S.6.1 β-catenin is a central mediator in SSc

Background. β-catenin is the central integrator of canonical Wnt signalling. Since recent evidence suggests a central role of Wnts in fibrosis, we examined the β-catenin/Wnt pathway in SSc and focused on the role of β-catenin in fibroblast activation. Methods. We performed qPCR for several Wnt ligands and axin-2 to examine Wnt expression in SSc skin. We further studied protein levels of Wnt-1, -4, -10b and β-catenin by IHC. To establish the effects of β-catenin/Wnt signalling on collagen release, we created mice with fibroblast-specific stabilization of β-catenin (dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER) as well as mice carrying fibroblast-specific deletion of β-catenin [Ctnnb1(fl/fl) × Col1a2; Cre-ER]. Summary of the results. We could demonstrate mRNA overexpression of Wnt-1, -2, -9a, -9b, -10a, -10b and -16 in SSc skin. Wnt-1, -4 and -10b consistently showed strong expression in SSc skin when compared with healthy skin. On protein level, however, Wnt-4 was indistinguishable between SSc patients and healthy controls, whereas Wnt-1 and Wnt-10b protein levels were increased in SSc skin. The overexpression of Wnt-1 and Wnt-10b resulted in a prominent nuclear accumulation of β-catenin in fibroblasts. Finally, increased mRNA levels of the target gene axin-2 confirmed the activation of canonical Wnt signalling. In dEx3 β-catenin (wt/ex) mice, we addressed the consequences of enhanced Wnt signalling and increased accumulation of β-catenin in SSc. We selectively targeted β-catenin in fibroblasts. Cre-activated dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER mice showed massive and spontaneous dermal thickening even 2 weeks after Cre activation. Eight weeks after Cre-activation, skin thickening cumulated at 102.6% (P < 0.001). In line with the dermal thickening, hydroxyproline content and myofibroblast counts showed strong increases. To test the therapeutic potential of targeting β-catenin/Wnt signaling, we created Ctnnb1(fl/fl) x Col1a2;Cre-ER mice to specifically delete β-catenin in fibroblasts. After Cre activation and β-catenin deletion in fibroblasts, mice were challenged with bleomycin subcutaneously for 4 weeks. We found that Cre-activated Ctnnb1(fl/fl) × Col1a2; Cre-ER mice were protected from bleomycin-induced dermal with a reduction of skin thickening by 71% (P < 0.05). Conclusions. We demonstrated a prominent activation of canonical Wnt signalling in SSc with nuclear accumulation of β-catenin in fibroblasts and activation of the target gene axin-2. Our results showed that fibroblast-specific stabilization of β-catenin resulted in enhanced collagen release, whereas deletion of β-catenin potently reduced collagen production. Together, our findings highlight a key role of β-catenin in fibroblast activation and fibrosis. Thus, β-catenin may be promising molecular target for anti-fibrotic therapie

Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis

Objectives Notch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice. Methods Notch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments. Results Notch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1. Conclusions Inhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA

Electrochemical determination of hydroquinone using hydrophobic ionic liquid-type carbon paste electrodes

Three types of carbon paste electrodes (CPEs) with different liquid binders were fabricated, and their electrochemical behavior was characterized via a potassium hexacyanoferrate(II) probe. 1-Octyl-3-methylimidazolium hexafluorophosphate ionic liquid (IL) as a hydrophobic conductive pasting binder showed better electrochemical performance compared with the commonly employed binder. The IL-contained CPEs demonstrated excellent electroactivity for oxidation of hydroquinone. A diffusion control mechanism was confirmed and the diffusion coefficient (D) of 5.05 × 10-4 cm2 s-1 was obtained. The hydrophobic IL-CPE is promising for the determination of hydroquinone in terms of high sensitivity, easy operation, and good durability

