destruction in patients with A type synovitis. Acknowledgements This work was supported by the Chinese National Natural Science Research Grant (grant no. 30972742 and 81001334), the Natural Science Research Grant of Guangdong Province, China (grant no. 9151008901000130 and 10451008901004542) and the Fundamental Research Funds for the Central Universities (no. 10ykpy19). Disclosure of Interest None Declared

Background Hepatitis B virus (HBV) reactivation, which refers to the abrupt rise in HBV replication in patients with inactive or resolved hepatitis B and is triggered typically by immunosuppressive chemotherapy in patients with cancer, can also occur in patients with RA undergoing DMARDs therapy, especially in endemic areas of HBV infection such as China. However, most related publications until now are based on case reports or case series. Objectives To investigate the clinical features of HBV reactivation associated with non-biological DMARDs therapy in a cohort of Chinese RA patients. Methods The follow-up records of 18 Chinese RA patients with positive serum HBsAg treated with non-biological DMARDs from March 2006 to September 2011 were analyzed. A multiple regression model was developed by stepwise method to screen the risk factor(s) for HBV reactivation from gender, age, non-biological DMARDs including glucocorticoid, MTX, leflunomide (LEF), HCQ, SSZ,and preventive antiviral therapy. Results 1. Among 18 patients, 15 (83%) were females and mean age was (47±12) years, with 2 to 61 months’ follow-up. Baseline load of HBV-DNA was determined in 11 patients and four (36%) was positive (≥10 3 copies/mL). Transaminases and bilirubins were normal before DMARDs therapy in all patients. All patients took combination therapy of non-biological DMARDs without biologic agents during follow-up. Glucocorticoid (equivalent to prednisone 10mg/d, except one overlapped with lupus and one complicated with rheumatoid vasculitis) was taken in 13 patients, and MTX 10∼20mg/week in sixteen, LEF 10∼20mg/d in ten, HCQ 0.2∼0.4g/d in five and SSZ 1.5∼2g/d in four. 2. Seven of 18 patients (39%) developed HBV reactivation 4 to 38 months after non-biological DMARDs therapy, of whom six cases showed more than 10-fold rise than baseline HBV-DNA and the other one showed reversion from negative to positive HBsAg. Five of 7 patients (71%) with HBV reactivation developed active hepatitis, of whom three (43%) with jaundice (one died of acute hepatic failure, one developed decompensated cirrhosis and one recovered), the other two (29%) without jaundice who recovered after therapy. 3. The multiple regression analysis showed LEF (p 5 copies/mL received preventive antiviral drug before DMARDs therapy, and the load of HBV-DNA decreased to 10 3 ∼10 4 copies/mL with normal liver function for more than one year. Conclusions Chinese RA patients should screen HBV infection including the load of HBV-DNA before non-biological DMARDs therapy. LEF, especially with glucocorticoid, should be used cautiously or even be avoided for RA patients with positive HBsAg and/or positive HBV-DNA. HBV reactivation and liver function should be monitored carefully during non-biological DMARDs therapy. Disclosure of Interest None Declared

