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Pesquisa de mutações no gene CFTR (Cystic Fribrosis Transmembrane Conductance Regulator) em homens brasileiros inférteis portadores de ausência congênita dos ductos deferentes (CAVD); Screening of mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene of Brazilian infertile men with congenital absence of vas deferens (CAVD)

Missaglia, Mariangela Tuzzolo
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 27/03/2009 PT
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146.51%
A Fibrose Cística (FC) é a doença autossômica recessiva mais freqüente em caucasianos e está associada, em seu amplo espectro de apresentação clínica, a mais de 1500 mutações no gene CFTR (Cystic Fibrosis Transmembrane conductance regulator). O papel de CFTR é especialmente relevante no desenvolvimento da porção reprodutiva dos ductos mesonéfricos. Em 98% dos pacientes masculinos portadores da FC, mutações em CFTR são responsáveis pela ausência bilateral congênita dos ductos deferentes (CBAVD), associada à anomalias variáveis das vesículas seminais, ductos ejaculatórios e da porção distal dos epidídimos. A ausência uni ou bilateral congênita dos ductos deferentes (CAVD), na ausência de outros sinais clínicos de FC, é conhecida causa de infertilidade masculina, presente em 1%-2% de todos os homens inférteis, e em cerca de 10% dos azoospérmicos. A reprodução assistida utilizando a injeção intracitoplasmática de espermatozóides (ICSI) obtidos preferencialmente por aspiração microcirurgica de espermatozóides do epidídimo (MESA) permite a paternidade biológica a esses pacientes. Em função da alta morbi-mortalidade da FC e da alta freqüência de portadores assintomáticos, estimada em 1:25...

Fibrose cística : avaliação diagnóstica através da diferença de potencial nasal e sua correlação com duas mutações genéticas; Cystic fibrosis diagnostic evaluation through nasal potential difference and its correlation with two genetic mutations

Ronny Tah Yen Ng
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 28/08/2013 PT
Relevância na Pesquisa
126.41%
A fibrose cística (FC) é uma doença genética autossômica recessiva, resultante da ausência total na proteína CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ou de alterações qualitativas ou quantitativas do gene que transcreve esta proteína, em células de diversos órgãos do corpo humano, resultando em inúmeros genótipos e fenótipos desta doença. Em muitos pacientes, o diagnóstico é difícil de ser definido, pelo método clássico de dosagem de sódio e cloro no suor, ou pelo sequenciamento genético, justificando a utilização de novas técnicas de auxílio diagnóstico, como a Diferença de Potencial Nasal (DPN). Este teste proporciona uma forma de avaliação direta e sensível, através do epitélio nasal, do transporte de sódio e cloro das membranas celulares, baseado nas propriedades bioelétricas transepiteliais. O objetivo deste trabalho foi verificar se existe diferença dos valores obtidos no exame de DPN em pacientes com FC em comparação com indivíduos controles saudáveis; e verificar se este teste permite diferenciar pacientes com FC das subclasses funcionais mais graves (I, II, III) das subclasses menos graves (IV, V, VI). Foram incluídos no estudo 15 pacientes FC, 10 com mutações mais graves (grupo A) e 5 com mutações menos graves (grupo B)...

Avaliação das correntes contínuas, constante e pulsada, pelo método de iontoforese por pilocarpina em indivíduos com e sem fibrose cística; Evaluation of direct constant and direct pulsed currents by pilocarpine iontophoresis in cystic fibrosis and healthy individuals

Carla Cristina Souza Gomez
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 20/08/2014 PT
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Introdução: O teste do suor clássico (TSC) é aceito como padrão-ouro para o diagnóstico da fibrose cística (FC). Objetivo: Comparara estimulação e peso do suor produzido, os efeitos colaterais associados ao uso das correntes, contínua pulsada (CCP) e contínua constante (CCC) e determinar o tempo ideal para a estimulação e para a coleta de suor em indivíduos com e sem FC. Método: Estudo de intervenção prospectivo de corte transversal. Experimento 1(braço direito): CCC e CCP. Tempo de estimulação (TE) de 10min e o de coleta do suor de 30min. Correntes de 0,5; 0,75; 1,0; e 1,5mA e frequências de 0; 200; 1000; e 5000Hz. Experimento 2 (braço esquerdo): Corrente de 1,0mA; TE: 5 e 10min e coleta de 15 e 30min com frequências de 0; 200; 1000; e 5000Hz. Ambos os experimentos foram testados com densidade de corrente (DC) de 0,07 a 0,21mA/cm2. Experimento 3: Avaliar a CCP e a CCC como métodos diagnósticos para a FC comparando com diagnósticos estabelecidos por estudos na biópsia retal e sequenciamento do gene CFTR(do inglês, Cystic Fibrosis Transmembrane Condutance Regulator). Resultados: Participaram do estudo48 sujeitos (79,16% do sexo feminino), com média de 29,54±8,87 anos de idade. Não houve diferença estatística entre a interação da frequência e da corrente no peso do suor (p=0...