Update of EULAR recommendations for the treatment of systemic sclerosis

The aim was to update the 2009 European League against Rheumatism (EULAR) recommendations for the treatment of systemic sclerosis (SSc), with attention to new therapeutic questions. Update of the previous treatment recommendations was performed according to EULAR standard operating procedures. The task force consisted of 32 SSc clinical experts from Europe and the USA, 2 patients nominated by the pan-European patient association for SSc (Federation of European Scleroderma Associations (FESCA)), a clinical epidemiologist and 2 research fellows. All centres from the EULAR Scleroderma Trials and Research group were invited to submit and select clinical questions concerning SSc treatment using a Delphi approach. Accordingly, 46 clinical questions addressing 26 different interventions were selected for systematic literature review. The new recommendations were based on the available evidence and developed in a consensus meeting with clinical experts and patients. The procedure resulted in 16 recommendations being developed (instead of 14 in 2009) that address treatment of several SSc-related organ complications: Raynaud's phenomenon (RP), digital ulcers (DUs), pulmonary arterial hypertension (PAH), skin and lung disease, scleroderma renal crisis and gastrointestinal involvement. Compared with the 2009 recommendations, the 2016 recommendations include phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of SSc-related RP and DUs, riociguat, new aspects for endothelin receptor antagonists, prostacyclin analogues and PDE-5 inhibitors for SSc-related PAH. New recommendations regarding the use of fluoxetine for SSc-related RP and haematopoietic stem cell transplantation for selected patients with rapidly progressive SSc were also added. In addition, several comments regarding other treatments addressed in clinical questions and suggestions for the SSc research agenda were formulated. These updated data-derived and consensus-derived recommendations will help rheumatologists to manage patients with SSc in an evidence-based way. These recommendations also give directions for future clinical research in SSc

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P &lt; 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P &lt; 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P &lt; 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P &lt; 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P &lt; 0.001; OR(BP) = 2.4, P &lt; 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P &lt; 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P &lt; 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

Sonoelectrochemistry in acoustically emulsified media: The detection of lead

The double extraction of target lead ions which are then voltammetrically quantified via acoustically assisted anodic stripping voltammetry is demonstrated. The technique involves first the extraction of lead from the aqueous volume into an organic phase containing the ligand dithizone (diphenylthiocarbazone). Second, the chelated lead is then stripped into a clean electrochemically clean aqueous solution where the lead from the original solution can be quantified. This method is shown to be applicable in electrochemically fouling media, providing a high sensitivity with a limit of detection for lead in the order of 10-7 mol dm-3. A theoretical extraction model is presented for the optimal conditions of extraction. The technique is applied to the analysis of lead in wine samples offering quantitative data, which is in good agreement with those obtained from an independent technique (AAS)

A self-catalytic carbon paste electrode for the detection of vitamin B12.

A simple, sensitive, and selective method for the detection of vitamin B12 (cyanocobalamin) is presented. A carbon paste electrode has been constructed with trans-1,2-dibromocyclohexane (DBCH) acting as both the paste binder and a reactive material facilitating the electrocatalytic detection of the target. The latter reaction is based on the electroreduction of the Co(III) center in vitamin B12 to Co(I), which specifically reacts with DBCH to produce electrocatalytic currents with the regeneration of Co (II); a detection limit of 8.5 x 10(-10) mol dm(-3) (based on 3sigma) was found. The electrode has been successfully used for the quantification of vitamin B12 in pharmaceutical products and biological matrix media

Sonoelectroanalysis in acoustically emulsified media: Zinc and cadmium

Dithizone (diphenylthiocarbazone) dissolved in chloroform was employed as a ligand for the solvent extraction of either cadmium or zinc from aqueous media. Acoustic emulsification was shown to be effective both in extraction and recovery or 'back extraction' with a fresh clean aqueous solution. Quantitative analysis of cadmium or zinc removed via double sono-extraction from aqueous media hostile to voltammetric analysis proceeded via sono-square wave anodic stripping voltammetry (sono-SWASV), resulting in high sensitivity in the relatively clean medium. This technique was shown to be viable for the quantification of cadmium from surfactant containing media where direct electroanalytical techniques fail. The method was also applied to the analysis of zinc in commercial shampoo and toothpaste, giving results in close agreement with those obtained from independent analysis. Biphasic sono-extraction synergistically coupled with sono-SWASV therefore presents an attractive technique for cadmium or zinc analysis in electrode passsivating media. The technique likely removes contaminants present in the test solution since these will 'prefer' to remain in the initial aqueous phase, or will transfer to the extracting organic phase, but are unlikely to be doubly transferred into the clean final aqueous phase

Determination of trace Mn(II) in pharmaceutical diet supplements by cathodic stripping voltammetry on bare carbon paste electrode

A simple electroanalytical method for quantification of Mn in pharmaceutical diet supplements has been developed. The method is based on the formation of insoluble MnO2 on the electrode during deposition step at the potential of +0.85V vs SCE, and its consecutive reductive dissolution back to Mn(II) during differential pulse voltammetry scan. Pure carbon paste was found to be a suitable material for this kind of determination because of its easy preparation, good accessibility, low cost, as well as its appropriate analytical properties. In 0.1 mol L-1 phosphate buffer (pH 7.4) supporting electrolyte and at optimised deposition time (120 s) the linear calibration plot was obtained in the concentration range 1 × 10 -6-12 × 10-6 mol L-1; the corresponding detection limit was 1 × 10-7 mol L-1. The method was applied to the analysis of pharmaceutical dosage forms containing manganese as an essential trace element