A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2.

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by Orientia tsutsugamushi) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Autophagy pathways in autoimmune diseases.

Autophagy comprises a growing range of cellular pathways, which occupy central roles in response to energy deprivation, organelle turnover and proteostasis. Over the years, autophagy has been increasingly linked to governing several aspects of immunity, including host defence against various pathogens, unconventional secretion of cytokines and antigen presentation. While canonical autophagy-mediated antigen processing in thymic epithelial cells supports the generation of a self-tolerant CD4+ T cell repertoire, mounting evidence suggests that deregulated autophagy pathways contribute to or sustain autoimmune responses. In animal models of multiple sclerosis (MS), non-canonical autophagy pathways such as microtubule-associated protein 1 A/1 B-light chain 3 (LC3)-associated phagocytosis can contribute to major histocompatibility complex (MHC) class II presentation of autoantigen, thereby amplifying autoreactive CD4+ T cell responses. In systemic lupus erythematosus (SLE), increased type 1 interferon production is linked to excessive autophagy in plasmacytoid dendritic cells (DCs). In rheumatoid arthritis (RA), autophagy proteins contribute to pathological citrullination of autoantigen. Immunotherapies effective in autoimmune diseases modulate autophagy functions, and strategies harnessing autophagy pathways to restrain autoimmune responses have been developed. This review illustrates recent insights in how autophagy, distinct autophagy pathways and autophagy protein functions intersect with the evolution and progression of autoimmune diseases, focusing on MS, SLE and RA.

Impaired systemic nucleocapsid antigen clearance in severe COVID-19.

The circulating nucleocapsid (NCP) antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detectable in coronavirus disease-2019 (COVID-19) patients. To better understand the biology of disease severity, we investigated NCP clearance kinetics in hospitalized COVID-19 patients. Serum NCP was quantified using a commercial NCP-specific enzyme-linked immunoassay in hospitalized COVID-19 patients (n = 63) during their hospital stay. Results were correlated to COVID-19 disease severity, inflammation parameters, antibody response, and results of SARS-CoV-2 PCR from nasopharyngeal swabs. We demonstrate that NCP antigen levels in serum remained elevated in 21/45 (46.7%) samples from patients in intensive care units (ICU) after >8 days postdiagnosis. The proportion of ICU patients with detectable antigenemia declined only gradually from 84.6% to 25.0% over several weeks. This was in contrast to complete NCP clearance in all non-ICU patients after 8 days, and also in contrast to mucosal clearance of the virus as measured by PCR. Antigen clearance was associated with higher IgG against S1 but not NCP. Clearance of NCP antigenemia is delayed in >40% of severely ill COVID-19 patients. Thus, NCP antigenemia detected after 8 days post COVID-19 diagnosis is a characteristic of patients requiring intensive care. Prospective trials should further investigate NCP antigen clearance kinetics as a mechanistic biomarker.

Impaired B Cell Expression of the Inhibitory Fcγ Receptor IIB in Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease in which pathogenic immunoglobulin G antibodies bind to acetylcholine receptors (or to functionally related molecules at the neuromuscular junction). B cell expression of the inhibitory immunoglobulin G receptor, Fc-gamma receptor (FcγR) IIB, maintains peripheral immune tolerance, and its absence renders B cells hyperresponsive to autoantigen. Here, we report that FcγRIIB expression levels are substantially reduced in B lineage cells derived from immunotherapy-naïve patients with acetylcholine receptor antibody-positive early-onset MG. In contrast, genetic variants associated with impaired FcγRIIB expression are not enriched in MG, indicating post-transcriptional dysregulation. FcγR-targeted therapies could have therapeutic benefits in MG. ANN NEUROL 2022;92:1046-1051.

Complement activation profiles in anti-acetylcholine receptor positive myasthenia gravis.

BACKGROUND AND PURPOSE: Complement component 5 (C5) targeting therapies are clinically beneficial in patients with acetylcholine receptor antibody+ (AChR-Ab+ ) generalized myasthenia gravis (MG). That clearly implicates antibody-mediated complement activation in MG pathogenesis. Here, classical and alternative complement pathways were profiled in patients from different MG subgroups. METHODS: In a case-control study, concentrations of C3a, C5a and sC5b9 were simultaneously quantified, indicating general activation of the complement system, whether via the classical and lectin pathways (C4a) or the alternative pathway (factors Ba and Bb) in MG patients with AChR or muscle-specific kinase antibodies (MuSK-Abs) or seronegative MG compared to healthy donors. RESULTS: Treatment-naïve patients with AChR-Ab+ MG showed substantially increased plasma levels of cleaved complement components, indicating activation of the classical and alternative as well as the terminal complement pathways. These increases were still present in a validation cohort of AChR-Ab+ patients under standard immunosuppressive therapies; notably, they were not evident in patients with MuSK-Abs or seronegative MG. Neither clinical severity parameters (at the time of sampling or 1 year later) nor anti-AChR titres correlated significantly with activated complement levels. CONCLUSIONS: Markers indicative of complement activation are prominently increased in patients with AChR-Ab MG despite standard immunosuppressive therapies. Complement inhibition proximal to C5 cleavage should be explored for its potential therapeutic benefits in AChR-Ab+ MG.

