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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development.

The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer

Normal development of thymic conventional αβ T cells, peripheral CD4⁺ and CD8⁺ T cells, and splenic B cells in TKO mice.

<p><b>(A)</b> Representative flow cytometry plots and <b>(B)</b> quantitation of single positive thymocytes. Data were pooled from 2 independent experiments, n = 3–8 mice/genotype. (<b>C)</b> Representative flow cytometry plots and <b>(D)</b> quantitation of splenic CD4⁺ and CD8⁺ T cells. Data were pooled from 2 independent experiments, n = 3–8 mice/genotype. (<b>E</b>) Representative flow plots and <b>(F)</b> quantitation of follicular (FO) and marginal zone (MZ) splenic B cells. Data were pooled from 2 independent experiments, n = 7–8 mice/genotype. Error bars show s.e.m., group means were compared by <i>t</i>-test, ns = not significant, *p<0.05.</p

One more step toward a warmer Arctic

This study was motivated by a strong warming signal seen in mooring-based and oceanographic survey data collected in 2004 in the Eurasian Basin of the Arctic Ocean. The source of this and earlier Arctic Ocean changes lies in interactions between polar and sub-polar basins. Evidence suggests such changes are abrupt, or pulse-like, taking the form of propagating anomalies that can be traced to higher-latitudes. For example, an anomaly found in 2004 in the eastern Eurasian Basin took ~1.5 years to propagate from the Norwegian Sea to the Fram Strait region, and additional ~4.5–5 years to reach the Laptev Sea slope. While the causes of the observed changes will require further investigation, our conclusions are consistent with prevailing ideas suggesting the Arctic Ocean is in transition towards a new, warmer state

TKO mice show mildly reduced responses to immunization with sheep RBC or NP-ova.

<p>(A) Representative flow cytometry plots and <b>(B)</b> quantitation of GC B cells, <b>(C)</b> representative flow cytometry plots and <b>(D)</b> quantitation of Tfh cells from spleen, day 8 post-immunization with sheep RBCs. Data were pooled from 3 independent experiments, n = 10–15 mice/group. <b>(E)</b> Representative flow cytometry plots and (<b>F)</b> quantitation of GC B cells, <b>(G)</b> representative flow cytometry plots and <b>(H)</b> quantitation of Tfh cells from spleen, day 7 post-immunization I.P. with NP-ova and Sigma Adjuvant System. Data were pooled from 4 independent experiments, n = 14–23 mice/group. GC B cells were gated on live CD19⁺B220⁺Fas⁺GL-7⁺ cells, Tfh cells were gated on live CD4⁺B220^-CXCR5^hiPD-1^hi cells. <b>(I)</b> Serum antibody titer ELISA, day 7 post-immunization with NP-ova and Sigma Adjuvant System. Concentration of antibodies for individual mice (NP-specific concentrations are relative to a pooled positive control), A.U. = arbitrary units. Data is representative of 1 of 2 independent experiments, n = 5–8 mice/group. Error bars show s.e.m., group means for (B, D, F, H) were compared by <i>t</i>-test, group means for (I) were compared by nonparametric Kruskal-Wallis test, followed by Dunn’s post hoc test, ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Impaired iNKT cell development in TKO mice.

<p><b>(A)</b> Representative flow cytometry plots and <b>(B)</b> quantitation of thymic iNKT cells. Data were pooled from 4 independent experiments, n = 7–15 mice/genotype. <b>(C)</b> Representative flow cytometry plots and <b>(D)</b> quantitation of mature CD44⁺NK1.1⁺ thymic iNKT cells. Data were pooled from 3 independent experiments, n = 7–11 mice/genotype. <b>(E)</b> Representative flow cytometry plots and <b>(F)</b> quantitation of hepatic and splenic iNKT cells. Data were pooled from 2 independent experiments, n = 3–7 mice/genotype for liver, n = 7–8 mice/genotype for spleen. <b>(G)</b> Representative flow cytometry plots and quantitation of 2B4 expression on liver iNKT cells. Representative data from 1 of 2 independent experiments is shown, n = 3–4 mice/genotype. Error bars show s.e.m., group means were compared by <i>t</i>-test, ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p