MiR-146a G/C rs2910164 variation in South African Indian and Caucasian patients with psoriatic arthritis.

BACKGROUND: Psoriasis and psoriatic arthritis (PsA) are inflammatory associated autoimmune disorders. MicroRNA (miR)-146a plays a crucial role in regulating inflammation. A single nucleotide polymorphism in the miR-146a gene (rs2910164), aberrantly alters its gene expression and linked with the pathogenesis of several disorders, including psoriasis and PsA. In South Africa, psoriasis and PsA are extremely rare in the indigenous African population and most common in both the Indian and Caucasian population. The aim of this study was to investigate whether the miR-146a rs2910164 contributes towards psoriasis and PsA development in South African Indian and Caucasian patients. METHODS: South African Indian (n = 84) and Caucasian (n = 32) PsA patients (total n = 116) and healthy control subjects (Indian: n = 62 and Caucasian: n = 38; total n = 100) were recruited in the study. DNA was extracted from whole blood taken from all subjects, and genotyped for the miR-146a rs2910164 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data for laboratory parameters were obtained from pathology reports. The consulting rheumatologist collected all other clinical data. RESULTS: Unstratified data (Caucasians + Indians): A significant decrease in C-reactive protein (CRP) levels in PsA patients was observed (CRP monitored at inclusion vs. after 6 months of treatment) (18.95 ± 2.81 mg/L vs. 9.68 ± 1.32 mg/L, p = 0.0011). The miR-146a rs2910164 variant C-allele frequency in PsA patients was significantly higher vs. healthy controls (35.78% vs. 26% respectively, p = 0.0295, OR = 1.59 95% CI 1.05-2.40). Stratified data (Indians): The variant C-allele frequency in Indian PsA patients was significantly higher vs. healthy Indian controls (35.71% vs. 22.58%, p = 0.0200, OR = 1.91 95% CI 1.13-3.22). Stratified data (Caucasians): The variant C-allele frequency distribution between Caucasian PsA patients and healthy Caucasian controls was similar. CONCLUSION: The rs2910164 variant C-allele may play a role in the progression of PsA in the South African Indian population. The main limitation in this study was the small sample size in the case-control cohorts, with a low overall statistical power (post-hoc power analysis = 19%).

Fibroblasts and Osteoblasts in Inflammation and Bone Damage.

This review discusses the important role fibroblasts play in the process of inflammation and the evidence that these cells may drive the persistence of inflammation. Fibroblasts are key components of the stroma normally involved in maintenance of extracellular matrix and tissue function; however, the term 'fibroblast' is used to describe a heterogeneous population of cells that vary in phenotype both between and within anatomical sites. Fibroblasts possess Toll-like receptors allowing them to respond to pathogen and damage-related signals by producing proinflammatory mediators such as IL-6, PGE2, and GM-CSF and can produce a range of chemokines such as CXCL12, CXCL13, and CXCL8 which attract B and T lymphocytes, monocytes, and neutrophils to sites of inflammation. Interactions between leukocytes and fibroblasts can facilitate increased survival of the leukocytes and modulate phenotypes leading to differential gene expression in the presence of mediators involved in inflammation. Fibroblasts also contribute to collateral tissue damage during inflammation through the production of members of the metalloproteinase family and cathepsins and also through induction of osteoclastogenesis leading to increased bone resorption rates. In persistent diseases, fibroblasts obtain an imprinted, aggressive phenotype leading to the production of higher basal levels of proinflammatory cytokines and the ability to damage tissue in the absence of continual stimuli. This aggressive phenotype offers an attractive new target for therapeutics that could help alleviate the burden of persistent inflammation.

The prolactin receptor is expressed in rheumatoid arthritis and psoriatic arthritis synovial tissue and contributes to macrophage activation.

