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Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied.ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers.We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables.Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

OBJECTIVE:To investigate whether exhaled breath analysis using an electronic nose can identify differences between inflammatory joint diseases and healthy controls. METHODS:In a cross-sectional study, the exhaled breath of 21 rheumatoid arthritis (RA) and 18 psoriatic arthritis (PsA) patients with active disease was compared to 21 healthy controls using an electronic nose (Cyranose 320; Smiths Detection, Pasadena, CA, USA). Breathprints were analyzed with principal component analysis, discriminant analysis, and area under curve (AUC) of receiver operating characteristics (ROC) curves. Volatile organic compounds (VOCs) were identified by gas chromatography and mass spectrometry (GC-MS), and relationships between breathprints and markers of disease activity were explored. RESULTS:Breathprints of RA patients could be distinguished from controls with an accuracy of 71% (AUC 0.75, 95% CI 0.60-0.90, sensitivity 76%, specificity 67%). Breathprints from PsA patients were separated from controls with 69% accuracy (AUC 0.77, 95% CI 0.61-0.92, sensitivity 72%, specificity 71%). Distinction between exhaled breath of RA and PsA patients exhibited an accuracy of 69% (AUC 0.72, 95% CI 0.55-0.89, sensitivity 71%, specificity 72%). There was a positive correlation in RA patients of exhaled breathprints with disease activity score (DAS28) and number of painful joints. GC-MS identified seven key VOCs that significantly differed between the groups. CONCLUSIONS:Exhaled breath analysis by an electronic nose may play a role in differential diagnosis of inflammatory joint diseases. Data from this study warrant external validation.

Abstract Background Recently, healing of a ruptured anterior cruciate ligament (ACL) is reconsidered. In a previous study, we have shown that the transected ACL can heal after treatment with the triple X locking suture alone or combined with small intestine submucosa (SIS). The first research question of this study was whether the healing ACLs in both groups show histological characteristics that are typical for ligament healing. Secondly, did the combined treatment with SIS lead to improved histological healing, in terms of the morphology of the fibrous synovial layer, the extracellular matrix (ECM), collagen fiber orientation, cellularity, ratio of myofibroblasts, and collagen type 3 staining. The hypothesis was that SIS enhances the healing by the scaffolding effect, endogenous growth factors, and chemoattractants. Methods In the Suture group, the left ACL was transected and sutured with the triple X locking suture repair technique. In the Suture‐SIS group, the left ACL underwent the same procedure with the addition of SIS. The right ACL served as internal control. Standard histology and immunostaining of α‐smooth muscle actin (SMA) and collagen type 3 were used. Results Microscopy showed that the fibrous synovial layer around the ACL was reestablished in both groups. The collagen fibers in the Suture‐SIS group stained denser, were more compactly arranged, and the ECM contained fewer voids and fat vacuoles. Neovasculature running between the collagen fibers was observed in both experimental groups. Collagen type 3 stained less in the Suture‐SIS group. The cellularity in the Suture group, Suture‐SIS group and Control was 1265 ± 1034 per mm2, 954 ± 378 per mm2, 254 ± 92, respectively; 49%, 26% and 20% of the cells stain positive for α‐SMA, respectively. Conclusion The healing ACL in both treated groups showed histological characteristics which are comparable to the spontaneously healing medial collateral ligament and showed that the ACL has a similar intrinsic healing response. Though, no definitive conclusions on the beneficial effects of the SIS scaffold on the healing process can be made.

Since high-density lipoprotein (HDL) has pro-endothelial and anti-thrombotic effects, a HDL recruiting stent may prevent restenosis. In the present study we address the functional characteristics of an apolipoprotein A-I (ApoA-I) antibody coating in vitro. Subsequently, we tested its biological performance applied on stents in vivo in rabbits.The impact of anti ApoA-I- versus apoB-antibody coated stainless steel discs were evaluated in vitro for endothelial cell adhesion, thrombin generation and platelet adhesion. In vivo, response to injury in the iliac artery of New Zealand white rabbits was used as read out comparing apoA-I-coated versus bare metal stents.ApoA-I antibody coated metal discs showed increased endothelial cell adhesion and proliferation and decreased thrombin generation and platelet adhesion, compared to control discs. In vivo, no difference was observed between ApoA-I and BMS stents in lumen stenosis (23.3±13.8% versus 23.3±11.3%, p=0.77) or intima surface area (0.81±0.62 mm2 vs 0.84±0.55 mm2, p=0.85). Immunohistochemistry also revealed no differences in cell proliferation, fibrin deposition, inflammation and endothelialization.ApoA-I antibody coating has potent pro-endothelial and anti-thrombotic effects in vitro, but failed to enhance stent performance in a balloon injury rabbit model in vivo.

