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CFTR
Terms modified by CFTR Selected AbstractsCFTR , a gatekeeper for duodenal HCO3, secretionACTA PHYSIOLOGICA, Issue 4 2008Jens Leipziger No abstract is available for this article. [source] Ceramide in Pseudomonas aeruginosa infectionsEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 10 2007Joachim Riethmüller Abstract Cystic fibrosis (CF), the most common autosomal recessive disorder, at least in western countries, is caused by mutations of the cystic fibrosis transmembranous conductance regulator (CFTR) molecule and affects approximately 80,000 patients in Europe and the USA. Most, if not all, CF patients develop a chronic pulmonary infection with Pseudomonas aeruginosa. At present it is unknown why CF patients are highly sensitive to P.,aeruginosa infections, and most importantly, no curative treatment for CF is available. P.,aeruginosa infection results in an activation of the enzyme acid sphingomyelinase which catalyzes the release of ceramide from sphingomyelin in the cell membrane. Ceramide forms large ceramide-enriched membrane domains that are required for internalization of bacteria, induction of cell death in infected cells and a controlled release of cytokines from infected cells. Ceramide-enriched membrane platforms seem to serve the reorganization of receptors and intracellular signaling molecules involved in the infection of mammalian cells with P.,aeruginosa. The significance of the acid sphingomyelinase and ceramide for the infection of mammalian cells with P.,aeruginosa was demonstrated on mice genetically deficient for the acid sphingomyelinase. Further studies with N.,gonorrhoeae, S.,aureus and rhinoviruses indicate that ceramide-enriched membrane domains are also important for the infection of mammalian cells with other bacterial and viral pathogens, suggesting a general role of these membrane domains in infectious biology. [source] Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR geneFEBS JOURNAL, Issue 2 2002Marios Phylactides The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhan- cer/reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell specificity of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in introns 10 and 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of regulation of expression of the CFTR gene in which multiple elements contribute to tightly co-ordinated expression in vivo. [source] Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulatorFEBS JOURNAL, Issue 17 2000Production of a suitable protein for structural studies Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure,function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation ,F508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N- 1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1. [source] Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger,HEPATOLOGY, Issue 2 2006Jesús M. Banales Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR),dependent Cl, efflux and subsequent biliary HCO3, secretion, possibly via Cl,/HCO3, anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl,/HCO3, exchange in secretin-stimulated HCO3, secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3, and Cl, excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl, channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl,/HCO3, exchange inhibitor 4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3, and Cl,. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3, excretion but not the increased Cl, excretion, revealing a role of biliary Cl,/HCO3, exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+ -independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl,/HCO3, exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride,bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266,275.) [source] Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineageHEPATOLOGY, Issue 3 2003Robbert G. E. Notenboom To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of ,-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes. [source] Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosisHEPATOLOGY, Issue 2 2000Nils Kinnman M.D. The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the ,F508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of ,F508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease. [source] Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci,HUMAN MUTATION, Issue 4 2010Sylvia Quemener Abstract Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95,bp) was used to analyze the entire length of the CFTR gene (189,kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2,3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of ±20,bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:1,8, 2010. © 2010 Wiley-Liss, Inc. [source] Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis,HUMAN MUTATION, Issue 8 2008Sarah Berwouts Abstract Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material. Hum Mutat 0, 1,8, 2008. © 2008 