Arthritis Research & Therapy

We and others have previously shown that antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and in a more recent study we reported that individuals who subsequently developed RA had increased concentrations of several cytokines and chemokines years before the onset of symptoms of joint disease. Here we aimed to evaluate the prevalence and predictive values of anti-CCP antibodies of IgG, IgM and IgA isotype in individuals who subsequently developed RA and also to relate these to cytokines and chemokines, smoking, genetic factors and radiographic score. A case-control study (1:4 ratio) was nested within the Medical Biobank and the Maternity cohorts of Northern Sweden. Patients with RA were identified from blood donors predating the onset of disease by years. Matched controls were selected randomly from the same registers. IgG, IgA and IgM anti-CCP2 antibodies were determined using EliA anti-CCP assay on ImmunoCAP 250 (Phadia AB, Uppsala, Sweden). Of 86 patients with RA identified as blood donors prior to the onset of symptoms, samples were available from 71 for analyses. The median (Q1 to Q3) predating time was 2.5 years (1.1 to 5.9 years). The sensitivity of anti-CCP antibodies in the pre-patient samples was 35.2% for IgG, 23.9% for IgA, and 11.8% for IgM. The presence of IgG and IgA anti-CCP antibodies was highly significant compared with controls. IgG and IgA anti-CCP2 predicted RA significantly in conditional logistic regression models odds ratio (OR) = 94.1, 95% confidence interval (CI) 12.7 to 695.4 and OR = 11.1, 95% CI 4.4 to 28.1, respectively, the IgM anti-CCP showed borderline significance OR = 2.5 95% CI 0.9 to 6.3. Concentrations of all anti-CCP isotypes increased the closer to the onset of symptoms the samples were collected with an earlier and higher increase for IgG and IgA compared with IgM anti-CCP. IgA and IgG anti-CCP positive individuals had different patterns of up-regulated chemokines and also, smoking brought forward the appearance of IgA anti-CCP antibodies in pre-RA individuals. Anti-CCP2 antibodies of both the IgG and IgA isotypes pre-dated the onset of RA by years; also, both IgG and IgA anti-CCP2 antibodies predicted the development of RA, with the highest predictive value for IgG anti-CCP2 antibodies.

There is currently no blood-based test for detection of early-stage osteoarthritis (OA) and the anti-cyclic citrullinated peptide (CCP) antibody test for rheumatoid arthritis (RA) has relatively low sensitivity for early-stage disease. Morbidity in arthritis could be markedly decreased if early-stage arthritis could be routinely detected and classified by clinical chemistry test. We hypothesised that damage to proteins of the joint by oxidation, nitration and glycation, and with signatures released in plasma as oxidized, nitrated and glycated amino acids may facilitate early-stage diagnosis and typing of arthritis. Patients with knee joint early-stage and advanced OA and RA or other inflammatory joint disease (non-RA) and healthy subjects with good skeletal health were recruited for the study (n = 225). Plasma/serum and synovial fluid was analysed for oxidized, nitrated and glycated proteins and amino acids by quantitative liquid chromatography-tandem mass spectrometry. Data-driven machine learning methods were employed to explore diagnostic utility of the measurements for detection and classifying early-stage OA and RA, non-RA and good skeletal health with training set and independent test set cohorts. Glycated, oxidized and nitrated proteins and amino acids were detected in synovial fluid and plasma of arthritic patients with characteristic patterns found in early and advanced OA and RA, and non-RA, with respect to healthy controls. In early-stage disease, two algorithms for consecutive use in diagnosis were developed: (1) disease versus healthy control, and (2) classification as OA, RA and non-RA. The algorithms featured 10 damaged amino acids in plasma, hydroxyproline and anti-CCP antibody status. Sensitivities/specificities were: (1) good skeletal health, 0.92/0.91; (2) early-stage OA, 0.92/0.90; early-stage RA, 0.80/0.78; and non-RA, 0.70/0.65 (training set). These were confirmed in independent test set validation. Damaged amino acids increased further in severe and advanced OA and RA. Oxidized, nitrated and glycated amino acids combined with hydroxyproline and anti-CCP antibody status provided a plasma-based biochemical test of relatively high sensitivity and specificity for early-stage diagnosis and typing of arthritic disease.

