Objective. To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients.Methods. Plasma proteins (n ‫؍‬ 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 ageand sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial leastsquares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor ␣ (anti-TNF␣).Results. The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colonystimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor ␣, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity ( ‫؍‬ 0.64). In anti-TNF␣-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by ‘third generation chemistries’ may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity 100 healthy human volunteers were polarized into the 10% most Gc-sensitive and 10% most Gc-resistant following a low dose dexamethasone (0.25 mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non-Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included bone morphogenetic protein receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.

SummaryBackground: Patients with severe asthma appear relatively corticosteroid resistant.Corticosteroid responsiveness is closely related to the degree of eosinophilic airway inflammation. The extent to which eosinophilic airway inflammation in severe asthma responds to treatment with systemic corticosteroids is not clear.Objective: To relate the physiological and inflammatory response to systemic corticosteroids in asthma to disease severity and the baseline extent of eosinophilic inflammation. Methods:Patients with mild/moderate and severe asthma were investigated before and after 2 weeks of oral prednisolone (Clintrials.gov NCT00331058 and NCT00327197). We pooled the results from two studies with common protocols.The US study contained two independent centres and the UK one independent centre. The effect of oral corticosteroids on FEV 1 , Pc20, airway inflammation and serum cytokines was investigated. Baseline measurements were compared with healthy subjects.Results: Thirty-two mild/moderate asthmatics, 50 severe asthmatics and 35 healthy subjects took part. At baseline, both groups of asthmatics had a lower FEV 1 and Pc20 and increased eosinophilic inflammation compared to healthy subjects. The severe group had a lower FEV 1 and more eosinophilic inflammation compared to mild/moderate asthmatics. Oral prednisolone caused a similar degree of suppression of eosinophilic inflammation in all compartments in both groups of asthmatics. There were small improvements in FEV 1 and Pc20 for both mild/ moderate and severe asthmatics with a correlation between the baseline eosinophilic inflammation and the change in FEV 1 . There was a~50% reduction in the serum concentration of CXCL10 (IP-10), CCL22 (MDC), CCL17(TARC), CCL-2 (MCP-1) and CCL-13 (MCP-4) in both asthma groups after oral corticosteroids.Conclusions and Clinical Relevance: Disease severity does not influence the response to systemic corticosteroids. The study does not therefore support the concept that severe asthma is associated with corticosteroid resistance. Only baseline