Wiley-Liss, Inc. [source] N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel,HUMAN MUTATION, Issue 5 2008G.G. Gené Abstract Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure,function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Hum Mutat 29(5), 738,749, 2008. © 2008 Wiley-Liss, Inc. [source] Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North-Eastern ItalyINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 3 2010L. Segat Summary Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5,-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients. [source] Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation associated with a congenital bilateral absence of vas deferensINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2008Hideo Sakamoto Abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations associated with cystic fibrosis have been reported to be rare in Japanese patients with congenital bilateral absence of vas deferens (CBAVD). A 28-year-old Japanese male was referred for infertility. Vas deferens and epididymis were not palpable bilaterally. Semen analyses showed azoospermia with volumes below 2.0 ml. Serum follicle-stimulating hormone value was slightly elevated. Seminal fructose concentration was also very low. Scrotal ultrasonography showed absence of the bodies and tails of the right and left epididymides. Imaging studies showed cystic dysplasia of the right seminal vesicle and agenesis of the left seminal vesicle. A CFTR gene mutation of I556V was found. Recent studies show that prevalence of CFTR gene mutation in Japanese CBAVD patients may be approximately equal to that of the Caucasian population. Genetic counselling may be recommended for any couple attempting assisted reproduction technology when the man has CBAVD. [source] Involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in the pathogenesis of hydrosalpinx induced by Chlamydia trachomatis infectionJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 6 2008Louis Chukwuemeka Ajonuma Abstract Background:, Genital Chlamydia (C) trachomatis infection has been recognized as the single most common cause of pelvic inflammatory disease leading to severe tubal damage, ectopic pregnancy, infertility and hydrosalpinx. However, the mechanism underlying the formation of hydrosalpinx induced by C. trachomatis infection remains largely unknown. We performed this study to determine the involvement of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel that regulates epithelial electrolyte and fluid secretion, in hydrosalpinx fluid formation. Methods:, Western blot analysis was used to determine CFTR expression in the hydrosalpinges that were seen on the ultrasound scans of infertile assisted reproduction treatment patients. Correlation with C. trachomatis infection was done by testing patients' sera for C. trachomatis immunoglobulin G antibody titer using a Capita enzyme-linked immunosorbent assay based kit. CFTR involvement was further verified in a rat C. trachomatis infection model and confirmed using CFTR mutant (CFTRtm1Unc) mice. Results:, Here we report on the up-regulated expression of CFTR in the hydrosalpinx tissues of infertile patients with detectable serum levels of C. trachomatis antibody (immunoglobulin G). In a rat model, increased CFTR expression and fluid accumulation could be observed in the uterine horns infected with C. trachomatis elementary bodies, which was reversed by antibiotics treatment. In C. trachomatis,infected CFTRtm1Unc mice, however, no detectable fluid accumulation was observed. Conclusion:, These findings suggest the involvement of CFTR in the pathogenesis of hydrosalpinx fluid formation and may provide grounds for a better treatment strategy to improve assisted reproduction treatment outcome in infertile patients with hydrosalpinx. [source] In vivo pharmacology and antidiarrheal efficacy of a thiazolidinone CFTR inhibitor in rodentsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2005N.D. Sonawane Abstract A small-molecule inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTRinh -172), reduces enterotoxin-induced intestinal fluid secretion in rodents. Here, we study CFTRinh -172 pharmacology and antidiarrheal efficacy in rodents using 14C-labeled CFTRinh -172, liquid chromatography/mass spectrometry, and a closed intestinal loop model of fluid secretion. CFTRinh -172 was cleared primarily by renal glomerular filtration without chemical modification. CFTRinh -172 accumulated in liver within 5 min after intravenous infusion in mice, and was concentrated fivefold in bile over blood. At 30,240 min, CFTRinh -172 was found mainly in liver, intestine, and kidney, with little detectable in the brain, heart, skeletal muscle, or lung. Pharmacokinetic analysis in rats following intravenous bolus infusion showed a distribution volume of 770 mL with redistribution and elimination half-times of 0.14 h and 10.3 h, respectively. CFTRinh -172 was stable in hepatic microsomes. Closed-loop studies in mice indicated that a single intraperitoneal injection of 20 ,g CFTRinh -172 inhibited fluid accumulation at 6 h after cholera toxin by >90% in duodenum and jejunum, ,60% in ileum and <10% in colon. No toxicity was seen after high-dose CFTRinh -172 administration (3 mg/kg/day in two daily doses) in mice over the first 6 weeks of life. The metabolic stability, enterohepatic recirculation, slow renal elimination, and intestinal accumulation of CFTRinh -172 account for its efficacy as an antidiarrheal. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:134,143, 2005 [source] Association Analyses of Genetic Polymorphisms of GSTM1, GSTT1, NQO1, NAT2, LPL, PRSS1, PSTI, and CFTR With Chronic Alcoholic Pancreatitis in JapanALCOHOLISM, Issue 2010Katsuya Maruyama Background:, Excessive consumption of alcohol is involved in the onset of pancreatitis. However, most of heavy drinkers do not always develop chronic pancreatitis. Various genetic factors appear to be involved in these individual differences in onset of chronic alcoholic pancreatitis. Here we investigated a possible association of alcoholic pancreatitis with polymorphisms of the various genes belong to the phase II detoxification enzymes responsible for metabolism of the oxidative compounds, and the several genes that have relevance to inherited pancreatitis. Methods:, The subjects consisted of 53 patients with chronic alcoholic pancreatitis, 54 alcoholic patients without pancreatic dysfunction, and 42 healthy individuals. DNA was extracted from the peripheral nucleated blood cells of all subjects and genetic mutations and subtypes were analyzed by the PCR and RFLP methods. We examined the correlation between chronic alcoholic pancreatitis and variants of the phase II detoxification enzymes such as Glutathione S-transferase M1 (GSTM1), glutathione S-transferase theta 1 (GSTT1), NADPH-quinone oxidoreductase 1 (NQO1), and N-acetyl transferase (NAT2). In addition, genes of lipoprotein lipase (LPL), cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (PSTI), and cystic fibrosis transmembrane conductance regulator (CFTR) were also analyzed. Results:, Frequencies of the gene deletion of GSTM1 and GSTT1 in addition to the C-allele frequency of NQO1 tended to be higher in the alcoholic patients with (AlCP) or without pancreatic dysfunction (Alc) than in the healthy controls although the difference was not significant. The NAT2 gene showed no relation with Alc and AlCP patients. PSTI, LPL, PRSS1, and CFTR genes presented no association with chronic alcoholic pancreatitis. Conclusions:, All genes analyzed in the present study lacked association with chronic alcoholic pancreatitis. However, the gene deletion of GSTM1 and GSTT1, and the C-allele of NQO1 cannot be ruled out for association with alcoholism. [source] Cystic fibrosis lung disease starts in the small airways: Can we treat it more effectively?PEDIATRIC PULMONOLOGY, Issue 2 2010Harm A.W.M. Tiddens MD Abstract The aims of this article are to summarize existing knowledge regarding the pathophysiology of small airways disease in cystic fibrosis (CF), to speculate about additional mechanisms that might play a role, and to consider the available or potential options to treat it. In the first section, we review the evidence provided by pathologic, physiologic, and imaging studies suggesting that obstruction of small airways begins early in life and is progressive. In the second section we discuss how the relationships between CF transmembrane conductance regulator (CFTR), ion transport, the volume of the periciliary liquid layer and airway mucus might lead to defective mucociliary clearance in small airways. In addition, we discuss how chronic endobronchial bacterial infection and a chronic neutrophilic inflammatory response increase the viscosity of CF secretions and exacerbate the clearance problem. Next, we discuss how the mechanical properties of small airways could be altered early in the disease process and how remodeling can contribute to small airways disease. In the final section, we discuss how established therapies impact small airways disease and new directions that may lead to improvement in the treatment of small airways disease. We conclude that there are many reasons to believe that small airways play an important role in the pathophysiology of (early) CF lung disease. Therapy should be aimed to target the small airways more efficiently, especially with drugs that can correct the basic defect at an early stage of disease. Pediatr Pulmonol. 