Objective: To investigate whether articular chondrocytes from rheumatoid arthritis (RA) patients have acquired a proinflammatory phenotype. Method: Articular cartilage explants from RA patients and healthy controls (HC) were cultured with or without interleukin (IL)-1 beta for two weeks. Protein levels of cytokines and metalloproteinases (MMPs) in the supernatant were measured by LUMINEX, mRNA with qPCR and nitrogen oxide (NO) levels with Griess assay. Results: Within 24 h after culture, cartilage explants from RA spontaneously produced MMP-1 and MMP-13, and matrix components (aggrecan and collagen type IV) were released. In addition, the RA explants released higher levels of tumor necrosis factor, interferon-gamma, IL-33, IL-18, vascular endothelial growth factor-A, IL-6 but not IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as compared with HC. During two weeks of incubation the higher levels did not diminish. IL-1 beta stimulation further increased the levels of IL-6, IL-8 and GMCSF, mainly in RA explants, and induced increased levels of NO in the supernatant from both HC and RA explants, as a result of chondrocyte activation. Conclusions: RA chondrocytes are activated with a proinflammatory profile involving the production of cytokines as well as MMP-1 and MMP-13, that can lead to release of matrix molecules after activation, which suggests that the chondrocytes have a proinflammatory phenotype and thereby an active role in the pathogenesis.

Abstract Objective Exercise can improve immune health and is beneficial for physical function in patients with rheumatoid arthritis (RA), but the immunological mechanisms are largely unknown. We evaluated the effect of moderate- to high intensity exercise with person-centred guidance on cells of the immune system, with focus on regulatory cell populations, in older adults with RA. Methods Older adults (≥65 years) with RA were randomized to either 20-weeks of moderate – to high intensity aerobic and resistance exercise (n = 24) or to an active control group performing home-based exercise of light intensity (n = 25). Aerobic capacity, muscle strength, DAS28 and CRP were evaluated. Blood samples were collected at baseline and after 20 weeks. The frequency of immune cells defined as adaptive regulatory populations, CD4 + Foxp3 + CD25 + CD127- T regulatory cells (Tregs) and CD19 + CD24hiCD38hi B regulatory cells (Bregs) as well as HLA-DR−/lowCD33 + CD11b + myeloid derived suppressor cells (MDSCs), were assessed using flow cytometry. Results After 20 weeks of moderate- to high intensity exercise, aerobic capacity and muscle strength were significantly improved but there were no significant changes in Disease Activity Score 28 (DAS28) or CRP. The frequency of Tregs and Bregs decreased significantly in the intervention group, but not in the active control group. The exercise intervention had no effect on MDSCs. The reduction in regulatory T cells in the intervention group was most pronounced in the female patients. Conclusion Moderate- to high intensity exercise in older adults with RA led to a decreased proportion of Tregs and Bregs, but that was not associated with increased disease activity or increased inflammation. Trial registration Improved Ability to Cope With Everyday Life Through a Person-centered Training Program in Elderly Patients With Rheumatoid Arthritis - PEP-walk Study, NCT02397798 . Registered at ClinicalTrials.gov March 19, 2015.

Background The mechanisms underlying tolerance induction and maintenance in autoimmune arthritis remain elusive. In a mouse model of rheumatoid arthritis, collagen type II (CII)-induced arthritis, we explore the contribution of B cells to antigen-specific tolerance. Methods To generate expression of the CII-peptide specifically on B-cell major histocompatibility complex type II, lentiviral-based gene therapy including a B-cell-specific Igk promoter was used. Results Presentation of the CII-peptide on B cells significantly reduced the frequency and severity of arthritis as well as the serum levels of CII -specific IgG antibodies. Further, both frequency and suppressive function of regulatory T cells were increased in tolerized mice. Adoptive transfer of regulatory T cells from tolerized mice to naïve mice ameliorated the development of CII-induced arthritis. Conclusion Our data suggest that endogenous presentation of the CII-peptide on B cells is one of the key contributors to arthritis tolerance induction and maintenance.

Random recombination of antibody heavy- and light-chain genes results in a diverse B-cell receptor (BCR) repertoire including self-reactive BCRs. However, tolerance mechanisms that prevent the development of self-reactive B cells remain incompletely understood. The absence of the surrogate light chain, which assembles with antibody heavy chain forming a pre-BCR, leads to production of antinuclear antibodies (ANAs). Here we show that the naive follicular B-cell pool is enriched for cells expressing prototypic ANA heavy chains in these mice in a non-autoimmune background with a broad antibody repertoire. This results in the spontaneous formation of T-cell-dependent germinal centres that are enriched with B cells expressing prototypic ANA heavy chains. However, peripheral tolerance appears maintained by selection thresholds on cells entering the memory B-cell and plasma cell pools, as exemplified by the exclusion of cells expressing the intrinsically self-reactive V(H)81X from both pools.

