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

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

A: Expression of <i>GSTCD</i> and <i>INTS12</i> mRNA in Lung and Airway cells.

<p>mRNA expression in human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC) and peripheral blood mononuclear cells (PBMC) is shown relative to mRNA from lung. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=3). Only the expression of <i>GSTCD</i> in HBEC relative to lung was statistically significant (* <i>P</i>=0.0494). <b>B</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> ΔCt values in HASM, HBEC, PBMC and lung</b>. mRNA expression levels as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone-0074630-g003" target="_blank">Figure 3A</a> from human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC), peripheral blood mononuclear cells (PBMC) and lung were correlated using a scatter plot. The correlation coefficient between these measures was r=0.8, <i>P</i><0.0001. <b>C</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> mRNA levels in the lung</b>. The scatter plot shows a positive correlation between the <i>GSTCD</i> and <i>INTS12</i> probe sets as investigated in the lung eQTL study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B20" target="_blank">20</a>].</p

<i>GSTCD</i> and <i>INTS12</i> gene expression is altered following exposure of HASM cells to TGFβ1.

<p>Human airway smooth muscle (HASM) cells were exposed to 10ng/ml TGFβ1 for 4 or 24 hours. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=5). Significant increases in both <i>GSTCD</i> and <i>INTS12</i> gene expression were observed following 24h exposure to TGFβ1 (<i>P</i><0.05 <i>GSTCD</i>, <i>P</i><0.01 <i>INTS12</i>) and after 4h TGFβ1 exposure in <i>INTS12</i> expression (<i>P</i><0.05).</p

Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see Table S5 for patient demographics).

<p>Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone.0074630.s009" target="_blank">Table S5</a> for patient demographics).</p

Regulatory motifs within the <i>GSTCD/INTS12</i> locus.

<p>The <i>GSTCD</i>/<i>INTS12</i> locus is shown, annotated with RNA sequencing, H3K27Ac histone marks, DNase hypersensitivity, transcription factor binding and CpG islands (UCSC Genome Browser (<a href="http://genome.ucsc.edu/" target="_blank"><u>http://genome.ucsc.edu/</u></a>)) on the Human Feb 2009 (GRCh37/hg19) assembly. For the H3K27Ac histone marks and RNA sequence tracks, peak height is proportional to signal amplitude, with colours representing datasets in different cell backgrounds (pale blue H3K27Ac histone trace = human umbilical vein endothelial cell (HUVEC); blue/grey = K562 erythroleukaemia cells). For the DNase hypersensitivity and transcription factor binding tracks, a grey band indicates the extent of the hypersensitive region and the intensity of the band is proportional to the maximum signal strength observed in any cell line.</p

Genetic architecture of the region containing both <i>GSTCD</i> and <i>INTS12</i> genes.

<p>The top panel depicts gene arrangements previously reported in NCBI, build 37, whereas the lower panel shows novel variants identified in lung. V1, 2, 3 refer to splice variants 1, 2 and 3 for each gene. Open boxes represent exons and connecting black lines represent introns. Also illustrated are the locations of Single Nucleotide Polymorphisms (SNPs) meeting genome-wide association (<i>P</i>≤5x10^-8) for FEV₁ in previously reported analyses of the SpiroMeta consortium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Highlighted in red is the sentinel SNP rs10516526 that was associated with FEV₁ (<i>P</i>=2.18 x 10^-23 in all stage analyses) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Translation start codons (ATG) are shown boxed in green and stop codons (TAA) boxed in red.</p