2010; 45:107,117. © 2010 Wiley-Liss, Inc. [source] Intrathoracic nontuberculous mycobacterial infections in otherwise healthy childrenPEDIATRIC PULMONOLOGY, Issue 11 2009Alexandra F. Freeman MD Abstract Background Nontuberculous mycobacterial (NTM) infection is typically associated with lymphadenitis in immune competent children, and disseminated disease in children with immune deficiencies. Isolated pulmonary NTM disease is seen in cystic fibrosis, and is increasingly recognized in immunocompetent elderly women, where it is associated with an increased incidence of cystic fibrosis transmembrane regulator (CFTR) mutations. Thoracic NTM infection has been reported rarely in otherwise healthy children. We aimed to determine whether otherwise healthy children with pulmonary NTM disease had immunologic abnormalities or CFTR mutations. Clinical presentations of five otherwise healthy children with pulmonary NTM were reviewed. Immunologic studies were performed including a complete blood cell count (CBC), flow cytometric lymphocyte phenotyping and IFN-gamma receptor expression, in vitro cytokine stimulation, and serum immunoglobulin levels. Mutational analysis was performed for CFTR. The children ranged in age from 12 months to 2.5 years at diagnosis. Four presented with new onset wheezing or stridor failing bronchodilator therapy. One child was asymptomatic. Endobronchial lesions and/or hilar lymph nodes causing bronchial obstruction were identified in all patients. Mycobacterium avium complex was cultured from four patients, and Mycobacterium abscessus from one patient. All patients were successfully treated with anti-mycobacterial therapy with or without surgery. No definitive immunologic abnormalities were identified. No clinically significant mutations were found in CFTR. Pulmonary NTM infection should be considered in otherwise healthy young children presenting with refractory stridor or wheezing with endobronchial lesions or hilar lymphadenopathy. It does not appear to be associated with recognized underlying immune deficiency or CFTR mutations. Pediatr Pulmonol. 2009; 44:1051,1056. ©2009 Wiley-Liss, Inc. [source] Assessment of body composition in pediatric patients with cystic fibrosisPEDIATRIC PULMONOLOGY, Issue 10 2008Greg D. Wells PhD Abstract Rationale Cystic fibrosis (CF) leads to pathological changes in organs that express the cystic fibrosis transmembrane conductance regulator (CFTR), including secretory cells of the digestive tract and the pancreas. Maintaining nutritional sufficiency is challenging for CF patients and therefore accurate monitoring is important for their clinical management. Purpose The objectives of this study were to evaluate the effectiveness of skinfold measurements as an accurate method for determining body composition (fat mass (FM) and lean body mass (LBM)) of this population, using dual-energy X-ray absorptiometry (DEXA) as a gold standard comparison and to determine the most accurate equation for this calculation in children with CF. Methods Fifty-five pediatric patients with CF participated in the study. FM and LBM calculated via four methods: Slaughter, Durnin, Durenberg (2-site and 4-site). The relationship between the methods and DEXA results were estimated by intraclass-correlation coefficient (ICC) and Bland and Altman analyses. Results The Slaughter method was the most accurate (ICC of 0.92 for FM and 0.99 for LBM) and displayed the least bias over the range of FM and LBM in CF patients. In addition, the results of Bland Altman analyses comparing each skinfold method to DEXA, revealed that the results were evenly distributed along the range of values for the Slaughter calculation, whereas the other three methods under and over estimated % fat results at the upper and lower ends of the range respectively. Conclusion We therefore conclude that the Slaughter method may be used for body composition assessment of pediatric CF patients. This provides clinical teams with a simple, accurate and non-invasive method that can be used to monitor nutritional status in pediatric patients with CF. Pediatr Pulmonol. 2008; 43:1025,1032. © 2008 Wiley-Liss, Inc. [source] The CFTR gene and regulation of its expressionPEDIATRIC PULMONOLOGY, Issue 1 2005Victoria A. McCarthy Abstract The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows clear temporal and developmental regulation of its expression. However, there are few well-defined regulatory elements that control this pattern of expression, and their mechanism of action is poorly understood. We review the structure and organization of the CFTR gene and what is known about its regulation. The CFTR gene promoter is clearly important for maintaining levels of CFTR gene expression, but apparently it does not contain any tissue-specific elements. Thus tissue-specificity is probably controlled by sequences lying elsewhere in this large gene. We discuss data from our group and others implicating additional regions of CFTR in regulatory functions, and evaluate candidate transcription factors that may be involved. Further, we summarize aspects of the regulation of the developmental expression of CFTR. Definition of CFTR gene regulatory elements could be of considerable therapeutic significance, since only a small increase in CFTR expression in the correct cell type could alleviate the disease phenotype. Pediatr Pulmonol © 2005 Wiley-Liss, Inc. [source] Processing of CFTR: Traversing the cellular maze,How much CFTR needs to go through