A pilot dose-finding study of Terazosin in humans.

Background: Parkinson's disease (PD) is a prevalent neurodegenerative disorder where progressive neuron loss is driven by impaired brain bioenergetics, particularly mitochondrial dysfunction and disrupted cellular respiration. Terazosin (TZ), an α-1 adrenergic receptor antagonist with a known efficacy in treating benign prostatic hypertrophy and hypertension, has shown potential in addressing energy metabolism deficits associated with PD due to its action on phosphoglycerate kinase 1 (PGK1). This study aimed to investigate the safety, tolerability, bioenergetic target engagement, and optimal dose of TZ in neurologically healthy subjects. Methods: Eighteen healthy men and women (60 - 85 years old) were stratified into two cohorts based on maximum TZ dosages (5 mg and 10 mg daily). Methods included plasma and cerebrospinal fluid TZ concentration measurements, whole blood ATP levels, 31 Phosphorous magnetic resonance spectroscopy for brain ATP levels, 18 F-FDG PET imaging for cerebral metabolic activity, and plasma metabolomics. Results: Our results indicated that a 5 mg/day dose of TZ significantly increased whole blood ATP levels and reduced global cerebral 18 F-FDG PET uptake without significant side effects or orthostatic hypotension. These effects were consistent across sexes. Higher doses did not result in additional benefits and showed a potential biphasic dose-response. Conclusions: TZ at a dosage of 5 mg/day engages its metabolic targets effectively in both sexes without inducing significant adverse effects and provides a promising therapeutic avenue for mitigating energetic deficiencies. Further investigation via clinical trials to validate TZ's efficacy and safety in neurodegenerative (i.e., PD) contexts is warranted.

Epithelial responses to CFTR modulators are improved by inflammatory cytokines and impaired by anti-inflammatory drugs.

Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well-understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ∆F508-CF epithelia with TNFα and IL-17, two inflammatory cytokines that are elevated in CF airways. TNFα+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl- (Na+/K+/2Cl- co-transport) and HCO3- (carbonic anhydrases and Na+/HCO3- co-transport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions, but prevented the rise in viscosity in epithelia treated with TNFα+IL-17. Lastly, anti-inflammatory drugs limited CFTR modulator responses in TNFα+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits versus limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.

Arteriovenous metabolomics in pigs reveals CFTR regulation ofmetabolism inmultiple organs.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a multiorgan disease that exhibits diverse metabolic defects. However, other than specific CFTR mutations, the factors that influence disease progression and severity remain poorly understood. Aberrant metabolite levels have been reported, but whether CFTR loss itself or secondary abnormalities (infection, inflammation, malnutrition, and various treatments) drive metabolic defects are uncertain. Here, we implemented comprehensive arteriovenous metabolomics in newborn CF pigs, and the results revealed CFTR as a bona fide regulator of metabolism. CFTR loss impaired metabolite exchange across organs, including disrupted lung uptake of fatty acids yet enhanced uptake of arachidonic acid, a precursor of pro-inflammatory cytokines. CFTR loss also impaired kidney reabsorption of amino acids and lactate and abolished renal glucose homeostasis. These and additional unexpected metabolic defects prior to disease manifestations reveal a fundamental role for CFTR in controlling multi-organ metabolism. Such discovery informs a basic understanding of CF, provides a foundation for future investigation, and has implications for developing therapies targeting only a single tissue.

A model for stimulation of enzyme activity by a competitive inhibitor based on the interaction of terazosin and phosphoglycerate kinase 1.

The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.

Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage.

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Injectable long-acting ivacaftor-loaded poly (lactide-co-glycolide) microparticle formulations for the treatment of cystic fibrosis: In vitro characterization and in vivo pharmacokinetics in mice.

Optimizing a sustained-release drug delivery system for the treatment of cystic fibrosis (CF) is crucial for decreasing the dosing frequency and improving patients' compliance with the treatment regimen. In the current work, we developed an injectable poly(D,L-lactide-co-glycolide) (PLGA) microparticle formulation loaded with ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases the open probability of the CFTR anion channel, using a single emulsion solvent evaporation technique. We aimed to study the effect of different parameters on the characteristics of the prepared formulations to select an optimized microparticle formulation to be used in an in vivo pharmacokinetic study in mice. First, a suite of ivacaftor-loaded microparticles were prepared using different formulation parameters in order to study the effect of varying these parameters on microparticle size, morphology, drug loading, encapsulation efficiency, and in vitro release profiles. Prepared microparticles were spherical with diameters ranging from 1.91-6.93 µm, percent drug loading (% DL) of 3.91-10.3%, percent encapsulation efficiencies (% EE) of 26.6-100%, and an overall slow cumulative release profile. We selected the formulation that demonstrated optimal combined % DL and % EE values (8.25 and 90.7%, respectively) for further studies. These microparticles had an average particle size of 6.83 µm and a slow tri-phasic in vitro release profile (up to 6 weeks). In vivo pharmacokinetic studies in mice showed that the subcutaneously injected microparticles resulted in steady plasma levels of ivacaftor over a period of 28 days, and a 6-fold increase in AUC 0 - t (71.6 µg/mL*h) compared to the intravenously injected soluble ivacaftor (12.3 µg/mL*h). Our results suggest that this novel ivacaftor-loaded microparticle formulation could potentially eliminate the need for the frequent daily administration of ivacaftor to people with CF thus improving their compliance and ensuring successful treatment outcomes.

