Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells

Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure

Clinical Remission in Severe Asthma : A Pooled Post hoc Analysis of the Patient Journey with Benralizumab

Funding This study, the Rapid Service Fee, and the Open Access Fee were funded by AstraZeneca (Gaithersburg, MD, USA).Peer reviewedPublisher PD

The Receptor for Urokinase Regulates TLR2 Mediated Inflammatory Responses in Neutrophils

The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role

A Response to : Letter to the Editor Regarding “Clinical Remission in Severe Asthma: A Pooled Post Hoc Analysis of the Patient Journey with Benralizumab”

Funding Information: No funding or sponsorship was received for the publication of this article. Medical writing support was provided by Dan Jackson, Ph.D., CMPP (CiTRUS Health Group), and was funded by AstraZeneca (Cambridge, UK) in accordance with Good Publication Practice (GPP3) guidelines. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. Andrew Menzies-Gow developed the outline and content of the response letter and commented on previous versions of the manuscript. All authors read and approved the final manuscript. Andrew Menzies-Gow has attended advisory boards for AstraZeneca, GlaxoSmithKline, Novartis, Sanofi, and Teva; has received speaker fees from AstraZeneca, Novartis, Sanofi, and Teva; has participated in research with AstraZeneca for which his institution has been remunerated and has attended international conferences with Teva; and has had consultancy agreements with AstraZeneca and Sanofi. Flavia L. Hoyte has attended advisory boards for AstraZeneca; has received speaker fees from AstraZeneca and GlaxoSmithKline; and has participated in research sponsored by AstraZeneca, GlaxoSmithKline, Genentech, Teva, Sanofi, and the National Institute of Allergy and Infectious Diseases (NIAID), for which her institution has been remunerated. David B. Price has board membership with AstraZeneca, Boehringer Ingelheim, Chiesi, Mylan, Novartis, Regeneron Pharmaceuticals, Sanofi Genzyme, and Thermofisher; consultancy agreements with Airway Vista Secretariat, AstraZeneca, Boehringer Ingelheim, Chiesi, EPG Communication Holdings Ltd, FIECON Ltd, Fieldwork International, GlaxoSmithKline, Mylan, Mundipharma, Novartis, OM Pharma SA, PeerVoice, Phadia AB, Spirosure Inc, Strategic North Limited, Synapse Research Management Partners S.L., Talos Health Solutions, Theravance, and WebMD Global LLC; grants and unrestricted funding for investigator-initiated studies (conducted through Observational and Pragmatic Research Institute Pte Ltd) from AstraZeneca, Boehringer Ingelheim, Chiesi, Mylan, Novartis, Regeneron Pharmaceuticals, Respiratory Effectiveness Group, Sanofi Genzyme, Theravance, and the UK National Health Service; received payment for lectures/speaking engagements from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, GlaxoSmithKline, Kyorin, Mylan, Mundipharma, Novartis, Regeneron Pharmaceuticals, and Sanofi Genzyme; received payment for travel/accommodation/meeting expenses from AstraZeneca, Boehringer Ingelheim, Mundipharma, Mylan, Novartis, and Thermofisher; stock/stock options from AKL Research and Development Ltd, which produces phytopharmaceuticals; ownership of 74% of the social enterprise Optimum Patient Care Ltd (Australia and UK) and 92.61% of Observational and Pragmatic Research Institute Pte Ltd (Singapore); 5% shareholding in Timestamp, which develops adherence monitoring technology; a peer reviewer role for grant committees of the UK Efficacy and Mechanism Evaluation programme and the Health Technology Assessment; and served as an expert witness for GlaxoSmithKline. David Cohen, Peter Barker, James Kreindler, Maria Jison, Chris Brooks, Peggy Papeleu, and Rohit Katial are employees of AstraZeneca. This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. Data sharing is not applicable to this article as no datasets were generated or analysed for this response letter. Funding Information: Andrew Menzies-Gow has attended advisory boards for AstraZeneca, GlaxoSmithKline, Novartis, Sanofi, and Teva; has received speaker fees from AstraZeneca, Novartis, Sanofi, and Teva; has participated in research with AstraZeneca for which his institution has been remunerated and has attended international conferences with Teva; and has had consultancy agreements with AstraZeneca and Sanofi. Flavia L. Hoyte has attended advisory boards for AstraZeneca; has received speaker fees from AstraZeneca and GlaxoSmithKline; and has participated in research sponsored by AstraZeneca, GlaxoSmithKline, Genentech, Teva, Sanofi, and the National Institute of Allergy and Infectious Diseases (NIAID), for which her institution has been remunerated. David B. Price has board membership with AstraZeneca, Boehringer Ingelheim, Chiesi, Mylan, Novartis, Regeneron Pharmaceuticals, Sanofi Genzyme, and Thermofisher; consultancy agreements with Airway Vista Secretariat, AstraZeneca, Boehringer Ingelheim, Chiesi, EPG Communication Holdings Ltd, FIECON Ltd, Fieldwork International, GlaxoSmithKline, Mylan, Mundipharma, Novartis, OM Pharma SA, PeerVoice, Phadia AB, Spirosure Inc, Strategic North Limited, Synapse Research Management Partners S.L., Talos Health Solutions, Theravance, and WebMD Global LLC; grants and unrestricted funding for investigator-initiated studies (conducted through Observational and Pragmatic Research Institute Pte Ltd) from AstraZeneca, Boehringer Ingelheim, Chiesi, Mylan, Novartis, Regeneron Pharmaceuticals, Respiratory Effectiveness Group, Sanofi Genzyme, Theravance, and the UK National Health Service; received payment for lectures/speaking engagements from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, GlaxoSmithKline, Kyorin, Mylan, Mundipharma, Novartis, Regeneron Pharmaceuticals, and Sanofi Genzyme; received payment for travel/accommodation/meeting expenses from AstraZeneca, Boehringer Ingelheim, Mundipharma, Mylan, Novartis, and Thermofisher; stock/stock options from AKL Research and Development Ltd, which produces phytopharmaceuticals; ownership of 74% of the social enterprise Optimum Patient Care Ltd (Australia and UK) and 92.61% of Observational and Pragmatic Research Institute Pte Ltd (Singapore); 5% shareholding in Timestamp, which develops adherence monitoring technology; a peer reviewer role for grant committees of the UK Efficacy and Mechanism Evaluation programme and the Health Technology Assessment; and served as an expert witness for GlaxoSmithKline. David Cohen, Peter Barker, James Kreindler, Maria Jison, Chris Brooks, Peggy Papeleu, and Rohit Katial are employees of AstraZeneca. Funding Information: Medical writing support was provided by Dan Jackson, Ph.D., CMPP (CiTRUS Health Group), and was funded by AstraZeneca (Cambridge, UK) in accordance with Good Publication Practice (GPP3) guidelines.Peer reviewedPublisher PD

PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

The PLUNC ("Palate, lung, nasal epithelium clone") protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family--the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP)--are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen

Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection

Real-World Assessment of Asthma Specialist Visits Among U.S. Patients with Severe Asthma.

BACKGROUND: U.S. guidelines recommend that patients with severe asthma be referred to specialists (allergists/immunologists or pulmonologists) for systematic assessment or comanagement; however, contemporary, real-world data on the frequency and impact of specialist care among U.S. severe asthma patients are lacking. OBJECTIVES: To quantify the frequency of asthma specialist visits among U.S. patients with severe asthma, identify patient demographic and clinical characteristics associated with specialist visits and describe health outcomes following specialist care. METHODS: Severe asthma patients aged 6 years or older were identified between January 1, 2015, and December 31, 2017, in the IQVIA PharMetrics® Plus database of commercially insured individuals, based on Healthcare Effectiveness Data and Information Set (HEDIS) criteria and Global Initiative for Asthma (GINA) step 4 or 5 treatment regimens. The frequency of asthma specialist (allergist/immunologist or pulmonologist) visits was described over 2 years. Patient characteristics associated with having 1 or more specialist visits were analyzed using multivariate regressions. Asthma exacerbations and health care resource utilization before and after specialist visit were compared. RESULTS: Of 54,332 patients identified, 38.2% had 1 or more specialist visits over 2 years. Patient characteristics predictive of specialist visits were asthma exacerbation frequency, younger age, and allergy/respiratory comorbidity burden (all P \u3c .001). Among patients with 1 or more specialist visits, a lower prevalence of asthma exacerbations and rescue inhaler use was observed following the first observed specialist visit. CONCLUSIONS: Specialist care was observed in fewer than half of U.S. patients with severe asthma and was least frequent among older adult patients and those with more nonrespiratory comorbidities. Increased specialist involvement in managing severe asthma may help improve care and patient outcomes

