An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer

Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.     

Decreased expression of the cystic fibrosis transmembrane conductance regulator protein in remodeled airway epithelium from lung transplanted patients

The absence or mislocalization of cystic fibrosis transmembrane conductance regulator (CFTR) is regarded as being specific for cystic fibrosis (CF). In principle, the supply of a non-CF lung transplant to a CF patient should bring up normal CFTR expression in the lower airways. Immunolocalization of CFTR and of epithelial differentiation markers (ie, cytokeratins 13, 14, and 18, and desmoplakins 1 and 2) was carried out on 21 mucosal biopsies from the upper lobe of grafts in non-CF (n = 12) and CF patients (n = 9) retrieved between days 23 and 1,608 after lung transplantation. Biopsy specimens from seven non-CF and four CF patients presented either a pseudostratified respiratory epithelium or slight basal cell hyperplasia. CFTR was distributed at the apical membrane of the ciliated cells. In remodeled epithelia with basal cell hyperplasia or squamous metaplasia, CFTR was either weakly expressed in the cytoplasm of the superficial epithelial cells or was undetectable. The extent of epithelium remodeling was significantly correlated with an impairment of lung function. The results suggest that posttransplant airway epithelium dedifferentiation of the graft leads to the loss of properly targeted CFTR irrespective of the underlying disease of the recipient.     

Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates

At present, it is conceivable that gene therapy of the cystic fibrosis airway epithelium is possible using the direct transfer of a functional human cystic fibrosis transmembrane conductance regulator (CFTR) gene to a wide variety of patients' tracheo-bronchial cells. Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways. Results obtained in vitro and in Rhesus monkeys suggest that the delivery of recombinant adenovirus as an aerosol is feasible and is not associated with severe toxicity after single or double administration depending on the Ad.CFTR dose. This study supports the concept of aerosolization as a delivery method for adenovirus-mediated lung gene therapy.     

Lentiviral vectors for gene therapy of cystic fibrosis

A replication-defective vector based on human immunodeficiency virus (HIV) was evaluated for gene transfer directed to the lung. The tropism of this vector has been expanded through the incorporation of the vesticular stomatitis virus G protein into its envelope. The HIV vector effectively transduced nondividing airway epithelial cells in vitro whereas a murine-based retroviral vector did not. Experiments in a human bronchial xenograft model demonstrated high-level gene transduction with a cystic fibrosis transmembrane conductance regulator (CFTR) HIV vector into undifferentiated, cystic fibrosis (CF)-derived cells of the xenograft. CFTR expression was stable and capable of functional correction of the CF defect after the graft matured. The HIV vector did not effectively transduce cells of the xenograft when instilled after the epithelium had differentiated. This block to transduction appears to be at the level of entry, although post entry restrictions cannot be ruled out. Further development of this vector system for CF gene therapy should focus on a better understanding of potential entry and post entry blocks.     

Role of secondary prevention in congestive heart failure due to coronary artery disease

BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.     

Mutations of the cystic fibrosis gene in patients with chronic pancreatitis

BACKGROUND: The pancreatic lesions of cystic fibrosis develop in utero and closely resemble those of chronic pancreatitis. Therefore, we hypothesized that mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may be more common than expected among patients with chronic pancreatitis. METHODS: We studied 134 consecutive patients with chronic pancreatitis (alcohol-related disease in 71, hyperparathyroidism in 2, hypertriglyceridemia in 1, and idiopathic disease in 60). We examined DNA for 22 mutations of the CFTR gene that together account for 95 percent of all mutations in patients with cystic fibrosis in the northwest of England. We also determined the length of the noncoding sequence of thymidines in intron 8, since the shorter the sequence, the lower the proportion of normal CFTR messenger RNA. RESULTS: The 94 male and 40 female patients ranged in age from 16 to 86 years. None had a mutation on both copies of the CFTR gene. Eighteen patients (13.4 percent), including 12 without alcoholism, had a CFTR mutation on one chromosome, as compared with a frequency of 5.3 percent among 600 local unrelated partners of persons with a family history of cystic fibrosis (P<0.001). A total of 10.4 percent of the patients had the 5T allele in intron 8 (14 of 134), which is twice the expected frequency (P=0.008). Four patients were heterozygous for both a CFTR mutation and the 5T allele. Patients with a CFTR mutation were younger than those with no mutations (P=0.03). None had the combination of sinopulmonary disease, high sweat electrolyte concentrations, and low nasal potential-difference values that are diagnostic of cystic fibrosis. CONCLUSIONS: Mutations of the CFTR gene and the 5T genotype are associated with chronic pancreatitis.     

