Background: Sepsis and acute respiratory distress syndrome are two heterogeneous acute illnesses with high risk of death and for which there are many ‘statistically negative’ randomised controlled trials. We hypothesised that negative randomised controlled trials occur because of between-participant differences in response to treatment, illness manifestation (phenotype) and risk of outcomes (heterogeneity). Objectives: To assess (1) heterogeneity of treatment effect, which tests whether or not treatment effect varies with a patient’s pre-randomisation risk of outcome; and (2) whether or not subphenotypes explain the treatment response differences in sepsis and acute respiratory distress syndrome demonstrated in randomised controlled trials. Study population: We performed secondary analysis of two randomised controlled trials in patients with sepsis [i.e. the Vasopressin vs Noradrenaline as Initial Therapy in Septic Shock (VANISH) trial and the Levosimendan for the Prevention of Acute oRgan Dysfunction in Sepsis (LeoPARDS) trial] and one acute respiratory distress syndrome multicentre randomised controlled trial [i.e. the Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trial], conducted in the UK. The VANISH trial is a 2 × 2 factorial randomised controlled trial of vasopressin (Pressyn AR^®; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and hydrocortisone sodium phosphate (hereafter referred to as hydrocortisone) (Efcortesol^TM; Amdipharm plc, St Helier, Jersey) compared with placebo. The LeoPARDS trial is a two-arm-parallel-group randomised controlled trial of levosimendan (Simdax^®; Orion Pharma, Espoo, Finland) compared with placebo. The HARP-2 trial is a parallel-group randomised controlled trial of simvastatin compared with placebo. Methods: To test for heterogeneity of the effect on 28-day mortality of vasopressin, hydrocortisone and levosimendan in patients with sepsis and of simvastatin in patients with acute respiratory distress syndrome. We used the total Acute Physiology And Chronic Health Evaluation II (APACHE II) score as the baseline risk measurement, comparing treatment effects in patients with baseline APACHE II scores above (high) and below (low) the median using regression models with an interaction between treatment and baseline risk. To identify subphenotypes, we performed latent class analysis using only baseline clinical and biomarker data, and compared clinical outcomes across subphenotypes and treatment groups. Results: The odds of death in the highest APACHE II quartile compared with the lowest quartile ranged from 4.9 to 7.4, across the three trials. We did not observe heterogeneity of treatment effect for vasopressin, hydrocortisone and levosimendan. In the HARP-2 trial, simvastatin reduced mortality in the low-APACHE II group and increased mortality in the high-APACHE II group. In the VANISH trial, a two-subphenotype model provided the best fit for the data. Subphenotype 2 individuals had more inflammation and shorter survival. There were no treatment effect differences between the two subphenotypes. In the LeoPARDS trial, a three-subphenotype model provided the best fit for the data. Subphenotype 3 individuals had the greatest inflammation and lowest survival. There were no treatment effect differences between the three subphenotypes, although survival was lowest in the levosimendan group for all subphenotypes. In the HARP-2 trial, a two-subphenotype model provided the best fit for the data. The inflammatory subphenotype was associated with fewer ventilator-free days and higher 28-day mortality. Limitations: The lack of heterogeneity of treatment effect and any treatment effect differences between sepsis subphenotypes may be secondary to the lack of statistical power to detect such effects, if they truly exist. Conclusions: We highlight lack of heterogeneity of treatment effect in all three trial populations. We report three subphenotypes in sepsis and two subphenotypes in acute respiratory distress syndrome, with an inflammatory phenotype with greater risk of death as a consistent finding in both sepsis and acute respiratory distress syndrome. Future work: Our analysis highlights the need to identify key discriminant markers to characterise subphenotypes in sepsis and acute respiratory distress syndrome with an observational cohort study. Funding: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a MRC and National Institute for Health Research (NIHR) partnership. This will be published in full in Efficacy and Mechanism Evaluation; Vol. 8, No. 10. See the NIHR Journals Library website for further project information.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22^phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.

Inflammation of the ileum, or ileitis, is commonly caused by Crohn's disease (CD) but can also accompany ulcerative colitis (backwash ileitis), infections or drug-related damage. Oxidative tissue injury triggered by reactive oxygen species (ROS) is considered part of the ileitis etiology. However, not only elevated ROS but also permanently decreased ROS are associated with inflammatory bowel disease (IBD). While very early onset IBD (VEO-IBD) is associated with a spectrum of NOX1 variants, how NOX1 inactivation contributes to disease development remains ill-defined. Besides propagating signaling responses, NOX1 provides superoxide for peroxynitrite formation in the epithelial barrier. Here we report that NOX4, an H₂O₂-generating NADPH oxidase with documented tissue protective effects in the intestine and other tissues, limits the generation of ileal peroxynitrite by NOX1/NOS2. Deletion of NOX4 leads to persistent peroxynitrite excess, hyperpermeability, villus blunting, muscular hypertrophy, chemokine/cytokine upregulation and dysbiosis. Conversely, SAMP1/YitFc mice, a CD-like ileitis model, showed age-dependent NOX1/NOS2 downregulation preventing ileal peroxynitrite formation in homeostasis and LPS-induced acute inflammation. Deficiency in NOX1 correlated with the upregulation of antimicrobial peptides, suggesting that ileal peroxynitrite acts as chemical barrier and microbiota modifier in the ileum.