to avoid cystic fibrosis?PEDIATRIC PULMONOLOGY, Issue 6 2005Margarida D. Amaral PhD Abstract Biosynthesis of the cystic fibrosis transmembrane conductance regulator (CFTR), like other proteins aimed at the cell surface, involves transport through a series of membranous compartments, the first of which is the endoplasmic reticulum (ER), where CFTR encounters the appropriate environment for folding, oligomerization, maturation, and export from the ER. After exiting the ER, CFTR has to traffic through complex pathways until it reaches the cell surface. Although not yet fully understood, the fine details of these pathways are starting to emerge, partially through identification of an increasing number of CFTR-interacting proteins (CIPs) and the clarification of their roles in CFTR trafficking and function. These aspects of CFTR biogenesis/degradation and by membrane traffic and CIPs are discussed in this review. Following this description of complex pathways and multiple checkpoints to which CFTR is subjected in the cell, the basic question remains of how much CFTR has to overcome these barriers and be functionally expressed at the plasma membrane to avoid CF. This question is also discussed here. Pediatr Pulmonol. © 2005 Wiley-Liss, Inc. [source] Beyond chloride transport: CFTR in the 21st century,introductory remarks to a new state of the art seriesPEDIATRIC PULMONOLOGY, Issue 4 2005Anil Mehta MB, FRCP (Edin), FRCPCH This new series of articles on cystic fibrosis provides an overview of the confusing plethora of problems that arise from the loss of function in a low abundance protein, the cystic fibrosis membrane conductance regulator CFTR. The references are designed to take the clinical reader into areas and journals that they might not normally read. In particular we have concentrated on recent advances that suggest CFTR has functions that do not relate to chloride transport alone. In forthcoming issues of the journal the topics covered range from prospects and difficulties in the translation of new therapies into clinical practice, the regulation of the defective gene (promoters, enhancers, silencers, etc.), regulation and interaction of the CFTR protein product with other proteins in the cell, to functional approaches using developmental and secretory paradigms. These themes have been chosen to bring controversies at the cutting edge of cystic fibrosis research to the practicing pulmonologist in order to stimulate lateral thinking, which we hope will ultimately benefit our patients. Pediatr Pulmonol. 2005; 39:289,291. © 2004 Wiley-Liss, Inc. [source] CFTR: More than just a chloride channelPEDIATRIC PULMONOLOGY, Issue 4 2005Anil Mehta MBBS, FRCP (Edin), FRCPCH Abstract This review examines the cystic fibrosis transmembrane conductance regulator (CFTR) protein. After summarizing the ion channels regulated by CFTR, the review focuses on the functions of CFTR that do not relate directly to a disease mechanism based on a channelopathy. The key concept is that newly synthesized CFTR has to enter lipid vesicles which bud from the endoplasmic reticulum. This is abnormally low in ,F508 CFTR. Normal wild type vesicular CFTR enters a recycling pool of lipid vesicles which transiently dock with the apical membrane only for CFTR to be retrieved shortly after into a sub-apical recycling compartment. This retrieval is abnormally fast in ,F508 CFTR. The review discusses the relationship between this process and the difficult topic of fat metabolism and then explores the possible links between abnormal fatty acid turnover and inflammatory cascades that are abnormal in cystic fibrosis. Finally the review concentrates on the emerging functions of a protein kinase (AMP-activated kinase) which is bound near the C terminus of the CFTR protein whose functions could intergrate some of the abnormalities in lipid metabolism that result from mislocalization of CFTR in clinical disease. Pediatr Pulmonol. 2005; 39:292,298. © 2004 Wiley-Liss, Inc. [source] Normal volume of distribution of tobramycin in a mother and daughter with a CFTR splice mutation (1717,,,1G,,,A)PEDIATRIC PULMONOLOGY, Issue 4 2002Dawn Simon MD Abstract A mother and daughter pair with CF who shared a splice mutation (1717,,,1G,,,A) had a normal volume of distribution of tobramycin. The literature on tobramycin pharmacokinetics, which was published before the genetic defect was identified, is discussed. The authors speculate on the role of CFTR in the distribution of aminoglycosides and recommend that CFTR mutations should be clarified in all future studies of tobramycin pharmacokinetics in patients with CF. Pediatr Pulmonol. 