Four adult patients with the missense mutation L206W and a mild cystic fibrosis phenotype

We report molecular and clinical analyses in four unrelated patients with cystic fibrosis (CF) with compound heterozygosity for the L206W mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). This uncommon missense mutation (frequency less than 1% in a sample of 336 CF chromosomes from Southern France) replaces a leucine by a tryptophan residue in the middle of the third transmembrane domain of CFTR. On the basis of the clinical features presented by the four patients, we postulate that the L206W might be associated with pancreatic sufficiency and residual transmembrane transport of chloride in lung.     

Interrelationship between the Na+/glucose cotransporter and CFTR in Caco-2 cells: relevance to cystic fibrosis

Both the Na+-dependent glucose cotransporter (SGLT1) and the cystic fibrosis transmembrane conductance regulator (CFTR) modulate Na+ and fluid movement, although in opposite directions. Yet few studies have investigated a possible interrelationship between these two transporters. By using the Caco-2 human colon carcinoma cell line, we confirmed that the activities of these transporters increased with spontaneous differentiation to the enterocytic phenotype. We showed that SGLT1 was positively regulated by Cl- and that optimal activity of CFTR was dependent on the presence of glucose. We also demonstrated that inhibition of CFTR by glibenclamide or diphenylamine-2-carboxylate did not modify the activity of SGLT1 and inhibition of SGLT1 by phlorizin did not modify the activity of CFTR, although it resulted in inhibition of glycoconjugate synthesis. These results point to positive substrate-cross regulation of SGLT1 and CFTR and suggest that NaCl and glucose are important for not only Na+ absorption and fluid movement, but also for cAMP-dependent Cl- efflux, and glycoconjugate synthesis, functions that are known to be anomalous in cystic fibrosis.     

Primary hyperoxaluria

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians and is caused by abnormalities in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR gene encodes a chloride channel that regulates secretion in many exocrine tissues especially pancreatic and pulmonary tissues. The clinical presentation of cystic fibrosis is highly variable with isolated CAVD (congenital absence of vas deferens) and/or typical pancreatic and pulmonary manifestations. Over 500 mutations in the CFTR gene have been described and vary among different geographic locations. The severity of clinical manifestations and specially the pulmonary disease is poorly correlated with genotype. It is interesting to collect clinical and genetical data by analysing a larger cohort of CF patients. These results are likely to improve our understanding of the physiopathology of CF and the genetic counselling; particular biochemical defect could lead to more specific treatments in the future. From our 110 patients selected in Champagne-Ardenne country, we analysed the entire coding sequence of CFTR gene and detected 95% of CF mutations and in fact, 89.5% if we include the CAVD patients; 59.4% of CF mutations were detected for these patients. Three new mutations have been here reported. We found numerous CF mutations with a large distribution throughout the gene. Nevertheless, three exons are mainly involved: 10, 11 and 21. Relationships between the genotype and phenotype are difficult to assess.     

More informative abstracts revisited

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.     

Signature current of SO2-induced bronchitis in rabbit

To investigate abnormalities of airway epithelial ion transport underlying chronic inflammatory airway diseases, we performed electrophysiological, histological, and molecular biological experiments using rabbits exposed to SO2 as a model of bronchitis. By comparison with control, the SO2-exposed trachea exhibited decreased short circuit current (Isc) and conductance associated with increased potential difference. In normal trachea, apical ATP induced a transient Isc activation followed by a suppression, whereas the bronchitis model exhibited a prolonged activation without suppression. This pathological ATP response was abolished by diphenylamine 2-carboxylate or Cl--free bath solution. A significant increase in net Cl- flux toward the lumen was observed after ATP in our bronchitis model. Isoproterenol or adenosine evoked a sustained Isc increase in SO2-exposed, but not in normal, tracheas. The Northern blot analysis showed a strong expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in SO2-exposed epithelium. The immunohistochemical study revealed a positive label of CFTR on cells located luminally only in SO2-exposed rabbits. We concluded that the prolonged ATP response in our bronchitis model was of a superimposed normal and adenosine-activated current. The latter current was also activated by isoproterenol and appeared as a signature current for the bronchitis model airway. This was likely mediated by CFTR expressed in the course of chronic inflammation.     

Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl > Br; R347P, Cl > Br; R347E, Br > Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Understanding how cystic fibrosis mutations cause a loss of Cl- channel function

Defective epithelial Cl- secretion is the hallmark of the lethal genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a regulated Cl- channel. Since the identification of the single gene encoding CFTR, several hundred disease-causing mutations, associated with a wide variety of clinical phenotypes, have been reported. To understand the relationship between genotype and clinical phenotype, researchers have investigated how mutations in CFTR disrupt its function. Here, we review the recent progress in understanding how CF-associated mutations in CFTR produce defective Cl- channels, and discuss the implications of this knowledge for the development of therapy for CF.     

Mucociliary clearance in cystic fibrosis knockout mice infected with Pseudomonas aeruginosa

In this study, we examined whether mucociliary clearance differed between cystic fibrosis (CF) knockout mice and wildtype controls. Additionally, we investigated whether infection with Pseudomonas aeruginosa, a common pathogen in the CF lung, affected this important host defence mechanism. Ciliary beat frequency (fcb) and particle transport (PT) were recorded using an in vitro lung explant preparation. Measurements were made from uninfected cystic fibrosis transmembrane conductance regulator (CFTR) knockout (-/-) mice and littermate controls (+/+) and compared to measurements from infected animals. While there were no differences detectable in fcb between CFTR -/- mice and their +/+ controls either in the presence or absence of P. aeruginosa, PT rates were different between these groups; interestingly, PT rates appeared dependent on both CFTR and infection status, with uninfected CFTR +/+ animals demonstrating higher rates of PT than their -/- littermates, while CFTR +/+ P. aeruginosa-infected mice demonstrated lower PT than knockout mice. These data demonstrate differences in mucociliary clearance between cystic fibrosis transmembrane conductance regulator knockout mice and controls, and further that Pseudomonas aeruginosa infection affects mucociliary clearance in the peripheral airways of mice. Additionally, the observed differences in particle transport suggest that cystic fibrosis transmembrane conductance regulator knockout mice demonstrate different mucociliary responses to infection.     

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

We have used the fluorescence in situ hybridization (FISH) technique to refine the localization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene on human chromosome 7. The result shows that the gene is most likely located within band q31.3.     

Chemokines and lymphocytes: novel receptors and HIV

Azoospermia due to an obstruction of the genital tract is one of numerous possible pathophysiologic mechanisms underlying male infertility. The blockage of the seminal ducts may be acquired or congenital. Only recently has the strong association between mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and various subtypes of obstructive azoospermia been elucidated. Most patients with congenital bilateral absence of the vas deferens or bilateral ejaculatory duct obstruction are carriers of such mutations. The relationship between abnormal CFTR alleles and unilateral absence of the vas deferens, isolated seminal vesicle anomalies, and Young syndrome is less well characterized and awaits further investigation.