Cadherin-11 regulates synovial fibroblast behavior in health and disease

Background IL-21 is secreted by activated T cells and modulates immune cell functions with both proinflammatory and anti-inflam-matory effects. IL-21 receptor (IL-21R), homologous to IL-2R β and IL-4R α , associates with the gamma common chain upon ligand binding. It was recently described that IL-21R is overexpressed in the inflamed synovial membrane and on leucocytes of rheumatoid arthritis patients. Objective Previously we have shown that blockade of the IL-21 pathway with soluble IL-21R-Fc resulted in a reduction of clinical signs of arthritis in rodent models. To understand potential mechanisms of IL-21 regulation in arthritis, we analyzed serum immunoglobulin levels, and cytokine expression in the paws, serum, and collagen-restimulated splenocytes, in response to IL-21 pathway blockade. Methods Arthritis was induced in DBA/1 male mice with bovine type II collagen. Animals were treated with either soluble mIL-21R-Fc, which neutralizes murine IL-21 bioactivity, with TNFRII-Fc or with control IgG. Spleens from each group of treated mice were cultured in vitro with collagen and assayed for cytokine secretion. Cytokines and anticollagen-specific IgG levels were also measured in the serum by ELISA. Cytokine mRNA levels in the paws were evaluated by quantitative PCR analysis. Results Treatment of mice with IL-21R-Fc or TNFRII-Fc reduced clinical and histological signs of collagen-induced arthritis. IL-6 mRNA in paws and serum IL-6 levels were decreased after IL-21R-Fc treatment. IFN γ mRNA was increased in paws of IL-21R-Fc-treated mice. Collagen-specific spleen cell responses from IL-21R-Fc-treated mice exhibited increased IFN γ and IL-2, and reduced IL-6 and IL-17 levels. Serum levels of total IgG 1 were also reduced in response to IL-21R-Fc treatment. Conclusion These data demonstrate a role for IL-21 in the modulation of collagen-specific T-cell responses and the pathology of arthritis, supporting a rationale for blockade of the IL-21 pathway in rheumatoid arthritis. linked to clinical disease phenotypes and subtraits in a cross between the CIA-susceptible DBA1/J and resistant FVB/NJ mouse strains. Additionally, gene expression profiling in target tissues and immune cells in parental strains and their F2 progeny is performed. Numerous classical QTL and expression QTL were identified. Master QTLs/expression QTLs controlling basic disease traits and subtraits were selected for further analysis using in silico tools; for example, haplotype sharing analysis, interspecies synteny analysis, congenic mapping, and pathway construction. suggesting the role of NO in regulating IFN γ synthesis. The cytoplasmic Ca 2+ concentration of unstimulated T cells and the T-cell activation-induced Ca 2+ signal were increased in T cells from the HDC-KO mice ( P = 0.02 and P = 0.04, respectively), while the T-cell activation-induced CD3 internalization was similar in both HDC-KO and WT animals. Our present data indicate that histamine deficiency does not protect against aggrecan-induced arthritis. The Th1 cytokine pattern and increased NO production may both contribute to the sensitivity of HDC-KO mice to aggrecan-induced arthritis. Furthermore, our data indicate that histamine, in addition to its direct effects on T-lymphocyte function, regulates cytokine production and T-cell signal transduction through regulating NO production. IL-21 promotes plasma cell (PC) differentiation while BAFF promotes B-cell survival. Here, we report that IL-21 synergizes with BAFF to elicit BLIMP-1 induction, PC differentiation and IgG production from a novel population of human splenic memory B cells. These human marginal zone analogue B cells are exquisitely sensitive to IL-21 and BAFF in the absence of further costimulation. The ability of IgG + marginal zone analogue to respond specifically and exclusively to IL-21 and BAFF demonstrates that they are uniquely poised to respond to antigen-independent signals and differentiate into IgG-producing PC, thereby replenishing serologic memory. Importantly, peripheral blood B cells from a portion of patients with systemic lupus erythematosus and rheumatoid arthritis were highly responsive to stimulation with IL-21 and BAFF. These data suggest that IL-21 and BAFF may be capable of inducing PC differentiation from memory B cells with autoreactive specificities and thereby contribute to autoimmunity. T1/ST2 IL-33 and T1/ST2 key functions T1/ST2. experimental the effect of IL-33 on P815 murine mastocytoma cells and mouse bone marrow-derived mast cells (BMMC). IL-33 dose-dependently and time-dependently stimulated IL-6 secretion by P815 mastocytoma cells and BMMC. Both cell types express T1/ST2 as assessed by RT-PCR and FACS analysis. This effect was dependent on T1/ST2 binding. The addition of ST2-Fc on BMMC inhibited the stimulatory effect of IL-33, whereas etanercept had no effect. IL-1RAcP-Fc alone or in combination with ST2-Fc inhibited the effect of IL-33, indicating that IL-33 binds to these soluble receptors. In addition, IL-33 also induced IL-1 β , TNF α , MCP-1, and PGD2 production in BMMC. By the RNase protection assay, we demonstrated that IL-33 increased IL-6 and IL-1 β ? mRNA expression. These effects of IL-33 appeared to occur independently of mast cell degranulation. the IL-1 of the of proinflammatory mediators by mast cells in addition effect on Th2 open new perspec-tives for the of diseases by IL-33. Objective To further investigate the role of eicosanoids in the pathogenesis of SSc through evaluation of: (1) the basal profile of eicosanoid synthesis, and (2) the pattern of eicosanoid biosynthesis in response to proinflammatory stimuli by peripheral blood mononuclear cells (PBMC) from patients with SSc in comparison with healthy people. Methods Mononuclear cells were isolated from peripheral blood using density gradient centrifugation on Lymphoprep and cultured in fetal calf serum-supplemented RPMI medium at 37°C under 5% CO Ionophore-stimulated production of 5-LOX-derived leuko- triene E 4 (LTE4) and 15-LOX-derived 15-HETE was evaluated by means of enzyme immunoassay at predefined time points in basal conditions (without stimuli) as well as in response to TNF α . Ten patients with SSc (five diffuse and five limited cutaneous SSc) as well as five healthy controls were studied. Results There were no significant differences in the basal production of LTE4 or 15-HETE between SSc patients and healthy controls. TNF α induced sequential changes in the production of eicosanoids, with an early (within 1 hour) increase in LTE4 followed by a delayed increase in 15-HETE in both SSc patients and healthy controls. PBMC from SSc patients responded to TNF α with significantly higher production of LTE4 in comparison with healthy controls ( P < 0.05 at 1 hour), while there were no significant differences in TNF α -induced production of 15-HETE between SSc patients and controls. Conclusions These preliminary results of our study indicate that the TNF α -induced eicosanoid synthesis is altered in PBMC from patients with SSc. Increased production of proinflammatory leukotrienes may contribute to the pathogenesis of SSc. To characterize the nature of other mutations, 316 genomic nonproductive V(D)J rearrangements amplified and sequenced from three AID –/– patients were analyzed. The mutation frequency was 20-fold less than normals (0.09% versus 2%) but ~5 x 10 4 -fold more than non-B cells. Reduced RGY (6% versus 27%) and WRC (6% versus 15%) motif mutations and decreased replacements in complementarity determining regions were attributed to the lack of AID. Reduced WA motif mutations (12% versus 23%) suggested POL η activity was decreased. Prominent G>C and A>T biases suggested that mutation and repair mechanisms occurred preferentially on one DNA strand. A high percentage of the mutations were G substitutions on the sense strand (43% versus 29% in normals), reflecting C mutational targeting on the opposite strand. These were primarily G to A transitions, suggesting that UNG activity was reduced even though UNG is upregulated in normal GC B cells and large GCs occur in AID –/– patients. The mutation pattern suggests that AID-independent C substitutions contribute a small proportion of SHM but lack the targeting provided by AID. Finally, a low level of SHM clearly can develop in the absence of AID; however, this mechanism is inefficient at altering the binding capacity of immunoglobulin heavy chain genes. demonstrated minor true lymphoneogenesis germinal (GCs), association with the ectopic expression of the lymphoid chemokines (CKs) CXCL13 and CCL21. In Sjogren’s syndrome major salivary glands (SS-MSGs) structures to clonal immunoglobulin heavy chain gene rearrangements and somatic hypermutation, favouring expansion and selection of autoreactive B-cell clones, clonally related to lymphomatous B-cell clones (responsible for lymphoma development in Sjogren’s syndrome (SS)). The involvement of lymphoid CKs and CXCL12 in the development of lymphoid malignancies been At however, no data on the expression of these CKs nor their cellular source within SS-MSGs and mucosal-associated lymphoid tissue lymphomas (MALT-Ls) from patients with SS have been described. Objective To assess the specific contribution of lymphoid CKs in the organization of lymphoid proliferation within SS-MSGs and SS-MALT-Ls. Methods We studied 12 SS-mSGs, four SS-MSGs with lympho-epithelial lesion and 20 SS-MSGs with non-Hodgkin B-cell MALT-Ls. In order to define the histological organization of the lymphoid infiltrate (reactive versus malignant areas) and identify the B-cell subpopulations infiltrating the glands, immunohistochemistry was carried out for cellular (CD21, bcl-2, bcl-6, IgD, CD20, CD3, CD68) and vascular markers (CD31, process within the malignancy. These findings support a contributory role for CXCL13 and CCL21 in the pathogenesis of MALT lymphomas in SS. These results imply that the CK/CK – receptor axis is functional in MALT-L malignant cells, and suggest that the glandular microenvironment, B-cell receptor signalling upon antigen engagement and autocrine signals from the same malignant population concur in MALT lymphomagenesis, triggering local activation of malignant B cells and favouring their survival and expansion. Objective In classic studies on the genetic background of antibody production, MHC has been shown to act as the most prominent immune-response gene that controls the magnitude and the specificity of antibody production. The strongest genetic risk factor for rheumatoid arthritis (RA), the human MHC, HLA-DRB1 shared epitope (SE) alleles, predisposes for antibodies against citrullinated proteins (ACPA). ACPA levels are higher in SE-positive than in SE-negative RA patients. The present study determines whether SE not only influences the magnitude, but also the specificity of the ACPA response. Methods In two independent cohorts of anti-CCP2-positive RA patients ( n = 206 and n = 214 patients, respectively), serum antibodies against a citrullinated peptide derived from vimentin (cVim) and antibodies against a citrullinated fibrinogen peptide (cFibr) were determined by ELISA. HLA-DRB1 genotyping was performed. Results In the first cohort, SE alleles were significantly associated with the presence of antibodies against cVim (OR = 4.55, 95% CI = 1.78–12.9) and not significantly with the presence of antibodies against cFibr (OR = 1.81, 95% CI = 0.78–4.13). These results were replicated in the second cohort (OR = 4.13, Single nucleotide polymorphisms (SNPs) of the TNF gene at positions and have earlier been associated with psoriasis vulgaris and psoriatic arthritis (PsA). However, a strong linkage disequilibrium at the chromosomal region 6p21 the interpretation of these findings difficult also other risk factors for psoriasis (PSORS1) than SNPs of the TNF gene have bee mapped to that particular region. in study several SNPs of the TNF gene and of its neighbouring lymphotoxin α ( LTA ) gene were analysed independently