OBJECTIVES: Prolactin (PRL) is a lactation-inducing hormone with immunomodulatory properties and is found at elevated levels in the serum of patients with RA and other rheumatic diseases. The PRL receptor (PRLR) has been shown to be expressed by macrophages in atherosclerotic plaques. The aim of this study was to examine PRLR expression by synovial macrophages and its role in the regulation of macrophage activation. METHODS: Serum monomeric 23 kDa PRL levels were measured in 119 RA patients using a fluoroimmunometric assay. PRLR expression was assessed in synovial tissue of 91 RA, 15 PsA and 8 OA patients by immunohistochemistry and digital image analysis. Double IF was used to identify PRLR-expressing cells. The effects of PRL on monocyte-derived macrophage gene expression were examined by quantitative real-time PCR and ELISA. RESULTS: Serum PRL levels were similar in female and male RA patients. Median (interquartile range) PRLR expression was significantly higher (P < 0.05) in RA and PsA synovial tissue compared with OA. PRLR colocalized with synovial CD68+ macrophages and von Willebrand factor+ endothelial cells. In vitro, PRLR was prominently expressed in IFN-γ-and IL-10-polarized macrophages compared with other polarizing conditions. PRL by itself had negligible effects on macrophage gene expression, but cooperated with CD40L and TNF to increase expression of pro-inflammatory genes including IL-6, IL-8 and IL-12ß. CONCLUSIONS: Synovial PRLR expression is enhanced in patients with inflammatory arthritis compared with OA, and PRL cooperates with other pro-inflammatory stimuli to activate macrophages. These results identify PRL and PRLR as potential new therapeutic targets in inflammatory arthritis.

Genome-wide association study and gene expression analysis identifies CD84 as a predictor of response to etanercept therapy in rheumatoid arthritis.

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (ΔDAS) in the etanercept subset of patients (P = 8 × 10(-8)), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3' UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1 × 10(-11) in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better ΔDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry.

A functional variant in microRNA-146a promoter modulates its expression and confers disease risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (P(meta) = 2.74×10(-8), odds ratio = 1.29 [1.18-1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.

The role of B lymphocytes in the progression from autoimmunity to autoimmune disease.

Autoimmunity, defined as the presence of autoreactive T and/or B lymphocytes in the periphery, is a frequent and probably even physiological condition. It is mainly caused by the fact that the central tolerance mechanisms, which are responsible for counter-selection of autoreactive lymphocytes, are not perfect and thus a limited number of these autoreactive cells can mature and enter the periphery. Nonetheless, autoreactive cells do not lead automatically to autoimmune disease as evidenced by a multitude of experimental and human data sets. Interestingly, the progression from autoimmunity to autoimmune disease is not only determined by the degree of central tolerance leakage and thus the amount of autoreactive lymphocytes in the periphery, but also by peripheral mechanism of activation and control of the autoreactive cells. In this review, we discuss the contribution of peripheral B lymphocytes in this process, ranging from activation of T cells and epitope spreading to control of the autoimmune process by regulatory mechanisms. We also discuss the parallels with the role of B cells in the induction and control of alloimmunity in the context of organ transplantation, as more precise knowledge of the pathogenic antigens and time of initiation of the immune response in allo- versus auto-immunity allows better dissection of the exact role of B cells. Since peripheral mechanisms may be easier to modulate than central tolerance, a more thorough understanding of the role of peripheral B cells in the progression from autoimmunity to autoimmune disease may open new avenues for treatment and prevention of autoimmune disorders.

Altered BANK1 expression is not associated with humoral autoimmunity in chronic joint inflammation.

