Killing without collateral damage: new hope for sepsis therapy.

Dr Proudfoot is a Wellcome Trust Clinical Research Training Fellow and Dr Summers is a Wellcome Trust Postdoctoral Clinical Training Fellow.This is the accepted manuscript version. The final version is available from NPG at http://www.nature.com/icb/journal/v92/n9/full/icb201473a.html

R-loop formation during S phase is restricted by PrimPol-mediated repriming

During DNA replication conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R- loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication

Cardiogenic Shock: Protocols, Teams, Centers, and Networks

The mortality of cardiogenic shock (CS) remains unacceptably high. Delays in the recognition of CS and access to disease-modifying or hemodynamically stabilizing interventions likely contribute to poor outcomes. In parallel to successful initiatives in other disease states, such as acute ST-elevation MI and major trauma, institutions are increasingly advocating the use of a multidisciplinary ‘shock team’ approach to CS management. A volume–outcome relationship exists in CS, as with many other acute cardiovascular conditions, and the emergence of ‘shock hubs’ as experienced facilities with an interest in improving CS outcomes through a hub-and-spoke ‘shock network’ approach provides another opportunity to deliver improved CS care as widely and equitably as possible. This narrative review outlines improvements from a networked approach to care, discusses a team-based and protocolized approach to CS management, reviews the available evidence and discusses the potential benefits, challenges, and opportunities of such systems of care

The pulmonary endothelium in acute respiratory distress syndrome: insights and therapeutic opportunities.

The pulmonary endothelium is a dynamic, metabolically active layer of squamous endothelial cells ideally placed to mediate key processes involved in lung homoeostasis. Many of these are disrupted in acute respiratory distress syndrome (ARDS), a syndrome with appreciable mortality and no effective pharmacotherapy. In this review, we consider the role of the pulmonary endothelium as a key modulator and orchestrator of ARDS, highlighting advances in our understanding of endothelial pathobiology and their implications for the development of endothelial-targeted therapeutics including cell-based therapies. We also discuss mechanisms to facilitate the translation of preclinical data into effective therapies including the application of biomarkers to phenotype patients with ARDS with a predominance of endothelial injury and emerging biotechnologies that could enhance delivery, discovery and testing of lung endothelial-specific therapeutics.This work was supported by the Royal Brompton and Harefield NHS Foundation Trust, NIHR Respiratory Biomedical Research Unit, London, United Kingdom COMPETING INTERESTS CS was a co-investigator on a project grant, funded GlaxoSmithKline, which undertook preclinical assessment of the effects of a potential ARDS therapy on human neutrophils (2012-14). MG & CS have received fees for consultancy from GSK. MG and AGP have had unrestricted Project Grant support from GlaxoSmithKline. FUNDING CS is a Wellcome Trust Postdoctoral Clinical Research Training Fellow. [WT101692MA].This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by the British Medical Journal

Validation of the CREST score for predicting circulatory-aetiology death in out-of-hospital cardiac arrest without STEMI

Aims: The CREST tool was recently developed to stratify the risk of circulatory-aetiology death (CED) in out-of-hospital cardiac arrest (OHCA) patients without ST-elevation myocardial infarction (STEMI). We aimed to validate the CREST score using an external cohort and determine whether it could be improved by the addition of serum lactate on admission. Methods: The study involved the retrospective analysis of consecutive patients admitted to a single tertiary centre with OHCA of presumed cardiac origin over a 51-month period. The CREST score was calculated by attributing points to the following variables: Coronary artery disease (CAD), non-shockable Rhythm, Ejection fraction <30%, cardiogenic Shock at presentation and ischaemic Time ≥25 minutes. The primary endpoint was CED vs neurological aetiology death (NED) or survival. Results: Of 500 patients admitted with OHCA, 211 did not meet criteria for STEMI and were included. 115 patients died in hospital (71 NED, 44 CED). When analysed individually, CED was associated with all CREST variables other than a previous diagnosis of CAD. The CREST score accurately predicted CED with excellent discrimination (C-statistic 0.880, 95% CI 0.813-0.946) and calibration (Hosmer and Lemeshow P=0.948). Although an admission lactate ≥7 mmol/L also predicted CED, its addition to the CREST score (the C-AREST score) did not significantly improve the predictive ability (CS 0.885, 0.815-0.954, HS P=0.942, X2 difference in -2 log likelihood =0.326, P=0.850). Conclusion: Our study is the first to independently validate the CREST score for predicting CED in patients presenting with OHCA without STEMI. Addition of lactate on admission did not improve its predictive ability.Publisher PDFPeer reviewe

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury.

BACKGROUND: Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects. METHODS: We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin. RESULTS: Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro (P<0.01 and P<0.001, respectively), and also significantly attenuated inflammation and signs of lung injury in non-human primates (P<0.01 in all cases). In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release (P<0.01 in all cases) and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples. CONCLUSION: These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01587807

The management of heart failure cardiogenic shock:an international RAND appropriateness panel

Background: Observational data suggest that the subset of patients with heart failure related CS (HF-CS) now predominate critical care admissions for CS. There are no dedicated HF-CS randomised control trials completed to date which reliably inform clinical practice or clinical guidelines. We sought to identify aspects of HF-CS care where both consensus and uncertainty may exist to guide clinical practice and future clinical trial design, with a specific focus on HF-CS due to acute decompensated chronic HF. Methods: A 16-person multi-disciplinary panel comprising of international experts was assembled. A modified RAND/University of California, Los Angeles, appropriateness methodology was used. A survey comprising of 34 statements was completed. Participants anonymously rated the appropriateness of each statement on a scale of 1 to 9 (1–3 as inappropriate, 4–6 as uncertain and as 7–9 appropriate). Results: Of the 34 statements, 20 were rated as appropriate and 14 were rated as inappropriate. Uncertainty existed across all three domains: the initial assessment and management of HF-CS; escalation to temporary Mechanical Circulatory Support (tMCS); and weaning from tMCS in HF-CS. Significant disagreement between experts (deemed present when the disagreement index exceeded 1) was only identified when deliberating the utility of thoracic ultrasound in the immediate management of HF-CS. Conclusion: This study has highlighted several areas of practice where large-scale prospective registries and clinical trials in the HF-CS population are urgently needed to reliably inform clinical practice and the synthesis of future societal HF-CS guidelines

Biomarkers of acute lung injury: worth their salt?

The validation of biomarkers has become a key goal of translational biomedical research. The purpose of this article is to discuss the role of biomarkers in the management of acute lung injury (ALI) and related research. Biomarkers should be sensitive and specific indicators of clinically important processes and should change in a relevant timeframe to affect recruitment to trials or clinical management. We do not believe that they necessarily need to reflect pathogenic processes. We critically examined current strategies used to identify biomarkers and which, owing to expedience, have been dominated by reanalysis of blood derived markers from large multicenter Phase 3 studies. Combining new and existing validated biomarkers with physiological and other data may add predictive power and facilitate the development of important aids to research and therapy

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB