Nitro-fatty acids decrease type I interferons and monocyte chemoattractant protein 1 in ex vivo models of inflammatory arthritis.

BACKGROUND: Inflammatory arthritis including rheumatoid arthritis (RA) and spondyloarthritis (SpA) is characterized by inflammation and destruction of the joints. Approximately one third of patients do not respond to first-line treatments. Nitro-fatty acids are bioactive lipids with anti-inflammatory properties and tissue-protective functions. The nitro-fatty acid 10-NO2-oleic acid (10-NO2-OA) is being tested in clinical trials for patients with fibrotic and inflammatory conditions. Here, we tested whether 10-NO2-OA could inhibit immune reactions involved in the inflammatory and joint destructive processes in inflammatory arthritis. METHODS: Synovial fluid and blood samples were obtained from 14 patients with active RA or SpA. The in vitro models consisted of synovial fluid mononuclear cells (SFMCs) cultured for 48 h, SFMCs cultured for 21 days, and fibroblast-like synovial cells (FLSs) co-cultured with peripheral blood mononuclear cells (PBMCs) for 48 h. Cells were treated with or without 10-NO2-OA or the tumor necrosis factor alpha (TNFα) inhibitor etanercept. Supernatants were analyzed for type I interferon, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase 3 (MMP3) and tartrate resistant acid phosphatase (TRAP). RESULTS: In SFMCs cultured for 48 h, 10-NO2-OA dose-dependently decreased the secretion of bioactive type I interferons and MCP-1 but not MMP3 (P = 0.032, P = 0.0001, and P = 0.58, respectively). Both MCP-1 and MMP3 were decreased by etanercept (P = 0.0031 and P = 0.026, respectively). In SFMCs cultured for 21 days, 10-NO2-OA significantly decreased the production of MCP-1 but not TRAP (P = 0.027 and P = 0.1523, respectively). Etanercept decreased the production of TRAP but not MCP-1 (P < 0.001 and P = 0.84, respectively). In co-cultures of FLSs and PBMCs, 10-NO2-OA decreased the production of MCP-1 (P < 0.0001). This decrease in MCP-1 production was not seen with etanercept treatment (P = 0.47). CONCLUSION: 10-NO2-OA decreased the release of MCP-1 in three models of inflammatory arthritis. Of particular interest, 10-NO2-OA inhibited type I interferon, and 10-NO2-OA was more effective in reducing MCP-1 production in cultures dominated by FLSs compared with etanercept. Our results encourage clinical investigations of 10-NO2-OA in patients with inflammatory arthritis.

Resveratrol displays anti-inflammatory properties in an ex vivo model of immune mediated inflammatory arthritis.

BACKGROUND: Resveratrol is a natural polyphenol found in berries, roots and wine that is well known to have anti-inflammatory and anti-oxidative properties. The anti-inflammatory effect has been reported for both immune cells and connective tissues, but only few studies have investigated effects on immune mediated inflammatory arthritis. None of which have studied this effect when combining resveratrol with methotrexate or adalimumab, two major drugs in the treatment of immune mediated inflammatory arthritis.We therefore aimed to investigate the anti-inflammatory effect of resveratrol alone and in combination with methotrexate or adalimumab in ex vivo models of immune mediated inflammatory arthritis. We furthermore aimed to describe any variations in this effect based on disease activity and cellular composition of the synovial fluid infiltrate. METHODS: Synovial fluid mononuclear cells from patients with rheumatoid arthritis (n = 7) and spondyloarthritis (n = 7) were cultured for either 48 h or 21 days. In both models, synovial fluid mononuclear cells were treated with resveratrol alone or in combination with methotrexate or adalimumab. Monocyte chemoattractant protein 1, matrix metalloproteinase 3 and tartrate resistant acidic phosphatase were measured to quantify inflammation, enzymatic degradation and osteoclast differentiation, respectively. RESULTS: Resveratrol reduced monocyte chemoattractant protein 1 production by synovial fluid mononuclear cells significantly (p = 0.005) compared to untreated controls. The effect of resveratrol was greatest in cultures from patients with low disease activity, i.e. DAS28CRP ≤ 3.2 (p = 0.022), and in cultures dominated by lymphocytes (p = 0.03). Further, the combination of methotrexate and resveratrol significantly reduced monocyte chemoattractant protein 1 levels compared with methotrexate alone in cultures from patients with low disease activity (p = 0.016), and in cultures with high lymphocyte count (p = 0.011). Resveratrol did not significantly affect matrix metalloproteinase 3 and tartrate resistant acidic phosphatase production. CONCLUSION: Resveratrol has anti-inflammatory properties in our ex vivo model of immune mediated inflammatory arthritis. Results show an additive effect of resveratrol, when combined with methotrexate in samples dominated by lymphocytes and samples from patients with low disease activity. This suggests further investigations in vitro and whether this effect may also be present in a clinical setting.

