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Abstract Biological sex differences exist for many airway diseases in which females have either worse or better health outcomes. Inflammatory airway diseases such as cystic fibrosis (CF) and asthma display a clear male advantage in post-puberty while a female benefit is observed in asthma during the pre-puberty years. The influence of menstrual cycle stage and pregnancy on the frequency and severity of pulmonary exacerbations in CF and asthma point to a role for sex steroid hormones, particularly estrogen, in underpinning biological sex differences in these diseases. There are many ways by which estrogen may aggravate asthma and CF involving disturbances in airway surface liquid (ASL) dynamics, inappropriate hyper-immune and allergenic responses, as well as exacerbation of pathogen virulence. The deleterious effect of estrogen on pulmonary function in CF and asthma contrasts with the female advantage observed in airway diseases characterised by pulmonary edema such as pneumonia, acute respiratory distress syndrome (ARDS) and COVID-19. Airway surface liquid hypersecretion and alveolar flooding are hallmarks of ARDS and COVID-19, and contribute to the morbidity and mortality of severe forms of these diseases. ASL dynamics encompasses the intrinsic features of the thin lining of fluid covering the airway epithelium which regulate mucociliary clearance (ciliary beat, ASL height, volume, pH, viscosity, mucins, and channel activating proteases) in addition to innate defence mechanisms (pathogen virulence, cytokines, defensins, specialised pro-resolution lipid mediators, and metabolism). Estrogen regulation of ASL dynamics contributing to biological sex differences in CF, asthma and COVID-19 is a major focus of this review. Graphical Abstract

Abstract Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

Abstract Positive feedbacks in climate processes can make it difficult to identify the primary drivers of climate phenomena. Some recent global climate model (GCM) studies address this issue by controlling the wind stress felt by the surface ocean such that the atmosphere and ocean become mechanically decoupled. Most mechanical decoupling studies have chosen to override wind stress with an annual climatology. In this study we introduce an alternative method of interannually varying overriding which maintains higher frequency momentum forcing of the surface ocean. Using a GCM (NCAR CESM1), we then assess the size of the biases associated with these two methods of overriding by comparing with a freely evolving control integration. We find that overriding with a climatology creates sea surface temperature (SST) biases throughout the global oceans on the order of ±1°C. This is substantially larger than the biases introduced by interannually varying overriding, especially in the tropical Pacific. We attribute the climatological overriding SST biases to a lack of synoptic and subseasonal variability, which causes the mixed layer to be too shallow throughout the global surface ocean. This shoaling of the mixed layer reduces the effective heat capacity of the surface ocean such that SST biases excite atmospheric feedbacks. These results have implications for the reinterpretation of past climatological wind stress overriding studies: past climate signals attributed to momentum coupling may in fact be spurious responses to SST biases.

The hyperexpression of human leukocyte antigen class I (HLA-I) molecules on pancreatic beta-cells is widely accepted as a hallmark feature of type 1 diabetes pathogenesis. This response is important clinically since it may increase the visibility of beta-cells to autoreactive CD8+ T-cells, thereby accelerating disease progression. In this review, key factors which drive HLA-I hyperexpression will be explored, and their clinical significance examined. It is established that the presence of residual beta-cells is essential for HLA-I hyperexpression by islet cells at all stages of the disease. We suggest that the most likely drivers of this process are interferons released from beta-cells (type I or III interferon; possibly in response to viral infection) or those elaborated from influent, autoreactive immune cells (type II interferon). In both cases, Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways will be activated to induce the downstream expression of interferon stimulated genes. A variety of models have highlighted that HLA-I expression is enhanced in beta-cells in response to interferons, and that STAT1, STAT2 and interferon regulatory factor 9 (IRF9) play key roles in mediating these effects (depending on the species of interferon involved). Importantly, STAT1 expression is elevated in the beta-cells of donors with recent-onset type I diabetes, and this correlates with HLA-I hyperexpression on an islet-by-islet basis. These responses can be replicated in vitro, and we consider that chronically elevated STAT1 may have a role in maintaining HLA-I hyperexpression. However, other data have highlighted that STAT2-IRF9 may also be critical to this process. Thus, a better understanding of how these factors regulate HLA-I under chronically stimulated conditions needs to be gathered. Finally, JAK inhibitors can target interferon signaling pathways to diminish HLA-I expression in mouse models. It seems probable that these agents may also be effective in patients; diminishing HLA-I hyperexpression on islets, reducing the visibility of beta-cells to the immune system and ultimately slowing disease progression. The first clinical trials of selective JAK inhibitors are underway, and the outcomes should have important implications for type 1 diabetes clinical management.

The recent biological redefinition of Alzheimer’s Disease (AD) has spurred the development of statistical models that relate changes in biomarkers with neurodegeneration and worsening condition linked to AD. The ability to measure such changes may facilitate earlier diagnoses for affected individuals and help in monitoring the evolution of their condition. Amongst such statistical tools, disease progression models (DPMs) are quantitative, data-driven methods that specifically attempt to describe the temporal dynamics of biomarkers relevant to AD. Due to the heterogeneous nature of this disease, with patients of similar age experiencing different AD-related changes, a challenge facing longitudinal mixed-effects-based DPMs is the estimation of patient-realigning time-shifts. These time-shifts are indispensable for meaningful biomarker modelling, but may impact fitting time or vary with missing data in jointly estimated models. In this work, we estimate an individual’s progression through Alzheimer’s disease by combining multiple biomarkers into a single value using a probabilistic formulation of principal components analysis. Our results show that this variable, which summarises AD through observable biomarkers, is remarkably similar to jointly estimated time-shifts when we compute our scores for the baseline visit, on cross-sectional data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Reproducing the expected properties of clinical datasets, we confirm that estimated scores are robust to missing data or unavailable biomarkers. In addition to cross-sectional insights, we can model the latent variable as an individual progression score by repeating estimations at follow-up examinations and refining long-term estimates as more data is gathered, which would be ideal in a clinical setting. Finally, we verify that our score can be used as a pseudo-temporal scale instead of age to ignore some patient heterogeneity in cohort data and highlight the general trend in expected biomarker evolution in affected individuals.