OBJECTIVE: The presence of disease-specific autoantibodies in RA but not spondyloarthritis (SpA) suggests that B-cell tolerance is preserved in the latter condition despite chronic joint inflammation. Which factors control B-cell tolerance vs autoimmunity in chronic arthritis remains incompletely understood. As single nucleotide polymorphisms in the B-cell scaffold protein with ankyrin repeats (BANK1) gene have recently been associated with various autoantibody-positive autoimmune diseases including RA, we explored whether altered expression of BANK1 was associated with humoral autoimmunity in arthritis. METHODS: Peripheral B-cell subsets and inflamed synovial tissue were obtained from active SpA and RA. Quantitative PCR was used to assess the expression of full-length BANK1 and delta2 BANK1, a splice variant lacking exon 2 that counteracts BANK1 function. B-cell subsets, autoantibody titres and clinical disease were monitored upon CIA induction in Bank1 knockout (KO) mice. RESULTS: Whereas full-length BANK1 was not differentially expressed, the BANK1 delta2 splicing variant was decreased in naïve peripheral B cells as well as in synovial tissue of SpA compared with RA. However, no differences were observed in seropositive vs seronegative RA. Performing functional analysis in mice, we found no differences in B-cell subsets and anti-collagen antibodies upon CIA induction between Bank1 KO mice and littermate controls. Accordingly, the incidence and severity of clinical disease were not altered in Bank1 KO mice. CONCLUSION: This study did not reveal a major role for BANK1 in humoral autoimmunity in chronic arthritis. The decreased levels of BANK1 delta2 in SpA, however, warrant more detailed analysis of the functional consequences of an altered BANK1/BANK1 delta2 balance.

Increased numbers of CD5+ B lymphocytes with a regulatory phenotype in spondylarthritis.

OBJECTIVE: Whether and how B lymphocytes contribute to the pathogenesis of spondylarthritis (SpA), a seronegative arthritis associated with gut inflammation, remains unknown. Because innate-like CD5+ B lymphocytes with regulatory functions have been identified in colitis models, we undertook the present study to analyze the presence and function of CD5+ B cells in human SpA. METHODS: Peripheral blood B cells from patients with SpA, patients with rheumatoid arthritis (RA), and healthy controls were analyzed by flow cytometry. Synovial biopsy samples were evaluated by immunohistochemistry analysis. Sorted CD5+ and CD5- B cells were analyzed for somatic hypermutation, expression of costimulatory molecules, and cytokine production. RESULTS: The naive, marginal zone-like, and to a lesser extent memory B cell compartments in patients with SpA exhibited a clear and specific increase of CD5+ B cells, which was not found in patients with RA. This increase was not due to either B cell activation or preferential migration of CD5- B cells to the inflamed synovium. Consistent with their phenotype and the low-affinity polyreactive immunoglobulins produced by their murine counterpart cells, CD5+ B cells from patients with SpA showed low levels of somatic hypermutation. With regard to antigen presentation, CD5+ B cells expressed slightly increased HLA-DR levels but low CD80 and CD86 levels. In vitro activation failed to up-regulate these costimulatory molecules but induced significant production of interleukin-10 and interleukin-6 by CD5+ B cells. CONCLUSION: CD5+ B cells are specifically increased in SpA. Analysis of somatic hypermutation, expression of antigen-presenting and costimulatory molecules, and cytokine production indicates that this B cell subset has regulatory capacities. Further investigation of the potential role of CD5+ cells in SpA is warranted.

SNAPIN: an endogenous Toll-like receptor ligand in rheumatoid arthritis.

OBJECTIVE: The mechanisms contributing to the persistent activation of macrophages in rheumatoid arthritis (RA) are not fully understood. Some studies suggest that endogenous toll-like receptor (TLR) ligands promote the chronic inflammation observed in RA. The objective of this study was to identify endogenous TLR ligands expressed in RA synovial tissue (ST) based on their ability to bind the extracellular domains of TLR2 or TLR4. METHODS: A yeast two-hybrid cDNA library was constructed from ST obtained by arthroscopy from patients with RA and screened using the extracellular domains of TLR2 and TLR4 as the bait. Interactions between TLRs and Snapin were demonstrated by reciprocal co-immunoprecipitation. ST was examined by histology, and single- and two-colour immunohistochemistry and quantitative reverse transcriptase PCR. Snapin (SNAP - associated protein) expression in macrophages was examined by Western Blot analysis and confocal microscopy. The ability of Snapin to activate through TLR2 was examined. RESULTS: Employing a yeast two-hybrid system, Snapin was the most frequently identified molecule that interacted with TLR2. These results were confirmed by pull-down of in vitro-expressed Snapin together with TLR2. By immunohistochemistry and quantitative reverse transcriptase PCR, Snapin was highly expressed in RA ST, and it was readily detected in macrophages, where it co-localised in the late endosomes. ST Snapin expression correlated with inflammation and was not disease specific. Finally, Snapin was capable of activating through TLR2. CONCLUSION: These observations identify Snapin as a novel endogenous TLR2 ligand in RA, and thus support a role for persistent TLR2 signalling in the pathogenesis of RA.

A randomized, phase II study of sequential belimumab and rituximab in primary Sjögren's syndrome.

BACKGROUNDPrimary Sjögren's syndrome (pSS) is characterized by B cell hyperactivity and elevated B-lymphocyte stimulator (BLyS). Anti-BLyS treatment (e.g., belimumab) increases peripheral memory B cells; decreases naive, activated, and plasma B cell subsets; and increases stringency on B cell selection during reconstitution. Anti-CD20 therapeutics (e.g., rituximab) bind and deplete CD20-expressing B cells in circulation but are less effective in depleting tissue-resident CD20+ B cells. Combined, these 2 mechanisms may achieve synergistic effects.METHODSThis 68-week, phase II, double-blind study (GSK study 201842) randomized 86 adult patients with active pSS to 1 of 4 arms: placebo, s.c. belimumab, i.v. rituximab, or sequential belimumab + rituximab.RESULTSOverall, 60 patients completed treatment and follow-up until week 68. The incidence of adverse events (AEs) and drug-related AEs was similar across groups. Infections/infestations were the most common AEs, and no serious infections of special interest occurred. Near-complete depletion of minor salivary gland CD20+ B cells and a greater and more sustained depletion of peripheral CD19+ B cells were observed with belimumab + rituximab versus monotherapies. With belimumab + rituximab, reconstitution of peripheral B cells occurred, but it was delayed compared with rituximab. At week 68, mean (± standard error) total EULAR Sjögren's syndrome disease activity index scores decreased from 11.0 (1.17) at baseline to 5.0 (1.27) for belimumab + rituximab and 10.4 (1.36) to 8.6 (1.57) for placebo.CONCLUSIONThe safety profile of belimumab + rituximab in pSS was consistent with the monotherapies. Belimumab + rituximab induced enhanced salivary gland B cell depletion relative to the monotherapies, potentially leading to improved clinical outcomes.TRIAL REGISTRATIONClinicalTrials.gov NCT02631538.FUNDINGFunding was provided by GSK.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Heat shock protein 96 is elevated in rheumatoid arthritis and activates macrophages primarily via TLR2 signaling.

Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased TLR2 and TLR4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared with peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNF-alpha, and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.

A randomised, placebo-controlled study of RIPK1 inhibitor GSK2982772 in patients with active ulcerative colitis.