Neutrophil extracellular traps (NETs) are critically involved in microscopic polyangiitis (MPA) pathogenesis, and some patients with MPA possess anti-NET antibody (ANETA). Anti-myosin light chain 6 (MYL6) antibody is an ANETA that affects NETs. This study aimed to determine the significance of anti-MYL6 antibody in MPA. The influence of anti-MYL6 antibody on NET formation and actin rearrangement necessary for NET formation was assessed by fluorescent staining. An enzyme-linked immunosorbent assay was established to detect serum anti-MYL6 antibody, and the prevalence of this antibody in MPA was determined. Furthermore, the disease activity and response to remission-induction therapy of MPA were compared between anti-MYL6 antibody-positive and anti-MYL6 antibody-negative MPA patients. Anti-MYL6 antibody disrupted G-actin polymerization into F-actin, suppressing phorbol 12-myristate 13-acetate-induced NET formation. Serum anti-MYL6 antibody was detected in 7 of 59 patients with MPA. The Birmingham vasculitis activity score (BVAS) of anti-MYL6 antibody-positive MPA patients was significantly lower than anti-MYL6 antibody-negative MPA patients. Among the nine BVAS evaluation items, the cutaneous, cardiovascular, and nervous system scores of anti-MYL6 antibody-positive MPA patients were significantly lower than anti-MYL6 antibody-negative MPA patients, although other items, including the renal and chest scores, were equivalent between the two groups. The proportion of patients with remission 6 months after initiation of remission-induction therapy in anti-MYL6 antibody-positive MPA patients was significantly higher than in anti-MYL6 antibody-negative MPA patients. Collective findings suggested that anti-MYL6 antibody disrupted actin rearrangement necessary for NET formation and could reduce the disease activity of MPA.

Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.

The purpose of this study was to evaluate and compare the serum levels and local expression of resistin in patients with idiopathic inflammatory myopathies to controls, and to determine the relationship between resistin levels, inflammation and disease activity. Serum resistin levels were determined in 42 patients with inflammatory myopathies and 27 healthy controls. The association among resistin levels, inflammation, global disease activity and muscle strength was examined. The expression of resistin in muscle tissues from patients with inflammatory myopathies and healthy controls was evaluated. Gene expression and protein release from resistin-stimulated muscle and mononuclear cells were assessed. In patients with inflammatory myopathies, the serum levels of resistin were significantly higher than those observed in controls (8.53 ± 6.84 vs. 4.54 ± 1.08 ng/ml, P < 0.0001) and correlated with C-reactive protein (CRP) levels (r = 0.328, P = 0.044) and myositis disease activity assessment visual analogue scales (MYOACT) (r = 0.382, P = 0.026). Stronger association was observed between the levels of serum resistin and CRP levels (r = 0.717, P = 0.037) as well as MYOACT (r = 0.798, P = 0.007), and there was a trend towards correlation between serum resistin and myoglobin levels (r = 0.650, P = 0.067) in anti-Jo-1 positive patients. Furthermore, in patients with dermatomyositis, serum resistin levels significantly correlated with MYOACT (r = 0.667, P = 0.001), creatine kinase (r = 0.739, P = 0.001) and myoglobin levels (r = 0.791, P = 0.0003) and showed a trend towards correlation with CRP levels (r = 0.447, P = 0.067). Resistin expression in muscle tissue was significantly higher in patients with inflammatory myopathies compared to controls, and resistin induced the expression of interleukins (IL)-1β and IL-6 and monocyte chemoattractant protein (MCP)-1 in mononuclear cells but not in myocytes. The results of this study indicate that higher levels of serum resistin are associated with inflammation, higher global disease activity index and muscle injury in patients with myositis-specific anti-Jo-1 antibody and patients with dermatomyositis. Furthermore, up-regulation of resistin in muscle tissue and resistin-induced synthesis of pro-inflammatory cytokines in mononuclear cells suggest a potential role for resistin in the pathogenesis of inflammatory myopathies.

The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-α were studied by stimulation with TNF-α alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-α reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-α in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.

Tetrathiomolybdate (TM), a drug developed for Wilson's disease, produces an anti-angiogenic and anti-inflammatory effect by reducing systemic copper levels. TM therapy has proved effective in inhibiting the growth of tumors in animal tumor models and in cancer patients. We have hypothesized that TM may be used for the therapy of rheumatoid arthritis and have examined the efficacy of TM on adjuvant-induced arthritis in the rat, which is a model of acute inflammatory arthritis and inflammatory cachexia. TM delayed the onset of and suppressed the severity of clinical arthritis on both paw volume and the arthritis score. Histological examination demonstrated that TM significantly reduces the synovial hyperplasia and inflammatory cell invasion in joint tissues. Interestingly, TM can inhibit the expression of vascular endothelial growth factor in serum synovial tissues, especially in endothelial cells and macrophages. Moreover, the extent of pannus formation, which leads to bone destruction, is correlated with the content of vascular endothelial growth factor in the serum. There was no mortality in TM-treated rat abnormalities. TM also suppressed inflammatory cachexia. We suggest that copper deficiency induced by TM is a potent approach both to inhibit the progression of rheumatoid arthritis with minimal adverse effects and to improve the well-being of rheumatoid arthritis patients.