Summary: Background: Type 1 diabetes is a complex heterogenous autoimmune disease without therapeutic interventions available to prevent or reverse the disease. This study aimed to identify transcriptional changes associated with the disease progression in patients with recent-onset type 1 diabetes. Methods: Whole-blood samples were collected as part of the INNODIA study at baseline and 12 months after diagnosis of type 1 diabetes. We used linear mixed-effects modelling on RNA-seq data to identify genes associated with age, sex, or disease progression. Cell-type proportions were estimated from the RNA-seq data using computational deconvolution. Associations to clinical variables were estimated using Pearson's or point-biserial correlation for continuous and dichotomous variables, respectively, using only complete pairs of observations. Findings: We found that genes and pathways related to innate immunity were downregulated during the first year after diagnosis. Significant associations of the gene expression changes were found with ZnT8A autoantibody positivity. Rate of change in the expression of 16 genes between baseline and 12 months was found to predict the decline in C-peptide at 24 months. Interestingly and consistent with earlier reports, increased B cell levels and decreased neutrophil levels were associated with the rapid progression. Interpretation: There is considerable individual variation in the rate of progression from appearance of type 1 diabetes-specific autoantibodies to clinical disease. Patient stratification and prediction of disease progression can help in developing more personalised therapeutic strategies for different disease endotypes. Funding: A full list of funding bodies can be found under Acknowledgments.

Microvascular integrity is a critical factor in myocardial fluid homeostasis. The subtle equilibrium between capillary filtration and lymphatic fluid removal is disturbed during pathological processes leading to inflammation, but also in hypoxia or due to alterations in vascular perfusion and coagulability. The degradation of the glycocalyx as the main component of the endothelial filtration barrier as well as pericyte disintegration results in the accumulation of interstitial and intracellular water. Moreover, lymphatic dysfunction evokes an increase in metabolic waste products, cytokines and inflammatory cells in the interstitial space contributing to myocardial oedema formation. This leads to myocardial stiffness and impaired contractility, eventually resulting in cardiomyocyte apoptosis, myocardial remodelling and fibrosis. The following article reviews pathophysiological inflammatory processes leading to myocardial oedema including myocarditis, ischaemia-reperfusion injury and viral infections with a special focus on the pathomechanisms evoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition, clinical implications including potential long-term effects due to viral persistence (long COVID), as well as treatment options, are discussed.

α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients.

Abstract Background Rodent and human β-cells are differentially susceptible to the “lipotoxic” effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent β–cells and present data that reveal new insights into the factors regulating β-cell lipotoxicity. Methods The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-βH1) β-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. Results Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-βH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-βH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. Conclusion In rodent β-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- βH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human β-cells to “lipotoxicity” since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Background Increasing awareness of milder presentations of cystic fibrosis (CF) and greater interest in non-CF bronchiectasis are likely to lead to more CF screening by respiratory clinicians. As a result, adults who may not strictly fulfil CF diagnostic criteria yet display evidence of abnormal CF transmembrane conductance regulator (CFTR) function are being identified. The degree of agreement on diagnosis and care needs in these cases between CF clinicians remains unknown, and has implications for patient care, including access to CFTR modulator therapies. Methods We surveyed adult CF physicians in Canada, the USA, the UK and Ireland, and presented them with anonymised vignettes of adult patients referred for assessment of possible CF. Diagnostic inter-rater agreement over diagnosis, ease of classifying cases and appropriate follow-up was assessed using Krippendorff's reliability coefficient (α). Results Agreement over diagnosis (α=0.282), ease of classification (α= −0.01) and recommended follow-up (α=0.054) was weak. Clinician experience (>10 and 5–10 years versus

Abstract The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

Aims/hypothesisThe Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls.MethodsRNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue.ResultsThe data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of β-cells displayed markedly increased PKR protein levels. These PKR+ β-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain β-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in β-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes β-cells.Conclusion/interpretationThe gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in β-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet β-cells contributes to the pathogenesis of type 1 diabetes.

Abstract Objective We aimed to characterize the effects of prone positioning on respiratory mechanics and oxygenation in invasively ventilated patients with SARS-CoV-2 ARDS. Results This was a prospective cohort study in the Intensive Care Unit (ICU) of a tertiary referral centre. We included 20 consecutive, invasively ventilated patients with laboratory confirmed SARS-CoV-2 related ARDS who underwent prone positioning in ICU as part of their management. The main outcome was the effect of prone positioning on gas exchange and respiratory mechanics. There was a median improvement in the PaO2/FiO2 ratio of 132 in the prone position compared to the supine position (IQR 67–228). We observed lower PaO2/FiO2 ratios in those with low ( median) static compliance (P

Abstract Obesity is a major risk factor for type 2 diabetes (T2D) although the causal links remain unclear. A feature shared by both conditions however is systemic inflammation and raised levels of circulating fatty acids (FFA). It is widely believed that in obese individuals genetically prone to T2D, elevated levels of plasma FFA may contribute towards the death and dysfunction of insulin-producing pancreatic β-cells in a process of (gluco)lipotoxicity. In support of this, in vitro studies have shown consistently that long-chain saturated fatty acids (LC-SFA) are toxic to rodent β-cells during chronic exposure (> 24 h). Conversely, shorter chain SFA and unsaturated species are well tolerated, suggesting that toxicity is dependent on carbon chain length and/or double bond configuration. Despite the wealth of evidence implicating lipotoxicity as a means of β-cell death in rodents, the evidence that a similar process occurs in humans is much less substantial. Therefore, the present study has evaluated the effects of chronic exposure to fatty acids of varying chain length and degree of saturation, on the viability of human β-cells in culture. We have also studied the effects of a combination of fatty acids and pro-inflammatory cytokines. Strikingly, we find that LC-FFA do not readily promote the demise of human β-cells and that they may even offer a measure of protection against the toxic effects of pro-inflammatory cytokines. Therefore, these findings imply that a model in which elevated circulating LC-FFA play a direct role in mediating β-cell dysfunction and death in humans, may be overly simplistic.

A 62-year-old male with symptomatic persistent atrial fibrillation underwent radiofrequency catheter ablation. During exchange of the saline irrigation bag, the patient developed sudden hypotension and bradycardia and was found to have a massive air embolism. Air was successfully aspirated with catheters, and the patient did not suffer any permanent sequelae. (Level of Difficulty: Intermediate.)

Summary: Background: Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection. Methods: Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab. Findings: AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samplesInduction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection. Interpretation: There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy.

The cytokine IFNα is expressed in the islets of individuals with type 1 diabetes and contributes to local inflammation and destruction of beta cells. Here, the authors provide a global multiomics view of IFNα-induced changes in human beta cells at the level of chromatin, mRNA and protein expression.

Autoimmune and paraneoplastic encephalitides represent an increasingly recognized cause of devastating human illness as well as an emerging area of neurological injury associated with immune checkpoint inhibitors. Two groups of antibodies have been detected in affected patients. Antibodies in the first group are directed against neuronal cell surface membrane proteins and are exemplified by antibodies directed against the N-methyl-D-aspartate receptor (anti-NMDAR), found in patients with autoimmune encephalitis, and antibodies directed against the leucine-rich glioma-inactivated 1 protein (anti-LGI1), associated with faciobrachial dystonic seizures and limbic encephalitis. Antibodies in this group produce non-lethal neuronal dysfunction, and their associated conditions often respond to treatment. Antibodies in the second group, as exemplified by anti-Yo antibody, found in patients with rapidly progressive cerebellar syndrome, and anti-Hu antibody, associated with encephalomyelitis, react with intracellular neuronal antigens. These antibodies are characteristically found in patients with underlying malignancy, and neurological impairment is the result of neuronal death. Within the last few years, major advances have been made in understanding the pathogenesis of neurological disorders associated with antibodies against neuronal cell surface antigens. In contrast, the events that lead to neuronal death in conditions associated with antibodies directed against intracellular antigens, such as anti-Yo and anti-Hu, remain poorly understood, and the respective roles of antibodies and T lymphocytes in causing neuronal injury have not been defined in an animal model. In this review, we discuss current knowledge of these two groups of antibodies in terms of their discovery, how they arise, the interaction of both types of antibodies with their molecular targets, and the attempts that have been made to reproduce human neuronal injury in tissue culture models and experimental animals. We then discuss the emerging area of autoimmune neuronal injury associated with immune checkpoint inhibitors and the implications of current research for the treatment of affected patients.

Oxidative stress from innate immune cells is a driving mechanism that underlies COPD pathogenesis. Individuals with α-1 antitrypsin (AAT) deficiency (AATD) have a dramatically increased risk of developing COPD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil NADPH oxidase activation, due to the specific lack of plasma AAT. Experiments were performed using circulating neutrophils isolated from healthy controls and individuals with AATD. Superoxide anion (O2−) production was determined from the rate of reduction of cytochrome c. Quantification of membrane NADPH oxidase subunits was performed by mass spectrometry and Western blot analysis. The clinical significance of our in vitro findings was assessed in patients with AATD and severe COPD receiving intravenous AAT replacement therapy. In vitro, AAT significantly inhibited O2− production by stimulated neutrophils and suppressed receptor stimulation of cyclic adenosine monophosphate and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. In addition, AAT reduced plasma membrane translocation of cytosolic phox components of the NADPH oxidase. Ex vivo, AATD neutrophils demonstrated increased plasma membrane-associated p67phox and p47phox and significantly increased O2− production. The described variance in phox protein membrane assembly was resolved post-AAT augmentation therapy in vivo, the effects of which significantly reduced AATD neutrophil O2− production to that of healthy control cells. These results expand our knowledge on the mechanism of neutrophil-driven airways disease associated with AATD. Therapeutic AAT augmentation modified neutrophil NADPH oxidase assembly and reactive oxygen species production, with implications for clinical use in conditions in which oxidative stress plays a pathogenic role.

Abstract Objectives The primary objective is to demonstrate that, in patients with PCR-confirmed SARS-CoV-2 resulting in Acute Respiratory Distress Syndrome (ARDS), administration of 120mg/kg of body weight of intravenous Prolastin®(plasma-purified alpha-1 antitrypsin) reduces circulating plasma levels of interleukin-6 (IL-6). Secondary objectives are to determine the effects of intravenous Prolastin® on important clinical outcomes including the incidence of adverse events (AEs) and serious adverse events (SAEs). Trial design Phase 2, randomised, double-blind, placebo-controlled, pilot trial. Participants The study will be conducted in Intensive Care Units in hospitals across Ireland. Patients with a laboratory-confirmed diagnosis of SARS-CoV-2-infection, moderate to severe ARDS (meeting Berlin criteria for a diagnosis of ARDS with a PaO2/FiO2 ratio 18 years of age and requiring invasive or non-invasive mechanical ventilation. All individuals meeting any of the following exclusion criteria at baseline or during screening will be excluded from study participation: more than 96 hours has elapsed from onset of ARDS; age < 18 years; known to be pregnant or breastfeeding; participation in a clinical trial of an investigational medicinal product (other than antibiotics or antivirals) within 30 days; major trauma in the prior 5 days; presence of any active malignancy (other than nonmelanoma skin cancer) which required treatment within the last year; WHO Class III or IV pulmonary hypertension; pulmonary embolism prior to hospital admission within past 3 months; currently receiving extracorporeal life support (ECLS); chronic kidney disease receiving dialysis; severe chronic liver disease with Child-Pugh score > 12; DNAR (Do Not Attempt Resuscitation) order in place; treatment withdrawal imminent within 24 hours; Prisoners; non-English speaking patients or those who do not adequately understand verbal or written information unless an interpreter is available; IgA deficiency. Intervention and comparator Intervention: Either a once weekly intravenous infusion of Prolastin® at 120mg/kg of body weight for 4 weeks or a single dose of Prolastin® at 120mg/kg of body weight intravenously followed by once weekly intravenous infusion of an equal volume of 0.9% sodium chloride for a further 3 weeks. Comparator (placebo): An equal volume of 0.9% sodium chloride intravenously once per week for four weeks. Main outcomes The primary effectiveness outcome measure is the change in plasma concentration of IL-6 at 7 days as measured by ELISA. Secondary outcomes include: safety and tolerability of Prolastin® in the respective groups (as defined by the number of SAEs and AEs); PaO2/FiO2 ratio; respiratory compliance; sequential organ failure assessment (SOFA) score; mortality; time on ventilator in days; plasma concentration of alpha-1 antitrypsin (AAT) as measured by nephelometry; plasma concentrations of interleukin-1β (IL-1β), interleukin-8 (IL-8), interleukin-10 (IL-10), soluble TNF receptor 1 (sTNFR1, a surrogate marker for TNF-α) as measured by ELISA; development of shock; acute kidney injury; need for renal replacement therapy; clinical relapse, as defined by the need for readmission to the ICU or a marked decline in PaO2/FiO2 or development of shock or mortality following a period of sustained clinical improvement; secondary bacterial pneumonia as defined by the combination of radiographic findings and sputum/airway secretion microscopy and culture. Randomisation Following informed consent/assent patients will be randomised. The randomisation lists will be prepared by the study statistician and given to the unblinded trial personnel. However, the statistician will not be exposed to how the planned treatment will be allocated to the treatment codes. Randomisation will be conducted in a 1:1:1 ratio, stratified by site and age. Blinding (masking) The investigator, treating physician, other members of the site research team and patients will be blinded to treatment allocation. The clinical trial pharmacy personnel and research nurses will be unblinded to facilitate intervention and placebo preparation. The unblinded individuals will keep the treatment information confidential. The infusion bag will be masked at the time of preparation and will be administered via a masked infusion set to maintain blinding. Numbers to be randomised (sample size) A total of 36 patients will be recruited and randomised in a 1:1:1 ratio to each of the trial arms. Trial status In March 2020, version 1.0 of the trial protocol was submitted to the local research ethics committee (REC), Health Research Consent Declaration Committee (HRCDC) and the Health Products regulatory Authority (HPRA). REC approval was granted on April 1st 2020, HPRA approval was granted on April 24th 2020 and the HRCDC provided a conditional declaration on April 17th 2020. In July 2020 a substantial amendment (version 2.0) was submitted to the REC, HRCDC and HPRA. Protocol changes in this amendment included: the addition of trial sites; extending the duration of the trial to 12 months from 3 months; removal of inclusion criteria requiring the need for vasopressors; amendment of randomisation schedule to stratify by age only and not BMI and sex; correction of grammatical error in relation to infusion duration; to allow for inclusion of subjects who may have been enrolled in a clinical trial involving either antibiotics or anti-virals in the past 30 days; to allow for inclusion of subjects who may be currently enrolled in a clinical trial involving either antibiotics or anti-virals; to remove the need for exclusion based on alpha-1 antitrypsin phenotype; removal of mandatory isoelectric focusing of plasma to confirm Pi*MM status at screening; removal of need for mandatory echocardiogram at screening; amendment on procedures around plasma analysis to reflect that this will be conducted at the central site laboratory (as trial is multi-site and no longer single site); wording amended to reflect that interim analysis of cytokine levels taken at 7 days may be conducted. HRCDC approved version 2.0 on September 14th 2020, and HPRA approved on October 22nd 2020. REC approved the substantial amendment on November 23rd. In November 2020, version 3.0 of the trial protocol was submitted to the REC and HPRA. The rationale for this amendment was to allow for patients with moderate to severe ARDS from SARS-CoV-2 with non-invasive ventilation. HPRA approved this amendment on December 1st 2020 and the REC approved the amendment on December 8th 2020. Patient recruitment commenced in April 2020 and the last patient will be recruited to the trial in April 2021. The last visit of the last patient is anticipated to occur in April 2021. At time of writing, patient recruitment is now complete, however follow-up patient visits and data collection are ongoing. Trial registration EudraCT 2020-001391-15 (Registered 31 Mar 2020). Full protocol The full protocol (version 3.0 23.11.2020) is attached as an additional file accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Altered microRNA expression patterns in bronchial brushings from people with versus without cystic fibrosis (CF) relate to functional changes and disease pathophysiology. The expression of microRNAs encoded on the X chromosome is also altered in peripheral blood monocytes of p. Phe508del homozygous versus non-CF individuals. Here we investigate whether levels of the top seven X-linked microRNAs (miR-224-5p, miR-452-5p, miR-450b-5p, miR-542-3p, miR-450a-5p, miR-424-5p, and miR-545-5p) that are significantly increased over 1.5 fold in CF versus non-CF monocytes correlate with lung function. CD14+ monocytes were isolated from males and females with (n = 12) and without cystic fibrosis (n = 12) and examined for the expression of X-linked microRNAs by qRT-PCR array. MicroRNA target mRNA levels were quantified using qRT-PCR. Clinical correlations with lung function data were analysed in the CF cohort. Increasing levels of miR-545-5p correlated moderately with FEV1% predicted (r = -0.4553, p > 0.05) and strongly with exacerbation rate (r = 0.5858, p = 0.0483). miR-224-5p levels were significantly higher in the severe (FEV1

