Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Alcohol-Induced Mucociliary Dysfunction: Role of Defective CFTR Channel Function.

Excessive alcohol use is thought to increase the risk of respiratory infections by impairing mucociliary clearance (MCC). In this study, we investigate the hypothesis that alcohol reduces the function of CFTR, the protein that is defective in individuals with cystic fibrosis, thus altering mucus properties to impair MCC and the airway's defense against inhaled pathogens. Methods: Sprague Dawley rats with wild type CFTR (+/+), matched for age and sex, were administered either a Lieber-DeCarli alcohol diet or a control diet with the same number of calories for eight weeks. CFTR activity was measured using nasal potential difference (NPD) assay and Ussing chamber electrophysiology of tracheal tissue samples. In vivo MCC was determined by measuring the radiographic clearance of inhaled Tc99 particles and the depth of the airway periciliary liquid (PCL) and mucus transport rate in excised trachea using micro-optical coherence tomography (µOCT). The levels of rat lung MUC5b and CFTR were estimated by protein and mRNA analysis. Results: Alcohol diet was found to decrease CFTR ion transport in the nasal and tracheal epithelium in vivo and ex vivo. This decrease in activity was also reflected in partially reduced full-length CFTR protein levels but not, in mRNA copies, in the lungs of rats. Furthermore, alcohol-fed rats showed a significant decrease in MCC after 8 weeks of alcohol consumption. The trachea from these rats also showed reduced PCL depth, indicating a decrease in mucosal surface hydration that was reflected in delayed mucus transport. Diminished MCC rate was also likely due to the elevated MUC5b expression in alcohol-fed rat lungs. Conclusions: Excessive alcohol use can decrease the expression and activity of CFTR channels, leading to reduced airway surface hydration and impaired mucus clearance. This suggests that CFTR dysfunction plays a role in the compromised lung defense against respiratory pathogens in individuals who drink alcohol excessively.

Nicotine aerosols diminish airway CFTR function and mucociliary clearance.

Electronic cigarettes (e-cigs) are often promoted as safe alternatives to smoking based on the faulty perception that inhaling nicotine is safe until other harmful chemicals in cigarette smoke are absent. Previously, others and we have reported that, similar to cigarette smoke, e-cig aerosols decrease CFTR-mediated ion transport across airway epithelium. However, it is unclear whether such defective epithelial ion transport by e-cig aerosols occurs in vivo and what the singular contribution of inhaled nicotine is to impairments in mucociliary clearance (MCC), the primary physiologic defense of the airways. Here, we tested the effects of nicotine aerosols from e-cigs in primary human bronchial epithelial (HBE) cells and two animal models, rats and ferrets, known for their increasing physiologic complexity and potential for clinical translation, followed by in vitro and in vivo electrophysiologic assays for CFTR activity and micro-optical coherence tomography (µOCT) image analyses for alterations in airway mucus physiology. Data presented in this report indicate nicotine in e-cig aerosols causes 1) reduced CFTR and epithelial Na+ channel (ENaC)-mediated ion transport, 2) delayed MCC, and 3) diminished airway surface hydration, as determined by periciliary liquid depth analysis. Interestingly, the common e-cig vehicles vegetable glycerin and propylene glycol did not affect CFTR function or MCC in vivo despite their significant adverse effects in vitro. Overall, our studies contribute to an improved understanding of inhaled nicotine effects on lung health among e-cig users and inform pathologic mechanisms involved in altered host defense and increased risk for tobacco-associated lung diseases.

HIV Increases the Risk of Cigarette Smoke-Induced Emphysema Through MMP-9.