and dependently on carrying the PSORS1 risk allele. SNPs in the promoter of the TNF gene one SNP of the LTA gene of the gene the gene were in the CCHCR1 gene CCHCR1*WWCC the genetic impact of the PSORS1 risk allele. blocked by anti-TNF therapy. This study aimed to correlate the expression of p38 δ in the rheumatoid synovium with clinical response and nonresponse to the anti-TNF biologic, infliximab (Remicade). Methods We examined the expression of p38 MAPK isoforms ( α and δ ) pre and post infliximab therapy in the synovium of RA patients, to compare clinical response with nonresponse. All patients entered into this study have failed at least two disease-modifying drugs, and fulfil the revised 1987 ACR criteria for RA. The effect of TNF blockade in five responders and three nonresponders on the p38 MAPK α and δ isotypes was studied; expression and activation (phospho-p38) in synovial biopsies of responders and nonresponders was measured by immunohistochemistry using a semiquantitative scoring system, at baseline and approximately week 16 of therapy. A therapeutic effect was determined by patients achieving an ACR20 response at week 14, and the Wilcoxon signed rank test was performed to assess significance of changes in expression levels post treatment. Results In responders, p38 α , p38 δ and phospho-p38 expression were all significantly decreased post infliximab treatment in the synovial sublining layer ( P < 0.05). A similar trend was seen in the synovial lining layer of responders, with a decrease in expression of p38 α and p38 δ and phospho-p38 post infliximab treatment in four of five responders, although these results did not reach statistical significance. p38 δ and phospho-p38 in the sublining layer and phospho-p38 in the lining layer increased, or remained high, in all three nonresponders tested, and this tendency was the opposite to that seen in the responders. Conclusion In the RA synovium, expression and activation of p38 α and p38 δ in the sublining layer correlated with response, and increased p38 δ expression and activation was associated with nonresponse. A flow cytometric-based investigation of p38 isoform agonists (oligodeoxy-nucleotides containing unmethylated CpG sequences on RA BM-derived B cells, and to estimate the presence of bacterial DNA in BM from RA and OA patients. BM samples, obtained from RA patients during joint replacement surgery, served for bone marrow mononuclear cell isolation. Mature (CD20 + ) B cells were next purified from bone marrow mononuclear cells using a magnetic cell separation technique (MACS) and cultured in vitro in the presence of agonistic CpG-ODN or control GpC-ODN alone or with addition of IL-15. mRNA was isolated from purified B cells using Trizol and served as the template for RT-PCR analysis. The presence of bacterial DNA in BM plasma and bone marrow mononuclear cells was evaluated by DNA extraction and PCR amplification using primers specific for eubacterial 16S-ribosomal RNA. Flow cytometry was applied to assess the expression of intracellular proteins (TLR9 and proliferation marker Ki-67) and surface activation markers (CD86, CD54) on B lymphocytes. B-cell populations were distinguished according to CD19, CD20, CD27 and CD138 molecule expression. Specific ELISAs were used to measure TNF α and IL-6 in supernatants from cultured cells. found that BM-derived B lymphocytes isolated from RA patients express TLR9 at the mRNA and protein levels. TLR9 are functional. of B cells by CpG-ODN, but not control GpC-ODN, in a dose-dependent of (CD86 and CD54), α Experimental and clinical evidence for T-cell involvement in the pathogenesis of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T-cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T-cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction in RA. NO has recently been recognized as a key signaling intermediate for T-cell activation and mitochondrial biogenesis. NO is synthesized from L -arginine by NO synthetases (NOS). Three distinct isoforms of NOS are known, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) enzymes. We previously detected the expression of eNOS and nNOS and the absence of iNOS in human PBL, while during inflammation macrophages and monocytes express iNOS. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown origin characterized by the involvement of multiple organs. Several studies carried out on patients with both RA and SLE have documented increased endogenous NO synthesis, but its contribution to T-lymphocyte mitochondrial biogenesis and T-cell dysregulation is not known. We investigated the role of NO in T-cell mitochondrial biogenesis in RA and SLE. Background Recent evidence suggests an important role for IL-7 in the pathogenesis of rheumatoid arthritis (RA) and a therapeutic potential for IL-7 blockade IL-7 is produced by synovial stromal cells (StrC) in RA but is barely detectable in osteoarthritis [2]. Our published data showed reduced production of IL-7 by bone marrow (BM) StrC and reduced levels of circulating IL-7 in RA compared with health [3]. IL-7 was also detected in synovial tissue in active RA but levels were below detection in clinical remission. These conflicting observations suggest that the regulation of IL-7 expression is tightly controlled at the level of tissue specificity. To support this hypothesis, we showed that several cytokines have a different effect on IL-7 production in BM StrC, epithelial cells from the and This work at identifying the factors that regulate IL-7 production in StrC isolated tissue of Background CD20-directed therapy is well established in oncology, and was recently demonstrated to be highly effective in the treatment of rheumatoid arthritis (RA) [1]. Small modular immunopharmaceutical drugs are smaller than antibodies yet are capable of utilizing effector functions of antibody-dependent cellular cytotoxicity, complement-mediated cytotoxicity and apoptotic signaling. TRU-015 is a small modular immunopharmaceutical drug candidate that is specific for CD20. TRU-015 has been demonstrated to effectively deplete B lymphocytes in cynomolgus monkeys in a dose-dependent manner, and to improve survival in mouse xenograft tumor models [2,3]. Objectives This phase 1 dose escalation study was performed to evaluate the safety, pharmacokinetics and pharmacodynamics of TRU-015 in subjects with RA. Methods Thirty-seven RA subjects receiving a stable background of methotrexate were enrolled in eight dosage groups (single dosages of TRU-015 of 0.015, 0.05, 0.15, 0.5, 1.5, 5, or 15 mg/kg, or two doses of 15 mg/kg given 1 week apart (30 mg/kg)). Safety was assessed at baseline, during infusion and at prespecified intervals after infusion by clinical (adverse events, physical examination, vital signs) and laboratory parameters. Serum samples were collected for pharmacokinetic analysis. Pharmacodynamic response was measured using B-cell counts (CD19 + cells by flow cytometry) at prespecified timepoints. Subjects were evaluated for a minimum of 4 weeks and until B-cell recovery. Results At least four subjects were exposed at each dose level. Peroxisome proliferator-activated receptor gamma (PPAR γ ) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPAR γ activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPAR γ expression in cartilage are poorly defined. This study was under-taken to investigate the quantitative expression and distribution of PPAR γ in normal and OA cartilage and to evaluate the effect of IL-1 β , a prominent cytokine in OA, on PPAR γ expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPAR γ protein expression were significantly lower in OA cartilage when compared with normal cartilage. Using real-time RT-PCR, we demonstrated that PPAR γ 1 mRNA levels were ~10-fold higher than PPAR γ 2 mRNA levels; and that only PPAR γ 1 was differentially expressed, its levels in OA cartilage being 2.4-fold lower than in normal cartilage ( P < 0.001). IL-1 treatment of OA chondrocytes downregulated PPAR γ 1 expression in a dose-dependent and time- dependent manner. This effect probably occurred at the trans-criptional level, since IL-1 decreases both PPAR γ 1 mRNA expression and PPAR γ 1 promoter activity. TNF α , IL-17, and prostaglandin E 2 , which are involved in the pathogenesis of OA, also downregulated PPAR γ 1 expression. Specific inhibitors of the mitogen-activated protein kinase (MAPK) p38 (SB203580) and JNK (SP600125), but not Erk (PD98059), prevented IL-1-induced downregulation of PPAR γ 1 expression. Similarly, inhibitors of NF- κ B signaling (PDTC, MG-132, and SN-50) abolished the suppressive effect of IL-1. In conclusion, our study has demonstrated for the first time that PPAR γ 1 is downregulated in OA cartilage. The proinflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF- κ B signaling pathways. The IL-1-induced downregulation of PPAR γ expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation. cartilage subchondral bone prominent role OA, osteoblast (Ob) differentiation. the differentiation of Obs; role for leptin in human OA Materials and methods We prepared primary cultures of normal and OA Obs from subchondral bone and OA chondrocytes from articular cartilage from tibial plateaus removed for knee replacement surgery of OA patients or at autopsy. We determined the expression and production of leptin using RT-PCR and ELISA. cell cell proliferation assay western blot Our results using two different sets of primers for RT-PCR experiments leptin was expressed only in Obs not also in normal using real-time PCR in articular cartilage is derived from bone tissue. The increase in leptin in OA bone tissue could also be responsible for increased ALP, OC and TGF β 1 in Obs. Since leptin can promote inflammation and cartilage loss in combination with cytokines, this suggests that subchondral bone production of leptin may be responsible, at least in part, for cartilage loss in OA. Background Nonsynonymous polymorphisms in the human FRZB gene have been associated with osteoarthritis (OA). In addition, a differential association between OA and osteoporosis (OP) has been reported. We have demonstrated that genetic deletion of the Frzb gene in mice increases cartilage damage in different models of OA. In addition, Frzb –/– mice show increased cortical bone density. Objective To study the underlying molecular mechanisms involved in cartilage damage and increased bone density in Frzb –/– mice. Methods Active Wnt signaling in the articular cartilage was studied in normal and methylated bovine serum albumin, collagenase and papain-induced arthritis using β -catenin (CTNNB1) immunohistochemistry. Gene expression patterns of components of the Wnt signaling pathway, its antagonist and target genes, were studied using multigene cDNA arrays (Superarray) in microdissected articular cartilage and soft tissues of healthy and affected knees from Frzb –/– and wild-type mice. Gene expression of tissue destructive enzymes in the cartilage was studied by real-time PCR. Matrix metalloproteinase-3 activity was tested of syndecan-4 in human OA cartilage both at the mRNA and the protein level. In the cartilage explant model, blocking antibodies to syndecan-4 substantially significantly reduced the IL-1-induced loss of proteoglycans in wild-type cartilage. Analysis of OA-like changes in mice revealed a strong and early induction of syndecan-4, and there was a significant reduction of proteoglycan loss in the syndecan-4 –/– mice compared with their wild-type controls. This was accompanied by a significantly reduced staining for ADAMTS-generated aggrecan neo-epitopes in syndecan-4 –/– mice. Our data show that syndecan-4 is induced in hypertrophic chondrocytes both during embryogenesis and in OA cartilage. By promoting ADAMTS-mediated cleavage of aggrecans, syndecan-4 facilitates enchondral ossification but is involved also in cartilage degradation by hypertrophic chondrocytes in OA. Inhibition of syndecan-4 may therefore constitute a promising strategy to interfere with osteoarthritic cartilage damage.