Novel hybrid silicon-lipid nanoparticles deliver a siRNA to cure autosomal dominant osteopetrosis in mice. Implications for gene therapy in humans.

Rare skeletal diseases are still in need of proper clinically available transfection agents as the major challenge for first-in-human translation relates to intrinsic difficulty in targeting bone without exacerbating any inherent toxicity due to used vector. SiSaf's silicon stabilized hybrid lipid nanoparticles (sshLNPs) constitute next-generation non-viral vectors able to retain the integrity and stability of constructs and to accommodate considerable payloads of biologicals, without requiring cold-chain storage. sshLNP was complexed with a small interfering RNA (siRNA) specifically designed against the human CLCN7G215R mRNA. When tested via single intraperitoneal injection in pre-puberal autosomal dominant osteopetrosis type 2 (ADO2) mice, carrying a heterozygous mutation of the Clcn7 gene (Clcn7G213R), sshLNP, this significantly downregulated the Clcn7G213R related mRNA levels in femurs at 48 h. Confirmatory results were observed at 2 weeks and 4 weeks after treatments (3 intraperitoneal injections/week), with rescue of the bone phenotype and demonstrating safety. The pre-clinical results will enable advanced preclinical development of RNA-based therapy for orphan and genetic skeletal disorders by safely and effectively delivering biologicals of interest to cure human systemic conditions.

CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

Implications of Pediatric Artificial Intelligence Challenges for Artificial Intelligence Education and Curriculum Development.

Several radiology artificial intelligence (AI) courses are offered by a variety of institutions and educators. The major radiology societies have developed AI curricula focused on basic AI principles and practices. However, a specific AI curriculum focused on pediatric radiology is needed to offer targeted education material on AI model development and performance evaluation. There are inherent differences between pediatric and adult practice patterns, which may hinder the application of adult AI models in pediatric cohorts. Such differences include the different imaging modality utilization, imaging acquisition parameters, lower radiation doses, the rapid growth of children and changes in their body composition, and the presence of unique pathologies and diseases, which differ in prevalence from adults. Thus, to enhance radiologists' knowledge of the applications of AI models in pediatric patients, curricula should be structured keeping in mind the unique pediatric setting and its challenges, along with methods to overcome these challenges, and pediatric-specific data governance and ethical considerations. In this report, the authors highlight the salient aspects of pediatric radiology that are necessary for AI education in the pediatric setting, including the challenges for research investigation and clinical implementation.

Inflammation as a Regulator of the Airway Surface Liquid pH in Cystic Fibrosis.

The airway surface liquid (ASL) is a thin sheet of fluid that covers the luminal aspect of the airway epithelium. The ASL is a site of several first-line host defenses, and its composition is a key factor that determines respiratory fitness. Specifically, the acid-base balance of ASL has a major influence on the vital respiratory defense processes of mucociliary clearance and antimicrobial peptide activity against inhaled pathogens. In the inherited disorder cystic fibrosis (CF), loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function reduces HCO3- secretion, lowers the pH of ASL (pHASL), and impairs host defenses. These abnormalities initiate a pathologic process whose hallmarks are chronic infection, inflammation, mucus obstruction, and bronchiectasis. Inflammation is particularly relevant as it develops early in CF and persists despite highly effective CFTR modulator therapy. Recent studies show that inflammation may alter HCO3- and H+ secretion across the airway epithelia and thus regulate pHASL. Moreover, inflammation may enhance the restoration of CFTR channel function in CF epithelia exposed to clinically approved modulators. This review focuses on the complex relationships between acid-base secretion, airway inflammation, pHASL regulation, and therapeutic responses to CFTR modulators. These factors have important implications for defining optimal ways of tackling CF airway inflammation in the post-modulator era.

Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction.

Pulmonary neuroendocrine cells (PNECs) are rare airway cells with potential sensory capacity linked to vagal neurons and immune cells. How PNECs sense and respond to external stimuli remains poorly understood. We discovered PNECs located within pig and human submucosal glands, a tissue that produces much of the mucus that defends the lung. These PNECs sense succinate, an inflammatory molecule in liquid lining the airway surface. The results indicate that succinate migrates down the submucosal gland duct to the acinus, where it triggers apical succinate receptors, causing PNECs to release ATP. The short-range ATP signal stimulates the contraction of myoepithelial cells wrapped tightly around the submucosal glands. Succinate-triggered gland contraction may complement the action of neurotransmitters that induce mucus release but not gland contraction to promote mucus ejection onto the airway surface. These findings identify a local circuit in which rare PNECs within submucosal glands sense an environmental cue to orchestrate the function of airway glands.