INTRODUCTION:The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice. METHODS:Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. RESULTS:Treatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system. CONCLUSIONS:These data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.

Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02).The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model.Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks.These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3 x 10(-21)), A928V (rs35018800, OR = 0.53, P = 1.2 x 10(-9)), and I684S (rs12720356, OR = 0.86, P = 4.6 x 10(-7)). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6 x 10(-18)), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; P(omnibus) = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.

We have previously shown that overweight may increase the risk of developing rheumatoid arthritis (RA) in autoantibody positive individuals. Adipose tissue could contribute to the development of RA by production of various bioactive peptides. Therefore, we examined levels of adipokines in serum and synovial tissue of subjects at risk of RA.Fifty-one individuals positive for immunoglobulin M rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA), without arthritis, were included in this prospective study. Levels of adiponectin, vaspin, resistin, leptin, chemerin and omentin were determined in baseline fasting serum samples (n = 27). Synovial tissue was obtained by arthroscopy at baseline and we examined the expression of adiponectin, resistin and visfatin by immunohistochemistry.The development of clinically manifest arthritis after follow-up was associated with baseline serum vaspin levels (HR1.5 (95% CI 1.1 to 2.2); p = 0.020), also after adjustment for overweight (HR1.7 (95% CI 1.1 to 2.5); p = 0.016). This association was not seen for other adipokines. Various serum adipokine levels correlated with BMI (adiponectin r = -0.538, leptin r = 0.664; chemerin r = 0.529) and systemic markers of inflammation such as CRP levels at baseline (adiponectin r = -0.449, omentin r = -0.557, leptin r = 0.635, chemerin r = 0.619, resistin r = 0.520) and ESR (leptin r = 0.512, chemerin r = 0.708), p-value

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and β chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and β chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

ObjectiveThe objective was to determine the safety of ocrelizumab (OCR) in patients with rheumatoid arthritis (RA).MethodsThis was an analysis of the double-blind, placebo-controlled periods and long-term follow-up of 4 OCR phase III trials in RA (SCRIPT, STAGE, FILM and FEATURE). Safety data per study and the results of a meta-analysis of serious infectious events (SIEs) are presented.ResultsOverall, 868 patients received placebo, 1064 patients OCR 200 mg×2 (or 400 mg×1) (OCR200), and 827 patients OCR 500 mg×2 (OCR500) plus background methotrexate (MTX) at baseline and 24 weeks. During the double-blind, placebo-controlled periods, the incidence of adverse events and serious adverse events was comparable between the OCR+MTX and placebo +MTX groups. Infusion-related reactions were more common with OCR+MTX and decreased in frequency with subsequent infusions. Serious infusion-related reactions were rare (0.1%). Serious infections occurred more frequently with OCR500+MTX. In the meta-analysis, a statistically significant difference from placebo +MTX in incidence of SIEs per 100 patient-years of 2.4 (95% CI, 0.3-4.5) was observed with OCR500+MTX, but not with OCR200+MTX (0.6; 95% CI, -1.3 to 2.4). Patients recruited in Asia exhibited a higher risk of serious infections (hazard ratio, 1.78; 95% CI, 1.03-3.06). The incidence of human anti-human antibodies was ConclusionsIn placebo-controlled clinical trials of RA, OCR500+MTX was associated with a higher risk of serious infections compared with placebo +MTX. The safety profile of OCR 200+MTX was comparable with placebo+MTX.Trial registrationSTAGE ClinicalTrials.gov NCT00406419 SCRIPT ClinicalTrials.gov NCT00476996 FILM ClinicalTrials.gov NCT00485589 FEATURE ClinicalTrials.gov NCT00673920.

OBJECTIVE:The change in number of CD68-positive sublining macrophages in serial synovial biopsies has been successfully used to discriminate on the group level between effective and ineffective treatment during early drug development in rheumatoid arthritis (RA) patients. Measurement of a soluble biomarker would clearly have practical advantages. Therefore, we investigated the sensitivity to change of myeloid related protein (MRP)8/14 in serum. METHODS:139 RA patients who received known effective biologics (infliximab, adalimumab and rituximab) and 28 RA patients who received placebo/ineffective therapies were included. MRP8/14 levels were analyzed in baseline and follow-up serum samples and the standardized response mean (SRM) was calculated to determine the sensitivity to change of MRP8/14 in comparison to C-reactive protein (CRP) levels and the disease activity score evaluated in 28 joints (DAS28). RESULTS:In patients treated with effective treatment, the SRM for MRP8/14 was moderate (0.56), but in patients treated with placebo/ineffective treatment the SRM was 0.06, suggesting that this biomarker is perhaps not susceptible to placebo effects in proof-of-concept studies of relatively short duration. In contrast, the SRM for DAS28 was high for effective treatment (1.07), but also moderate for ineffective treatment (0.58), representing the placebo effect. The SRM for CRP was low in the effective (0.33) and ineffective (0.23) treatment groups. CONCLUSION:These data support the notion that quantification of changes in MRP8/14 serum levels could be used to predict potential efficacy of novel antirheumatic drugs in an early stage of drug development. A positive result would support the rationale for larger, conventional clinical trials to determine whether the effects are clinically relevant.

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 -differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 -differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

Integrating genetic data from families with highly penetrant forms of disease together with genetic data from outbred populations represents a promising strategy to uncover the complete frequency spectrum of risk alleles for complex traits such as rheumatoid arthritis (RA). Here, we demonstrate that rare, low-frequency and common alleles at one gene locus, phospholipase B1 (PLB1), might contribute to risk of RA in a 4-generation consanguineous pedigree (Middle Eastern ancestry) and also in unrelated individuals from the general population (European ancestry). Through identity-by-descent (IBD) mapping and whole-exome sequencing, we identified a non-synonymous c.2263G>C (p.G755R) mutation at the PLB1 gene on 2q23, which significantly co-segregated with RA in family members with a dominant mode of inheritance (P = 0.009). We further evaluated PLB1 variants and risk of RA using a GWAS meta-analysis of 8,875 RA cases and 29,367 controls of European ancestry. We identified significant contributions of two independent non-coding variants near PLB1 with risk of RA (rs116018341 [MAF = 0.042] and rs116541814 [MAF = 0.021], combined P = 3.2 × 10(-6)). Finally, we performed deep exon sequencing of PLB1 in 1,088 RA cases and 1,088 controls (European ancestry), and identified suggestive dispersion of rare protein-coding variant frequencies between cases and controls (P = 0.049 for C-alpha test and P = 0.055 for SKAT). Together, these data suggest that PLB1 is a candidate risk gene for RA. Future studies to characterize the full spectrum of genetic risk in the PLB1 genetic locus are warranted.

BACKGROUND: Chemerin is a specific chemoattractant for macrophages and dendritic cells (DC). In addition, it can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules and is able to activate fibroblast-like synoviocytes (FLS), suggesting a role in the pathogenesis of rheumatoid arthritis (RA). Chemerin is also an adipocytokine that has been related to the inflammatory state of endothelial cells and as such could be involved in the changes in endothelial cells in RA and perhaps increased cardiovascular morbidity. We investigated whether anti-Tumor Necrosis Factor (TNF) treatment affects chemerin levels. MATERIALS AND METHODS: 49 patients with active RA (disease activity score evaluated in 28 joints (DAS28) ≥3.2) were started on adalimumab therapy. Blood was drawn from patients while fasting at baseline and 16 weeks after initiation of treatment. Chemerin serum levels were measured by ELISA and related to disease activity, mediators of inflammation and known risk factors for cardiovascular disease. RESULTS: Adalimumab therapy reduced chemerin serum levels, which was correlated with the reduction in DAS28 (r = 0.37, p = 0.009). In addition, the decrease in chemerin serum levels after anti-TNF treatment was associated with the decrease in serum levels of IL-6 (r = 0.39, p = 0.033) and macrophage migration inhibitory factor (MIF) (r = 0.31, p = 0.049). Baseline chemerin serum levels were not related to traditional risk factors for atherosclerosis, except perhaps for smoking (p = 0.07). CONCLUSIONS: This exploratory study shows that adalimumab therapy lowers chemerin levels, which is associated with the reduction in disease activity parameters, and inflammatory mediators IL-6 and MIF. This suggests a possible involvement of chemerin in the migration/retention of macrophages in the synovium. TRIAL REGISTRATION NEDERLANDS TRIAL REGISTER: NTR 857.