Background: In septic shock, cardiovascular resuscitation using catecholamine vasopressors and inotropes is standard therapy, but catecholamines have important side effects. Levosimendan (Simdax^®; Orion Pharma, Newbury, UK) is a calcium-sensitising drug with inotropic and other properties that may have a role in sepsis.Objectives: To determine, in adult septic shock, whether or not levosimendan reduces the incidence and severity of acute organ dysfunction, the effect of levosimendan on individual organ function and the safety profile of levosimendan.Design: Multicentre, randomised, double-blind, parallel-group, placebo-controlled study.Setting: UK intensive care units.Participants: Adult patients with sepsis and cardiovascular failure requiring vasopressors to maintain blood pressure despite adequate fluid resuscitation.Intervention: Levosimendan, at a dosage of 0.05–0.2 µg/kg/minute, compared with placebo for 24 hours, in addition to standard care, within 24 hours of meeting inclusion criteria.Main outcome measure: The primary outcome was mean Sequential Organ Failure Assessment (SOFA) score on the intensive care unit after randomisation to a maximum of 28 days. Secondary outcomes were time to extubation, survival up to 6 months and serious adverse events.Results: In total, 2382 patients were screened at 34 centres, of whom 516 were randomised to treatment, 259 to levosimendan and 257 to placebo. Baseline characteristics were well balanced across treatment arms. There was no significant difference in mean ± standard deviation (SD) SOFA score between the levosimendan group (6.7, SD 4.0) and the placebo group (6.1, SD 3.9) [mean difference 0.61, 95% confidence interval (CI) –0.07 to 1.29]. The 28-day mortality rate was 34.5% and 30.9% in the levosimendan and placebo groups, respectively (absolute difference 3.6%, 95% CI –4.5% to 11.7%). Patients in the levosimendan group were less likely to be successfully extubated over 28 days than patients in the placebo group (hazard ratio 0.77, 95% CI 0.60 to 0.97). More patients in the levosimendan group had supraventricular tachyarrhythmias (3.1% vs. 0.4%; absolute difference 2.7%, 95% CI 0.1% to 5.3%), but there was no overall difference in serious adverse events.Conclusions: In the population of septic shock patients randomised to treatment in this study, the addition of levosimendan to standard medical care did not reduce organ dysfunction or mortality. Levosimendan was associated with a reduced likelihood of successful extubation and an increased risk of supraventricular tachyarrhythmias.Limitations: This was a trial of levosimendan added to standard care rather than a comparison against an alternative inotrope such as dobutamine. No echocardiographic analyses were performed to provide detailed information about changes in myocardial function; therefore, this trial cannot provide guidance as to which inotrope (if any) is best to use in the management of sepsis if a very low cardiac index is present.Future work: Levosimendan could be compared against dobutamine and placebo in patients with a very low cardiac output in sepsis to test which, if any, inotrope should be used in this select group.Trial registration: Current Controlled Trials ISRCTN12776039.Funding: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership. Study drugs were provided by Orion Pharma and additional research funds were provided by Tenax Therapeutics. The study was supported by the NIHR Biomedical Research Centre based at Imperial College, London, and the UK Intensive Care Foundation.

Messenger RNA (mRNA) therapeutics are redefining treatment approaches in vaccines, cancer immunotherapy, protein replacement, and gene editing. Lipid nanoparticles have enabled early clinical successes, but they can be limited by liver-dominant biodistribution, long-term storage stability, and systemic tolerability. Polymeric delivery systems offer a versatile alternative, with tunable physicochemical properties enabling precise control over mRNA complexation, protection, release, and targeting. This review examines recent progress across polyethyleneimine derivatives, poly(β-amino ester)s, poly(amino acid)s, polyesters, dendrimers, charge-altering releasable transporters, and lipid-polymer hybrids. We highlight strategies such as structural modification, stimuli-responsive designs, and high-throughput polymer screening that enhance stability, reduce cytotoxicity, and enable organ- or cell-specific delivery. Addressing challenges in immunogenicity, biodistribution, and manufacturing scalability will be pivotal to translating these innovations into safe and effective mRNA therapeutics.

Dystrophic epidermolysis bullosa (DEB) is a severe monogenic skin disorder resulting from mutations in the COL7A1 gene, which disrupts the synthesis of type VII collagen. This leads to impaired anchoring fibrils and results in dermoepidermal separation. The clinical manifestation of DEB varies significantly, ranging from localized blistering in milder forms to extensive blistering with subsequent severe complications such as vision loss and squamous cell carcinoma in more severe cases. Despite the recent approval of the first gene replacement therapy by the U.S. Food and Drug Administration, the majority of DEB patients still depend on palliative care, an indication of the continued unmet therapeutic needs. In the past two decades, there has been a rapid advancement of gene therapy techniques, extensive research efforts, and pre-clinical studies focusing on the correction of DNA, RNA, and protein defects specific to DEB. In this review, we provide a comprehensive update on the current state of gene engineering strategies for DEB, including gene replacement, pre-mRNA regulatory therapies, and gene editing techniques. In addition, this review critically evaluates the role and development of animal models in DEB research, which are crucial for the progression of therapeutic strategies. Our discussion aims to delineate the existing challenges and emphasize ongoing advancements in the gene therapy landscape for DEB, providing insights that may guide future research and clinical approaches.