Altered intestinal chloride transport in cystic fibrosis

Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients.     

Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands

We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.     

Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and cGMP-regulated chloride channel critical to the regulation of intestinal fluid, chloride, and bicarbonate secretion. In cystic fibrosis (CF), mutations in CFTR result in downregulation of CFTR function and small intestinal obstruction. Unlike the human CF intestine, severe gastrointestinal disease and lethal obstruction is common in transgenic mice deficient in CFTR. The relevance of the physiology of CFTR and pathophysiology of CF in genetically altered mice to that of human CF disease remains incompletely understood. We hypothesized that the expression and distribution of CFTR in mouse intestine may differ from that of human and may contribute to the variation in disease expression between the two species. Using immunocytochemical and immunoblot techniques and well-characterized anti-rodent anti-CFTR antibodies, we examined the cellular distribution of CFTR in the mouse intestinal tract. We identified significant differences in villus distribution for CFTR in the mouse proximal small intestine compared to those previously reported for human and rat. These observations are important to the understanding of CFTR pathophysiology in transgenic CF mouse model systems and bear relevance to the different phenotypic expression of disease in mice compared to human.     

Analysis of fetal intestinal enzymes in amniotic fluid for the prenatal diagnosis of cystic fibrosis

Amniotic-fluid intestinal alkaline phosphatase activity, gamma-glutamyltranspeptidase activity, and leucine-aminopeptidase activity were quantitated to assess their reliability for the prenatal diagnosis of cystic fibrosis. The results indicate that for each of these enzymes an arbitrary cutoff point could be chosen that would enable one to correctly predict the outcome for the majority of at-risk pregnancies. However, some false positives and false negatives occurred with each enzyme. To obtain optimal diagnostic discrimination, the three enzyme values obtained for each sample were combined into a single linear discriminant function that proved to be a more accurate indicator of the outcome of the pregnancy. This was especially important for those cases in which the predicted outcome as based on the individual enzyme results was in disagreement. From the cases studied here, it appears that this method can be expected to give a correct prediction in approximately 96.5% of all 25%-at-risk pregnancies. False positives can be expected in approximately 1.4% of the pregnancies and false negatives in approximately 2.2%.     

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.     

What genetic risk should suggest prenatal diagnosis for cystic fibrosis?

Prenatal diagnosis of cystic fibrosis is today possible by chemical study of amniotic fluid during the 18th week of pregnancy. We have, among 90 families seen for genetic counseling between 1972 and 1985, estimated the risk of recurrence; it was of 0.25 in 55 cases; between 0.05 and 0.16 in 30 cases, greater than 0.01 in 16 cases. Before suggesting a prenatal diagnosis, it is necessary to take as basis the risk of abortion and the reliability of the method. The first risk is well known, less 0.005, in these young women; reliability can be estimated: almost 0.01 of false wrong negative, and this number is probably overvalued if all technical conditions are perfect. In front of these risks, it seems possible to propose a prenatal diagnosis from a risk of 0.01, the families being informed of all risks. We think so to hearten some families and to make possible for them to live quietly these pregnancies, probably non undertaken without our help.     

Pharmacogenomics of cystic fibrosis

Pharmacogenomics is becoming a frontline instrument of drug discovery, where the drug-dependent patterns of global gene expression are employed as biologically relevant end points. In the case of cystic fibrosis (CF), cells and tissues from CF patients provide the starting points of genomic analysis. The end points for drug discovery are proposed to reside in gene expression patterns of CF cells that have been corrected by gene therapy. A case is made here that successful drug therapy and gene therapy should, hypothetically, converge at a common end point. In response to a virtual tidal wave of genomic data, bioinformatics algorithms are needed to identify those genes that truly reveal drug efficacy. As examples, we describe the hierarchical clustering, GRASP, and GENESAVER algorithms, particularly within a hypothesis-driven context that focuses on data for a CF candidate drug. Pharmacogenomic approaches to CF, and other similar diseases, may eventually give us the opportunity to create drugs that work in a patient- or mutation-specific manner.     

Terminal glycosylation in cystic fibrosis

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.