Background TNFα is a key cytokine in rheumatoid arthritis (RA) physiopathology. We recently demonstrated that a new cationic liposome formulation allowed intravenous delivery of a small interfering RNA (siRNA) targeting TNFα and efficiently restoring the immunological balance in an experiment model of RA. Since 30% of patients do not respond to anti-TNF biotherapies, however, there is a need to develop alternative therapeutic approaches. Objective Strong association of other proinflammatory cytokines with the pathogenesis of RA prompted us to investigate which cytokine other than TNFα could be targeted for therapeutic benefit using RNA interference. Methods Two siRNA sequences were designed for IL-1β, IL-6 and IL-18 proinflammatory cytokines, and their efficacy and specificity were validated in vitro on J774.1 mouse macrophage cells, measuring both mRNA and protein levels following a lipopolysaccharide challenge. For in vivo administration, siRNAs were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA, and were injected intravenously in DBA/1 mice having collagen-induced arthritis. The clinical course of the disease was assessed by paw thickness over time, and radiological and histological scores were obtained at euthanasia. The cytokine profiles were measured by ELISA in sera and knee-conditioned media. The immunological balance was assessed using antitype II collagen assays. The distribution of siRNAs was evaluated by fluorometry in GFP transgenic mice over time after anti-GFP siRNA lipoplex injections. Results The designed siRNA sequences silenced 70-75% of the lipopolysaccharide-induced IL-1β, IL-6 and IL-18 mRNA expression in macrophages compared with a control siRNA. Each siRNA affected the targeted cytokine specifically, without modifying other proinflammatory cytokine mRNAs. In the collagen-induced arthritis model, weekly injections of siRNA lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling, and destruction of cartilage and bone, in both preventive and curative settings. The most striking therapeutic effect was observed when Introduction Rheumatoid arthritis is a chronic inflammatory disorder that primarily affects the joints and results in the destruction of cartilage and subchondral bone by the inflamed synovium. Investigating the time course of cartilage destruction is of importance to better understand whether structural damage is reversible and when therapeutic intervention may be most effective. Methods To study the time course and mechanisms of inflammatory destruction of articular cartilage, we used the human TNFα transgenic (hTNFtg) mouse model of rheumatoid arthritis. hTNFtg mice were sacrificed from weeks 2 to 14 in 2-week intervals, and sections of the hind paws were analyzed for histopathological changes. Specifically, the cartilage area and thickness were determined by histomorphometry, and proteoglycan loss was assessed by toluidin-blue staining. Inflammation was quantified by measuring the area of hyperplastic synovial tissue. The length of adhesion zones between pannus and cartilage were taken as measures for synovial attachment. To study the effects of proteoglycan loss on the attachment of synoviocytes in vitro, synovial fibroblasts were seeded onto isolated and IL-1treated (2 ng/ml and 10 ng/ml, 24 hours) murine femoral heads, and the attachment was quantified by light microscopy. Results In hTNFtg mice, synovial inflammation increased over time (0.025 mm 2 at week 2 versus 0.65 mm 2 at week 14; P < 0.05), with the most prominent increase between weeks 4 and 6 (0.029 mm 2 and 0.78 mm 2 , respectively; P < 0.05). Loss of cartilage was seen only between weeks 2 and 4. After week 4, the cartilage area and thickness remained stable, arguing for a dissociation between cartilage degradation and pannus formation. As seen in toluidin-blue staining, a loss of proteoglycans occurred over time, which started at week 4 and peaked at week 8. There was a prominent attachment of pannus tissue to articular cartilage. Attachment started after week 4, peaked at week 10 and showed a decrease thereafter. This observation was supported by in vitro attachment data showing that early moderate loss of proteoglycans (as induced by 1 ng/ml IL-1) strongly enhanced attachment of synovial fibroblasts while an almost complete loss of proteoglycans (as induced by 10 ng/ml IL-1) did not facilitate fibroblast attachment.
cartilage erosion. Mast cell activation has been reported for a significant proportion of rheumatoid specimens. Because the mast cell contains potent mediators, including histamine, its potential contributions to the processes of inflammation and matrix degradation have recently become evident; thereafter, we investigated the potential protective effect of histamine deficiency against aggrecan-induced arthritis. To study the role of histamine in rheumatoid arthritis we investigated cartilage proteoglycan (aggrecan)-induced arthritis in histidine decarboxylase knockout (HDC-KO) mice, with complete lack of endogenously produced histamine. Aggrecan-induced arthritis was similar in HDC-KO and wild-type (WT) mice. Arthritis was even more severe in HDC-KO mice than in the WT animals 10 weeks following the immunization with aggrecan (arthritis score 1.2 ± 1.5 and 0 ± 0, respectively; P = 0.01). At later time points, arthritis scores were similar in both HDC-KO and WT mice. Since T-lymphocyte dysfunction has an important role in the pathogenesis of rheumatoid arthritis, next we investigated T-cell signal transduction and cytokine production in HDC-KO and WT mice. In the absence of histamine, elevated INFγ mRNA and protein levels of splenocytes (P < 0.001 and P = 0.001, respectively) were associated with a markedly increased (2.5-fold, P = 0.0009) nitric oxide (NO) production, compared with WT animals. Furthermore, histamine treatment decreased the NO production of splenocytes from both WT and HDC-KO mice (P = 0.001 and P = 0.0004, respectively). NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolatediethylenetriamine (NOC-18) elicited IFNγ production (P = 0.0002), suggesting the role of NO in regulating IFNγ synthesis. The cytoplasmic Ca 2+ concentration of unstimulated T cells and the Tcell activation-induced Ca 2+ signal were increased in T cells from the HDC-KO mice (P = 0.02 and P = 0.04, respectively), while the T-cell activation-induced CD3 internalization was similar in both HDC-KO and WT animals. Our present data indicate that histamine deficiency does not protect against aggrecan-induced arthritis. The Th1 cytokine pattern and increased NO production may both contribute to the sensitivity of HDC-KO mice to aggrecan-induced arthritis. Furthermore, our data indicate that histamine, in addition to its direct effects on T-lymphocyte function, regulates cytokine production and T-cell signal transduction through regulating NO production.
Mast cell-deficient W/Wv mice are resistant to K/BxN serum transfer arthritis, and this resistance may be overcome by engraftment with mast cells. However, the pathways by which mast cells participate in arthritis remain unknown. Using a candidate mediator approach, we explored IL-1 as a potentially key mediator by which mast cells promote arthritis. As expected, IL-1α/β-deficient mice were completely resistant to arthritis. Short-term administration of exogenous IL-1 restored an attenuated arthritis course in these animals, consistent with an ongoing requirement for IL-1. Surprisingly, deficient W/Wv mice treated with IL-1 at disease induction displayed a full normal course of arthritis, demonstrating that exogenous IL-1 can bypass the need for mast cells. TNF proved unable to exert a similar effect. We therefore engrafted IL-1 -/bone marrow-derived mast cells (BMMC) into W/Wv animals and found that these animals displayed resistance to arthritis equivalent to nonengrafted W/Wv mice, consistent with an obligate role for IL-1 of mast cell origin. Exploring further the mechanisms by which mast cells may become activated in this IgG 1 -driven model, we found that BMMC stimulated in vitro via FcγRIII elaborated IL-1, while BMMC lacking this receptor were unable to mediate arthritis upon engraftment into W/Wv recipients. While BMMC engrafted into W/Wv animals disproportionately populate the spleen, we excluded a contribution from this aphysiologic mast cell population via splenectomy and by documenting that systemic levels of IL-1 were not detectable during arthritis initiation in engrafted animals. We conclude that mast cells local to the joint and activated via FcγRIII promote K/BxN serum transfer arthritis by production of IL-1, an activity that appears delimited to the initiation of disease (the 'jump start').
The Mer receptor tyrosine kinase mediates apoptotic cell phagocytosis and modulates macrophage cytokine production. Mer knockout mice (Mer-KO) have defective clearance of apoptotic debris and develop systemic lupus erythematosus-like autoimmune disease. Because of their spontaneous autoimmune propensity, we wondered whether Mer-KO mice (backcrossed 10 generations onto C57BL/6) might be particularly susceptible to the systemic lupus erythematosus-like autoimmunity induced by chronic graftversus-host disease (GVHD). Using the well-established cGVH model (bm12→B6 mice), we were surprised to observe that the Mer-KO mice were protected from the development of GVHD. Mer-KO recipients of bm12 spleen cells failed to develop detectable anti-dsDNA and antichromatin autoantibodies, while the B6 hosts produced significant amounts of these antibodies that peaked at week 2. Slightly increased levels of rheumatoid factor were found in Mer-KO mice, although much lower than B6 control hosts. Mer-KO mice did not develop splenomegaly. The lack of autoantibody formation was not due to an absence of alloreactivity, because spleen cells from bm12 mice proliferated equally in response to irradiated wild-type and Mer-KO irradiated spleen cells. GVHD developed normally in F1 (Mer-KO x B6) hosts given bm12 donor cells, thus indicating that the failure to develop GVHD was not due to mer-KO-derived minor MHC genes. These findings indicate a hitherto unrecognized requirement for Mer in the genesis of alloreactivity-induced autoimmunity. Future experiments will determine whether this reflects the role of this kinase in recognition and phagocytosis of apoptotic cells, its function in regulating cytokine production, or perhaps a new function in the immune response. IL-21 promotes plasma cell (PC) differentiation while BAFF promotes B-cell survival. Here, we report that IL-21 synergizes with BAFF to elicit BLIMP-1 induction, PC differentiation and IgG production from a novel population of human splenic memory B cells. These human marginal zone analogue B cells are exquisitely sensitive to IL-21 and BAFF in the absence of further costimulation. The ability of IgG + marginal zone analogue to respond specifically and exclusively to IL-21 and BAFF demonstrates that they are uniquely poised to respond to antigen-independent signals and differentiate into IgG-producing PC, thereby replenishing serologic memory. Importantly, peripheral blood B cells from a portion of patients with systemic lupus erythematosus and rheumatoid arthritis were highly responsive to stimulation with IL-21 and BAFF. These data suggest that IL-21 and BAFF may be capable of inducing PC differentiation from memory B cells with autoreactive specificities and thereby contribute to autoimmunity.

IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from human marginal zone B cells as well as from circulating peripheral blood B cells from autoimmune patients
Introduction IL-33 (or IL-1F11) was recently identified as a ligand for the previously orphaned IL-1 family receptor T1/ST2. IL-33 belongs to the IL-1 family of cytokines and upon binding to T1/ST2 induces intracellular signals similar to those of IL-1. Previous studies have established that IL-33 and T1/ST2 exert key functions in Th2 responses. Mast cells are known to express T1/ST2. Besides their well-known function in allergy and host defense, mast cells have recently been shown to play an important role in experimental models of arthritis. Objective To examine the effect of IL-33 on P815 murine mastocytoma cells and primary mouse bone marrow-derived mast cells (BMMC). Results IL-33 dose-dependently and time-dependently stimulated IL-6 secretion by P815 mastocytoma cells and BMMC. Both cell types express T1/ST2 as assessed by RT-PCR and FACS analysis. This effect was dependent on T1/ST2 binding. The addition of ST2-Fc on BMMC inhibited the stimulatory effect of IL-33, whereas etanercept had no effect. IL-1RAcP-Fc alone or in combination with ST2-Fc inhibited the effect of IL-33, indicating that IL-33 binds to these soluble receptors. In addition, IL-33 also induced IL-1β, TNFα, MCP-1, and PGD2 production in BMMC. By the RNase protection assay, we demonstrated that IL-33 increased IL-6 and IL-1β? mRNA expression. These effects of IL-33 appeared to occur independently of mast cell degranulation.

Conclusion
The results of this study show for the first time that IL-33, a novel member of the IL-1 family of cytokines, stimulates the production of proinflammatory mediators by mast cells in addition to its effect on Th2 responses. These findings open new perspectives for the treatment of inflammatory diseases by targeting IL-33. Background Dendritic cells (DC) can be alternatively activated to induce tolerogenic DC, making them a promising therapy for autoimmunity. Because naïve and memory T cells have different requirements for tolerisation, we tested the modulatory activity of tolerogenic DC on both subsets of T cells. Methods Human monocyte-derived DC were matured with lipopolysaccharide (LPS-DC) or treated with dexamethasone, vitamin D3 and lipopolysaccharide (LPS-DexD3 DC) to obtain tolerogenic DC. DC were cocultured with allogeneic naïve or memory CD4 + T cells. Primed T cells were rested and restimulated with LPS-DC or CD3/CD28 beads. Results and conclusions LPS-DexD3 DC have reduced stimulatory capacity for both naïve and memory T cells. However, restimulation of T cells revealed a distinct difference between naïve and memory T cells that had been primed by LPS-DexD3 DC. Naïve T cells did not become anergic but were skewed to a regulatory phenotype (low IFNγ and high IL-10 production), whereas memory T cells were rendered hyporesponsive, with low proliferation and cytokine production. Thus, naïve and memory T cells are differently regulated by LPS-DexD3 DC. These data have implications for the use of tolerogenic DC vaccines as immunomodulators. Human monocyte-derived dendritic cells (DC) treated with dexamethasone and vitamin D3 (DexVitD3 DC) have tolerogenic properties and phenotype. DexVitD3 DC induce limited T-cell proliferation in an allogeneic mixed lymphocyte reaction and inhibit the T-cell proliferation induced by lipopolysaccharide and/or cytokine (TNFα/IL-1β) matured DC. Furthermore, T cells primed with DexVitD3 DC proliferate in response to restimulation with a distinct cytokine profile including significantly reduced production of IFNγ. DexVitD3 DC have characteristically low expression of the costimulatory molecules CD80/83/86 with high HLA-DR. Upon stimulation with lipopolysaccharide, DexVitD3 DC maintain their surface phenotype and produce large quantities of IL-10. We are currently characterising the cytokine profiles of DexVitD3 DC and the T cells they prime, as well as investigating whether these T cells have regulatory properties.

Generation of dexamethasone and vitamin D3-treated human monocyte-derived dendritic cells with tolerogenic properties
Cellular therapies are being explored as treatment for autoimmune diseases including rheumatoid arthritis. DexVitD3 DC have potential for future use in this field as well as being a useful tool to elucidate mechanisms of immune regulation. The antigens that trigger the pathogenic immune response in rheumatoid arthritis remain unknown. Until recently it was assumed that joint-specific antigens were the targets of arthritogenic T lymphocytes and B lymphocytes in rheumatoid arthritis. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/BxN T-cell receptor transgenic mouse model, arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I or SJL mice [1,2]. T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4 cells. In contrast, antibodies and FcγR effector cells are necessary but not sufficient for G6PIinduced arthritis in genetically unaltered mice [1]. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.

Regulation of eicosanoid production in peripheral blood mononuclear cells from patients with systemic sclerosis
Otylia Background Eicosanoids are arachidonic acid-derived mediators that play a key role in the regulation of inflammatory response. 5-Lipoxygenase (5-LOX)-derived leukotrienes are considered proinflammatory while 15-lipoxygenase (15-LOX)-derived products such as 15-hydroxyeicosatetraenoic acid (15-HETE) and lipoxins inhibit proinflammatory mediators including leukotrienes, and actively participate in the resolution of inflammation. It has been demonstrated that a temporal switch of arachidonic acid metabolism from predominant 5-LOX-derived to 15-LOX-derived products is crucial for the resolution of inflammation. We have recently shown that there is an imbalance between proinflammatory leukotrienes and anti-inflammatory lipoxins in the lungs of patients with systemic sclerosis (SSc)-related interstitial lung disease, which may favour chronic inflammation and fibrosis [1,2]. Objective To further investigate the role of eicosanoids in the pathogenesis of SSc through evaluation of: (1) the basal profile of eicosanoid synthesis, and (2) the pattern of eicosanoid biosynthesis in response to proinflammatory stimuli by peripheral blood mononuclear cells (PBMC) from patients with SSc in comparison with healthy people. Methods Mononuclear cells were isolated from peripheral blood using density gradient centrifugation on Lymphoprep and cultured in fetal calf serum-supplemented RPMI medium at 37°C under 5% CO 2 . Ionophore-stimulated production of 5-LOX-derived leukotriene E 4 (LTE4) and 15-LOX-derived 15-HETE was evaluated by means of enzyme immunoassay at predefined time points in basal conditions (without stimuli) as well as in response to TNFα. Ten patients with SSc (five diffuse and five limited cutaneous SSc) as well as five healthy controls were studied. Results There were no significant differences in the basal production of LTE4 or 15-HETE between SSc patients and healthy controls. TNFα induced sequential changes in the production of eicosanoids, with an early (within 1 hour) increase in LTE4 followed by a delayed increase in 15-HETE in both SSc patients and healthy controls. PBMC from SSc patients responded to TNFα with significantly higher production of LTE4 in comparison with healthy controls (P < 0.05 at 1 hour), while there were no significant differences in TNFα-induced production of 15-HETE between SSc patients and controls. Conclusions These preliminary results of our study indicate that the TNFα-induced eicosanoid synthesis is altered in PBMC from patients with SSc. Increased production of proinflammatory leukotrienes may contribute to the pathogenesis of SSc. (POLη). To characterize the nature of other mutations, 316 genomic nonproductive V(D)J rearrangements amplified and sequenced from three AID -/patients were analyzed. The mutation frequency was 20-fold less than normals (0.09% versus 2%) but 5 x 10 4 -fold more than non-B cells. Reduced RGY (6% versus 27%) and WRC (6% versus 15%) motif mutations and decreased replacements in complementarity determining regions were attributed to the lack of AID. Reduced WA motif mutations (12% versus 23%) suggested POLη activity was decreased. Prominent G>C and A>T biases suggested that mutation and repair mechanisms occurred preferentially on one DNA strand. A high percentage of the mutations were G substitutions on the sense strand (43% versus 29% in normals), reflecting C mutational targeting on the opposite strand. These were primarily G to A transitions, suggesting that UNG activity was reduced even though UNG is upregulated in normal GC B cells and large GCs occur in AID -/patients. The mutation pattern suggests that AIDindependent C substitutions contribute a small proportion of SHM but lack the targeting provided by AID. Finally, a low level of SHM clearly can develop in the absence of AID; however, this mechanism is inefficient at altering the binding capacity of immunoglobulin heavy chain genes. Background We previously demonstrated in minor salivary glands of patients with Sjogren's syndrome (SS-mSGs) that a true phenomenon of ectopic lymphoneogenesis takes places, with the formation of germinal centres (GCs), in association with the ectopic expression of the lymphoid chemokines (CKs) CXCL13 and CCL21. In Sjogren's syndrome major salivary glands (SS-MSGs) these structures are known to support clonal immunoglobulin heavy chain gene rearrangements and somatic hypermutation, favouring expansion and selection of autoreactive B-cell clones, clonally related to lymphomatous B-cell clones (responsible for lymphoma development in Sjogren's syndrome (SS)). The involvement of lymphoid CKs and CXCL12 in the development of lymphoid malignancies has been demonstrated. At present, however, no data on the expression of these CKs nor their cellular source within SS-MSGs and mucosal-associated lymphoid tissue lymphomas (MALT-Ls) from patients with SS have been described. Objective To assess the specific contribution of lymphoid CKs in the organization of lymphoid proliferation within SS-MSGs and SS-MALT-Ls. Methods We studied 12 SS-mSGs, four SS-MSGs with lymphoepithelial lesion and 20 SS-MSGs with non-Hodgkin B-cell MALT-Ls. In order to define the histological organization of the lymphoid infiltrate (reactive versus malignant areas) and identify the B-cell subpopulations infiltrating the glands, immunohistochemistry was carried out for cellular (CD21, bcl-2, bcl-6, IgD, CD20, CD3, CD68) and vascular markers (CD31, PNAd). On sequential sections, digital images for CXCL13, CCL21 and CXCL12 were analysed by thresholding positive staining in detected areas of interest. Digital image analysis estimated CK's volume fraction areas as a ratio between CXCL13, CCL21 and CXCL12 positive areas over malignant or reactive areas. CK's producing cells were identified by double staining on sequential sections. Mononuclear cells were isolated from two SS-MALT-L parotids following enzymatic digestion and stained for CD19, CD38, CD27, CD24, IgM, IgD, CD10, CD5, CD3, CD4, CD69, CD45RO, CXCR5, CXCR4 and CCR7. Finally, mRNA from total SS-MSGs and MALT-Ls and isolated B cells and T cells from MALT-Ls were analysed by RT-PCR for the transcript levels of CXCL13, CCL21 and CXCL12. Results Reactive areas, characterized by T-cell/B-cell segregation, CD20 + IgD + bcl-2 + follicular B cells, presence of follicular dendritic cell networks in GCs and high endothelial venule formation were detected in 100% of MALT-Ls. A B-cell population (CD20 + IgDbcl-2 + ) characterized by nuclear abnormalities and monocytoid appearance was consistently observed within the proliferating ducts and identified as the B-cell malignant component. FACS analysis on isolated MALT-L mononuclear cells showed B cells in diverse maturative stages (transitional, mature and memory B cells) and a B-cell population CD19 pos IgD low CD24 neg CD27 low/neg CD5 neg CD10 neg not detectable in the controls. Ectopic expression of CXCL13 and CCL21 was observed in 100% of SS-MSGs and MALT-Ls. A significant increase in CXCL13's volume fraction analysis was observed both in SS-MSGs and SS-MALT-L reactive areas, as compared with SS-mSG follicular areas (P < 0.05 and P < 0.05, respectively). A strong difference in CXCL13 within the MALT-L reactive area compared with malignant areas was detected (P < 0.001). CCL21 was significantly increased in MALT-Ls compared with both SS-mSGs and SS-MSGs and was mainly confined to the T-cell area. CXCL12 was strongly expressed by ductal epithelial cells, vessels and reactive areas in SS-mSGs, SS-MSGs and MALT-Ls. Interestingly a significant increase in CXCL12 expression on MALT-L malignant areas as compared with reactive areas in SS-mSGs and SS-MSGs was detected (P < 0.01 and P < 0.05). RT-PCR analysis showed increased CXCL13 and CCL21 level in SS-MSGs compared with MALT-Ls and SS-mSGs, while strong upregulation in CXCL12 transcript in MALT-Ls as compared with SS-mSGs and SS-MSGs was detected. CXCL13, CCL21 and CXCL12 were detected on CD68 + cells by immunohistochemistry, while CD20 and CD3, CXCL13 and CCL21double staining and mRNA analysis on MALT-extracted T and B lymphocytes showed negligible expression of the two CKs in MALT-L-extracted lymphocytes. Interestingly we identified strong CXCL12 expression on CD19 + MALT-L isolated cells both at the protein and mRNA level and on ductal epithelial cells in close contact with the malignant B-cell infiltration. In agreement with this increase, we demonstrated a significant downregulation of CXCR4 on MALT-L-isolated B cells, likely to be the result of CXCR4 internalization upon CXCL12 ligation. Conclusion In SS-MALT-Ls a strong upregulation of the lymphoid CKs CXCL13 and CCL21 takes place and is associated with the organization of the reactive areas involved in the maintenance of the autoimmune process within the malignancy. These findings support a contributory role for CXCL13 and CCL21 in the pathogenesis of MALT lymphomas in SS. These results imply that the CK/CKreceptor axis is functional in MALT-L malignant cells, and suggest that the glandular microenvironment, B-cell receptor signalling upon antigen engagement and autocrine signals from the same malignant population concur in MALT lymphomagenesis, triggering local activation of malignant B cells and favouring their survival and expansion.
an elevated expression of stromelysin-1 (MMP-3). We therefore investigated the regulation of additional matrix metalloproteinases (MMPs) and their specific tissue inhibitors of matrix metalloproteinases (TIMPs) by attachment to LM-111 in the presence of transforming growth factor beta (TGFβ). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative RT-PCR. Production of MMPs and cytokines was monitored by a multiplexed immunoarray or by ELISA. Signal transduction pathways were studied by immunoblotting. Attachment of SF to LM-111 in the presence of TGFβ induced significant increases in stromelysin-1 mRNA (12.35-fold, P < 0.001) and protein (mean 62 ng/ml, sixfold, P < 0.008). Expression of stromelysin-2 (MMP-10) mRNA (11.68-fold, P < 0.05) and protein (54 ng/ml, 20-fold, P ≥ 0.02) was significantly activated as well. All other TIMPs and MMPs investigated failed to show this LM-111facilitated TGFβ response. Induction of stromelysin-1 and stromelysin-2 was associated with the activation of transcription factors c-fos and Egr-1, but phosphorylation of NF-κB was not observed. Further, LM-111-activated and TGFβ-activated SF failed to produce remarkable amounts of IL-1β or TNFα. We conclude that costimulation of synovial fibroblasts by LM-111 together with TGFβ suffices to induce significant expression of MMP-3 and MMP-10 by SF and that this induction is independent of phosphorylation of NF-κB.
Background Single nucleotide polymorphisms (SNPs) of the TNF gene at positions -238 and -308 have earlier been associated with psoriasis vulgaris and psoriatic arthritis (PsA). However, a strong linkage disequilibrium at the chromosomal region 6p21 renders the interpretation of these findings difficult since also other risk factors for psoriasis (PSORS1) than SNPs of the TNF gene have bee mapped to that particular region. Therefore, in this study several SNPs of the TNF gene and of its neighbouring lymphotoxin α (LTA) gene were analysed independently and dependently on carrying the PSORS1 risk allele. Methods SNPs in the promoter of the TNF gene (-238G/A, -308G/A, -857C/T, -1031T/C), and one SNP of the LTA gene (+252A/G), of the TNLFRSF1A gene (+36A/G) and of the TNLFRSF1B gene (+676T/G), respectively, were genotyped in 375 psoriasis patients, 375 PsA patients, and 376 controls. The tryptophan-tryptophan-cysteine-cysteine haplotype of the CCHCR1 gene (CCHCR1*WWCC) was used to estimate the genetic impact of the PSORS1 risk allele. Results Whereas an earlier-described association of allele TNF*-238A with psoriasis could be confirmed, our study revealed that this association was completely dependent on concomitant carriage of the PSORS1 risk allele. For PsA, but not psoriasis vulgaris without joint manifestations, strong association with the allele TNF*-857T was detected (OR = 1.956; P value corrected for multiple testing, P corr = 0.0025) also in patients negative for the PSORS1 risk allele. Conclusions Our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. While the previously reported association between TNF*-238A and psoriasis seems to primarily reflect linkage disequilibrium with PSORS1, TNF*-857T may represent a risk factor for PsA independent of PSORS1. A potential pathophysiologic relevance of the elucidated genetic association is further suggested by previously reported experimental evidence for a functional impact of the respective TNF polymorphism on TNFα expression levels. Background There are few rheumatologists who now use the technique of arthroscopy; in most centers, referral is made to an orthopedic surgeon who specializes in this technique. It has become clear in recent years that the synovium is the primary site of inflammation and the major effecter organ in a variety of joint diseases including rheumatoid arthritis [1]. The present retrospective study was primarily carried out to assess the safety of this procedure in the hands of a rheumatologist and also to assess the utility of the procedure in various joint diseases. Objective To evaluate the safety and diagnostic value of medical arthroscopy performed by a rheumatologist. Methods Decisions for performing arthroscopy were taken when a detailed clinical history and relevant rheumatological investigations failed to arrive at a definite diagnosis. Initial assessment included detailed clinical history and relevant diagnostic tests for rheumatic diseases. Routine blood count, bleeding time, clotting time and biochemical parameters were also determined. After obtaining written consent, arthroscopies were performed as an outpatient department procedure using a 4 mm arthroscope with 0° and 30°v iewing. All the procedures of arthroscopy were performed by a single rheumatologist. Procedures were done in a minor operating theatre under local anesthesia using strict aseptic precautions. Xylocaine 2% and bupivacaine 0.5% were used as local anesthetic agents for skin portal and intraarticular anesthesia, respectively. A tourniquet was not used during the procedures and the skin portal was not stitched. Synovial biopsies taken during procedures were subjected to only histopathological examination. All the procedures were recorded on a video-cassette. Results See Tables 1 and 2. None of the patients developed any complications during the procedure or after 6 months of follow-up. None of the patients experienced any discomfort during the procedure except for a mild stretching sensation experienced by male patients. Females tolerated the procedure better than males. Conclusion Arthroscopy using a 4 mm scope in the hands of rheumatologists is a safe daycare procedure. It can be performed without a tourniquet. The local anesthetic agent bupivacaine provides an excellent local anesthesia. Females tolerate the procedure better than males. In a few cases arthroscopy helped in arriving at a final diagnosis but many patients remained undiagnosed. Both the rheumatologists and the pathologists require further experience in this field. Blockade of TNF, using current biologics, has had profound therapeutic effects; nevertheless, between 30% and 40% of RA patients do not respond to this treatment. The mechanisms of response and nonresponse to biologic therapy remain unclear. p38 MAPK is present in the rheumatoid synovium and thought to play a role in the pathogenesis of RA, suggested by evidence that p38 is required for TNF-induced inflammatory processes. There are four p38 MAPK isoforms, α, β, γ and δ, which are found in varying levels in inflammatory cells. Although most research has to date focused on p38α, it has been demonstrated in the synovium that the functional protein produced from the L1 retrotransposable element can specifically induce p38δ. This suggests a possible second p38-mediated pathway of joint destruction in RA that may not be S9 blocked by anti-TNF therapy. This study aimed to correlate the expression of p38δ in the rheumatoid synovium with clinical response and nonresponse to the anti-TNF biologic, infliximab (Remicade).
Methods We examined the expression of p38 MAPK isoforms (α and δ) pre and post infliximab therapy in the synovium of RA patients, to compare clinical response with nonresponse. All patients entered into this study have failed at least two diseasemodifying drugs, and fulfil the revised 1987 ACR criteria for RA. The effect of TNF blockade in five responders and three nonresponders on the p38 MAPK α and δ isotypes was studied; expression and activation (phospho-p38) in synovial biopsies of responders and nonresponders was measured by immunohistochemistry using a semiquantitative scoring system, at baseline and approximately week 16 of therapy. A therapeutic effect was determined by patients achieving an ACR20 response at week 14, and the Wilcoxon signed rank test was performed to assess significance of changes in expression levels post treatment.

Results
In responders, p38α, p38δ and phospho-p38 expression were all significantly decreased post infliximab treatment in the synovial sublining layer (P < 0.05). A similar trend was seen in the synovial lining layer of responders, with a decrease in expression of p38α and p38δ and phospho-p38 post infliximab treatment in four of five responders, although these results did not reach statistical significance. p38δ and phospho-p38 in the sublining layer and phospho-p38 in the lining layer increased, or remained high, in all three nonresponders tested, and this tendency was the opposite to that seen in the responders. Conclusion In the RA synovium, expression and activation of p38α and p38δ in the sublining layer correlated with response, and increased p38δ expression and activation was associated with nonresponse. A flow cytometric-based investigation of p38 isoform expression/activation in peripheral blood mononuclear cells has been initiated.