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

Complement Activation Is a Prominent Feature of MOGAD.

Myelin oligodendrocyte glycoprotein (MOG)-antibody (Ab)-associated diseases (MOGADs) account for a substantial proportion of pediatric and adult patients who present with acquired demyelinating disorders. Its pathogenesis and optimal therapy are incompletely understood. We profiled systemic complement activation in adult and pediatric patients with MOGAD compared with patients with relapse-onset multiple sclerosis, patients with neuromyelitis optica spectrum disorder, and pediatric control and adult healthy donors. Proteins indicative of systemic classical and alternative complement activation were substantially increased in patients with MOGAD compared to control groups. Elevated levels were detected in both adult and pediatric cases and across all clinical syndromes. Complement inhibition should be explored for its therapeutic merit in patients with MOGAD. ANN NEUROL 2021;90:976-982.

Allograft discard risk index for heart transplantation.

BACKGROUND: The numberof patients awaiting heart transplantation (HTx) substantially exceeds the number of donor hearts transplanted each year, yet nearly 65% of eligible donor hearts are discarded rather than transplanted. METHODS: Deceased organ donors listed within the UNOS Deceased Donor Database between 2010 and 2020 were reviewed. Those greater than 10 years old and consented for heart donation were included and randomly separated into training (n = 48 435) and validation (n = 24 217) cohorts. A discard risk index (DSRI) was created using the results of univariable and multivariable analyses. Discard data were assessed at DSRI value deciles, and stratum-specific likelihood ratio (SSLR) analysis and Kaplan-Meier survival function were used for mortality data. RESULTS: Factors associated with higher DSRI values included donor age > 45, LVEF, HBV-core antibodies, hypertension, and diabetes. The DSRI C-statistic was .906 in the training cohort and .904 in the validation cohort. The DSRI did not reliably predict 30-day or 1-year mortality after transplantation (C-statistic .539 and .532, respectively). CONCLUSIONS: The factors leading to heart allograft discard are not correlated to the same degree with post-transplant outcomes. This suggests that optimizing utilization of certain allografts with slightly higher risk of discard could increase the heart donor pool with limited impact on posttransplant mortality.

NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients.

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Salivary gland hyalinizing clear-cell carcinoma with cutaneous metastasis: A rare and deceptive tumor.

Clear-cell carcinoma (CCC) is an uncommon malignant tumor of minor salivary glands. It characteristically has a low-grade morphology and a favorable outcome by most reports. An EWSR1-ATF1 fusion can be detected in the majority of cases. We present a rare case of CCC, which had an aggressive course with the development of cutaneous metastases. Practicing dermatopathologists should be aware of this tumor given its low-grade appearance and histopathologic resemblance to other primary cutaneous adnexal and metastatic neoplasms.

Fc-Receptor Targeted Therapies for the Treatment of Myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease in which immunoglobulin G (IgG) antibodies (Abs) bind to acetylcholine receptors (AChR) or to functionally related molecules in the postsynaptic membrane at the neuromuscular junction. IgG crystallizable fragment (Fc)-mediated effector functions, such as antibody-dependent complement deposition, contribute to disease development and progression. Despite progress in understanding Ab-mediated disease mechanisms, immunotherapy of MG remained rather unspecific with corticosteroids and maintenance with immunosuppressants as first choice drugs for most patients. More specific therapeutic IgG Fc-based platforms that reduce serum half-life or effector functions of pathogenic MG-related Abs are currently being developed, tested in clinical trials or have recently been successfully translated into the clinic. In this review, we illustrate mechanisms of action and clinical efficacies of emerging Fc-mediated therapeutics such as neonatal Fc receptor (FcRn)-targeting agents. Furthermore, we evaluate prospects of therapies targeting classical Fc receptors that have shown promising therapeutic efficacy in other antibody-mediated conditions. Increased availability of Fc- and Fc receptor-targeting biologics might foster the development of personalized immunotherapies with the potential to induce sustained disease remission in patients with MG.