OBJECTIVE: Tumour necrosis factor signalling via the receptor-interacting protein kinase 1 (RIPK1) pathway regulates colonic inflammation suggesting that RIPK1 inhibition may be a potential therapeutic target in ulcerative colitis (UC). This phase IIa, randomised, double-blind experimental medicine study investigated the safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary efficacy of the RIPK1 inhibitor GSK2982772 in patients with active UC. DESIGN: In part A, prior to a protocol amendment, one patient was randomised to receive GSK2982772 60 mg twice daily for 42 days. After the amendment, patients were randomised 2:1 to receive GSK2982772 60 mg or placebo three times daily for 42 days. In part B, all patients switched to open-label GSK2982772 60 mg three times daily for 42 days. Safety, PK, PD biomarkers, histological disease activity, clinical efficacy and quality of life were assessed at days 43 and 85. RESULTS: Thirty-six patients were randomised (n=12, placebo/open-label GSK2982772; n=24, GSK2982772/open-label GSK2982772). Most adverse events were mild, with headache reported the most frequently across groups (placebo/open-label GSK2982772, n=2 (17%); GSK2982772/open-label GSK2982772, n=8 (33%)). GSK2982772 was well distributed into colonic tissue, with generally higher concentrations in colonic biopsy samples versus plasma. No apparent differences between treatment groups were observed for PD, histological disease activity, clinical disease activity or quality-of-life measures. At screening, all patients had Mayo endoscopic scores of 2 or 3. At day 43, no patients in the placebo/open-label GSK2982772 group achieved Mayo endoscopic scores of 0 or 1 vs 3/24 (13%) for GSK2982772/open-label GSK2982772. At day 85, 1/9 (11%) achieved scores of 0 or one for placebo/open-label GSK2982772 vs 3/22 (14%) for GSK2982772/open-label GSK2982772. CONCLUSION: GSK2982772 was generally well tolerated, with no treatment-related safety concerns identified. However, no significant differences in efficacy were observed between treatment groups, suggesting that GSK2982772 as monotherapy is not a promising treatment for patients with active UC. TRIAL REGISTRATION NUMBER: NCT02903966.

A randomized, placebo-controlled experimental medicine study of RIPK1 inhibitor GSK2982772 in patients with moderate to severe rheumatoid arthritis.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of inflammation through cell death and proinflammatory cytokine production. This multicenter, randomized, double-blind (sponsor-unblinded), placebo-controlled, experimental medicine study evaluated the safety, pharmacokinetics (PK), and preliminary efficacy of GSK2982772, a RIPK1 inhibitor, in moderate to severe rheumatoid arthritis (RA). METHODS: Patients with moderate to severe RA who had received ≥12 weeks' stable-dose conventional synthetic disease-modifying antirheumatic drug (csDMARD) therapy were randomized (2:1) to GSK2982772 60 mg or placebo orally 2 or 3 times daily for 84 days. Safety, PK, disease activity, joint damage, and pharmacodynamic (PD) biomarkers were assessed at days 43 and 85. RESULTS: A total of 52 patients were randomized (placebo, 18; GSK2982772, 34). Adverse events (AEs) were reported in 13 (72%) in patients in the placebo group (n = 3 b.i.d; n = 10 t.i.d.) and 20 (61%) in the GSK2982772 group (n = 3 b.i.d; n = 17 t.i.d.). All treatment-related AEs were mild/moderate, except one severe case of alopecia areata at day 49 and retinal vein thrombosis at day 66 (which led to withdrawal from the study) in patients receiving GSK2982772 t.i.d. Disease Activity Score in 28 Joints-C-reactive protein (DAS28-CRP) scores, ACR20/50/70 response, and rates of low disease activity and remission were similar between placebo and GSK2982772 arms. CONCLUSIONS: These results suggest that inhibition of RIPK1 activity at the GSK2982772 exposure levels evaluated do not translate into meaningful clinical improvement of RA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02858492 . Registered 8 August 2016.

Continued inhibition of structural damage over 2 years in patients with rheumatoid arthritis treated with rituximab in combination with methotrexate.

BACKGROUND: Rituximab inhibited structural damage at 1 year in patients with rheumatoid arthritis (RA) who had had a previous inadequate response to tumour necrosis factor (TNF) inhibitors. OBJECTIVE: To assess structural damage progression through 2 years. METHODS: Intention-to-treat patients with one post-baseline radiograph (rituximab n=281; placebo n=187) received background methotrexate (MTX) and were randomised to rituximab (2 x 1000 mg infusions, 2 weeks apart) or placebo; patients were eligible for rituximab re-treatment every 6 months. By week 104, 82% of the placebo population had received > or = 1 dose of rituximab. Radiographic end points included the change in total Sharp score (TSS), erosion and joint space narrowing scores at week 104. RESULTS: At week 104, significantly lower changes in TSS (1.14 vs 2.81; p<0.0001), erosion score (0.72 vs 1.80; p<0.0001) and joint space narrowing scores (0.42 vs 1.00; p<0.0009) were observed with rituximab plus MTX vs placebo plus MTX. Within the rituximab group, 87% who had no progression of joint damage at 1 year remained non-progressive at 2 years. CONCLUSIONS: Rituximab plus MTX demonstrated significant and sustained effects on joint damage progression in patients with RA and a previously inadequate response to TNF inhibitors.

The prothrombotic state in rheumatoid arthritis: an additive risk factor for adverse cardiovascular events.

Rheumatoid arthritis (RA) has been recognized to increase cardiovascular morbidity and mortality independent of established risk factors. The chronic inflammatory state, a hallmark of RA, is considered an autonomous risk factor, whereas components of innate and adaptive immunity are believed to contribute to the onset of acute cardiovascular events. Several studies have suggested that RA confers a prothrombotic state featured by abnormalities in coagulation and fibrinolytic systems together with an altered state of platelet reactivity. It is conceivable that these findings may be partly instrumental for the observed increased risk for adverse cardiovascular events in RA. Therapeutic strategies aimed at attenuating the inflammatory disease activity and intervening at the point of cross-talk between mediators of inflammation and thrombogenesis may help reduce cardiovascular disease burden in patients with RA.

cDC1 are required for the initiation of collagen-induced arthritis.

Rheumatoid arthritis (RA) is chronic autoimmune disease which etiology remains unknown. Several cell types have been described to potentiate/aggravate the arthritic process however the initiating event in synovial inflammation is still elusive. Dendritic cells (DCs) are essential for the initiation of primary immune responses and thus we hypothesized that these cells might be crucial for RA induction. DCs are a heterogeneous population of cells comprising different subsets with distinct phenotype and function. Here we investigated which DC subset(s) is/are crucial for the initiation of the arthritic process. We have previously demonstrated that Flt3-/- mice, with reduced DCs, were protected from collagen induced arthritis (CIA). Here we have shown that GM-CSF derived DCs in Flt3L-/- mice are functional but not sufficient to induce arthritis. Batf3-/- mice lacking both CD103+ and CD8α+ cDC1 were resistant to collagen induced arthritis (CIA), demonstrating that this DC subset is crucial for arthritis development. CEP-701 (a Flt3L inhibitor) treatment prevented CIA induction, and reduced dramatically the numbers CD103+ cDC1s present in the lymph nodes and synovium. Hence this study identified cDC1 as the main subset orchestrating the initiation of cell-mediated immunity in arthritis.

Incidence and risk factors for adalimumab and infliximab anti-drug antibodies in rheumatoid arthritis: A European retrospective multicohort analysis.

OBJECTIVES: To evaluate the incidence of anti-drug antibody (ADA) occurrences and ADA-related risk factors under adalimumab and infliximab treatment in rheumatoid arthritis (RA) patients. METHODS: The study combined retrospective cohorts from the ABIRISK project totaling 366 RA patients treated with adalimumab (n = 240) or infliximab (n = 126), 92.4% of them anti-TNF naive (n = 328/355) and 96.6% of them co-treated with methotrexate (n = 341/353) with up to 18 months follow-up. ADA positivity was measured by enzyme-linked immunosorbent assay. The cumulative incidence of ADA was estimated, and potential bio-clinical factors were investigated using a Cox regression model on interval-censored data. RESULTS: ADAs were detected within 18 months in 19.2% (n = 46) of the adalimumab-treated patients and 29.4% (n = 37) of the infliximab-treated patients. The cumulative incidence of ADA increased over time. In the adalimumab and infliximab groups, respectively, the incidence was 15.4% (5.2-20.2) and 0% (0-5.9) at 3 months, 17.6% (11.4-26.4) and 0% (0-25.9) at 6 months, 17.7% (12.6-37.5) and 34.1% (11.4-46.3) at 12 months, 50.0% (25.9-87.5) and 37.5% (25.9-77.4) at 15 months and 50.0% (25.9-87.5) and 66.7% (37.7-100) at 18 months. Factors associated with a higher risk of ADA development were: longer disease duration (1-3 vs. < 1 year; adalimumab: HR 3.0, 95% CI 1.0-8.7; infliximab: HR 2.7, 95% CI 1.1-6.8), moderate disease activity (DAS28 3.2-5.1 vs. < 3.2; adalimumab: HR 6.6, 95% CI 1.3-33.7) and lifetime smoking (infliximab: HR 2.7, 95% CI 1.2-6.3). CONCLUSIONS: The current study focusing on patients co-treated with methotrexate for more than 95% of them found a late occurrence of ADAs not previously observed, whereby the risk continued to increase over 18 months. Disease duration, DAS28 and lifetime smoking are clinical predictors of ADA development.

Induction of sustained remission in early inflammatory arthritis with the combination of infliximab plus methotrexate: the DINORA trial.

BACKGROUND: In the present study, we explored the effects of immediate induction therapy with the anti-tumour necrosis factor (TNF)α antibody infliximab (IFX) plus methotrexate (MTX) compared with MTX alone and with placebo (PL) in patients with very early inflammatory arthritis. METHODS: In an investigator-initiated, double-blind, randomised, placebo-controlled, multi-centre trial (ISRCTN21272423, http://www.isrctn.com/ISRCTN21272423 ), patients with synovitis of 12 weeks duration in at least two joints underwent 1 year of treatment with IFX in combination with MTX, MTX monotherapy, or PL randomised in a 2:2:1 ratio. The primary endpoint was clinical remission after 1 year (sustained for at least two consecutive visits 8 weeks apart) with remission defined as no swollen joints, 0-2 tender joints, and an acute-phase reactant within the normal range. RESULTS: Ninety patients participated in the present study. At week 54 (primary endpoint), 32% of the patients in the IFX + MTX group achieved sustained remission compared with 14% on MTX alone and 0% on PL. This difference (p < 0.05 over all three groups) was statistically significant for IFX + MTX vs PL (p < 0.05), but not for IFX + MTX vs MTX (p = 0.10), nor for MTX vs PL (p = 0.31). Remission was maintained during the second year on no therapy in 75% of the IFX + MTX patients compared with 20% of the MTX-only patients. CONCLUSIONS: These results indicate that patients with early arthritis can benefit from induction therapy with anti-TNF plus MTX compared with MTX alone, suggesting that intensive treatment can alter the disease evolution. TRIAL REGISTRATION: The trial was registered at http://www.isrctn.com/ISRCTN21272423 on 4 October 2007 (date applied)/12 December 2007 (date assigned). The first patient was included on 24 October 2007.

Quantitative image analysis of synovial tissue.

Quantitative image analysis is a form of imaging that includes microscopic histological quantification, video microscopy, image analysis, and image processing. Hallmarks are the generation of reliable, reproducible, and efficient measurements via strict calibration and step-by-step control of the acquisition, storage and evaluation of images with dedicated hardware and software. Major advantages of quantitative image analysis over traditional techniques include sophisticated calibration systems, interaction, speed, and control of inter- and intraobserver variation. This results in a well controlled environment, which is essential for quality control and reproducibility, and helps to optimize sensitivity and specificity. To achieve this, an optimal quantitative image analysis system combines solid software engineering with easy interactivity with the operator. Moreover, the system also needs to be as transparent as possible in generating the data because a "black box design" will deliver uncontrollable results. In addition to these more general aspects, specifically for the analysis of synovial tissue the necessity of interactivity is highlighted by the added value of identification and quantification of information as present in areas such as the intimal lining layer, blood vessels, and lymphocyte aggregates. Speed is another important aspect of digital cytometry. Currently, rapidly increasing numbers of samples, together with accumulation of a variety of markers and detection techniques has made the use of traditional analysis techniques such as manual quantification and semi-quantitative analysis unpractical. It can be anticipated that the development of even more powerful computer systems with sophisticated software will further facilitate reliable analysis at high speed.