Relation of exaggerated cytokine responses of CF airway epithelial cells to PAO1 adherence

In many model systems, cystic fibrosis (CF) phenotype airway epithelial cells in culture respond to P. aeruginosa with greater interleukin (IL)-8 and IL-6 secretion than matched controls. In order to test whether this excess inflammatory response results from the reported increased adherence of P. aeruginosa to the CF cells, we compared the inflammatory response of matched pairs of CF and non CF airway epithelial cell lines to the binding of GFP-PAO1, a strain of pseudomonas labeled with green fluorescent protein. There was no clear relation between GFP-PAO1 binding and cytokine production in response to PAO1. Treatment with exogenous aGM1 resulted in greater GFP-PAO1 binding to the normal phenotype compared to CF phenotype cells, but cytokine production remained greater from the CF cell lines. When cells were treated with neuraminidase, PAO1 adherence was equalized between CF and nonCF phenotype cell lines, but IL-8 production in response to inflammatory stimuli was still greater in CF phenotype cells. The polarized cell lines 16HBEo-Sense (normal phenotype) and Antisense (CF phenotype) cells were used to test the effect of disrupting tight junctions, which allows access of PAO1 to basolateral binding sites in both cell lines. IL-8 production increased from CF, but not normal, cells. These data indicate that increased bacterial binding to CF phenotype cells cannot by itself account for excess cytokine production in CF airway epithelial cells, encourage investigation of alternative hypotheses, and signal caution for therapeutic strategies proposed for CF that include disruption of tight junctions in the face of pseudomonas infection

Chronic E-cigarette use increases neutrophil elastase and matrix metalloprotease levels in the lung

Rationale: Proteolysis is a key aspect of the lung's innate immune system. Proteases, including neutrophil elastase and MMPs (matrix metalloproteases), modulate cell signaling, inflammation, tissue remodeling, and leukocyte recruitment via cleavage of their target proteins. Excessive proteolysis occurs with chronic tobacco use and is causative for bronchiectasis and emphysema. The effect of e-cigarettes (vaping) on proteolysis is unknown. Objectives: We used protease levels as biomarkers of harm to determine the impact of vaping on the lung. Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers), and determined protease levels in BAL. In parallel, we studied the effects of e-cigarette components on protease secretion in isolated human blood neutrophils and BAL-derived macrophages. We also analyzed the nicotine concentration in induced sputum and BAL. Measurements and Main Results: Neutrophil elastase, MMP-2, and MMP-9 activities and protein levels were equally elevated in both vapers' and smokers' BAL relative to nonsmokers. In contrast, antiprotease levels were unchanged. We also found that exposure of isolated neutrophils and macrophages to nicotine elicited dose-dependent increases in protease release. After vaping, measurable levels of nicotine were detectable in sputum and BAL, which corresponded to the half-maximal effective concentration values for protease release seen in immune cells. Conclusions:We conclude that vaping induces nicotine-dependent protease release from resident pulmonary immune cells. Thus, chronic vaping disrupts the protease-antiprotease balance by increasing proteolysis in lung, which may place vapers at risk of developing chronic lung disease. These data indicate that vaping may not be safer than tobacco smoking

Different expression ratio of S100A8/A9 and S100A12 in acute and chronic lung diseases

Calgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of ARDS patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls (p< 0.001). Based on the hypothesis that the increased expression of S100A12 relative to the S100A8/A9 heterodimer was a characteristic of respiratory diseases with neutrophilic inflammation, we measured calgranulin expression in BALF of cystic fibrosis (CF) patients. Despite similarly elevated levels of S100A8/A9, S100A12 was significantly higher in ARDS compared to CF BALF (p<0.001). The differential expression of calgranulins was unique for inflammatory markers, as an array of cytokines did not differ between CF and ARDS patients

Plasma TGF-β1 in pediatric cystic fibrosis: Potential biomarker of lung disease and response to therapy

Transforming growth factor beta-1 (TGF-β1) is an important genetic modifier of lung disease severity in cystic fibrosis (CF), yet the mechanism behind this disease association remains unknown. Initial steps in the investigation of the relationship between TGF-β1 and CF lung disease include determining the most appropriate available biospecimen for TGF-β1 protein measurement

A highly immunogenic and effective measles virus-based Th1-biased COVID-19 vaccine

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1- biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+and CD4+T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines

Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ΔF508 Mice

Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Inter-α-inhibitor (IαI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IαI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IαI decreased amiloride-sensitive (IENaC) but not cAMP-activated Cl− currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in IENaC by IαI in ATII cells were accompanied by increased levels of uncleaved (immature) surface α-ENaC. IαI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (∆F508), or CF transmembrane conductance regulator knockout mice with IαI for 3 hours decreased the open probability of their ENaC channels by 50%. ∆F508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IαI in their BALF. A single intranasal instillation of IαI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IαI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of ∆F508 mice

Parenteral provision of micronutrients to pediatric patients: an international expert consensus paper

© 2020 The Authors. Published by Wiley. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1002/jpen.1990INTRODUCTION:Micronutrients (vitamins and trace elements) are essential to all nutrition. For children and neonates who are dependent upon nutrition support therapies for growth and development, the prescribed regimen must supply all essential components. This paper aims to facilitate interpretation of existing clinical guidelines into practical approaches for the provision of micronutrients in pediatric parenteral nutrition. METHODS:An international, interdisciplinary expert panel was convened to review recent evidence-based guidelines and published literature to develop consensus- based recommendation on practical micronutrient provision in pediatric parenteral nutrition. RESULTS:The guidelines and evidence have been interpreted as answers to 10 commonly asked questions around the practical principles for provision and monitoring of micronutrients in pediatric patients CONCLUSION: Micronutrients are an essential part of all parenteral nutrition and should be included in the pediatric nutrition therapy care plan.Published versio

Assessment of stability and fluctuations of cultured lower airway bacterial communities in people with cystic fibrosis

Background: Routine clinical culture detects a subset of the cystic fibrosis (CF) airways microbiota based on culture-independent (molecular) methods. This study aimed to determine how extended sputum culture of viable bacteria changes over time in relation to clinical status and predicts exacerbations. Methods: Sputa from patients at a baseline stable and up to three subsequent time-points were analysed by extended-quantitative culture; aerobe/anaerobe densities, ecological indexes and community structure were assessed together with clinical outcomes. Results: Eighty patients were prospectively recruited. Sputa were successfully collected and cultured at 199/267 (74.5%) study visits. Eighty-two sputa from 25 patients comprised a complete sample-set for longitudinal analyses. Bacterial density, ecological indexes and clinical outcomes were unchanged in 18 patients with three sequential stable visits. Conversely, in 7 patients who had an exacerbation, total bacterial and aerobe densities differed over four study visits (P < .001) with this difference particularly apparent between the baseline visit and completion of acute antibiotic treatment where a decrease in density was observed. Bacterial communities were more similar within than between patients but stable patients had the least variation in community structure over time. Using logistic regression in a further analysis, baseline features in 37 patients without compared to 15 patients with a subsequent exacerbation showed that clinical measures rather than bacterial density or ecological indexes were independent predictors of an exacerbation. Conclusions: Greater fluctuation in the viable bacterial community during treatment of an exacerbation than between stable visits was observed. Extended-quantitative culture did not provide prognostic information of a future exacerbation

Diagnosing Hunter syndrome in pediatric practice: practical considerations and common pitfalls

Mucopolysaccharidosis II (MPS II), or Hunter syndrome, is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme iduronate-2-sulfatase. Affected patients suffer progressive damage to multiple organ systems and early mortality. Two thirds of patients also manifest cognitive impairment and developmental delays. MPS II can be extremely difficult to diagnose before irreversible organ and tissue damage has occurred because of an insidious onset and the overlap in signs and symptoms with common childhood complaints. This is particularly true of patients without cognitive impairment (attenuated phenotype). Although not curative, early treatment with enzyme replacement therapy before irreversible organ damage has occurred may result in the greatest clinical benefit. Here, the signs, symptoms, and surgical history that should trigger suspicion of MPS II are described, and the diagnostic process is reviewed with a focus on practical considerations and the avoidance of common diagnostic pitfalls. Once a diagnosis is made, multidisciplinary management with an extended team of pediatric specialists is essential and should involve the pediatrician or family practice physician as facilitator and medical home for the patient and family. Conclusion: Because routine newborn screening is not yet available for MPS II, the involvement and awareness of pediatricians, family practice physicians, and pediatric specialists is critical for early identification, diagnosis, and referral in order to help optimize patient outcomes