BACKGROUND: HIV is associated with an increased risk for emphysema. Matrix metalloproteinase 9 (MMP-9) is a lung tissue remodeling enzyme associated with emphysema. We previously found MMP-9 activity increases with increases in oxidative stress and that HIV increases alveolar oxidative stress. We hypothesized that HIV proteins would increase the risk of cigarette smoke-induced emphysema due to MMP-9. METHODS: HIV-1 transgenic rats and wild-type littermates were exposed to cigarette smoke or sham for 8 weeks. Lung compliance and histology were assessed. Bronchoalveolar lavage (BAL), primary alveolar macrophages (AM), and serum samples were obtained. A rat alveolar macrophage cell line was exposed to the HIV protein Tat, and MMP-9 levels were assessed by Western immunoblotting. MMP-9 protein expression and activity were assessed in AM from the HIV rat model by ELISA and cytoimmunofluoresence, respectively. Serum from human subjects with and without HIV and tobacco dependence was assessed for MMP-9 levels. RESULTS: MMP-9 expression was significantly increased in rat alveolar macrophages after Tat exposure. HIV-1 transgenic rats developed emphysema while wild-type littermates did not. MMP-9 expression was also increased in the serum, BAL, and AM of HIV-1 transgenic rats after exposure to cigarette smoke compared with wild-type rats. In parallel, serum samples from HIV+ smokers had higher levels of MMP-9 than subjects without HIV and those who did not smoke. CONCLUSION: The combination of HIV and cigarette smoke increases MMP-9 expression in experimental rat HIV models and human subjects. HIV and cigarette smoke both induce alveolar oxidative stress and thereby increase MMP-9 activity.

Comparative transcriptomics in human COPD reveals dysregulated genes uniquely expressed in ferrets.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with poor treatment options. However, most mouse models of COPD produce a primarily emphysematous disease not recapitulating clinically meaningful COPD features like chronic bronchitis. METHODS: Wild-type ferrets (Mustela putorius furo) were divided randomly into two groups: whole body cigarette smoke exposure and air controls. Ferrets were exposed to smoke from 1R6F research cigarettes, twice daily for six months. RNA-sequencing was performed on RNA isolated from lung tissue. Comparative transcriptomics analyses of COPD in ferrets, mice, and humans were done to find the uniquely expressed genes. Further, Real-time PCR was performed to confirmed RNA-Seq data on multiple selected genes. RESULTS: RNA-sequence analysis identified 420 differentially expressed genes (DEGs) that were associated with the development of COPD in ferrets. By comparative analysis, we identified 25 DEGs that are uniquely expressed in ferrets and humans, but not mice. Among DEGs, a number were related to mucociliary clearance (NEK-6, HAS1, and KL), while others have been correlated with abnormal lung function (IL-18), inflammation (TREM1, CTSB), or oxidative stress (SRX1, AHRR). Multiple cellular pathways were aberrantly altered in the COPD ferret model, including pathways associated with COPD pathogenesis in humans. Validation of these selected unique DEGs using real-time PCR demonstrated > absolute 2-fold changes in mRNA versus air controls, consistent with RNA-seq analysis. CONCLUSION: Cigarette smoke-induced COPD in ferrets modulates gene expression consistent with human COPD and suggests that the ferret model may be uniquely well suited for the study of aspects of the disease.

Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

RATIONALE: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. OBJECTIVES: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. METHODS: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. MEASUREMENTS AND MAIN RESULTS: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. CONCLUSIONS: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the ß2 -adrenergic receptor.

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.

Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporation mediated delivery and embryo culture.

Successful use of the CRISPR-Cas9 system for gene manipulation relies on identifying effective and efficient guide RNA sequences (gRNAs). When the goal is to create transgenic animal/rodent models by knocking-in desired sequences using homology-directed repair (HDR), selecting effective guides becomes even more critical to minimize developmental time and resources. Currently, validation experiments for gRNAs for generating rat models are carried out using immortalized rat cells. However, there are several limitations with using such cell lines, including ploidy of the genome, non-predictive transfection efficiency, and the ability to identify gene modifications efficiently within diverse cell populations. Since embryos are authentic representatives of live animals compared to cell lines, validating CRISPR guides for their nuclease activity in freshly isolated embryos will provide greater accuracy of in vivo gene editing efficiency. In contrast to microinjections, delivery by electroporation is a more accessible method that can be simple and does not require special skills and equipment. We demonstrate an accessible workflow to either delete or edit target genes in vivo in rats using the efficient editing of a human mutation in alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) ortholog using electroporation as a delivery method for CRISPR-Cas9 ribonucleoprotein complexes in rat embryos.•Upon identifying CRISPR targets at the desired genetic alteration site, we designed homologydriven repair (HDR) templates for effective and easy identification of gene editing by Restriction Fragment Length Polymorphism (RFLP).•Cultured rat embryos can be genotyped to assess CRISPR activity as seen by either presence of indels resulting from NHEJ or knock-in of repair template resulting from homology driven repair.•Heteroduplex mobility assay (HMA) and Restriction Fragment Length Polymorphism (RFLP) of PCR products can be performed reliably and reproducibly at a low-cost.

Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD.

RATIONALE: Non-typeable Haemophilus influenzae (NTHi) is a common inhabitant of the human nasopharynx and upper airways that can cause opportunistic infections of the airway mucosa including bronchopulmonary infections in patients with chronic obstructive pulmonary disease (COPD). It is clear that opportunistic infections contribute significantly to inflammatory exacerbations of COPD; however, there remains much to be learned regarding specific host and microbial determinants of persistence and/or clearance in this context. METHODS: In this study, we used a recently described ferret model for COPD, in which animals undergo chronic long-term exposure to cigarette smoke, to define host-pathogen interactions during COPD-related NTHi infections. RESULTS: NTHi bacteria colonised the lungs of smoke-exposed animals to a greater extent than controls, and elicited acute host inflammation and neutrophilic influx and activation, along with a significant increase in airway resistance and a decrease in inspiratory capacity consistent with inflammatory exacerbation; notably, these findings were not observed in air-exposed control animals. NTHi bacteria persisted within multicellular biofilm communities within the airway lumen, as evidenced by immunofluorescent detection of bacterial aggregates encased within a sialylated matrix as is typical of NTHi biofilms and differential bacterial gene expression consistent with the biofilm mode of growth. CONCLUSIONS: Based on these results, we conclude that acute infection with NTHi initiates inflammatory exacerbation of COPD disease. The data also support the widely held hypothesis that NTHi bacteria persist within multicellular biofilm communities in the lungs of patients with COPD.

Airway remodeling in ferrets with cigarette smoke-induced COPD using µCT imaging.

Structural changes to airway morphology, such as increased bronchial wall thickness (BWT) and airway wall area, are cardinal features of chronic obstructive pulmonary disease (COPD). Ferrets are a recently established animal model uniquely exhibiting similar clinical and pathological characteristics of COPD as humans, including chronic bronchitis. Our objective was to develop a microcomputed tomography (µCT) method for evaluating structural changes to the airways in ferrets and assess whether the effects of smoking induce changes consistent with chronic bronchitis in humans. Ferrets were exposed to mainstream cigarette smoke or air control twice daily for 6 mo. µCT was conducted in vivo at 6 mo; a longitudinal cohort was imaged monthly. Manual measurements of BWT, luminal diameter (LD), and BWT-to-LD ratio (BWT/LD) were conducted and confirmed by a semiautomated algorithm. The square root of bronchial wall area (√WA) versus luminal perimeter was determined on an individual ferret basis. Smoke-exposed ferrets reproducibly demonstrated 34% increased BWT (P < 0.001) along with increased LD and BWT/LD versus air controls. Regression indicated that the effect of smoking on BWT persisted despite controlling for covariates. Semiautomated measurements replicated findings. √WA for the theoretical median airway luminal perimeter of 4 mm (Pi4) was elevated 4.4% in smoke-exposed ferrets (P = 0.015). Increased BWT and Pi4 developed steadily over time. µCT-based airway measurements in ferrets are feasible and reproducible. Smoke-exposed ferrets develop increased BWT and Pi4, changes similar to humans with chronic bronchitis. µCT can be used as a significant translational platform to measure dynamic airway morphological changes.

Excess mucus viscosity and airway dehydration impact COPD airway clearance.