The 18th germinal centre conference Uddevalla, Sweden September 11–14, 2014

Objective: S100A4 is a Ca-binding protein that regulates cell growth, survival, and motility. The abundant expression of S100A4 in rheumatiod arthritis contributes to the invasive growth of joint tissue and to bone damage. In the present study, we analysed the role of S1 00A4 in bone homeostasis. Methods: Peripheral quantitative computed tomography and histomorphometric analysis were performed in mice lacking the entire S100A4 protein (S100A4K0) and in wild-type (WT) counterparts treated with shRNA-lentiviral constructs targeting S100A4 (S100A4-5hRNA). Control groups consisted of sex-matched WT counterparts and WT mice treated with a non-targeting RNA construct. Results: S100A4 deficiency was associated with higher trabecular and cortical bone mass, increased number and thickness of trabeculi combined with larger periosteal circumference and higher predicted bone strength. Si 00A4 inhibition by shRNA led to an increase in cortical bone in WT mice, S100A4-deficieny was associated with a reduced number of functional osteodasts. S100A4K0 and S100A4-shRNA-treated bone marrow progenitors gave rise to a large number of small TRAP+ cells with few nuclei and few pseudopodial processes. Poor osteoclastogenesis and the low resorptive capacity in S100A4Ko mice may be linked to low levels of surface integrins, impaired adhesion capacity, and poor multinucleation in S100A4-deficient osteodasts, as well as a low content of proteolytic enzymes cathepsin K and MMP3 and MMP9 to break down the organic matrix. Conclusion: S100A4 emerges as a negative regulator of bone metabolism potentially responsible for the excessive bone turnover in conditions marked by high levels of S100A4 protein, such as inflammation and rheumatoid arthritis. (c) 2013 Elsevier B.V. All rights reserved.

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.

OBJECTIVETo assess the potential of metastasin S100A4 as a biological marker in patients with RA.METHODSA total of 87 unselected patients with established RA (disease duration 2-44 years) and treated with MTX and infliximab at a single rheumatology centre were included in a cross-sectional study. Radiographs of hands and feet were taken prior to infliximab treatment and at inclusion (time interval 48 ± 27 months) and scored for the radiographic damage. S100A4 levels were analysed in relation to radiographic damage, clinical disease activity (DAS-28), inflammation (IL-6, CRP, ESR), bone and cartilage markers [MMP-3, COMP, C-telopeptide of type I collagen (CTX-I)] and proto-oncogenes [survivin, insulin-like growth factor 1 (IGF-1), Flt3 ligand].RESULTSHigh levels of S100A4 were associated with severe radiographic damage (OR = 3.40, P = 0.025), non-response to infliximab (OR = 4.63, P = 0.003), presence of antibodies to infliximab (OR = 6.24, P = 0.003) and high levels of Flt3 ligand (OR = 2.73, P = 0.04). Regression analysis showed that high S100A4 was predictive for radiographic progression during infliximab treatment [positive predictive value (PPV) 0.68, P = 0.05]. Low levels of S100A4 were associated with response to infliximab (OR = 2.67, P = 0.049), clinical remission (OR = 4.01, P = 0.0047) and negative RF (OR = 9.22, P = 0.0047). S100A4 correlated with survivin (r = 0.71, P > 0.0001).CONCLUSIONS100A4 levels are increased in proportion to radiographic damage and its further progression in RA patients. High S100A4 levels were associated with a poor clinical response to infliximab and high rate of anti-infliximab antibodies. The finding of a correlation between S100A4 and survivin and Flt3 ligand suggests that these proteins may represent a new cluster of biomarkers predicting radiographic progression and poor treatment response in RA patients.

Survivin is known as an inhibitor of apoptosis and a positive regulator of cell division. We have recently identified survivin as a predictor of joint destruction in patients with rheumatoid arthritis (RA). Flt3 ligand (Flt3L) is expressed in the inflamed joints and has adjuvant properties in arthritis. Studies on 90 RA patients (median age 60.5 years [range, 24-87], disease duration 10.5 years [range, 0-35]) show a strong positive association between the levels of survivin and Flt3L in blood. Here, we present experimental evidence connecting survivin and Flt3L signaling. Treatment of BALB/c mice with Flt3L led to an increase of survivin in the bone marrow and in splenic dendritic cells. Flt3L changed the profile of survivin splice variants, increasing transcription of the short survivin40 in the bone marrow. Treatment with an Flt3 inhibitor reduced total survivin expression in bone marrow and in the dendritic cell population in spleen. Inhibition of survivin transcription in mice, by shRNA lentiviral constructs, reduced the gene expression of Flt3L. We conclude that expression of survivin is a downstream event of Flt3 signaling, which serves as an essential mechanism supporting survival of leukocytes during their differentiation, and maturation of dendritic cells, in RA.

