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

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

STRIP1, a core component of STRIPAK complexes, is essential for normal mesoderm migration in the mouse embryo

Regulated mesoderm migration is necessary for the proper morphogenesis and organ formation during embryonic development. Cell migration and its dependence on the cytoskeleton and signaling machines have been studied extensively in cultured cells; in contrast, remarkably little is known about the mechanisms that regulate mesoderm cell migration in vivo. Here, we report the identification and characterization of a mouse mutation in striatin-interacting protein 1 (Strip1) that disrupts migration of the mesoderm after the gastrulation epithelial-to-mesenchymal transition (EMT). STRIP1 is a core component of the biochemically defined mammalian striatin-interacting phosphatases and kinase (STRIPAK) complexes that appear to act through regulation of protein phosphatase 2A (PP2A), but their functions in mammals in vivo have not been examined. Strip1-null mutants arrest development at midgestation with profound disruptions in the organization of the mesoderm and its derivatives, including a complete failure of the anterior extension of axial mesoderm. Analysis of cultured mesoderm explants and mouse embryonic fibroblasts from null mutants shows that the mesoderm migration defect is correlated with decreased cell spreading, abnormal focal adhesions, changes in the organization of the actin cytoskeleton, and decreased velocity of cell migration. The results show that STRIPAK complexes are essential for cell migration and tissue morphogenesis in vivo

2001 Research Honors Program Abstracts

Faculty in the College of Agriculture and Life Sciences at Cornell University mentor and guide undergraduate students who have chosen to pursue a research project and graduate with honors. These abstracts reflect the depth of their scholarship and intellectual ability. The research projects encompass work in animal science, biological science, entomology, natural resources, physical science, plant science, and social science

EGF repeat 6 in the purified full-length CRUMBS2 protein is O-glucosylated.

<p>(A) The domain organization of mammalian CRUMBS proteins. All three proteins have a short highly conserved cytoplasmic domain and a large variable extracellular domain. CRUMBS1 has 19 EGF repeats, 13 of which could be modified by POGLUT1, and CRUMBS2 has 15 EGF repeats, 8 of which could be modified by POGLUT1 based on its consensus recognition sequence [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005551#pgen.1005551.ref013" target="_blank">13</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005551#pgen.1005551.ref048" target="_blank">48</a>]. (B) Western blot of wild-type, <i>Poglut1</i>^<i>wsnp</i> and <i>Crumbs2</i>^<i>-/-</i> E8.5 embryos probed with a pan-CRUMBS antibody, showing the more rapid migration of CRUMBS2 in <i>Poglut1</i>^<i>wsnp</i> embryos and the absence of this band in <i>Crumbs2</i> mutants. (C) Western blot for expression of endogenous CRUMBS2 in embryoid bodies at day 6 following differentiation from ES cells probed with pan-CRUMBS antibody. As in embryos, CRUMBS2 in <i>Poglut1</i>^<i>wsnp</i> embryoid bodies migrated more rapidly than the protein in wild-type embryos. (D-E) Mass spectral analysis of O-glucosylation of full-length tagged CRUMBS2 purified from WT and <i>Poglut1</i>^<i>wsnp</i> EBs differentiated for 2 days. Relative amounts of different glycoforms of the peptide ²⁵⁸SGERCEVDEDECA<u>S</u>GPCQNGGQCL²⁸¹ from EGF repeat 6 of CRUMBS2 were compared by generating Extracted Ion Chromatograms (EICs) from the mass spectral data. (D) The peptide from wild-type EBs was modified with O-glucose in the trisaccharide form (green line). The same peptide from <i>Poglut1</i>^<i>wsnp</i> EBs (E) was unmodified by O-glucose (black line). Glucose is represented by a blue circle, xylose by a yellow star, and the peptide by a black line. Full mass spectra for the peptides from each sample are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005551#pgen.1005551.s003" target="_blank">S3B and S3C Fig</a>.</p

POGLUT1 is required for cell-surface localization of CRUMBS2.

<p>(A) Western blot of V5-tagged CRUMBS2 expressed in WT or <i>Poglut1</i>^<i>wsnp</i> (<i>ws</i>) EBs after being subjected to PNGase F or Endo H digestions (Materials and Methods). EBs were differentiated for 5–6 days. Wild-type CRUMBS2 protein is modified with N-glycans sensitive to PNGase F, whereas CRUMBS2 from <i>Poglut1</i>^<i>wsnp</i> EBs is modified with Endo H-sensitive N-glycans, consistent with trapping of the protein in the ER. (B-E) Immunolocalization of CRUMBS2 in transverse sections of E8.5 wild-type neural plate (B) and primitive streak (C). CRUMBS2 in transverse sections of the E8.5 <i>Poglut1</i>^<i>wsnp</i> neural plate (D) primitive streak (E) is not localized to the apical cell surface (up). Scale bars: 20 μm.</p

Indistinguishable phenotypes of early <i>Poglut1</i>^<i>wsnp</i> and <i>Crumbs2</i> mutant embryos.

<p>(A) <i>Meox1</i> expression is reduced and unsegmented in the paraxial mesoderm of both mutants at E8.5 (dorsal views). (B) <i>Brachyury</i> expression at E8.5 shows a smaller primitive streak at the posterior end and a discontinuous midline in both mutants (ventral view, anterior up). (C) <i>Nkx2</i>.<i>5</i> expression shows the abnormal shape of the heart fields in the mutants at E8.5, ventral views. Anterior is up in all panels. Scale bars: 150 μm.</p