In the lung, glycosaminoglycans (GAGs) are dispersed in the extracellular matrix (ECM) occupying the interstitial space between the capillary endothelium and the alveolar epithelium, in the sub-epithelial tissue and in airway secretions. In addition to playing key structural roles, GAGs contribute to a number of physiologic processes ranging from cell differentiation, cell adhesion and wound healing. Cytokine and chemokine–GAG interactions are also involved in presentation of inflammatory molecules to respective receptors leading to immune cell migration and airway infiltration. More recently, pathophysiological roles of GAGs have been described. This review aims to discuss the biological roles and molecular interactions of GAGs, and their impact in the pathology of chronic airway diseases, such as cystic fibrosis and chronic obstructive pulmonary disease. Moreover, the role of GAGs in respiratory disease has been heightened by the current COVID-19 pandemic. This review underlines the essential need for continued research aimed at exploring the contribution of GAGs in the development of inflammation, to provide a better understanding of their biological impact, as well as leads in the development of new therapeutic agents.

Significant progress has been made in understanding the phenotypes of circulating immune cell sub-populations in human type 1 diabetes but much less is known about the equivalent populations that infiltrate the islets to cause beta-cell loss. In particular, considerable uncertainties remain about the phenotype and role of B-lymphocytes in the pancreas. This gap in understanding reflects both the difficulty in accessing the gland to study islet inflammation during disease progression and the fact that the number and proportion of islet-associated B-lymphocytes varies significantly according to the disease endotype. In very young children (especially those

Purpose: The purpose of this study was to investigate UHb-rDWI signal in white matter tracts of the cervical spinal cord (CSC) and compare quantitative values between healthy control WM with both MS NAWM and MS WM lesions. Methods: UHb-rDWI experiments were performed on (a) 7 MS patients with recently active or chronic lesions in CSC and on (b) 7 healthy control of similar age range and gender distribution to MS subjects. All MRI data were acquired using clinical 3T MRI system. Axial high-b diffusion images were acquired using 2D single-shot DW stimulated EPI with reduced FOV and a CSC-dedicated 8 channel array coil. High-b diffusion coefficient DH was estimated by fitting the signal-b curve to a double or single-exponential function. Results: The high-b diffusivity DH values were measured as (0.767 ± 0.297) × 10−3 mm2/s in the posterior column lesions, averaged over 6 MS patients, and 0.587 × 10−3 mm2/s in the corticospinal tract for another patient. The averaged DH values of the 7 healthy volunteers from the posterior and lateral column were (0.0312 ± 0.0306) × 10−3 and (0.0505 ± 0.0205) × 10−3 mm2/s, respectively. UHb-rDWI signal-b curves of the MS patients revealed to noticeably behave differently to that of the healthy controls. The patient signal-b curves decayed with greater high-b decay constants to reach lower signal intensities relative to signal-b curves of the healthy controls. Conclusion: UHb-DWI of the CSC reveals a marked difference in signal-b-curves and DH values in MS lesions compared to NAWM and healthy control WM. Based on physical principles, we interpret these altered observations of quantitative diffusion values to be indicative of demyelination. Further studies in animal models will be required to fully interpret UHb-DWI quantitative diffusion values during demyelination and remyelination.

Coronavirus disease (COVID)-19, as a result of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, has been the direct cause of over 2.2 million deaths worldwide. A timely coordinated host-immune response represents the leading driver for restraining SARS-CoV-2 infection. Indeed, several studies have described dysregulated immunity as the crucial determinant for critical illness and the failure of viral control. Improved understanding and management of COVID-19 could greatly reduce the mortality and morbidity caused by SARS-CoV-2. One aspect of the immune response that has to date been understudied is whether lipid mediator production is dysregulated in critically ill patients. In the present study, plasma from COVID-19 patients with either severe disease and those that were critically ill was collected and lipid mediator profiles were determined using liquid chromatography tandem mass spectrometry. Results from these studies indicated that plasma concentrations of both pro-inflammatory and pro-resolving lipid mediator were reduced in critically ill patients when compared with those with severe disease. Furthermore, plasma concentrations of a select group of mediators that included the specialized pro-resolving mediators (SPM) Resolvin (Rv) D1 and RvE4 were diagnostic of disease severity. Interestingly, peripheral blood SPM concentrations were also linked with outcome in critically ill patients, where we observed reduced overall concentrations of these mediators in those patients that did not survive. Together the present findings establish a link between plasma lipid mediators and disease severity in patients with COVID-19 and indicate that plasma SPM concentrations may be linked with survival in these patients.

Mexico has experienced one of the highest COVID-19 mortality rates in the world. A delayed implementation of social distancing interventions in late March 2020 and a phased reopening of the country in June 2020 has facilitated sustained disease transmission in the region. In this study we systematically generate and compare 30-day ahead forecasts using previously validated growth models based on mortality trends from the Institute for Health Metrics and Evaluation for Mexico and Mexico City in near real-time. Moreover, we estimate reproduction numbers for SARS-CoV-2 based on the methods that rely on genomic data as well as case incidence data. Subsequently, functional data analysis techniques are utilized to analyze the shapes of COVID-19 growth rate curves at the state level to characterize the spatiotemporal transmission patterns of SARS-CoV-2. The early estimates of the reproduction number for Mexico were estimated between Rt ~1.1-1.3 from the genomic and case incidence data. Moreover, the mean estimate of Rt has fluctuated around ~1.0 from late July till end of September 2020. The spatial analysis characterizes the state-level dynamics of COVID-19 into four groups with distinct epidemic trajectories based on epidemic growth rates. Our results show that the sequential mortality forecasts from the GLM and Richards model predict a downward trend in the number of deaths for all thirteen forecast periods for Mexico and Mexico City. However, the sub-epidemic and IHME models perform better predicting a more realistic stable trajectory of COVID-19 mortality trends for the last three forecast periods (09/21-10/21, 09/28-10/27, 09/28-10/27) for Mexico and Mexico City. Our findings indicate that phenomenological models are useful tools for short-term epidemic forecasting albeit forecasts need to be interpreted with caution given the dynamic implementation and lifting of social distancing measures.

Background Cardiac sympathetic denervation (CSD) has been used as a bailout strategy for refractory ventricular tachycardia (VT). Risk of VT recurrence in patients with scar‐related monomorphic VT referred for CSD and the extent to which CSD can modify this risk is unknown. We aimed to quantify arrhythmia recurrence risk and impact of CSD in this population. Methods and Results Adjusted competing risk time to event models were developed to adjust for risk of VT recurrence and sustained VT/implantable cardioverter–defibrillator shocks after VT ablation based on patient comorbidities at the time of VT ablation. Adjusted VT and implantable cardioverter–defibrillator shock recurrence rates were estimated for the subgroup who subsequently required CSD after ablation. The expected adjusted recurrence rates were then compared with the observed rates after CSD. Data from 381 patients with scar‐mediated monomorphic VT who underwent VT ablation were analyzed, excluding patients with polymorphic VT. Sixty eight patients underwent CSD for recurrent VT. CSD reduced the expected adjusted VT recurrence rate by 36% (expected rate of 5.61 versus observed rate of 3.58 per 100 person‐months, P=0.01) and the sustained VT/implantable cardioverter–defibrillator shock rates by 34% (expected rate of 4.34 versus observed 2.85 per 100 person‐months, P=0.03). The median number of sustained VT/implantable cardioverter–defibrillator shocks in the year before versus the year after CSD was reduced by 90% (10 versus 1, P

Studies have endeavored to understand the cause for impaired antimicrobial killing by neutrophils of people with cystic fibrosis (PWCF). The aim of this study was to focus on the bacterial phagosome. Possible alterations in degranulation of cytoplasmic granules and changes in pH were assessed. Circulating neutrophils were purified from PWCF (n = 28), PWCF receiving ivacaftor therapy (n = 10), and healthy controls (n = 28). Degranulation was assessed by Western blot analysis and flow cytometry. The pH of phagosomes was determined by use of BCECF-AM-labelled Staphylococcus aureus or SNARF labelled Candida albicans. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Bacterial killing by CF and healthy control neutrophils were found to differ (p = 0.0006). By use of flow cytometry and subcellular fractionation the kinetics of intraphagosomal degranulation were found to be significantly altered in CF phagosomes, as demonstrated by increased primary granule CD63 (p = 0.0001) and myeloperoxidase (MPO) content (p = 0.03). In contrast, decreased secondary and tertiary granule CD66b (p = 0.002) and decreased hCAP-18 and MMP-9 (p = 0.02), were observed. After 8 min phagocytosis the pH in phagosomes of neutrophils of PWCF was significantly elevated (p = 0.0001), and the percentage of viable bacteria was significantly increased compared to HC (p = 0.002). Results demonstrate that the recorded alterations in phagosomal pH generate suboptimal conditions for MPO related peroxidase, and α-defensin and azurocidine enzymatic killing of Staphylococcus aureus and Pseudomonas aeruginosa. The pattern of dysregulated MPO degranulation (p = 0.02) and prolonged phagosomal alkalinization in CF neutrophils were normalized in vivo following treatment with the ion channel potentiator ivacaftor (p = 0.04). Our results confirm that alterations of circulating neutrophils from PWCF are corrected by CFTR modulator therapy, and raise a question related to possible delayed proton channel activity in CF.

α1-antitrypsin deficiency (AATD) is a rare and under-recognised genetic condition. Owing to its low prevalence, international initiatives are key for conducting high-quality research in the field. From July 2018 to December 2019, the European Alpha-1 Research Collaboration (EARCO) developed and conducted two surveys, one for healthcare providers and one for patients and caregivers, aiming to identify research priorities and barriers in access to treatment for AATD. A survey on 164 research questions was electronically sent to 230 AATD experts in Europe, and 94 completed surveys from 24 countries were received. The top research areas identified by healthcare providers were causes of variable progression and poor outcomes, improvement in diagnosis, initiation and optimal dosing of augmentation therapy and effectiveness of self-management interventions. During the same period, 438 surveys were completed by patients and caregivers from 26 countries. The top research areas identified were improving knowledge about AATD, in particular among general practitioners, access to AATD specialised centres and access to reliable, easy to understand information about living with AATD. Regarding barriers to treatment, participants from countries where augmentation therapy was reimbursed prioritised improving knowledge in AATD, while respondents in non-reimbursed countries regarded access to AATD augmentation therapy and to specialised centres as the most relevant. The main research and management priorities identified by healthcare providers and patients included understanding the natural history of AATD, improving information to physicians, improving access to specialised reference centres, personalising treatment and having equal opportunities for access to existing therapies.