Prevalence of self-reported diabetes risk factors and integration of diabetes screening and referral at two urban HIV care and treatment clinics in Zambia.

People living with HIV (PLWH) on antiretroviral therapy (ART) are living longer and are at risk of HIV co-morbidities including non-communicable diseases (NCDs), particularly in low-resource settings. However, the evidence base for effectively integrating HIV and NCD care is limited. The Chronic Health Care (CHC) checklist, designed to screen for multiple NCDs including a 6-item diabetes self-report screener, was implemented at two PEPFAR-supported HIV clinics in Kabwe and Kitwe, Zambia. Study objectives were to describe the HIV care and treatment population and their self-reported diabetes-related symptoms, and to evaluate provider-initiated screening and referral post-training on the CHC checklist. This cross-sectional study enrolled 435 adults receiving combination ART services. Clinic exit interviews revealed 46% self-reported at least one potential symptom, and 6% self-reported three or more symptoms to the study team, indicating risk for diabetes and need for further diagnostic testing. In comparison, only 8% of all participants reported being appropriately screened for diabetes by their health provider, with less than 1% referred for further testing. This missed opportunity for screening and referral indicates that HIV-NCD integration efforts need more fully resourced and multi-pronged approaches in order to ensure that PLWH who are already accessing ART receive the comprehensive, holistic care they need.

Use of Glycolysis-Enhancing Drugs and Risk of Parkinson's Disease.

BACKGROUND: Terazosin (TZ) and closely related α1-adrenergic receptor antagonists (doxazosin [DZ] and alfuzosin [AZ]) enhance glycolysis and reduce neurodegeneration in animal models. Observational evidence in humans from several databases supports this finding; however, a recent study has suggested that tamsulosin, the comparator medication, increases the risk of Parkinson's disease. AIMS: We consider a different comparison group of men taking 5α-reductase inhibitors (5ARIs) as a new, independent comparison allowing us to both obtain new estimates of the association between TZ/DZ/AZ and Parkinson's disease outcomes and validate tamsulosin as an active comparator. METHODS: Using the Truven Health Analytics Marketscan database, we identified men without Parkinson's disease, newly started on TZ/DZ/AZ, tamsulosin, or 5ARIs. We followed these matched cohorts to compare the hazard of developing Parkinson's disease. We conducted sensitivity analyses using variable duration of lead-in to mitigate biases introduced by prodromal disease. RESULTS: We found that men taking TZ/DZ/AZ had a lower hazard of Parkinson's disease than men taking tamsulosin (hazard ratio (HR) = 0.71, 95% CI [confidence interval]: 0.65-0.77, n = 239,888) and lower than men taking 5ARIs (HR = 0.84, 95% CI: 0.75-0.94, n = 129,116). We found the TZ/DZ/AZ versus tamsulosin HR to be essentially unchanged with up to 5 years of lead-in time; however, the TZ/DZ/AZ versus 5ARI effect became attenuated with longer lead-in durations. CONCLUSIONS: These data suggest that men using TZ/DZ/AZ have a somewhat lower risk of developing Parkinson's disease than those using tamsulosin and a slightly lower risk than those using 5ARIs. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs.

SignificanceIn many lung diseases, increased amounts of and/or abnormal mucus impair mucociliary clearance, a key defense against inhaled and aspirated material. Submucosal glands lining cartilaginous airways secrete mucus strands that are pulled by cilia until they break free from the duct and sweep upward toward the larynx, carrying particulates. In cystic fibrosis (CF) pigs, progressive clearance of insufflated microdisks was repeatedly interrupted as microdisks abruptly recoiled. Aerosolizing a reducing agent to break disulfide bonds linking mucins ruptured mucus strands, freeing them from submucosal gland ducts and allowing cilia to propel them up the airways. These findings highlight the abnormally increased elasticity of CF mucus and suggest that agents that break disulfide bonds might have value in lung diseases with increased mucus.

Perspectives on Vascular Regulation of Mechanisms Controlling Selective Immune Cell Function in the Tumor Immune Response.

The vasculature plays a major role in regulating the tumor immune cell response although the underlying mechanisms explaining such effects remain poorly understood. This review discusses current knowledge on known vascular functions with a viewpoint on how they may yield distinct immune responses. The vasculature might directly influence selective immune cell infiltration into tumors by its cell surface expression of cell adhesion molecules, expression of cytokines, cell junction properties, focal adhesions, cytoskeleton and functional capacity. This will alter the tumor microenvironment and unleash a plethora of responses that will influence the tumor's immune status. Despite our current knowledge of numerous mechanisms operating, the field is underexplored in that few functions providing a high degree of specificity have yet been provided in relation to the enormous divergence of responses apparent in human cancers. Further exploration of this field is much warranted.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.