Guided vs. non-guided insertion of Ambu AuraGain™ in edentulous patients.

BACKGROUND: Supraglottic airway devices perform more poorly and have lower oropharyngeal leak pressure in edentulous patients than in patients with teeth. The Ambu Aura Gain is a newer second generation supraglottic airway device. OBJECTIVE: This randomized clinical trial assessed the oropharyngeal leak pressure in edentulous patients using the Ambu Aura Gain with a gastric tube for insertion guidance and without insertion guidance. MATERIAL AND METHODS: Patients with ASA (American Society of Anesthesiology) physical status I-III were recruited. Primary outcome was oropharyngeal leak pressure after insertion. Secondary outcome parameters were oropharyngeal leak pressure 15 min and 30 min after insertion, insertion time, insertion attempts and glottis view through flexible fiberscope. RESULTS: In this study 72 patients aged between 51 and 90 years (mean 73 years) were randomly allocated to the "with guidance" (n = 36) or the "without guidance" group (n = 36). Mean (SD) oropharyngeal leak pressure in "with guidance" and "without guidance" group was 24 cm H2O and 24 cm H2O (ns), respectively. A difference was found in mean insertion time with guidance versus without guidance group 52 s (45 s) vs. 26 s (15 s) (p < 0.001). No difference was found in any of the other secondary outcome parameters. CONCLUSION: A guided insertion technique does not improve oropharyngeal leak pressure of the Ambu AuraGain™ in edentulous patients. As the only difference is an increase in insertion time this technique is of no benefit for this population.

Global molecular diversity of RSV - the "INFORM RSV" study.

BACKGROUND: Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. METHODS: The INFORM study is performed in 17 countries spanning all inhabited continents and will provide insight into the molecular epidemiology of circulating RSV strains worldwide. Sequencing of > 4000 RSV-positive respiratory samples is planned to detect temporal and geographical molecular patterns on a molecular level over five consecutive years. Additionally, RSV will be cultured from a subset of samples to study the functional implications of specific mutations in the viral genome including viral fitness and susceptibility to different monoclonal antibodies. DISCUSSION: The sequencing and functional results will be used to investigate susceptibility and resistance to novel RSV preventive or therapeutic interventions. Finally, a repository of globally collected RSV strains and a database of RSV sequences will be created.

ATG-dependent phagocytosis in dendritic cells drives myelin-specific CD4+ T cell pathogenicity during CNS inflammation.

Although reactivation and accumulation of autoreactive CD4+ T cells within the CNS are considered to play a key role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the mechanisms of how these cells recognize their target organ and induce sustained inflammation are incompletely understood. Here, we report that mice with conditional deletion of the essential autophagy protein ATG5 in classical dendritic cells (DCs), which are present at low frequencies in the nondiseased CNS, are completely resistant to EAE development following adoptive transfer of myelin-specific T cells and show substantially reduced in situ CD4+ T cell accumulation during the effector phase of the disease. Endogenous myelin peptide presentation to CD4+ T cells following phagocytosis of injured, phosphatidylserine-exposing oligodendroglial cells is abrogated in the absence of ATG5. Pharmacological inhibition of ATG-dependent phagocytosis by the cardiac glycoside neriifolin, an inhibitor of the Na+, K+-ATPase, delays the onset and reduces the clinical severity of EAE induced by myelin-specific CD4+ T cells. These findings link phagocytosis of injured oligodendrocytes, a pathological hallmark of MS lesions and during EAE, with myelin antigen processing and T cell pathogenicity, and identify ATG-dependent phagocytosis in DCs as a key regulator in driving autoimmune CD4+ T cell-mediated CNS damage.

Oropharyngeal leak pressure of the LMA Protector™ vs the LMA Supreme™; a prospective, randomized, controlled clinical trial.

