Nicotinamide adenine dinucleotide biosynthesis enzymes in rheumatoid arthritis

Introduction: Synovial fibroblasts (SF) display a ‘hyperactive’ phenotype in patients with rheumatoid arthritis (RA). Nicotinamide adenine dinucleotide (NAD+) plays a role in cell metabolism, but may also be a key molecule in maintaining this ‘activated’ phenotype. NAD+ can be synthesised from precursor vitamin molecules, nicotinamide (Nam), nicotinic acid (NA) and Tryptophan (TRP); with their respective phosphoribosyl transferases (NAMPT, NAPRT, QAPRT) and Indoleamine (IDO) being the rate limiting enzymes involved in these pathways. NAMPT and IDO are known to be elevated in RA synovial tissue (ST). However, the expression and regulation of other NAD+ biosynthesis enzymes are unknown. Methods: RA, OA and normal ST were obtained from joints of patients undergoing surgery and expression of NAD+ biosynthesis enzymes were quantified using qPCR. Synovial fibroblasts were cultured and stimulated with 10ng/ml of TNF-α, IL-1β, OSM & IFN- and the expression of NAD+ biosynthesis enzymes were quantified using qPCR. Results: qPCR analyses showed that all NAD+ biosynthesis enzymes tested were constitutively expressed in synovial tissue ex vivo and in vitro, with the exception of NMN adenyltransferase (NMNAT)-3. NAMPT, IDO, QAPRT, NADSYN and NMNAT-2 were all upregulated in RA ST compared to normal tissue, however only NAMPT was significantly upregulated in RA compared to OA and normal, (NAMPT reached statistical significance when patients on anti-TNF therapy were excluded). Moreover, NAMPT was found to be upregulated in ST of young actively developing individuals, decreasing with age. Expression of NAD salvage enzymes, NAMPT and NMNAT-2 in ST correlated with each other and de novo NAD enzymes, IDO, QAPRT, NADSYN and NMNAT-2 were also correlated with each other in ST. NAMPT and IDO were both significantly upregulated in vitro following stimulation with OSM & IFN- but only NAMPT and NMNAT-2 were upregulated following stimulation with TNF-α & IL-1β. NAPRT expression was found to be low in RA ST and there was no upregulation following stimulation by OSM, IFN-, TNF-α & IL-1β. Conclusion: The data presented in this thesis emphasises NAMPT and IDO as a potential therapeutic target in rheumatoid arthritis

Does the external remote controller’s reading correspond to the actual lengthening in magnetic-controlled growing rods?

Purpose Magnetic-controlled growing rods (MCGRs) are now routinely used in many centres to treat early-onset scoliosis (EOS). MCGR lengthening is done non-invasively by the external remote controller (ERC). Our experience suggests that there may be a discrepancy between the reported rod lengthening on the ERC and the actual rod lengthening. The aim of this study was to investigate this discrepancy. Methods This was a prospective series. Eleven patients who were already undergoing treatment for EOS using MCGRs were included in this study. Results One hundred and ninety-two sets of ultrasound readings were obtained (96 episodes of rod lengthening on dual-rod constructs) and compared to their ERC readings. Only 15/192 (7.8%) readings were accurate; 27 readings (14.9%) were false positive; and 8 readings (4.2%) were an underestimation while 142 readings (74.0%) were an overestimation by the ERC. Average over-reporting by the ERC was 5.31 times of the actual/ultrasound reading. When comparing interval radiographs with lengthening obtained on ultrasound, there was a discrepancy with an average overestimation of 1.35 times with ultrasound in our series. There was a significant difference between ERC and USS (p = 0.01) and ERC and XR (p = 0.001). However, there was no significant difference between USS and XR (p > 0.99). Conclusion The reading on the ERC does not equate to the actual rod lengthening. The authors would recommend that clinicians using the MCGR for the treatment of early-onset scoliosis include pre- and post-extension imaging (radiographs or ultrasound) to confirm extension lengths at each outpatient extension. In centres with ultrasound facilities, we would suggest that patients should have ultrasound to monitor each lengthening after distraction but also 6-month radiographs

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes