Effect of Anti-Rheumatic Treatment on the Periodontal Condition of Rheumatoid Arthritis Patients.

Periodontitis, a bacterial-induced infection of the supporting soft and hard tissues of the teeth (the periodontium), is common in patients with rheumatoid arthritis (RA). As RA and periodontitis underlie common inflammatory pathways, targeting the progression of RA might mediate both periodontitis and RA. On the other hand, patients with RA on immunosuppressive medication have an increased risk of infection. Therefore, the objective of this longitudinal observation study was to assess the effect of methotrexate (MTX) and anti-tumor necrosis factor-α (anti-TNF, etanercept) treatment on the periodontal condition of RA patients. Overall, 14 dentate treatment-naive RA patients starting with MTX and 12 dentate RA patients starting with anti-TNF therapy in addition to MTX were included. Follow-up was scheduled matching the routine protocol for the respective treatments. Prior to the anti-rheumatic treatment with MTX or the anti-TNF therapy in addition to MTX, and during follow-up, i.e., 2 months for MTX, and 3 and 6 months for the anti-TNF therapy in addition to MTX, the periodontal inflamed surface area (PISA) was measured. The efficacy of the anti-rheumatic treatment was assessed by determining the change in RA disease activity (DAS28-ESR). Furthermore, the erythrocyte sedimentation rates were determined and the levels of C-reactive protein, IgM-rheumatoid factor, anti-cyclic citrullinated protein antibodies, and antibodies to the periodontal pathogen Porphyromonas gingivalis, were measured. Subgingival sampling and microbiological characterization of the subgingival microflora was done at baseline. MTX or anti-TNF treatment did not result in an improvement of the periodontal condition, while both treatments significantly improved DAS28 scores (both p < 0.01), and reduced C-reactive protein levels and erythrocyte sedimentation rates (both p < 0.05). It is concluded that anti-rheumatic treatment (MTX and anti-TNF) has negligible influence on the periodontal condition of RA patients.

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.

An improved cyan fluorescent protein variant useful for FRET.

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.