Background: Prognostic tools are required to guide clinical decision-making in COVID-19. Methods: We studied the relationship between the ratio of interleukin (IL)-6 to IL-10 and clinical outcome in 80 patients hospitalized for COVID-19, and created a simple 5-point linear score predictor of clinical outcome, the Dublin-Boston score. Clinical outcome was analysed as a three-level ordinal variable (“Improved”, “Unchanged”, or “Declined”). For both IL-6:IL-10 ratio and IL-6 alone, we associated clinical outcome with a) baseline biomarker levels, b) change in biomarker level from day 0 to day 2, c) change in biomarker from day 0 to day 4, and d) slope of biomarker change throughout the study. The associations between ordinal clinical outcome and each of the different predictors were performed with proportional odds logistic regression. Associations were run both “unadjusted” and adjusted for age and sex. Nested cross-validation was used to identify the model for incorporation into the Dublin-Boston score. Findings: The 4-day change in IL-6:IL-10 ratio was chosen to derive the Dublin-Boston score. Each 1 point increase in the score was associated with a 5.6 times increased odds for a more severe outcome (OR 5.62, 95% CI -3.22–9.81, P = 1.2 × 10−9). Both the Dublin-Boston score and the 4-day change in IL-6:IL-10 significantly outperformed IL-6 alone in predicting clinical outcome at day 7. Interpretation: The Dublin-Boston score is easily calculated and can be applied to a spectrum of hospitalized COVID-19 patients. More informed prognosis could help determine when to escalate care, institute or remove mechanical ventilation, or drive considerations for therapies. Funding: Funding was received from the Elaine Galwey Research Fellowship, American Thoracic Society, National Institutes of Health and the Parker B Francis Research Opportunity Award.

In people with cystic fibrosis (PWCF), inflammation with concurrent infection occurs from a young age and significantly influences lung disease progression. Studies indicate that neutrophils are important effector cells in the pathogenesis of CF and in the development of anti-neutrophil cytoplasmic autoantibodies (ANCA). ANCA specific for bactericidal permeability increasing protein (BPI-ANCA) are detected in people with CF, and correlate with infection with Pseudomonas aeruginosa. The aim of this study was to determine the signaling mechanism leading to increased BPI release by CF neutrophils, while identifying IgG class BPI-ANCA in CF airways samples as the cause for impaired antimicrobial activity of BPI against P. aeruginosa. Plasma and/or bronchoalveolar lavage fluid (BAL) was collected from PWCF (n = 40), CF receiving ivacaftor therapy (n = 10), non-CF patient cohorts (n = 7) and healthy controls (n = 38). Plasma and BAL BPI and BPI-ANCA were measured by ELISA and GTP-bound Rac2 detected using an in vitro assay. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Levels of BPI are significantly increased in plasma (p = 0.007) and BALF (p < 0.0001) of PWCF. The signaling mechanism leading to increased degranulation and exocytosis of BPI by CF neutrophils (p = 0.02) involved enhancement of Rac2 GTP-loading (p = 0.03). The full-length BPI protein was detectable in all CF BAL samples and patients displayed ANCA with BPI specificity. IgG class autoantibodies were purified from CF BAL complexed to BPI (n=5), with IgG autoantibody cross-linking of antigen preventing BPI induced P. aeruginosa killing (p < 0.0001). Results indicate that the immune-mediated diminished antimicrobial defense, attributed to anti-BPI-IgG, necessitates the formation of a drug/immune complex intermediate that can maintain cytotoxic effects of BPI towards Gram-negative pathogens, with the potential to transform the current treatment of CF airways disease.

Summary: Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.

Background: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. Methods: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-βH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. Findings: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1β, induced PDL1 expression in vitro in human islet cells and EndoC-βH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs – a proposed therapeutic strategy for T1D – or IRF1 prevented PDL1 induction. Interpretation: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1. Keywords: Type 1 diabetes, PDL1, PDL-1, Pancreatic beta cells, Pancreatic islets, IRF1, Immune checkpoint inhibitor, CD274

Summary: Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. : Here, Clancy et al. show that proteases released by activated neutrophils into the extracellular space exhibited poor antimicrobial activity but were potent modulators of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ activation states. Thus, neutrophils play a key role in modulating inflammatory responses through processing of multiple IL-1 family cytokines. Keywords: IL-1 family, inflammation, IL-33, IL-36, neutrophil, cathepsin G, elastase, cell death, necrosis, microbial killing

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.

Background: Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. Methodology: PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n = 48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays. Findings: Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. Interpretation: Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.

Summary: Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression. : Saarimäki-Vire et al. use iPSCs derived from a patient with permanent neonatal diabetes to demonstrate that an activating STAT3 mutation leads to premature NEUROG3 expression and concomitant differentiation of pancreatic progenitors through increased nuclear shuttling of the mutant protein. Keywords: monogenic diabetes, STAT3, NEUROG3, iPSC, stem cells, CRISPR, genome editing, pancreatic differentiation, beta cell, endocrine cells

Heart failure is an increasingly prevalent condition, which is associated with ventricular arrhythmias. The reduction in cardiac pumping efficiency leads to the activation of several compensatory mechanisms. These mechanisms eventually lead to cardiac remodelling and a decline in haemodynamic status, contributing to the formation of a substrate conducive to arrhythmias, including increased automaticity, triggered activity, and, most commonly, re-entry circuits. In turn, ventricular arrhythmias can lead to the worsening of heart failure. A diagnosis of heart failure and ventricular arrhythmias is obtained using the patient’s history, examination findings, and investigation results. A key tool in this is echocardiogram imaging, which visualises the cardiac chambers, determines ventricular ejection fraction, and identifies structural abnormalities. A reduction in ejection fraction is a significant risk factor for the development of ventricular arrhythmias. Arrhythmias are diagnosed by ECG, Holter monitoring, and telemetry or event monitoring, and should initially be treated by optimising the medical management of heart failure. Anti-arrhythmic drugs, including beta-blockers, are usually the first-line therapy. Sudden cardiac death is a significant cause of mortality in heart failure patients, and implantable cardioverter defibrillator devices are used in both primary and secondary prevention. Anti-arrhythmic drugs and catheter ablation are important adjunctives for minimising shock therapy. In addition, autonomic modulation may offer a novel method of controlling ventricular arrhythmias. The objective of this review is to provide a practical overview of this rapidly developing field in relation to current evidence regarding the underlying pathophysiology, burden of disease, and management strategies available.