Unexplained high BMD in DXA-scanned patients is generalized throughout the skeleton and characterized by thicker cortical and trabecular bone.

Unexplained high bone mineral density (BMD) is a rare condition and the mechanisms responsible are yet to be described in detail. The aim of the study was to identify patients with unexplained high BMD from a local DXA database and compare their radiological phenotype with an age- and a gender-matched group of population-based controls. We defined high BMD as a DXA Z-score ≥ + 2.5 at the total hip and lumbar spine. We characterized the findings as "unexplained" if no osteodegenerative changes, bone metabolic disease, or arthritis at the hip or lumbar spine was observed. All participants were investigated with high-resolution peripheral quantitative computed tomography (HR-pQCT), QCT, DXA, fasting blood samples, a 24-h urine sample, and questionnaires. The DXA database contained data on 25,118 patients. Initially, 138 (0.55%) potential participants with high BMD were identified, and during the study ten additional cases were identified from new DXA scans. Sixty-seven patients accepted to participate in the study, and among these we identified 15 women and one man with unexplained high BMD. These 15 women had higher BMD throughout the skeleton relative to controls, similar area/volume at the hip and the distal extremities, a higher number of trabeculae, which was thicker than in the controls, and a higher finite element estimated bone strength. The 15 women were heavier and had a higher fat mass then controls. We conclude that patients with unexplained high BMD have a generalized high BMD phenotype throughout their skeleton, which is characterized with a denser microarchitecture.

Standardized reaction times used to describe the mechanism of enzyme-induced gelation in whey protein systems.

Whey protein isolate (WPI), either untreated or pretreated at 80 degrees C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed. Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v. reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition). This provided a connection between the gelation profile and the degree of hydrolysis. In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk. This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration. In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation. Results obtained at 40-60 degrees C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed. The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease.

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.

Effect of partial hydrolysis with an immobilized proteinase on thermal gelation properties of beta-lactoglobulin B.

We have investigated the influence of partial hydrolysis with an immobilized proteinase from Bacillus licheniformis on the thermal gelation of isolated beta-lactoglobulin B. Gelation behaviour was determined by dynamic rheological measurements (small deformation) and the gels were characterized with respect to microstructure and water-holding properties. A fine-stranded gel with a complex modulus of approximately 2000 Pa was formed from beta-lactoglobulin (50 g/l in 75 mM-Tris-HCl, pH 7.5). Limited hydrolysis prior to thermal gelation resulted in coarser gels with thicker protein strands and larger pores. Gel structure correlated with its permeability, proton mobility and water-holding capacity. Total stiffness gel increased with low degrees of hydrolysis, but decreased after prolonged hydrolysis. Maximal gel stiffness was 1.5-fold that gels made from of unhydrolysed beta-lactoglobulin. This was much lower than the stiffening effect obtained after partial hydrolysis of whey protein isolate, showing that the gel strengthening effect of partial hydrolysis was depedent on the protein composition and/or the hydrolysis and gelatin conditions. A mechanism to explain the observed effects of hydrolysis on gelation and gel properties is presented.

A Novel Approach to Turbidimetry of Dense Systems: An Investigation of the Enzymatic Gelation of Casein Micelles.

A novel approach to turbidimetry enabling the extraction of structural information about highly turbid systems has been developed. Turbidimetric spectra have been obtained in the wavelength region 500-1100 nm using an acceptance angle of 1 degrees for detecting the transmitted light. It is demonstrated that the influence of multiple scattering can be eliminated by measurement of turbidimetric spectra at several sample thicknesses and subsequent extrapolation to zero thickness. The validity of this method is demonstrated by Monte Carlo simulations of multiple scattering of light using simple Rayleigh-Debye-Gans theory. The simulations demonstrate that turbidimetric spectra are very insensitive to multiple scattering measured with an acceptance angle of 1 degrees when the particles are smaller than about 1 µm. It was further shown that no shape information can be derived from turbidimetric spectra under our condition for objects having diameters up to 6 µm. The turbidimetric spectra of casein aggregation/gelation have been fitted by modeling the aggregates either as homogeneous spheres or as fractals. The turbidimetric averaged mean radii found using the sphere model are at all stages of the process consistently smaller than radii found by either static or dynamic light scattering. This is found to be a consequence of the angular integration involved in turbidimetry which weights larger radii less than in the case of light scattering. Copyright 1998 Academic Press.