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 regular (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 delta F508 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.

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.

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia.

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.

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 delta F508 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.

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.

Genotypic analysis of respiratory mucous sulfation defects in cystic fibrosis.

Intracellular dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to alter endosomal acidification. The most widely studied consequence of this defect has been alterations in the biochemical properties of cystic fibrosis (CF) respiratory mucus glycoproteins. However, studies confirming the existence of mucous processing defects in CF have been hindered by the lack of in vivo animal models by which to test these hypotheses in the absence of secondary effects of chronic bacterial infection. The human bronchial xenograft model has been useful in evaluating the pathophysiologic differences between CF and non-CF airway epithelium, in the absence of secondary disease effects such as goblet cell hyperplasia. In this study we sought to compare the extent of sulfation within secreted mucus glycoproteins from CF and non-CF human bronchial xenografts. Cumulative results of xenografts generated from 13 independent CF tissue samples demonstrated a statistically significant higher level of sulfation (1.7 +/- 0.18, P < 0.026) as compared to non-CF paired controls. Such findings add to the growing body of knowledge that primary defects in sulfation exist in CF respiratory mucin. Correlation of genotype with the extent of mucus sulfation revealed two categories of CF tissues with statistically different mucus sulfation profiles. Results from these studies demonstrated a 2.0 +/- 0.15-fold higher level of mucus sulfation produced from xenografts of five defined CF genotypes as compared to non-CF controls (P < 0.004, n= 10). Interestingly, three CF samples for which one mutant allele remained undefined (deltaoff8/unknown or G551D/unknown) demonstrated no statistical difference in the level of sulfation as compared with matched non-CF controls (n= 3). This as yet unknown allele was not identified within a screen for the 26 most common CF mutations. These results provide preliminary evidence for allelic variation within the CF population which may begin to elucidate the structure-function of CFTR with regards to intracellular mucus processing defects.

Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis.

Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface.

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.

Gut epithelial cells as targets for gene therapy of hemophilia.

Gut epithelium is an attractive target for gene therapy of hemophilia due to the large number of rapidly dividing cells that should be readily accessible to a wide range of vectors by a noninvasive route of administration. We have performed in vitro tests to determine the suitability of gut epithelial cells for gene transfer, protein synthesis, and secretion of coagulation factors VIII and IX. The results with retroviral vectors indicate that transduced epithelial cells from human, rat, or porcine small or large intestine can synthesize significant amounts of factor VIII or factor IX and that two-thirds or more of the recombinant protein is secreted in a basolateral direction (i.e., away from the lumen and toward underlying capillaries and lymphatics). Furthermore, we have demonstrated that intestinal epithelial cells are susceptible to efficient gene transfer by lipofection and adenovirus vectors. In the case of factor IX, we have produced a high-titer adenovirus vector capable of transducing gut epithelial cells resulting in synthesis of factor IX. The results of our in vitro studies indicate that gene transfer targeting gut epithelium as a new approach to hemophilia gene therapy is rational and merits in vivo studies in hemophilia animal models.

Vector-specific complementation profiles of two independent primary defects in cystic fibrosis airways.

Cystic fibrosis (CF) lung disease has been linked to multiple primary defects in airway epithelia caused by a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) gene. These defects include altered Cl- and Na+ permeability as well as intracellular defects in glycoprotein processing. This apparent diversity in CFTR function is reflected in the complex patterning of CFTR expression in airway epithelia. Such complexities present challenges in the design of CF gene therapies that are capable of reconstituting the endogenous patterns of CFTR gene expression in appropriate target cells. Using a human bronchial xenograft model of the CF airway, we have evaluated the efficacy of recombinant adenoviral and cationic liposome-mediated gene transfer to correct Cl- permeability and mucous sulfation defects found in CF lung disease. Results from these studies demonstrated a clear vector-specific complementation profile for these two defects that was dependent on the type of cell transduced and the level of transgene expression. Single-dose administration of recombinant adenovirus effectively transduced high levels of CFTR transgene expression in 11 +/- 1% of epithelial cells and was capable of correcting cAMP-induced changes in Cl- permeability to 91 +/- 14% that seen in non-CF airways. However, this level of transgene expression was incapable of reversing defects in mucous sulfation due to the lack of efficient targeting to goblet cells. In contrast, cationic liposome-mediated delivery of CFTR encoding plasmids to CF airways achieved extremely low levels of transgene expression with insignificant correction (7.4 +/- 2.4%) of cAMP-induced Cl- permeability. This low level of transgene expression, however, efficiently reduced mucous sulfation to levels seen in non-CF airways. Differences in the complementation profiles of these two vectors in correcting Cl- permeability and mucous sulfation defects mirror the ability of recombinant adenovirus and liposomes to reconstitute only certain features of the endogenous distribution and abundance of CFTR protein expression. Such findings suggest that the level of intracellular CFTR required to facilitate proper glycoprotein processing may be much lower than that needed to mediate bulk Cl- flow across the airway epithelium. In summary, these data present the first example by which two different vector systems can efficiently complement independent primary defects associated with a single dysfunctional gene.

Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer.

We are studying the introduction and expression of the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into cultured human airway epithelial cells as a model for gene therapy of cystic fibrosis. In this paper, we show that the chloride transport defect at the apical membrane is corrected in vitro in differentiated ion-transporting CF airway epithelial cells that exhibit polarized properties similar to those found in vivo. Using a retroviral vector containing a copy of the normal CFTR cDNA, we infected cultures of proliferating, cystic fibrosis CFT1 cells and found that correction was maintained following differentiation into a polarized epithelial sheet. At least partial correction of the Cl- transport defect was preserved in CFT1 cells for periods of up to 6 months without selection for maintenance of the retroviral provirus. These results suggest that it may be feasible to target proliferating cells in the lung using retroviral vectors for treatment of CF lung disease.

Transformation of airway epithelial cells with persistence of cystic fibrosis or normal ion transport phenotypes.

These studies demonstrate the feasibility of transforming human airway epithelial cells while inducing only modest changes in function. Features central to the pathophysiology of cystic fibrosis, i.e., abnormal regulation of a chloride permeability in the apical cell membrane, appear to be preserved in the CF/T43 transformed cell line. This work indicates that additional cystic fibrosis and normal cell lines may be developed, as well as epithelial cell lines from other diseases of interest. In addition to SV40T gene, temperature-sensitive viral genes, or genes driven by inducible promoters (e.g., glucocorticoids, heavy metals) may produce cell lines in which proliferation or differentiation can be controlled. For example, the temperature-selective SV40A gene is expressed in cells cultured at the permissive (33 degrees) temperature but is degraded at the nonpermissive (40 degrees) temperature. Thus, the transfected gene may induce proliferation to increase cell number, and then be suppressed to permit expression of a differentiated phenotype. Out strategy of initially selecting clones by G418 resistance and then selecting clones that develop functional tight junctions (and a transepithelial resistance) was useful in identifying a cell line with highly differentiated phenotypic properties. Cell lines that do not form transepithelial resistances may be valuable for studies that do not depend on cell polarization. Initial evidence suggests that some of the differentiated properties of CF/T43, i.e., formation of functional tight junctions and a transepithelial resistance, are lost at late passages.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung transplantation in cystic fibrosis: consensus conference statement.

The first successful heart-lung and lung transplant operations in cystic fibrosis (CF) patients were performed in 1983 and 1987, respectively. Lung transplantation is now available at dozens of centers in North America, Europe, and Australia. Recent technical developments and the major limitations of donor organ availability prompted the CF Foundation to sponsor a meeting of 37 experts to evaluate the state of the art in lung transplantation for CF, highlighting areas of consensus, practice variations, and controversy. This document summarizes the work of that group.

Nontuberculous mycobacteria in adult patients with cystic fibrosis.

Because patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual microorganisms, we screened the sputum of adult CF patients for mycobacterial organisms. Acid-fast bacilli (AFB) smears and mycobacterial culture were performed on 297 sputum specimens from 87 patients. Cultures for mycobacteria were frequently overgrown with other bacteria; 22.6 percent of cultures were contaminated. Despite this limitation of mycobacterial culture, 17 patients had at least one positive culture for a Mycobacterium other than tuberculosis (MOTT). Eleven patients were positive for Mycobacterium avium-intracellulare (MAI), two for MAI and M chelonei, three for M chelonei, and one for M fortuitum. None was positive for M tuberculosis. Patients with CF with MOTT were similar to patients with CF without MOTT; only a slightly different (older) age distribution was recognized. The clinical significance of MOTT was difficult to determine in any individual patient, but patients with positive AFB smears appeared more likely to suffer pathogenic effects. We conclude that MOTT is frequently recovered from adult CF patients in the southeastern United States. A specific risk factor for colonization and/or pathogenic infection in this patient group was not evident. The general prevalence and clinical pathogenesis in CF patients in the United States remains to be determined.

