James R. Yankaskas

Submucosal glands are the predominant site of CFTR expression in the human bronchus

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regulator (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non–CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with ΔF508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.

An Official American Thoracic Society Clinical Policy Statement: Palliative Care for Patients with Respiratory Diseases and Critical Illnesses

Executive Summary Introduction Methods Goals, Timing, and Settings for Palliative Care Decision-making Process Advance Directives Care Planning and Delivery Hospice Care Alternative End-of-Life Decisions Symptom Management Dyspnea Management Pain Management Management of Psychological and Spiritual Distress and Suffering Withdrawal of Mechanical Ventilation Process of Decision Making Process of Withdrawing Mechanical Ventilation Bereavement Care Barriers to Palliative Care Program Development, Education, Training, and Research in Palliative Care

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses.

We describe the use of a human bronchial xenograft model for studying the efficiency and biology of in vivo gene transfer into human bronchial epithelia with recombinant E1 deleted adenoviruses. All cell types in the surface epithelium except basal cells efficiently expressed the adenoviral transduced recombinant genes, lacZ and CFTR, for 3–5 weeks. Stable transgene expression was associated with high level expression of the early adenoviral gene, E2a, in a subset of transgene expressing cells and virtually undetectable expression of the late adenoviral genes encoding the structural proteins, hexon and fiber. These studies begin to address important issues that relate to safety and in vivo efficacy of recombinant adenoviruses for gene delivery into the human airway.

A Pilot Study of Aerosolized Amiloride for the Treatment of Lung Disease in Cystic Fibrosis

Excessive active absorption of sodium is a unique abnormality of the airway epithelium in patients with cystic fibrosis. This defect is associated with thickened mucus and poor clearance of airway secretions and may contribute to the pulmonary disease in these patients. To study whether the inhibition of excessive absorption of sodium might affect the course of lung disease in cystic fibrosis, we performed a double-blind, crossover trial comparing aerosolized amiloride (5 mmol per liter; 3.5 ml four times daily), a sodium-channel blocker, with vehicle alone. Fourteen of the 18 adult patients initially enrolled in the study completed the one-year trial (25 weeks for each treatment). The mean (+/- SEM) loss of forced vital capacity (FVC) was reduced from 3.39 +/- 1.13 ml per day during treatment with vehicle alone to 1.44 +/- 0.67 ml per day during treatment with amiloride (P less than 0.04). A measured index of sputum viscosity and elasticity was abnormal during treatment with vehicle alone and improved during treatment with amiloride. Calculated indexes of mucociliary and cough clearance also improved during amiloride treatment. No systemic, respiratory, or subjective toxic effects of amiloride were noted. We conclude from this preliminary study that aerosolized amiloride can be safely administered to adults with cystic fibrosis. The slowing of the loss of FVC and the improvement in sputum viscosity and elasticity suggest a beneficial clinical effect. Aerosolized amiloride deserves further evaluation in the treatment of lung disease in patients with cystic fibrosis.

Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures.

1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl‐ pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (‐27 +/‐ 4 mV, mean +/‐ S.E.M.; n = 28) exceeded the PD of normal epithelia (‐10 +/‐ 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/‐ 9 microA cm‐2) as compared with normal epithelia (23 +/‐ 3 microA cm‐2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl‐), the apical membrane of normal cells exhibits an apical membrane Cl‐ conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride‐sensitive Ieq and amiloride‐sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride‐sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl‐ permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of this disease.

Expression of the cystic fibrosis gene in adult human lung.

Critical to an understanding of the pulmonary disease in cystic fibrosis (CF) and the development of effective gene therapies is a definition of the distribution and regulation of CF gene expression in adult human lung. Previous studies have detected the product of the CF gene, the CF transmembrane conductance regulator (CFTR), in submucosal glands of human bronchi. In this report, we have characterized the distribution of CFTR RNA and protein in the distal airway and alveoli of human lungs. Samples from eight human lungs were analyzed for CFTR expression by in situ hybridization and immunocytochemistry. CFTR was detected in a subpopulation of epithelial cells at every level of the distal lung, including proximal, terminal, and respiratory bronchioles, and the alveoli. However, there was substantial variation in the level of CFTR expression between samples. In bronchioles, CFTR protein localized to the apical plasma membrane and was found primarily in a subpopulation of nonciliated cells. CFTR was expressed in the same distribution as the Clara cell marker CC10 in proximal bronchioles, however, expression was discordant in the more distal bronchioles and alveoli where CC10 was not detected. These studies suggest that epithelial cells of the distal lung may play a primary role in the pathogenesis of CF as well as expand the spectrum of target cells that should be considered in the development of gene therapies.

Abnormal glutathione transport in cystic fibrosis airway epithelia

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), ΔF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or β-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells ( P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. γ-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

Improved results of lung transplantation for patients with cystic fibrosis

Patients with cystic fibrosis pose particular challenges for lung transplant surgeons. Earlier reports from North American centers suggested that patients with cystic fibrosis were at greater risk for heart-lung or isolated lung transplantation than other patients with end-stage pulmonary disease. During a 3 1/2 year period, 44 patients with end-stage lung disease resulting from cystic fibrosis underwent double lung transplantation at this institution. During the same interval, 18 patients with cystic fibrosis died while waiting for lung transplantation. The ages of the recipients ranged from 8 to 45 years, and mean forced expiratory volume in 1 second was 21% predicted. Seven patients had Pseudomonas cepacia bacteria before transplantation. Bilateral sequential implantation with omentopexy was used in all patients. There were no operative deaths, although two patients required urgent retransplantation because of graft failure. Cardiopulmonary bypass was necessary in six procedures in five patients and was associated with an increased blood transfusion requirement, longer postoperative ventilation, and longer hospital stay. Actuarial survival was 85% at 1 year and 67% at 2 years. Infection was the most common cause of death within 6 months of transplantation (Pseudomonas cepacia pneumonia was the cause of death in two patients), and bronchiolitis obliterans was the most common cause of death after 6 months. Actuarial freedom from development of clinically significant bronchiolitis obliterans was 59% at 2 years. Results of pulmonary function tests improved substantially in survivors, with forced expiratory volume in 1 second averaging 78% predicted 2 years after transplantation. Double lung transplantation can be accomplished with acceptable morbidity and mortality in patients with cystic fibrosis.

In vivo retroviral gene transfer into human bronchial epithelia of xenografts.

Cystic fibrosis (CF) is the most common lethal inherited disease in the Caucasian population with an incidence of approximately 1 in 2,500 live births. Pulmonary complications of CF, which are the most morbid aspects of the disease, are caused by primary abnormalities in epithelial cells that lead to impaired mucociliary clearance. One potential therapeutic strategy is to reconstitute expression of the CF gene in airway epithelia by somatic gene transfer. To this end, we have developed an animal model of the human airway using bronchial xenografts and have tested the efficiency of in vivo retroviral gene transfer. Using the LacZ reporter gene, we find the efficiency of in vivo retroviral gene transfer to be dramatically dependent on the regenerative and mitotic state of the epithelium. Within an undifferentiated regenerating epithelium in which 40% of nuclei labeled with BrdU, 5-10% retroviral gene transfer was obtained. In contrast, no gene transfer was noted in a fully differentiated epithelium in which 1% of nuclei labeled with BrdU. These findings suggest that retroviral mediated gene transfer to the airway in vivo may be feasible if the proper regenerative state can be induced.