P21
The synovial cadherin (cadherin-11) promotes intercellular motility Cadherin-11 is a homophilic adhesion molecule that is expressed on fibroblast-like synoviocytes. Cadherins mediate tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. To determine the molecular mechanisms that contribute to cadherin-11-dependent cell migration, we generated cadherin-11 mutants. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of α-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 ΔJMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these studies implicate the cadherin-11 cytoplasmic JMD as a regulator of intercellular motility and cellular rearrangement in multicellular clusters and provide insight into a critical pathway that determines the behavior of fibroblast-like synoviocytes in arthritis, especially rheumatoid arthritis.

P22
Cadherin-11 regulates synovial fibroblast behavior in health and disease Cadherin adhesion molecules, structurally typified by their immunoglobulin-like extracellular (cadherin) domains rigidified as an extended chain by interdomain calcium binding, mediate cellular adhesion by binding a cadherin of the same type on an adjacent cell (homophilic adhesion). During embryonic development, cadherins provide a basis for cell sorting, aggregation and resultant tissue morphogenesis and they are thought to contribute to tissue maintenance postnatally. In tumors, cadherins have been implicated in tissue extension, migration and invasion. Knowing these processes are active in the synovium both at baseline and in the context of the hyperplastic and invasive synovial tissue in inflammatory arthritis, we hypothesized that a cadherin may play a role in regulating this tissue behavior. Using mouse models, we demonstrate that synovial tissue fibroblasts express cadherin-11. We find that cadherin-11 null mice display a hypoplastic synovial lining and display attenuated arthritic responses to K/BxN serum transfer arthritis. Moreover, we find a remarkable decrease in synovial pannus invasion into cartilage. These observations reveal a role for cadherin-11 in the molecular regulation of the organized behavior of the synovial fibroblast both in health and disease.

P23
Toll-like receptor 9 agonists and IL-15 promote activation, proliferation, secretion of proinflammatory cytokines and differentiation of B cells isolated from bone marrow of rheumatoid arthritis patients Recent studies have identified CD74, the cell surface form of the class II-associated invariant chain, as a binding site for the cytokine macrophage migration inhibitory factor (MIF). MIF is implicated in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus, but a functional relationship between MIF and CD74 in inflammation has not been demonstrated. We used CD74 -/mice to examine the role of this molecule in cellular and in vivo inflammatory responses, using innate immune responsedependent models to avoid confounding by the role of MHC Ii in adaptive immunity. MIF has a demonstrated role in macrophage responses to endotoxin. LPS induced IL-1, IL-6, TNF and MIF release from WT bone-marrow macrophages. Bone-marrow macrophages lacking CD74 released significantly lower amounts of IL-1 and IL-6, but no difference in TNF release was evident. LPS-induced MIF release by WT and CD74 -/bone-marrow macrophages was also similar. Murine K/BxN serum-transfer arthritis is MIF dependent [1], and also requires the induction of IL-1. Compared with WT mice, arthritis severity (clinical score) was significantly lower in mice lacking CD74 (P < 0.05). These data imply but do not demonstrate a role of CD74 in MIF-dependent inflammatory responses.
To confirm the role of CD74 in responses to MIF in vivo, we examined a recently described action of MIF -the induction of leukocyte trafficking [2]. rhMIF was injected into mouse cremaster tissue and leukocyte-endothelial interactions examined using intravital microscopy. MIF induced leukocyte adhesion and emigration in vivo in WT mice. In CD74 -/mice, in contrast, this response could not be elucidated. These data demonstrate for the first time the role of CD74 in innate immune responses and inflammatory arthritis, and demonstrate for the first time in vivo the requirement for CD74 for the action of MIF. A functional role for CD74 in MIF responses in inflammation is strongly supported. References The mitochondrial mass, NO production and cytoplasmic Ca 2+ levels were measured by flow cytometry. Mitochondria were visualized using transmission electron microscope. T cells from RA patients produce >2.5 times more NO than T cells from healthy donors (P < 0.001). Unexpectedly, the mitochondrial mass was found to be similar in RA and control T cells (P = 0.65), whilst increased NO production was associated with increased cytoplasmic Ca 2+ concentrations in RA T cells (P < 0.001). We observed that T-cell NO production decreased in most RA patients following anti-TNF treatment. Although lupus T cells produced comparable amounts of NO to normal T cells, lupus monocytes produced twice as much NO as normal monocytes (P = 0.015). We also observed increased mitochondrial mass (47.7 ± 2.8%; P = 0.00017) and increased cytoplasmic (38 ± 6.4%; P = 0.0023) Ca 2+ content in T cells from SLE patients when compared with control donors. Electron microscopy revealed that T cells of lupus patients contained 8.76 ± 1 mitochondria, while control donors contained 3.18 ± 0.28 mitochondria per cell (P = 0.0009). In addition, lupus lymphocytes harbor several-fold enlarged megamitochondria. These data suggest that monocytes are the primary source of NO in SLE, while T lymphocytes are the primary source of NO in RA. Although the iNOS pathway is not as rapid as eNOS or nNOS, it is thought to be capable of generating much larger quantities of NO (nanomolar range) than the constitutive NOS isoforms (picomolar range), explaining the differences in T-cell mitochondrial biogenesis in SLE and RA. Since mitochondria can take up, store and release Ca 2+  We have previously demonstrated reduced circulating levels of IL-7 in active rheumatoid arthritis (RA) compared with health [1]. Controversy exist as to whether IL-7 correlates or not with C-reactive protein (CRP) in active disease or with other disease activity markers. Normal IL-7 levels were, however, found in 50% of patients in clinical remission and correlated with the recovery of thymic activity [1]. Patients in clinical remission may represent a heterogeneous group, and the aim of this work was to identify predictors of IL-7 recovery in demographic, clinical, imaging and functional data and to compare them with IL-7 in active disease. One hundred and six patients deemed to be in clinical remission were recruited: stable disease for the preceding 6 months, previous disease duration of at least 12 months, no clinically significant synovitis, CRP below 15 mg/l for the preceding 6 months. Blood and serum were collected from these patients (n = 106). Clinical data and imaging data were gathered at the time of sampling. High-sensitivity ELISA was used for cytokine analysis, proliferation assays for T-cell function (mitogen, T-cell receptor stimulation, IL-2, recall antigens); real-time PCR to quantify T-bet and GATA3 expression, and DNA sequencing was carried out to investigate two genetic polymorphisms in the IL-7 gene. Several studies in healthy controls indicate that normal levels of circulating IL-7 are between 10 and 25 pg/ml. In remission, circulating levels of IL-7 vary between 2.47 and 23.85 pg/ml. Recovery of T-cell function was directly related to levels of circulating IL-7 in vivo (P < 0.001, R = 0.873 for phytohaemagglutinin, R = 0.786 for T-cell receptor stimulation and R = 0.821 for recall antigen). Age and sex had no effect on IL-7. Combining active (n = 35) and remission patients (n = 106) showed no indication of a relationship between IL-7 and routine measures of disease activity (CRP, DAS28, erythrocyte sedimentation rate, plasma viscosity and joint counts), suggesting that recovery of IL-7 is not an indicator of remission. To confirm this observation, we used imaging data (MRI and US assessment of hand and wrist) to address whether subclinical disease could predict lack of IL-7 recovery. Evidence of subclinical synovitis was found in 96% of patients in remission [2] despite no evidence of clinically significant synovitis and there was no relationship between IL-7 and imaging scores. However, we found that levels of IL-7 in remission and the age of the patient at disease onset correlate (P < 0.001, R = 0.498, n = 86). A family history of RA and smoking at the time of onset (both self-reported) were also strongly associated with lower levels of IL-7 (both P < 0.001, n = 66). We analysed two polymorphisms in the promoter and enhancer regions of the IL-7 gene. We found no allele frequency difference between RA patients and healthy controls, remission patients with or without family history, or in relation to IL-7 levels. Finally we analysed circulating cytokines known to regulate IL-7 expression, and found a strong correlation between IL-7 and IFNγ (P < 0.03, R = 0.650, n = 10). Since IL-7 is a co-activator of Th1 polarisation, we analysed the expression of T-bet and found a direct relationship between the two (P < 0.01, R = 0.601, n = 15) but not with GATA3, which is a regulator of Th2 polarisation. Recent evidence suggests an important role for IL-7 in the pathogenesis of RA [3]. Our data demonstrate that circulating IL-7 is not an indicator of disease activity. Despite the relationship with age at onset (genetic anticipation) and the family history association, levels of IL-7 in the circulation are apparently not driven by these two polymorphisms. Our results suggest that Th1 polarisation and IL-7 are related and that the mechanism by which IL-7 is recovered in remission may be associated with the recovery of Th1 polarisation. Background Recent evidence suggests an important role for IL-7 in the pathogenesis of rheumatoid arthritis (RA) and a therapeutic potential for IL-7 blockade [1]. IL-7 is produced by synovial stromal cells (StrC) in RA but is barely detectable in osteoarthritis [2]. Our published data showed reduced production of IL-7 by bone marrow (BM) StrC and reduced levels of circulating IL-7 in RA compared with health [3]. IL-7 was also detected in synovial tissue in active RA but levels were below detection in clinical remission. These conflicting observations suggest that the regulation of IL-7 expression is tightly controlled at the level of tissue specificity. To support this hypothesis, we showed that several cytokines have a different effect on IL-7 production in BM StrC, epithelial cells from the liver and gut. This work aims at identifying the factors that regulate IL-7 production in StrC isolated from synovial tissue of RA patients.