Objective: To evaluate if the measurement of survivin in the blood of patients with rheumatoid arthritis (RA) undergoing infliximab treatment has predictive value for treatment response.Methods: The study included 87 consecutive RA patients (age 24-89 years, disease duration 18-526 months) treated with regular infusions of influximab. Survivin levels were measured by enzyme-linked immunosorbent assay and evaluated in relation to the total dose of infliximab, disease activity (DAS28), response to infliximab treatment (change in DAS28 > 1.2), and radiographic damage (vdH-Sharp score).Results: Thirty-seven percent of patients were survivin-positive (survivin > 0.9 ng/mL) and showed severe radiographic damage at the start of infliximab treatment compared with survivin-negative (P = 0.027). Patients with high survivin levels were unlikely to respond to infliximab treatment (OR 4.02 [1.22-14.61], P = 0.022) and achieve remission (OR 4.32[1.01-30.11], P = 0.048) compared with patients with low survivin levels.Conclusions: High survivin levels are associated with severe radiographic damage at the start of treatment and a poor response to infliximab. Survivin measurement should be considered an additional tool for aiding the selection and follow-up of antirheumatic treatment. (C) 2012 Elsevier Inc. All rights reserved. Semin Arthritis Rheum 41:652-657

OBJECTIVE:Human resistin has proinflammatory properties that activate NF-κB-dependent pathways, whereas its murine counterpart is associated with insulin resistance. The aim of this study was to examine potential cross-talk between resistin and insulin/insulin-like growth factor (IGF) signaling in rheumatoid arthritis (RA).METHODS:Levels of IGF-1, IGF binding protein 3, and resistin were measured in the blood and synovial fluid of 60 patients with RA and 39 healthy control subjects. Human RA synovium was implanted subcutaneously into SCID mice, and the mice were treated with resistin-targeting small interfering RNA. Primary synovial fibroblasts from patients with RA, as well as those from patients with osteoarthritis, and the human fibroblast cell line MRC-5 were stimulated with resistin. Changes in the IGF-1 receptor (IGF-1R) signaling pathway were evaluated using histologic analysis, immunohistochemistry, and reverse transcription-polymerase chain reaction.RESULTS:Resistin and IGF-1R showed different expression profiles in RA synovia. Low levels of IGF-1 in RA synovial fluid were associated with systemic inflammation and inversely related to the levels of resistin. Stimulation of synovial fibroblasts with resistin induced phosphorylation of IGF-1R to a degree similar to that with insulin, and also induced phosphorylation of transcription factor Akt. This was followed by gene expression of GLUT1, IRS1, GSK3B, and the Akt inhibitors PTPN and PTEN. Abrogation of resistin expression in vivo reduced the expression of IGF-1R, the phosphorylation of Akt, and the expression of PTPN and PTEN messenger RNA in RA synovium implanted into SCID mice.CONCLUSION:Resistin utilizes the IGF-1R pathway in RA synovia. Abrogation of resistin synthesis in the RA synovium in vivo leads to reductions in the expression of IGF-1R and level of phosphorylation of Akt.

ABSTRACTTKs are intracellular signaling molecules essential for cell homeostasis. Inhibition of TKs is used in treatment of malignancies and diabetes mellitus. The present study evaluated the role of Flt3 in antigen-induced arthritis. Mice were immunized with mBSA, and arthritis was induced by an i.a. injection of mBSA. Treatment with the Flt3 inhibitor sunitinib was started together with mBSA immunization or together with the induction of arthritis. The mBSA-injected joints were evaluated morphologically for signs of synovitis and bone/cartilage destruction. Markers of bone metabolism and antibody responses were measured by ELISA. Maturation of DCs in the bone marrow and spleen was evaluated by flow cytometry. Sunitinib treatment reduced the intensity of synovitis and the incidence of bone destruction. The reduction in bone destruction was seen when the treatment was started at the time of immunization or at the time of arthritis induction. The antiarthritic effect was achieved by inhibition of DCs, reduction of antibody production, and bone metabolism. Inhibition of Flt3 is a potent antiarthritic mechanism reducing antigen presentation, synovial inflammation, and bone resorption. Down-regulation of TKs may be a useful tool in the treatment of human RA.