The mechanisms by which cigarette smoking impairs airway mucus clearance are not well understood. We recently established a ferret model of cigarette smoke-induced chronic obstructive pulmonary disease (COPD) exhibiting chronic bronchitis. We investigated the effects of cigarette smoke on mucociliary transport (MCT).Adult ferrets were exposed to cigarette smoke for 6 months, with in vivo mucociliary clearance measured by technetium-labelled DTPA retention. Excised tracheae were imaged with micro-optical coherence tomography. Mucus changes in primary human airway epithelial cells and ex vivo ferret airways were assessed by histology and particle tracking microrheology. Linear mixed models for repeated measures identified key determinants of MCT.Compared to air controls, cigarette smoke-exposed ferrets exhibited mucus hypersecretion, delayed mucociliary clearance (-89.0%, p<0.01) and impaired tracheal MCT (-29.4%, p<0.05). Cholinergic stimulus augmented airway surface liquid (ASL) depth (5.8±0.3 to 7.3±0.6 µm, p<0.0001) and restored MCT (6.8±0.8 to 12.9±1.2 mm·min-1, p<0.0001). Mixed model analysis controlling for covariates indicated smoking exposure, mucus hydration (ASL) and ciliary beat frequency were important predictors of MCT. Ferret mucus was hyperviscous following smoke exposure in vivo or in vitro, and contributed to diminished MCT. Primary cells from smokers with and without COPD recapitulated these findings, which persisted despite the absence of continued smoke exposure.Cigarette smoke impairs MCT by inducing airway dehydration and increased mucus viscosity, and can be partially abrogated by cholinergic secretion of fluid secretion. These data elucidate the detrimental effects of cigarette smoke exposure on mucus clearance and suggest additional avenues for therapeutic intervention.

Summary of the 2018 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On January 26, 2018, the 23rd annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado. The meeting consisted of plenary sessions with oral presentations and a poster presentation session. There were four plenary sessions that covered a wide range of topics relating to alcohol use: Alcohol and Liver Disease; Alcohol, Inflammation and Immune Response; Alcohol and Organ Injury; Heath Consequences and Alcohol Drinking. The meeting provided a forum for the presentation and discussion of novel research findings regarding alcohol use and immunology.

A Neutralizing Aptamer to TGFBR2 and miR-145 Antagonism Rescue Cigarette Smoke- and TGF-ß-Mediated CFTR Expression.

Transforming growth factor ß (TGF-ß), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-ß and CS (via TGF-ß) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-ß alters the microRNAome of primary human bronchial epithelium. TGF-ß and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-ß in bronchial epithelial cells following transfection, an aptamer to block TGF-ß signaling rescues CS- and TGF-ß-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-ß signaling in the airway and rescue CFTR biogenesis and function.

Vaporized E-Cigarette Liquids Induce Ion Transport Dysfunction in Airway Epithelia.

Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 µA/cm2 control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.

Heme scavenging reduces pulmonary endoplasmic reticulum stress, fibrosis, and emphysema.

Pulmonary fibrosis and emphysema are irreversible chronic events after inhalation injury. However, the mechanism(s) involved in their development remain poorly understood. Higher levels of plasma and lung heme have been recorded in acute lung injury associated with several insults. Here, we provide the molecular basis for heme-induced chronic lung injury. We found elevated plasma heme in chronic obstructive pulmonary disease (COPD) (GOLD stage 4) patients and also in a ferret model of COPD secondary to chronic cigarette smoke inhalation. Next, we developed a rodent model of chronic lung injury, where we exposed C57BL/6 mice to the halogen gas, bromine (Br2) (400 ppm, 30 minutes), and returned them to room air resulting in combined airway fibrosis and emphysematous phenotype, as indicated by high collagen deposition in the peribronchial spaces, increased lung hydroxyproline concentrations, and alveolar septal damage. These mice also had elevated pulmonary endoplasmic reticulum (ER) stress as seen in COPD patients; the pharmacological or genetic diminution of ER stress in mice attenuated Br2-induced lung changes. Finally, treating mice with the heme-scavenging protein, hemopexin, reduced plasma heme, ER stress, airway fibrosis, and emphysema. This is the first study to our knowledge to report elevated heme in COPD patients and establishes heme scavenging as a potential therapy after inhalation injury.