CD40-CD154 (CD40 ligand) interaction in the co-stimulatory pathway is involved in many (auto)immune processes and both molecules are upregulated in salivary glands of Sjögren's syndrome (SS) patients. Interference within the CD40 pathway has ameliorated (auto)inflammation in a number of disease models. To test the potential role of the CD40 pathway in loss of gland function and inflammation in SS, an inhibitor of CD40-CD154 interaction was overexpressed in the salivary glands (SGs) of a spontaneous murine model of SS; the Non-Obese Diabetic (NOD) mouse.At different disease stages an adeno associated viral vector encoding CD40 coupled to a human Fc domain (CD40:Fc) was injected locally into the SGs of NOD mice. Delivery was confirmed by PCR. The overall effect on local inflammation was determined by assessment of the focus score (FS), quantification of infiltrating cell types, immunoglobulin levels, and microarray analysis. The effect on SG function was determined by measuring stimulated salivary flow.CD40:Fc was stably expressed in the SG of NOD mice, and the protein was secreted into the blood stream. Microarray analysis revealed that expression of CD40:Fc affected the expression of many genes involved in regulation of the immune response. However, FS, infiltrating cell types, immunoglobulin levels, and salivary gland output were similar for treated and control mice.Although endogenous CD40 is expressed in SG inflammatory foci in the SG of NOD mice, the expression of soluble CD40:Fc did not lead to reduced overall inflammation and/or improved salivary gland function. These data indicate possible redundancy of the CD40 pathway in the SG and suggests that targeting CD40 alone may not be sufficient to alter the disease phenotype.

BACKGROUND:CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. METHODS:FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. RESULTS:CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. CONCLUSIONS:Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4). In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

OBJECTIVES: It has been shown in early arthritis cohorts that the 2010 ACR/EULAR criteria for rheumatoid arthritis (RA) enable an earlier diagnosis, perhaps at the cost of a somewhat more heterogeneous patient population. We describe the features of synovial inflammation in RA patients classified according to these new criteria. METHODS: At baseline, synovial tissue biopsy samples were obtained from disease-modifying antirheumatic drug (DMARD)-naïve early RA patients (clinical signs and symptoms

Intercellular adhesion molecule-1 (ICAM-1) is involved in migration and co-stimulation of T and B cells. Membrane bound ICAM-1 is over expressed in the salivary glands (SG) of Sjögren's syndrome (SS) patients and has therefore been proposed as a potential therapeutic target. To test the utility of ICAM-1 as a therapeutic target, we used local gene therapy in Non Obese Diabetic (NOD) mice to express soluble (s)ICAM-1 to compete with membrane bound ICAM-1 for binding with its receptor. Therapy was given prior to and just after the influx of immune cells into the SG.A recombinant serotype 2 adeno associated virus (rAAV2) encoding ICAM-1/Fc was constructed and its efficacy tested in the female NOD mice after retrograde instillation in SG at eight (early treatment) and ten (late treatment) weeks of age. SG inflammation was evaluated by focus score and immunohistochemical quantification of infiltrating cell types. Serum and SG tissue were analyzed for immunoglobulins (Ig).Early treatment with ICAM-1/Fc resulted in decreased average number of inflammatory foci without changes in T and B cell composition. In contrast, late treated mice did not show any change in focus scores, but immunohistochemical staining showed an increase in the overall number of CD4+ and CD8+ T cells. Moreover, early treated mice showed decreased IgM within the SGs, whereas late treated mice had increased IgM levels, and on average higher IgG and IgA.Blocking the ICAM-1/LFA-1 interaction with sICAM-1/Fc may result in worsening of a SS like phenotype when infiltrates have already formed within the SG. As a treatment for human SS, caution should be taken targeting the ICAM-1 axis since most patients are diagnosed when inflammation is clearly present within the SG.

INTRODUCTION: Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögren's syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved. METHODS: Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts. RESULTS: Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity. CONCLUSIONS: Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.

BACKGROUND: The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either anti-CCR1, anti-CCR2, or anti-CCR5 blocking antibodies (or medium or isotype controls). In addition, a small molecule CCR1 antagonist (BX471) was tested. Chemotaxis was induced by CCL2/MCP-1 (CCR2 ligand), CCL5/RANTES (CCR1 and CCR5 ligand), or by a mix of 5 RA synovial fluids (SFs), and cellular responses compared to chemotaxis in the presence of medium alone. Anti-CCR2 antibody treatment blocked CCL2/MCP-1-induced chemotaxis of both HD and RA monocytes compared to isotype control. Similarly, anti-CCR5 antibody treatment blocked CCL5/RANTES-induced chemotaxis of RA monocytes. While neither CCR2 nor CCR5 blocking antibodies were able to inhibit SF-induced monocyte chemotaxis, even when both receptors were blocked simultaneously, both anti-CCR1 antibodies and the CCR1 antagonist were able to inhibit SF-induced monocyte chemotaxis. CONCLUSIONS/SIGNIFICANCE: The RA synovial compartment contains several ligands for CCR1, CCR2, and CCR5 as well as other chemokines and receptors involved in monocyte recruitment to the site of inflammation. The results suggest that CCR2 and CCR5 are not critical for the migration of monocytes towards the synovial compartment in RA. In contrast, blockade of CCR1 may be effective. Conceivably, CCR1 blockade failed in clinical trials, not because CCR1 is not a good target, but because very high levels of receptor occupancy at all times may be needed to inhibit monocyte migration in vivo.

BACKGROUND: There is growing interest in soluble biomarkers that could be used on the group level for screening purposes in small proof of principle studies during early drug development. We investigated early changes in serum levels of several candidate biomarkers involved in cartilage and bone metabolism following the initiation of adalimumab as a prototypic active treatment in psoriatic arthritis (PsA) compared to placebo. MATERIALS AND METHODS: Twenty-four PsA patients were randomized to receive either adalimumab 40 mg s.c. every other week or placebo for 4 weeks, followed by an open label extension phase. Serum samples were obtained at baseline and after 4 and 12 weeks of treatment and analyzed for levels of CPII and PINP (synthesis of type II and type I procollagen), melanoma inhibitory activity (MIA) (chondrocyte anabolism), matrix metalloproteinase (MMP)-3, C2C and cartilage oligomeric matrix protein (COMP) (type II collagen degradation), osteocalcin (OC) (bone formation), NTX-I and ICTP (both type I collagen degradation). RESULTS: After 4 weeks, there was a significant decrease in serum MMP-3 levels in adalimumab-treated patients (P

BACKGROUND: Although the use of TNF inhibitors has fundamentally changed the way rheumatoid arthritis (RA) is treated, not all patients respond well. It is desirable to facilitate the identification of responding and non-responding patients prior to treatment, not only to avoid unnecessary treatment but also for financial reasons. In this work we have investigated the transcriptional profile of synovial tissue sampled from RA patients before anti-TNF treatment with the aim to identify biomarkers predictive of response. METHODOLOGY/PRINCIPAL FINDINGS: Synovial tissue samples were obtained by arthroscopy from 62 RA patients before the initiation of infliximab treatment. RNA was extracted and gene expression profiling was performed using an in-house spotted long oligonucleotide array covering 17972 unique genes. Tissue sections were also analyzed by immunohistochemistry to evaluate cell infiltrates. Response to infliximab treatment was assessed according to the EULAR response criteria. The presence of lymphocyte aggregates dominated the expression profiles and a significant overrepresentation of lymphocyte aggregates in good responding patients confounded the analyses. A statistical model was set up to control for the effect of aggregates, but no differences could be identified between responders and non-responders. Subsequently, the patients were split into lymphocyte aggregate positive- and negative patients. No statistically significant differences could be identified except for 38 transcripts associated with differences between good- and non-responders in aggregate positive patients. A profile was identified in these genes that indicated a higher level of metabolism in good responding patients, which indirectly can be connected to increased inflammation. CONCLUSIONS/SIGNIFICANCE: It is pivotal to account for the presence of lymphoid aggregates when studying gene expression patterns in rheumatoid synovial tissue. In spite of our original hypothesis, the data do not support the notion that microarray analysis of whole synovial biopsy specimens can be used in the context of personalized medicine to identify non-responders to anti-TNF therapy before the initiation of treatment.

BACKGROUND:Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man. METHODS/PRINCIPAL FINDINGS:We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4 x 10(-3) (0.95-5.1 x 10(-3)) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion. CONCLUSIONS/SIGNIFICANCE:The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.

The adaptive immune system recognizes billions of unique antigens using highly variable T-cell receptors. The alphabeta T-cell receptor repertoire includes an estimated 10(6) different rearranged beta chains per individual. This paper describes a novel micro-array based method that monitors the beta chain repertoire with a resolution of a single T-cell clone. These T-arrays are quantitative and detect T-cell clones at a frequency of less than one T cell in a million, which is 2 logs more sensitive than spectratyping (immunoscope), the current standard in repertoire analysis. Using T-arrays we detected CMV-specific CD4+ and CD8+ T-cell clones that expanded early after viral antigen stimulation in vitro and in vivo. This approach will be useful in monitoring individual T-cell clones in diverse experimental settings, and in identification of T-cell clones associated with infectious disease, autoimmune disease and cancer.