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.

Lung transplantation for mechanically ventilated patients.

As lung transplantation has become more successful, the selection criteria have broadened; however, some relative contraindications to lung transplantation are controversial. Some programs consider mechanical ventilation to be a major contraindication to lung transplantation because airway colonization with bacteria may lead to nosocomial infection and respiratory muscle deconditioning may necessitate prolonged postoperative ventilatory support. We report our experience of seven double lung transplant procedures on six patients requiring mechanical ventilation. Five patients with cystic fibrosis required preoperative mechanical ventilation for 7 to 19 days (mean, 10.7 days). One patient with acute lung injury required 115 days of preoperative mechanical ventilatory support. Only the latter patient required prolonged (27 days) postoperative mechanical ventilation because of respiratory muscle weakness; the others were extubated in 1 to 19 days (mean, 7.8 days). No early complications related to bacterial infection were seen. Two patients required temporary hemodialysis for transient kidney failure. Three patients had postoperative neurologic residua; one patient had a transient hemiparesis, and seizures developed in two patients. One patient died 3 months after transplantation from severe central nervous system complications with no evidence of pulmonary problems; and two patients died 17 months after transplantation, one of them receiving a second double lung transplant for obliterative bronchiolitis. Except for the patient who required prolonged preoperative ventilatory support, mechanical ventilation did not appear to play a role in the outcome of these patients. The posttransplantation hospital stay and hospital charges for patients requiring pretransplantation ventilatory support were not significantly different from those for other lung transplant recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of raised osmolarity on canine tracheal epithelial ion transport function.

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.

Abnormal respiratory epithelial ion transport in cystic fibrosis.

The respiratory epithelium of cystic fibrosis patients exhibits excessive sodium (and volume) absorption and an absence of chloride (and volume) secretion in response to usual stimuli. These abnormalities likely contribute to the pathophysiology of cystic fibrosis lung disease, and provide a rationale for a novel therapeutic approach to this problem.

Increased ion transport in cultured nasal polyp epithelial cells.

OBJECTIVE: To measure transepithelial bioelectric properties of cultured human nasal polyp and turbinate epithelial cells to test the hypothesis that nasal polyps have increased rates of ion transport. DESIGN: Cohort study. SETTING: Private referral center. PATIENTS: Individuals undergoing surgery for symptomatic nasal obstruction due to polyps caused by cystic fibrosis, nonatopic rhinosinusitis, or allergic rhinosinusitis. METHODS: Epithelial cells were removed from separated polyp and turbinate samples by protease disaggregation and cultured on permeable collagen matrix supports. Transepithelial potential difference and resistance were measured daily. At the time of maximal transepithelial potential difference, the cultures were mounted in modified Ussing chambers and exposed to a sodium-positive channel blocker (amiloride hydrochloride) and to selected chloride-negative channel agonists (isoproterenol bitartrate, adenosine triphosphate). OUTCOME MEASURES: Maximal transepithelial potential difference, resistance, and equivalent short-circuit current. Bioelectric responses to amiloride, isoproterenol, and adenosine triphosphate. RESULTS: Polyp cultures had higher transepithelial potential difference and equivalent short-circuit current than turbinate cultures. The mediator responses were greater for polyp than for turbinate cultures. CONCLUSIONS: Polyp epithelia have increased Na+ absorption and Cl- permeability relative to turbinate epithelia. These results are consistent with the hypothesis that increased epithelial fluid absorption contributes to the development of nasal polyps.

Methods for the Use of Retroviral Vectors for Transfer of the CFTR Gene to Airway Epithelium.

Cystic fibrosis (CF) is a recessive genetic disease that affects the regulation of ion transport in the epithelia of various organs in the body including the lungs, pancreas, intestine, salivary glands, and urogenital tract. The protein encoded by the CF gene is an integral plasma membrane protein called the cystic fibrosis transmembrane conductance regulator (CFTR) and has been shown to function as a chloride channel (1). In the lungs, CFTR dysfunction affects electrolyte and fluid transport across the apical membrane of airway epithelial cells. There, sodium hyperabsorption and defective chloride secretion lead to dehydration of the fluids on the airway surface and, in turn, this leads to chronic infections and severe damage. The severity of CF lung disease and the potential accessibility of the airways to gene transfer vectors has led to proposals that gene therapy be applied for the treatment of CF lung disease (2).