Background Experiments and clinical trial have shown that peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist could inhibit inflammation in rheumatoid arthritis (RA) [1,2] . Recent reports also showed that PPAR-γ agonist could prevent inflammatory periodontal bone loss by inhibiting receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis [3] . However, whether PPAR-γ agonist can inhibit bone erosion by RANKL-mediated osteoclastogenesis in RA is not clearly understood. Objectives To investigate the effects and underlying mechanism of rosiglitazone (RSG), a high-affinity synthetic agonist for PPAR-γ, on rheumatoid synovial fibroblasts (RA-FLS) induced osteoclastogenesis. Methods RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone (0, 15μM) for 14d. Osteoclast formation was assayed by tartrate-resistant acid phosphatase (TRAP) staining, and the RANKL/OPG mRNA was detected by Real-time PCR, the protein expression of RANKL, OPG, p-ERK, p-p38 and p-JNK was measured by Western blot. RA-FLS were cocultured with normal monocytes on bone slices for 14d, and rosiglitazone (0, 15μM) was then added from day 15, resorption lacunae area was identified by toluidine blue staining at day 21 and quantitated by Image Analysis Software. Results 15μM rosiglitazone could significantly decrease the number of osteoclasts [(38±6) vs (85±10) per well,P 0.05). Conclusions Our results showed that PPAR-γ agonist rosiglitazone could inhibit RA-FLS induced osteoclastogenesis and bone resorption activity by down-regulating RANKL expression and suppression of ERK phosphorylation. Funding This work is supported by Chinese National Natural Science Research Grant (no. 30572290 and 81001334), Guangdong Natural Science Research Grant (no. 9151008901000130 and 10451008901004542). References Cuzzocrea S, Mazzon E, Dugo L, et al. Reduction in the evolution of murine type II collagen-induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma. Arthritis Rheum, 2003, 48 (12): 3544-3556. Dina Shahin, Ehab El Toraby, Hala Abdel-Malek, et al. Effect of Peroxisome Proliferator-Activated Receptor Gamma Agonist (Pioglitazone) and Methotrexate on Disease Activity in Rheumatoid Arthritis (Experimental and Clinical Study). Clinical Medicine Insights: Arthritis and Musculoskeletal Disorders, 2011, 4: 1-10. Hassumi MY, Silva-Filho VJ, Campos-Junior JC, et al. PPAR-gamma agonist rosiglitazone prevents inflammatory periodontal bone loss by inhibiting osteoclastogenesis. Int Immunopharmacol, 2009, 9 (10): 1150-1158. Disclosure of Interest None Declared

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and disability. Focal bone erosion is due to excess bone resorption of osteoclasts. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the critical mediators both in inflammatory signal pathway and differentiation and resorption activity of osteoclasts. Here we aimed to investigate TRAF6 expression in RA synovium and its correlation with histological synovitis severity and radiological joint destruction in RA. Methods Synovitis score was determined in needle biopsied synovium from 44 patients with active RA. Synovium from nine patients with osteoarthritis (OA) and seven with orthopedic arthropathies (Orth.A) were enrolled as "less inflamed" disease controls. Serial sections were stained immunohistochemically for TRAF6 as well as CD68 (macrophage), CD3 (T cell), CD20 (B cell), CD38 (plasmocyte), CD79a (B lineage cells from pre-B cell to plasmocyte stage), and CD34 (endothelial cell). Double immunofluorescence staining of TRAF6 and CD68 were tested. Densities of positive staining cells were determined and correlated with histological disease activity (synovitis score) and radiographic joint destruction (Sharp score). Results TRAF6 expression was found in the intimal and subintimal area of RA synovium, with intense staining found in the endochylema and nucleus of intimal synoviocytes and subintimal inflammatory cells. Double immunofluorescence staining showed TRAF6 was expressed in most of the intimal cells and obviously expressed in CD68+ cells and some other CD68- cells in subintimal area. Synovial TRAF6 was significantly over-expressed in the RA group compared with the OA and Orth.A group (2.53 ± 0.94 vs. 0.72 ± 0.44 and 0.71 ± 0.49, P < 0.0001). Synovial TRAF6 expression in RA correlated significantly with synovitis score (r = 0.412, P = 0.006), as well as the inflammatory cell infiltration (r = 0.367, P = 0.014). Significant correlation was detected between synovial TRAF6 expression and intimal CD68+ cells, as well as the cell density of subintimal CD68+ cells, CD3+ cells, CD20+ cells, CD38+ cells, and CD79a+ cells (all P < 0.05). Conclusions Elevated synovial TRAF6 expression correlated with synovitis severity and CD68+ cell density in RA. It is, therefore, hypothesized that synovial TRAF6 is involved in the pathogenesis of synovial inflammation and osteoclast differentiation in RA.