The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.

RHO family proteins are important for the function of inflammatory cells.They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I).Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins.Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders.However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis.The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments.The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane.Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines.The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

Persistence and competition of denitrifying biofilms subjected to a natural wastewater flora

Research performed at the Division of Environmental Microbiology has over the last years resulted in the isolation of possible bacterial key-organisms with efficient nutrient removal properties (Comamonas denitrificans, Brachymonas denitrificans, Aeromonas hydrophila). Effective use of these organisms for enhanced nutrient removal in wastewater treatment applications requires the strains to be retained, to proliferate and to maintain biological activity within theprocess. This can be achieved by immobilization of the organisms using an appropriate system.Two putative immobilization systems, agar entrapment and biofilm formation, wereassessed. Surface attached biofilm growth provided better results with respect to cell retention,proliferation and microbial activity than immobilization in agar beads. Thus, biofilm physiology was further characterized using simplified systems of single, dual or multi strain bacterial consortia containing the key-organisms as well as other wastewater treatment isolates. Mechanisms for initial adherence, biofilm formation over time, dynamics and characteristics of extracellular polymeric substances (EPS) and exopolysaccharides, nutrient removal activity as well as the effect of bacterial interactions were investigated. The results showed that all theassessed bacterial strains could form single strain biofilm providing that a suitable nutrientsupply was given. Production of EPS was found to be critical for biofilm development and both EPS and polysaccharide residue composition varied with bacterial strain, culture conditions and biofilm age. Denitrification and phosphorus removal activity of the keyorganisms was maintained in biofilm growth. Co-culturing of two or more strains resulted in both synergistic and antagonistic effects on biofilm formation as well as the microbial activitywithin the biofilm. Bacterial interactions also induced the synthesis of new polysaccharideswhich were not produced in pure strain biofilms.The complexity of single and mixed strain biofilm development and the implications of interactions on biofilm performance were underlined in this study. The data presented can be useful for modeling of biofilm systems, serve as a tool for selection of bacterial strain combinations to use for bioaugmentation/bioremediation or provide a base for further experiment design.

Proto-oncogene survivin has recently been identified as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (RA). In the present material of 132 RA patients and 82 controls, the levels of survivin correlated to urokinase (uPA) (r= 0.46), a plasminogen activator over-expressed in inflamed joints and known to exhibit potent arthritogenic properties. Here we evaluate the functional relationship between these proteins using primary synovial fibroblasts and leucocytes of RA patients, human monocytic (THP-1) and fibroblast (MRC-5) cell lines. Using inhibitors of intracellular signalling, we show that uPA and survivin share common transduction pathways in synovial fibroblasts being dependent on the activity of tyrosine kinases, phosphatidylinositide 3 kinase and mitogen effector kinase. Moreover, uPA production is significantly reduced in fibroblasts if survivin synthesis has been silenced by siRNA. Importantly, silencing of survivin in fibroblasts prevented their invasive growth in knee joints of severe combined immune deficient mice. Interaction of uPA with receptor up-regulates survivin expression in leucocytes. In turn, survivin is required for the up-regulation of uPA receptor on the cell surface. These findings indicate that survivin is an essential mediator of arthritogenic properties of uPA regulating its synthesis in synovial fibroblasts and uPAR expression in leucocytes. Close correlation between survivin and uPA levels in patients with RA supports the importance of this connection for the pathogenesis of arthritis.

Bioaugmentation for enhanced wastewater treatment performance depends on survival, maintained activity and retention of the inoculated microorganisms in the reactor. Biofilms could be suitable mediators of bio­ augmentation due to the retention of the immobilized cells and the protection offered by the biofilm matrix. The overall objective of this study was to investigate the potential of biofilm­mediated bioaugmentation for enhanced denitrification in the presence of a natural wastewater flora. In order to select a suitable inoculum, single and mixed strain biofilms were investigated in sterile wastewater with respect to biofilm formation, denitrification activity and bacterial interactions. The dual strain biofilm formed by the denitrifying organisms Comamonas denitrificans and Brachymonas denitrificans displayed good biofilm formation and high denitrifica­ tion activity and was thus further investigated. When exposed to a natural wastewater flora, the relative number of C. denitrificans and B. denitrificans cells in the biofilm decreased over time until it was stabilized at just below 20 % of the total population after 4 weeks. The selected strains were thereby shown to persist in the competition with natural wastewater flora, highlighting the potential of biofilm mediated bioaugmentation.