We present the case of a 19-year-old female with a mild form of Autosomal Dominant Hyper IgE syndrome (HIES) associated with a loss-of-function mutation in STAT3. Within the first years of life she developed multiple, Staphylococcus aureus associated abscesses in the neck and face requiring frequent incision and drainage. Respiratory tract infections were not a feature of the clinical phenotype and a high resolution thoracic CT scan was unremarkable. Retained dentition was noted but fungal nail disease and recurrent thrush were absent. The total IgE was 970 IU/L, Lymphocyte counts and immunoglobulin levels were normal (IgG borderline 18.5 gr/L). There was suboptimal response to test immunisation with Pneumovax II vaccine. Th17 cell phenotyping revealed low levels of IL-17 expressing cells (0.3% of total CD4 T Cells numbers). Genetic analysis identified a missense mutation, N567D, in a conserved region of the linker domain of STAT3. Functional studies in HEK293 cells reveal that this mutation potently inhibits STAT3 activity when compared to the wildtype protein. This is consistent with other reported mutations in STAT3 associated with HIES. However, surprisingly, the magnitude of inhibition was similar to another STAT3 mutation (V637M) which causes a much more severe form of the disease.

Chronic lithium administration for the treatment of bipolar disorder leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, natriuresis, kaliuresis, and collecting duct remodeling and cell proliferation among other features. Previously, using a 2-week lithium-induced NDI model, we reported that P2Y2 receptor (R) knockout mice are significantly resistant to polyuria, natriuresis, kaliuresis, and decrease in AQP2 protein abundance in the kidney relative to wild type mice. Here we show this protection is long-lasting, and is also associated with significant amelioration of lithium-induced collecting duct remodeling and cell proliferation. Age-matched wild type and knockout mice were fed regular (n = 5/genotype) or lithium-added (40 mmol/kg chow; n = 10/genotype) diet for 5 months and euthanized. Water intake, urine output and osmolality were monitored once in every month. Salt blocks were provided to mice on lithium-diet to prevent sodium loss. At the end of 5 months mice were euthanized and serum and kidney samples were analyzed. There was a steady increase in lithium-induced polyuria, natriuresis and kaliuresis in wild type mice over the 5-month period. Increases in these urinary parameters were very low in lithium-fed knockout mice, resulting in significantly widening differences between the wild type and knockout mice. Terminal AQP2 and NKCC2 protein abundances in the kidney were significantly higher in lithium-fed knockout vs. wild type mice. There were no significant differences in terminal serum lithium or sodium levels between the wild type and knockout mice. Confocal immunofluorescence microscopy revealed that lithium-induced marked remodeling of collecting duct with significantly increased proportion of [H+]-ATPase-positive intercalated cells and decreased proportion of AQP2-positive principal cells in the wild type, but not in knockout mice. Lithium-induced collecting duct cell proliferation (indicated by Ki67 labeling), was significantly lower in knockout vs. wild type mice. This is the first piece of evidence that purinergic signaling is potentially involved in lithium-induced collecting duct remodeling and cell proliferation. Our results demonstrate that genetic deletion of P2Y2-R protects against the key structural and functional alterations in Li-induced NDI, and underscore the potential utility of targeting this receptor for the treatment of NDI in bipolar patients on chronic lithium therapy.

Background: Hospitalisation is an ideal time to implement smoking cessation interventions. However, little is known about the extent to which inpatients receive such advice, or the impact it has on motivation to quit and quitting behaviours post-hospitalisation. Aims: This study aimed to determine the prevalence of smoking and cessation advice received by inpatients in two teaching hospitals in Ireland, and the impact of cessation advice on smoking at 3 months post discharge. Methods: We surveyed 1001 inpatients across two hospital sites, over a six-month period. Demographic details, clinical history, smoking history, motivation to quit, cigarette dependence, and recent quitting history were assessed. Results: Prevalence of smoking within the sample was 23.4% (235/1001). Only 32% (75/235) of smokers reported that smoking cessation was discussed during admission. Smokers’ mean Fagerström nicotine-dependence score was 3.7 (SD = 2.7), indicating low dependence levels. At 3 months, 17% (25/146) of smokers reported smoking cessation. Provision of smoking cessation advice during hospitalisation was associated with higher motivation to quit (OR = 2.79, 95% CI 2.12–3.68), and successful quit behaviour for confirmed (OR = 1.98, 95% CI 1.55–2.53) and self-reported quitters (OR = 1.47, 95% CI 1.3–1.66) Conclusions: This observational study finds that provision of brief cessation advice and smoking status documentation was suboptimal. Where advice was given, it was associated with enhanced motivation to quit and increased quit rates. These findings, along with low dependence scores, suggest that systematic provision of low-intensity cessation interventions could significantly enhance quit rates in hospitalised smokers.

Enteroviruses (EVs) have long been implicated in the pathogenesis of type 1 diabetes (T1D), and accumulating evidence has associated virus-induced autoimmunity with the loss of pancreatic beta cells in T1D. Inflammatory cytokines including interferons (IFN) form a primary line of defence against viral infections, and their chronic elevation is a hallmark feature of many autoimmune diseases. IFNs play a key role in activating and regulating innate and adaptive immune responses, and to do so they modulate the expression of networks of genes and transcription factors known generically as IFN stimulated genes (ISGs). ISGs in turn modulate critical cellular processes ranging from cellular metabolism and growth regulation to endoplasmic reticulum (ER) stress and apoptosis. More recent studies have revealed that IFNs also modulate gene expression at an epigenetic as well as post-transcriptional and post-translational levels. As such, IFNs form a key link connecting the various genetic, environmental and immunological factors involved in the initiation and progression of T1D. Therefore, gaining an improved understanding of the mechanisms by which IFNs modulate beta cell function and survival is crucial in explaining the pathogenesis of virally-induced T1D. This should provide the means to prevent, decelerate or even reverse beta cell impairment.