Resting and Post Bronchial Challenge Testing Carbon Dioxide Partial Pressure in Individuals with and without Asthma

Objective: There is conflicting evidence about resting carbon dioxide levels in asthmatic individuals. We wanted to determine if transcutaneously measured carbon dioxide levels prior and during bronchial provocation testing differ according to asthma status reflecting dysfunctional breathing. Methods: We investigated active firefighters and policemen by means of a validated questionnaire on respiratory symptoms, spirometry, bronchial challenge testing with methacholine (MCT) and measurement of transcutaneous blood carbon dioxide partial pressure (PtcCO 2) at rest prior performing spirometry, one minute and five minutes after termination of MCT. A respiratory physician blinded to the PtcCO2 results assigned a diagnosis of asthma after reviewing the available study data and the files of the workers medical screening program. Results: The study sample consisted of 128 male and 10 female individuals. Fifteen individuals (11%) had physiciandiagnosed asthma. There was no clinically important difference in median PtcCO 2 at rest, one and five minutes after recovery from MCT in asthmatics compared to non-asthmatics (35.6 vs 35.7 mmHg, p = 0.466; 34.7 vs 33.4 mmHg, p = 0.245 and 37.4 vs 36.4 mmHg, p = 0.732). The median drop in PtcCO2 during MCT and the increase after MCT was lower in asthmatics compared to non-asthmatics (0.1 vs 3.2 mmHg, p = 0.014 and 1.9 vs 2.9 mmHg, p = 0.025). Conclusions: PtcCO2 levels at rest prior and during recovery after MCT do not differ in individuals with or without physicia

Tobacco Smoke Mediated Induction of Sinonasal Microbial Biofilms

Cigarette smokers and those exposed to second hand smoke are more susceptible to life threatening infection than non-smokers. While much is known about the devastating effect tobacco exposure has on the human body, less is known about the effect of tobacco smoke on the commensal and commonly found pathogenic bacteria of the human respiratory tract, or human respiratory tract microbiome. Chronic rhinosinusitis (CRS) is a common medical complaint, affecting 16% of the US population with an estimated aggregated cost of $6 billion annually. Epidemiologic studies demonstrate a correlation between tobacco smoke exposure and rhinosinusitis. Although a common cause of CRS has not been defined, bacterial presence within the nasal and paranasal sinuses is assumed to be contributory. Here we demonstrate that repetitive tobacco smoke exposure induces biofilm formation in a diverse set of bacteria isolated from the sinonasal cavities of patients with CRS. Additionally, bacteria isolated from patients with tobacco smoke exposure demonstrate robust in vitro biofilm formation when challenged with tobacco smoke compared to those isolated from smoke naïve patients. Lastly, bacteria from smoke exposed patients can revert to a non-biofilm phenotype when grown in the absence of tobacco smoke. These observations support the hypothesis that tobacco exposure induces sinonasal biofilm formation, thereby contributing to the conversion of a transient and medically treatable infection to a persistent and therapeutically recalcitrant condition

NGF Is an Essential Survival Factor for Bronchial Epithelial Cells during Respiratory Syncytial Virus Infection

Background: Overall expression of neurotrophins in the respiratory tract is upregulated in infants infected by the respiratory syncytial virus (RSV), but it is unclear where (structural vs. inflammatory cells, upper vs. lower airways) and why, these changes occur. We analyzed systematically the expression of neurotrophic factors and receptors following RSV infection of human nasal, tracheal, and bronchial epithelial cells, and tested the hypothesis that neurotrophins work as innate survival factors for infected respiratory epithelia. Methodology: Expression of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF) and receptors (trkA, trkB, p75) was analyzed at the protein level by immunofluorescence and flow cytometry and at the mRNA level by real-time PCR. Targeted siRNA was utilized to blunt NGF expression, and its effect on virus-induced apoptosis/ necrosis was evaluated by flow cytometry following annexin V/7-AAD staining. Principal Findings: RSV infection was more efficient in cells from more distal (bronchial) vs. more proximal origin. In bronchial cells, RSV infection induced transcript and protein overexpression of NGF and its high-affinity receptor trkA, with concomitant downregulation of the low-affinity p75 NTR. In contrast, tracheal cells exhibited an increase in BDNF, trkA and trkB, and nasal cells increased only trkA. RSV-infected bronchial cells transfected with NGF-specific siRNA exhibited decreased trkA and increased p75 NTR expression. Furthermore, the survival of bronchial epithelial cells was dramaticall