Santos LL, Dacumos
Results Synovial fluid and tissue biopsy were obtained from RA (n = 6) and osteoarthritis (n = 4) patients (documented with inflammation scores, visual analogue score) and BM aspirates from healthy donors (n = 6). Skin fibroblasts were used as negative control. We quantified IL-7 in freshly isolated StC (using an advanced cell-sorting strategy [4]) from healthy BM and showed that IL-7 expression is high. However expression declined rapidly with tissue culture (20-fold by passage 2), suggesting that environmental factors are required to sustain IL-7 expression in vitro. IL-7 expression was threefold lower in expanded StrC from RA synovial tissue compared with healthy BM; however, levels were also directly correlated with a visual analogue score of inflammation performed during arthroscopy (ρ = 0.930, P < 0.001). IL-7 expression is regulated by several cytokines. IFNγ induced IL-7 by ninefold in health but this regulation is lost in RA in direct relationship with levels of inflammation (ρ = 0.842, P < 0.001). In contrast, TNFα and IL-1β induced IL-7 by an average of threefold and 15-fold, respectively, in RA but had no effect on healthy BM StrC. The effect of these cytokines was again correlated with exposure to inflammation (ρ = 0.883, P < 0.001 for TNFα and ρ = 0.964, P < 0.001 for IL-1β). TGFβ 1 reduced IL-7 expression in health (fivefold) but had no effect in RA. Therefore, the promoter of IL-7 is only sensitive to regulation by other cytokine at low levels of exposure to inflammation. In contrast, IL-7 expression is maximal at high levels of inflammation and cannot be further increased by other cytokines. Conclusion The expression of IL-7 is tightly controlled in health, the accessibility of the IL-7 promoter to regulation by other cytokines probably being restricted by tissue specificity. In contrast, in RA joints this regulation is abrogated in direct relation with exposure to inflammation. The specific effect of cytokine is therefore only observed when inflammation is low, suggesting promiscuity on the promoter of the IL-7 gene. This may be resulting from an alteration of the IL-7 promoter at the epigenetic level possibly through the effect of Line 1-mediated hypomethylation of chromatin [5]. Background The potential value of autologous mesenchymal stem cells (MSCs) derived from a patient's joint is now well recognized in osteoarthritis (OA) and rheumatoid arthritis (RA) for the repair of bone and cartilage damage. However, the fact that MSCs have been directly exposed to inflammation in the joint has not been given sufficient attention. We have shown recently that chondrogenesis was qualitatively diminished in MSCs derived from the synovial fluid of RA patients compared with OA [1]. In this report, we investigate the effect of the joint microenvironment on MSCs' repair capability in vitro and the possible molecular mechanism by which cells become deficient. Results Synovial tissue was obtained during arthroscopy from 15 patients with RA and OA, fully documented with visual analogue scores, which were validated as measure of inflammation locally. High and low levels of inflammation were observed for both diseases. We observed an inverse relationship between levels of inflammation in the joint (VAS) and the chondrogenic potential of MSCs (R = -0.750, P < 0.01) using an in vitro differentiation assay. Such a relationship was not observed for bone differentiation. The induction of two specific transcription factors (TF) is necessary for differentiation to take place: Sox9 for chondrogenesis and Runx2 for osteogenesis. We measured the levels of expression of these TF in MSCs isolated from nine synovial fluids of RA and OA patients and compared them with MSCs isolated from healthy bone marrow donors (n = 6) and skin fibroblasts (negative control, n = 4). Sox9 expression was higher in OA than in bone marrow but was reduced to negative control levels in RA. Runx2 expression was reduced in both RA and OA. Sox9 and Runx2 expression was also investigated in synovial tissue MSCs from six RA patients. Sox9 and Runx2 expression were inversely correlated with VAS scores for inflammation (R = -0.737, P < 0.01 and R = -0.843, P < 0.01, respectively).
To investigate the effect of proinflammatory cytokines on the ability of MSCs to differentiate we quantified the expression of Sox9 and Runx2 following treatment with TNFα, TGF-β 3 , and IFNγ. TGF-β 3 is a trigger of chondrogenesis and accordingly increases the expression of Sox9 and Runx2 in bone marrow MSCs (n = 4), whereas TNFα and INFγ reduce the expression of both TF. In RA (n = 6), TGF-β 3 had lost the ability to induce the expression of Sox9 and Runx2 whereas TNFα and INFγ increased it. The cytokine effects were all directly correlated with previous exposure to inflammation (P < 0.05). Therefore, in vivo the joint 'milieu' (which includes these cytokines and several others) is unlikely to allow the induction of a differentiation pathway where TF must be expressed at the right time and amounts and in the right order. Finally, using tissues collected pre and post biologic treatment, we also demonstrated that this deficit can be corrected in vivo in a controlled joint milieu (but so far not in vitro) -raising the hope that one day cell-based therapies for cartilage repair will be available. Conclusion Altogether, these data demonstrate that exposure to inflammation has a profound effect on the ability of MSCs to respond to differentiation triggers. Our data also suggest that cytokine(s) are likely to be involved in the acquired deficit in chondrogenesis in RA. Controlling joint inflammation with anti-TNF is associated with the downregulation of inflammation-related parameters (including bone damage [2]) and would therefore be necessary, but may not be sufficient to allow MSCs to undertake cartilage repair. We also determined rheumatoid factor (RF) and compared it with anti-CCP antibodies for the area under the curve (AUC), sensitivity, specificity and likelihood ratios. In RA patients we assessed the activity score, and radiographic joint score. Extra-articular manifestations were recorded in an additional 75 RA patients.

Results
The sensitivity of anti-CCP antibodies for RA was 63.2% and the specificity was 95.0%. The respective values of RF were 59.1% and 91.2%. When considered simultaneously, the AUC for anti-CCP antibodies was 0.90 with a 95% CI of 0.87-0.93 and the AUC for RF was 0.71 with a 95% CI of 0.64-0.77. The presence of both antibodies increased specificity to 98.2%. Anti-CCP antibodies were positive in 34.9% of RF-negative RA patients. Anti-CCP antibodies exhibited a correlation with the radiographic joint damage (Spearman correlation coefficient r = 0.27, P = 0.001). Anti-CCP-positive RA patients had increased the swollen joint count and serum CRP concentration compared with anti-CCP-negative RA patients (Mann-Whitney U test, P = 0.01 and P < 0.001, respectively). However, no correlation was found between anti-CCP antibodies and the DAS28 score (r = 0.13, P = 0.12). RA patients with high anti-CCP2 antibodies (>100 IU/ml) were more likely to have extra-articular manifestations. Conclusion In Greek patients with RA, anti-CCP2 antibodies exhibit a better diagnostic value than RF. Anti-CCP2 antibodies show a correlation with joint damage whereas high levels of anti-CCP2 antibodies are associated with extra-articular manifestations. Anti-CCP2 antibodies are therefore useful in rheumatology practice.
Background CD20-directed therapy is well established in oncology, and was recently demonstrated to be highly effective in the treatment of rheumatoid arthritis (RA) [1]. Small modular immunopharmaceutical drugs are smaller than antibodies yet are capable of utilizing effector functions of antibody-dependent cellular cytotoxicity, complement-mediated cytotoxicity and apoptotic signaling. TRU-015 is a small modular immunopharmaceutical drug candidate that is specific for CD20. TRU-015 has been demonstrated to effectively deplete B lymphocytes in cynomolgus monkeys in a dose-dependent manner, and to improve survival in mouse xenograft tumor models [2,3]. Objectives This phase 1 dose escalation study was performed to evaluate the safety, pharmacokinetics and pharmacodynamics of TRU-015 in subjects with RA. Methods Thirty-seven RA subjects receiving a stable background of methotrexate were enrolled in eight dosage groups (single dosages of TRU-015 of 0.015, 0.05, 0.15, 0.5, 1.5, 5, or 15 mg/kg, or two doses of 15 mg/kg given 1 week apart (30 mg/kg)). Safety was assessed at baseline, during infusion and at prespecified intervals after infusion by clinical (adverse events, physical examination, vital signs) and laboratory parameters. Serum samples were collected for pharmacokinetic analysis. Pharmacodynamic response was measured using B-cell counts (CD19 + cells by flow cytometry) at prespecified timepoints. Subjects were evaluated for a minimum of 4 weeks and until B-cell recovery.
Results At least four subjects were exposed at each dose level. TRU-015 was generally well tolerated. No dose-limiting toxicities were observed, and no serious adverse events have been reported. Decreases in peripheral B-cell counts were observed in all dose groups at the first time-point after drug administration (24 hours), with doses ≥0.5 mg/kg causing depletion of peripheral B cells. The degree, duration, and recovery of B-cell depletion were dose dependent. Pharmacokinetic parameters of TRU-015 were calculated for cohorts receiving 0.5 mg/kg and higher, and are presented in Table 1. The overall mean half-life of TRU-015 was 295 hours. Conclusion TRU-015, a novel CD20-directed therapy, was generally well tolerated and resulted in dose-dependent B-lymphocyte depletion. Following intravenous infusion, pharmacokinetic properties of TRU-015 (area under the curve, maximum concentration of drug) were approximately dose proportional. Further evaluation of TRU-015 is warranted for the treatment of RA, and other autoimmune diseases.
Background Protein therapeutics directed toward CD20 antigen on B lymphocytes have been demonstrated to be highly effective in the treatment of rheumatoid arthritis (RA) [1][2][3]. Small modular immunopharmaceutical drugs are single-chain polypeptides that are smaller than antibodies. TRU-015 is a CD20-directed small modular immunopharmaceutical drug candidate that effectively depletes B lymphocytes in cynomolgus monkeys in a dose-dependent manner, and improves survival in mouse xenograft tumor models [4,5].
Objective Previously, a dose-escalation study in subjects with RA demonstrated that TRU-015 was generally well tolerated and resulted in dose-dependent B-lymphocyte depletion [6]. The present study was designed to further assess the safety and pharmacokinetics of TRU-015 and to additionally evaluate clinical responses in RA subjects with active disease treated with TRU-015. Methods Thirty-six RA subjects with active disease despite background methotrexate were enrolled in this randomized, double-blind, multicenter, placebo-controlled study. Subjects were enrolled into one of three cohorts in a 10:2 (active:placebo) ratio to receive TRU-015 as a single intravenous infusion of 5 mg/kg, two infusions of 2.5 mg/kg, or two infusions of 7.5 mg/kg. Subjects in cohorts with two infusions received the infusions 1 week apart. Subjects were premedicated with steroids, but only peri-infusional doses were used.  Objective To assess the performance of the QFT-G test for screening before the initiation and also during anti-TNF treatment.
Methods The QFT-G test (intube method) was used to determine IFNγ production after stimulation with MT-specific antigens (ESAT-6, CFP-10 and TB7.7(p4) proteins) as well as with nonspecific mitogen (PHA). Altogether, 317 patients were investigated (rheumatoid arthritis (RA) = 117, ankylosing spondylitis (AS) = 95, adult juvenile idiopathic arthritis (JIA) = 54, Crohn's disease (CD) = 30, and psoriatic arthritis (PsA) = 21). Fifteen AS patients completed QFT-G assessments prior to anti-TNF and also after 2 and 14 weeks of treatment (before the second and fourth infliximab infusions). One hundred and eight patients had the QFT-G only before they started anti-TNF, six patients before and during therapy, and 203 patients only during the treatment. Tuberculin 2TU was used for the TST and results were read after 48-72 hours.
Results Out of the total 317 tests, 12 (3.8%) were indeterminate (5 x CD, 4 x RA, 2 x JIA, 1 x AS) (two high spontaneous IFNγ production, 10 low mitogen response). Eleven out of these 12 patients were on combined immunosuppression. Twelve patients (10.5%) (5 x RA, 4 x AS, 2 x CD, 1 x JIA) were positive in the QFT-G in the screening. Four had a negative TST, five a positive, in two the TST was unavailable. From 209 patients investigated during the treatment, 12 were QFT-G-positive. In three of them the initial pretreatment status was known and the QFT-G was negative; however, all three were positive in the TST at 16, 7 and 10 mm. Two of them became QFT-G-positive before the second infliximab infusion, one before the fourth infusion. The remaining nine positive patients had longstanding treatment with different anti-TNF biologicals.