BACKGROUND: Clinical characteristics such as oropharyngeal leak pressure (OLP) and ventilation peak pressure are important factors for successful use of supraglottic airway devices in general anaesthesia. We hypothesized that the LMA Protector™ compared to the LMA Supreme™ may develop a higher OLP, which could be of clinical significance. METHODS: Ninety-six patients were randomized to the LMA Protector™ or LMA Supreme™ groups. We measured oropharyngeal leak pressure within 5 minutes after insertion of the supraglottic airway device with a standardized cuff pressure at 60 cm H2 O. Secondary parameters, such as insertion time of the supraglottic airway device, the number of attempts inserting the supraglottic airway device and the gastric tube, volume of gastric contents, and maximum airway pressure, as well as pulse oximetry throughout the operation were measured. Further, blood staining after removal of the supraglottic airway device and postoperative airway morbidity 3 hours after surgery were determined. RESULTS: The mean difference of oropharyngeal leak pressure was 5.2 (95% CI 2.8-7.6), ie, 30.9 (7.4) cmH2 O for the LMA Protector™ vs 25.6 (4.4) cmH2 O for the LMA Supreme™ (P < 0.001; mean(SD)). Similarly, there was a mean difference between OLP and maximal ventilation peak pressure 5.6 (95% CI 3.1-8.2) ie 19.6 (7.7) cmH2 O for the LMA Protector™ vs 14.0 (4.4) cmH2 O for the LMA Supreme™ (P < 0.001). No difference was found between the groups for other secondary parameters, as well as postoperative airway morbidity. CONCLUSION: The LMA Protector™ enabled a higher OLP compared to the LMA Supreme™. This finding may be important for patients requiring a higher peak pressure for sufficient supraglottic airway device ventilation.

Fiberoptic intubation of severely obese patients through supraglottic airway: A prospective, randomized trial of the Ambu® AuraGain™ laryngeal mask vs the i-gel™ airway.

INTRODUCTION: Airway management in severely obese patients remains a challenging issue for anaesthetists and may lead to life-threatening situations. Supraglottic airway devices, such as the i-gel™ or the AuraGain™, were developed, with the possibility to ventilate the patient or use them as a conduit for endotracheal intubation. METHODS: In our randomized prospective trial, we hypothesized a 10 seconds faster fiberoptic trans-device intubation time through the AuraGain™ laryngeal mask compared to the i-gel™ laryngeal mask in severely obese patients. We randomly assigned 44 patients to the AuraGain or i-gel group and measured trans-device intubation time after 5 minutes of successful ventilation through the device. Secondary parameters relating to the trans-device intubation success, oropharyngeal leak pressure, and parameters regarding insertion of the supraglottic airway devices were measured. Postoperative airway morbidity was determined 5 hours after surgery. RESULTS: Mean (SD) intubation time was 55.7 (5.8) seconds for the AuraGain™ vs 54.1 (8.5) for i-gel™ mask (95% CI -2.7 to 5.9; P = 0.474), respectively, on a mean body mass index (BMI) of 39.4 kg/m2 in the AuraGain™ group vs 38.9 kg/m2 in i-gel™ group. No difference could be found in the other studied parameters. CONCLUSIONS: Time for intubation through both supraglottic airway devices was similar. Attributed to fast possibility of securing the airway with both supraglottic airway devices, we believe that both, AuraGain™ and i-gel™, can be a good alternative in the airway management in obese patients.

Ambu® Aura Gain™ versus Ambu® Aura Once™ in children: a randomized, crossover study assessing oropharyngeal leak pressure and fibreoptic position.

PURPOSE: The Ambu® Aura Gain™ is a new second-generation supraglottic airway device that-because of a wider curvature and a wide airway tube-allows fibreoptic intubation. The purpose of this study was to assess the oropharyngeal leak pressure of the Ambu® Aura GainTM compared with the Ambu® Aura Once™. METHODS: In this randomized non-blinded crossover trial with 50 patients aged 18 months to six years (10-20 kg), we compared the Ambu® Aura Gain™ and the Ambu® Aura Once™ for airway maintenance in anesthetized, non-paralyzed participants. Our primary outcome was oropharyngeal leak pressure. Time of insertion, success rates for each device, evaluation of fibreoptic view and ventilation quality during anesthesia, as well as possible complications (e.g., blood staining) were assessed. RESULTS: There were no differences in first and overall insertion attempt rates (Ambu® Aura Once™ 50/50 (100%) vs Ambu® Aura Gain™ 50/50 (100%). Mean (standard deviation) oropharyngeal leak pressure was found to be significantly higher for Ambu® Aura Gain™ than it was for Ambu® Aura Once™ [21 (7) vs 19 (6) cmH2O, respectively; mean difference [MD] - 2 cmH2O; 95% confidence interval [CI], - 3.8 to - 1.0; P = 0.001]. Mean (SD) insertion time was faster for Ambu® Aura Once™ than for Ambu® Aura Gain™ [8 (3) vs 10 (4) seconds, respectively; MD, - 2 sec; 95% CI, - 2.9 to - 1.2; P < 0.001]. There were no differences in ventilation quality, fibreoptic view, or blood staining. CONCLUSION: We conclude that Ambu® Aura Gain™ is a good alternative to the Ambu® Aura Once™ and an efficient device for children in this age group. TRIAL REGISTRATION: www.clincaltrials.gov (NCT02811042). Registered 23 June 2016.