2002; 33:315-317. © 2002 Wiley-Liss, Inc. [source] Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosisPROTEIN SCIENCE, Issue 10 2010Chi Wang Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source] Single-dose lentiviral gene transfer for lifetime airway gene expressionTHE JOURNAL OF GENE MEDICINE, Issue 10 2009Alice G. Stocker Abstract Background Cystic fibrosis (CF) is caused by a defect in cystic fibrosis transmembrane conductance regulator (CFTR) activity, often resulting in an incurable airway disease. Gene therapy into the conducting airway epithelium is a potential cure for CF; however, most gene vectors do not result in long-lived expression, and require re-dosing. Perversely, intrinsic host immune responses can then block renewed gene transfer. Methods To investigate whether persistent gene expression could be achieved after a single dosing event, thus avoiding the issue of blocking host responses, we used a gene transfer protocol that combined an airway pretreatment using lysophosphatidylcholine with a human immunodeficiency virus type-1 (vesicular stomatitis virus G pseudotype) derived lentiviral vector to test whether an integrating vector could produce gene expression able to last for a substantial part of the lifetime of the laboratory mouse. Results We found that a single dose of LV-LacZ produced immediate as well as lifetime mouse airway expression, confirming our hypothesis that use of an integrating vector extends transgene expression. Importantly, LV-CFTR dosing achieved at least 12 months of CFTR expression, representing partial functional correction of the CFTR defect in CF-null mice. Conclusions These findings validate the potential of this methodology for developing a gene transfer treatment for CF airway disease. Copyright © 2009 John Wiley & Sons, Ltd. [source] Enhancing rAAV vector expression in the lungTHE JOURNAL OF GENE MEDICINE, Issue 7 2005Isabel Virella-Lowell Abstract Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (C,) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the C, promoter, and the C, promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the C,-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 × 106 relative light units (RLU)/g tissue and 2.7 × 106 RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful. Copyright © 2005 John Wiley & Sons, Ltd. [source] A new role for bicarbonate secretion in cervico-uterine mucus releaseTHE JOURNAL OF PHYSIOLOGY, Issue 13 2010Ruth W. Muchekehu Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO3,) secretion. Prostaglandin E2 (PGE2)- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO3,, HCO3, transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE2 - and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis ,F508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis. [source] CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005G. Nagel The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR,ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. [source] CFTR gene mutations and male infertilityANDROLOGIA, Issue 2 2000M. Stuhrmann Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are a relatively frequent cause of male infertility. Depending on their molecular consequences, CFTR mutations may either result in typical cystic fibrosis (CF), one of the most common autosomal recessive disorders, which is characterized by chronic lung disease, pancreatic exocrine insufficiency, an increase in the concentration of sweat electrolytes and male infertility, due to obstructive azoospermia, or in atypical (often monosymptomatic) forms of CF such as congenital absence of the vas deferens (bi- or unilateral), bilateral ejaculatory duct obstruction or bilateral obstructions within the epididymides. All males with idiopathic obstructive azoospermia bear an increased risk for CF offspring. Couples requesting microsurgical epididymal sperm aspiration and in vitro fertilization, e.g. intracytoplasmic sperm injection, should be offered genetic counselling and molecular genetic analysis of the CFTR gene, if male infertility due to obstructive azoospermia is the underlying cause. [source] Identification and Characterization of CFTR Gene Mutations in Indian CF PatientsANNALS OF HUMAN GENETICS, Issue 1 2009N. Sharma Summary Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study was performed on Indian CF patients (n = 50) to investigate the spectrum of mutations in the CFTR gene and their association with intragenic and extragenic marker haplotypes. We report identification of 14 previously known and eight novel mutations, namely 3986-3987delC, 876-6del4, 1792InsA, L69H, S158N, Q493L, I530L and E1329Q. The frequency of delta F508 was found to be 27%. Absolute linkage between delta F508 and the KM.19-GATT-TUB9-M470V-T854T haplotype (2-2-1-1-1) predicts a relatively recent appearance of delta F508 in Indian CF patients. Low frequency of delta F508 mutation and detection of eight novel and thirteen rare mutations reflect a heterogeneous spectrum of mutations in Indian CF patients. Failure to detect mutations in 34% of alleles indicates the possible presence of gross deletions involving one or more exons or may indicate the location of the molecular defects in either the noncoding parts of the gene or in the promoter region, which warrants analysis of those regions. [source] | ||||||||||||||||