In patients with a positive TST (n = 64), 30 had the QFT-G done before treatment -five had positive results and one an indeterminate result. The remaining 34 TST-positive patients were QFT-G-positive in nine cases and one result was indeterminate. In those who were TST-negative (n = 100), five were positive for the QFT-G. One patient developed TB, TST-negative, QFT-G-positive before therapy. In patients who were on infliximab and repeatedly investigated, IFNγ production after the nonspecific mitogen stimulation increased: 6.7 ± 3.8 IU/ml, 8.4 ± 2.6 IU/ml, and 8.9 ± 3.4 IU/ml (P = 0.001) before treatment and after 2, and 12 weeks, respectively. No difference was found in IFNγ in longitudinal samples after stimulation with TB antigens. Conclusion There is scarce information about QFT-G use before and during anti-TNF treatment. Our first experience shows that the QFT-G may be more sensitive and specific for LTBI detection than the TST, although its real usefulness for screening before anti-TNF treatment needs to be assessed in long-term studies. The QFT-G is valuable once anti-TNF has been initiated as we detected a relatively high number of positive patients who may be in danger of TB development. No decrease of the mitogen-induced capacity in IFNγ production shows that test can be meaningfully used during anti-TNF treatment.  Alkaline phosphatase activity was determined by p-nitrophenyl phosphate hydrolysis, osteocalcin release by enzyme immunoassay and collagen production by the release of the CICP propeptide. TGFβ 1 production was determined by ELISA. Inactivating antibodies raised against leptin receptors and inhibitors of leptin signaling, typhostin (Tyr) and piceatannol (Pce) were used to determine their effect on Ob cell markers. Cellular proliferation was assessed using the BrdU cell proliferation assay whereas activation of the Erk1/2 pathway was determined by western blot analysis. Results Our results indicated using two different sets of primers for RT-PCR experiments that leptin was expressed only in Obs not in chondrocytes. The expression of leptin was also higher in OA Obs compared with normal using real-time PCR and was responsible for the increase in leptin levels noted in conditionedmedia from OA Obs. Although leptin was not expressed by chondrocytes, it was present in articular cartilage -suggesting that leptin produced in bone tissue reached the overlaying cartilage. The long-form leptin receptor mRNA levels were slightly reduced in OA Obs compared with normal Obs. Since leptin can promote the differentiation of Obs, we next questioned whether the observed increase in alkaline phosphatase activity (ALP) and osteocalcin release (OC) observed in OA Obs was linked with their endogenous leptin production. Inactivating antibodies against the leptin receptor reduced both ALP and OC in OA Obs about 35%, a situation reproduced with Tyr and Pce. Likewise, Tyr and Pce also reduced ALP, CICP release, and TGFβ 1 production in OA Obs. Last, leptin dose-dependently (1 ng/ml to 10 mg/ml) stimulated cellular proliferation in OA Obs and this was reflected by an increase in phosphorylation of Erk1/2 signaling. Discussion These results indicate that OA Obs produce more leptin than normal, and suggest that leptin found in articular cartilage is derived from bone tissue. The increase in leptin in OA bone tissue could also be responsible for increased ALP, OC and TGFβ 1 in Obs. Since leptin can promote inflammation and cartilage loss in combination with cytokines, this suggests that subchondral bone production of leptin may be responsible, at least in part, for cartilage loss in OA. Objective To study the underlying molecular mechanisms involved in cartilage damage and increased bone density in Frzb -/mice. Methods Active Wnt signaling in the articular cartilage was studied in normal and methylated bovine serum albumin, collagenase and papain-induced arthritis using β-catenin (CTNNB1) immunohistochemistry. Gene expression patterns of components of the Wnt signaling pathway, its antagonist and target genes, were studied using multigene cDNA arrays (Superarray) in microdissected articular cartilage and soft tissues of healthy and affected knees from Frzb -/and wild-type mice. Gene expression of tissue destructive enzymes in the cartilage was studied by realtime PCR. Matrix metalloproteinase-3 activity was tested in vitro.

Molecular mechanisms involved in a differential association of Frzb biology with osteoarthritis and osteoporosis
Cortical bone stiffness was estimated by compression of the ulnae. Mechanical loading-induced bone adaptation was studied by compression of the ulnae of 17-week-old mice, followed by microcomputed tomography analysis at a resolution of 5 ìm. Subchondral bone properties were studied with histomorphometry, peripheral quantitative computed tomography and microcomputed tomography.
Results In healthy cartilage of Frzb -/mice, four genes were consistently expressed at lower levels than in the wild-type mice: Ctnnb1, Ctbp1, Fosl1 and Myc. In contrast, Wnt8b expression was upregulated in healthy cartilage in 2/3 samples from Frzb -/versus wild-type mice and in all samples in affected versus healthy wild-type cartilage. Ctbp1 and Fosl1 were also downregulated in all samples from arthritic wild-type mice versus healthy ones. In addition, Wnt8b appears to be downregulated in arthritic versus healthy joints of Frzb -/mice, suggesting a distinct regulation of Wnt ligand expression in the genetic model as compared with the wild-type mice. Cartilage damage in Frzb -/mice is associated with increased canonical Wnt signaling, matrix metalloproteinase-3 expression and activity. In addition, the Frzb -/mice have an increased cortical bone thickness and density, resulting in stiffer bones as demonstrated by a different stress-strain relationship in Frzb -/mice. Moreover, the periosteal anabolic response to mechanical loading is significantly greater in Frzb -/mice than in wild-type mice. FRZB is expressed in the periosteum. Conclusion The preinduction gene expression profile in Frzb -/mice shows similarities with arthritic cartilage in wild-type mice. Loss of Frzb may contribute to cartilage damage by increased expression and activity of matrix metalloproteinase in both a Wntdependent and Wnt-independent manner. FRZB deficiency also results in thicker cortical bone with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to OA by producing increased strain on the articular cartilage during locomotion. Increased cortical density of long bones in Frzb -/mice supports our earlier observation that polymorphisms in the human FRZB gene are differentially associated with hip OA and OP hip fractures. The role of FRZB in cartilage and bone biology may therefore provide a mechanistic basis for the longstanding clinical observation that OA and OP show an inverse relationship.

P38
Matrix remodeling during enchondral ossification and aggrecan cleavage in osteoarthritic cartilage depends on syndecan-4 heparan sulfate proteoglycans in matrix turnover and cell differentiation, we analyzed the role of syndecan-4 during limb development in mice and studied its expression and function in OA. Syndecan-4 promoter activity was detected in whole embryos by staining for β-galactosidase in syndecan-4 -/-LacZ knockin mice. For cellular localization of syndecan-4 expression within cartilage, immunohistochemistry with antibodies against syndecan-4 and type X collagen was performed. Alizarin red S staining was carried out to analyze the mineralization of bones in wild-type and syndecan-4 -/littermates at days E13.5 and E14.5. To study syndecan-4 in OA, we compared its expression in normal and OA articular cartilage by Northern blot analysis and immunohistochemistry. Blocking antibodies against syndecan-4 were generated and used to analyze the role of syndecan-4 in IL-1-mediated proteoglycan loss in vitro. For functional analysis in vivo, osteoarthritic changes were induced in syndecan-4 -/mice and in wildtype controls by surgically achieved joint instability, and the loss of proteoglycans was assessed by safranin-orange staining. Staining for syndecan-4 and ADAMTS-generated aggrecan neo-epitopes was performed in the knees of these mice. β-Galactosidase-staining of syndecan-4 -/mice at E12.0 showed a strong activity of the syndecan-4 promoter at sites of cartilage condensations. In later stages, syndecan-4 was detected in the growth plates of long bones. In wild-type embryos, syndecan-4 protein was also found mainly in chondrocytes of the hypertrophic zone, where it colocalized with type X collagen. The loss of syndecan-4 was associated with a significant retardation in the mineralization of axial and appendicular bones. Interestingly, there was a clear upregulation of syndecan-4 in human OA cartilage both at the mRNA and the protein level. In the cartilage explant model, blocking antibodies to syndecan-4 substantially significantly reduced the IL-1-induced loss of proteoglycans in wild-type cartilage. Analysis of OA-like changes in mice revealed a strong and early induction of syndecan-4, and there was a significant reduction of proteoglycan loss in the syndecan-4 -/mice compared with their wild-type controls. This was accompanied by a significantly reduced staining for ADAMTS-generated aggrecan neo-epitopes in syndecan-4 -/mice. Our data show that syndecan-4 is induced in hypertrophic chondrocytes both during embryogenesis and in OA cartilage. By promoting ADAMTS-mediated cleavage of aggrecans, syndecan-4 facilitates enchondral ossification but is involved also in cartilage degradation by hypertrophic chondrocytes in OA. Inhibition of syndecan-4 may therefore constitute a promising strategy to interfere with osteoarthritic cartilage damage. signalling pathway activation and their role in collagenase induction was analysed by western blotting and selective pathway inhibitors and siRNA. TLR ligand expression was compared in total RNA from femoral head cartilage of normal (neck of femur fracture) and OA patients undergoing joint replacement surgery. Results All the ligands upregulated MMP-1 and MMP-13, although to differing degrees, indicating differential regulation of the collagenases and indicating that chondrocytes express most TLRs. MALP-2 (TLR6/2 ligand) and poly(IC) (a dsRNA mimicking TLR3 ligand) most robustly upregulated MMP-1 and MMP-13, respectively. Using siRNA the activation of MMP-13 by TLR3 and TLR6/2 ligands were confirmed to be via the adaptor Trif and MyD88. The induction of both MMP-1 and MMP-13 by TLR1/2, TLR3 and TLR6/2 ligands were dependent upon the NF-κB pathway. Interestingly, MMP-1 and MMP-13 induction by the three TLR ligands were differentially inhibited by various MAPK inhibitors, with MMP-13 induction sensitive to ERK pathway inhibition. In a cartilage resorption assay, TLR1/2 and TLR6/2 ligands, but not TLR3 ligand, led to significant collagen release. The expression profile of TLRs in neck of femur and OA cartilage revealed a highly significant (P < 0.001) downregulation of TLR2 and upregulation of TLR3. Conclusion Our gene expression profile reveals that chondrocytes express a large repertoire of TLRs, which upon activation leads to collagenase gene activation. The TLR1/2 and TLR6/2 ligands, which resulted in cartilage collagen resorption, require TLR2, whose expression is significantly repressed in end-stage OA. These data suggest modulation of TLR-mediated signalling, particularly via TLR2, as a potential therapeutic strategy for OA prevention.