p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil

Vidigal,P.V.T.; Reis,F.J.C.; Boson,W.L.M.; De Marco,L.A.; Brasileiro-Filho,G.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2008 EN
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126.3%
Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7%) of 230 unrelated CF alleles. Fifteen (13.0%) patients were homozygous for this mutation, while 20 (17.4%) were heterozygous; the remaining 80 (69.6%) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2%) patients, 21 (18.3%) had the sequence variation 4521G/A, 11 (9.6%) had a not yet described sequence variation 4407T/A and 8 (7.0%) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid...

Adeno-associated virus for cystic fibrosis gene therapy

Martini,S.V.; Rocco,P.R.M.; Morales,M.M.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2011 EN
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126.33%
Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR) to the affected organ (lung). Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

Screening for F508del as a first step in the molecular diagnosis of cystic fibrosis

Marson,Fernando Augusto de Lima; Bertuzzo,Carmen Silvia; Ribeiro,Maria Angela Goncalves de Oliveira; Ribeiro,Antonio Fernando; Ribeiro,Jose Dirceu
Fonte: Sociedade Brasileira de Pneumologia e Tisiologia Publicador: Sociedade Brasileira de Pneumologia e Tisiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2013 EN
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OBJECTIVE: To determine the relevance of screening for the F508del mutation of the cystic fibrosis transmembrane conductance regulator gene as a first step in the genetic diagnosis of cystic fibrosis (CF) by associating the genotype with various clinical variables. METHODS: We evaluated 180 CF patients regarding the F508del mutation. The clinical data were obtained from the medical records of the patients and from interviews with their parents or legal guardians. RESULTS: Of the 180 patients studied, 65 (36.1%) did not carry the F508del mutation (group 0 [G0]), 67 (37.2%) were F508del heterozygous (G1), and 48 (26.7%) were F508del homozygous (G2). All three groups showed associations with the clinical variables. Homozygosis was associated with younger patients, younger age at CF diagnosis, and younger age at the first isolation of Pseudomonas aeruginosa (PA), as well as with higher prevalence of pancreatic insufficiency (PI) and non-mucoid PA (NMPA) colonization. In comparison with G1+G2 patients, G0 patients were older; first experienced clinical symptoms, digestive disease, and pulmonary disease at an older age; were older at CF diagnosis and at first PA isolation; and had a lower prevalence of PI and meconium ileus, as well as of colonization by NMPA...

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis

Coutinho,Cyntia Arivabeni de Araujo Correia; Marson,Fernando Augusto de Lima; Ribeiro,Antonio Fernando; Ribeiro,Jose Dirceu; Bertuzzo,Carmen Silvia
Fonte: Sociedade Brasileira de Pneumologia e Tisiologia Publicador: Sociedade Brasileira de Pneumologia e Tisiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2013 EN
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126.3%
OBJECTIVE: To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. METHODS: This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. RESULTS: All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease...

The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways.

Mall, M; Bleich, M; Greger, R; Schreiber, R; Kunzelmann, K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1998 EN
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106.35%
Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl- conductance, are characterized by an enhanced Na+ conductance. In this study we have examined the Na+ conductance in human respiratory tract by measuring transepithelial voltage and resistance (Vte, Rte) and by assessing membrane voltages (Vm) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 micromol/liter) equivalent short circuit current (Isc = Vte/Rte) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 micromol/liter) a significant depolarization of Vm corresponding to the cAMP-dependent activation of a Cl- conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N-methyl-D-glucamine on Vm were attenuated in the presence of forskolin. Also the amiloride-inhibitable Isc was significantly reduced by forskolin (1 micromol/liter) and isobutylmethylxanthine (IBMX; 100 micromol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na+ channels in human airways. This downregulation of epithelial Na+ channels is absent in CF airways...

Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia

Sun, Fei; Mi, Zhibao; Condliffe, Steven B.; Bertrand, Carol A.; Gong, Xiaoyan; Lu, Xiaoli; Zhang, Ruilin; Latoche, Joseph D.; Pilewski, Joseph M.; Robbins, Paul D.; Frizzell, Raymond A.
Fonte: The Federation of American Societies for Experimental Biology Publicador: The Federation of American Societies for Experimental Biology
Tipo: Artigo de Revista Científica
Publicado em /09/2008 EN
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106.39%
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, ΔF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin-proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of ΔF508 CFTR (ΔFRD) restores the biogenesis of mature ΔF508 CFTR protein. In addition, ΔFRD elicited a cAMP-stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous ΔF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (∼90%) airway epithelial cells. When fused to a PTD, direct addition of the ΔFRD peptide conferred a dose-dependent, cAMP-stimulated anion efflux to ΔF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and ΔF508 CFTR, whereas nearly twice as much of the Hsp90 cochaperone, Aha1, associated with ΔF508 CFTR. Expression of ΔFRD produced a dose-dependent removal of Aha1 from ΔF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone...

Detection of Cystic Fibrosis Transmembrane Conductance Regulator Activity in Early-Phase Clinical Trials

Rowe, Steven M.; Accurso, Frank; Clancy, John P.
Fonte: American Thoracic Society Publicador: American Thoracic Society
Tipo: Artigo de Revista Científica
Publicado em 01/08/2007 EN
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Advances in our understanding of cystic fibrosis pathogenesis have led to strategies directed toward treatment of underlying causes of the disease rather than treatments of disease-related symptoms. To expedite evaluation of these emerging therapies, early-phase clinical trials require extension of in vivo cystic fibrosis transmembrane conductance regulator (CFTR)–detecting assays to multicenter trial formats, including nasal potential difference and sweat chloride measurements. Both of these techniques can be used to fulfill diagnostic criteria for the disease, and can discriminate various levels of CFTR function. Full realization of these assays in multicenter clinical trials requires identification of sources of nonbiological intra- and intersite variability, and careful attention to study design and statistical analysis of study-generated data. In this review, we discuss several issues important to the performance of these assays, including efforts to identify and address aspects that can contribute to inconsistent and/or potentially erroneous results. Adjunctive means of detecting CFTR including mRNA expression, immunocytochemical localization, and other methods are also discussed. Recommendations are presented to advance our understanding of these biomarkers and to improve their capacity to predict cystic fibrosis outcomes.

Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel*

El Hiani, Yassine; Linsdell, Paul
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
106.34%
Opening of the cystic fibrosis transmembrane conductance regulator Cl− channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl− channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C...

Activation of the Cystic Fibrosis Transmembrane Conductance Regulator by the Flavonoid Quercetin: Potential Use as a Biomarker of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator Rescue

Pyle, Louise C.; Fulton, Jennifer C.; Sloane, Peter A.; Backer, Kyle; Mazur, Marina; Prasain, Jeevan; Barnes, Stephen; Clancy, J. P.; Rowe, Steven M.
Fonte: American Thoracic Society Publicador: American Thoracic Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
106.34%
Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β2 adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o− cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o− cells. High concentrations inhibited Cl− conductance. In CFBE41o− airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β2 adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr+ mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr−/− mice. Finally...

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis*

Thibodeau, Patrick H.; Richardson, John M.; Wang, Wei; Millen, Linda; Watson, Jarod; Mendoza, Juan L.; Du, Kai; Fischman, Sharon; Senderowitz, Hanoch; Lukacs, Gergely L.; Kirk, Kevin; Thomas, Philip J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
106.43%
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

Interleukin-13 (IL-13)/IL-13 Receptor α1 (IL-13Rα1) Signaling Regulates Intestinal Epithelial Cystic Fibrosis Transmembrane Conductance Regulator Channel-dependent Cl− Secretion*

Wu, David; Ahrens, Richard; Osterfeld, Heather; Noah, Taeko K.; Groschwitz, Katherine; Foster, Paul S.; Steinbrecher, Kris A.; Rothenberg, Marc E.; Shroyer, Noah F.; Matthaei, Klaus I.; Finkelman, Fred D.; Hogan, Simon P.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
106.37%
Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl− ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl− conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1−/−, iIL-13Tg/Il13rα2−/−, and iIL-13Tg/Stat6−/− mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl− conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl− secretion via up-regulation of cystic fibrosis transmembrane conductance regulator...

Lack of Correlation Between Pulmonary Disease and Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in Cystic Fibrosis: A Case Report

Levy, Hara; Cannon, Carolynn L; Asher, Daniel; García, Christopher; Knowles, Michael R; Colin, Andrew A; Cleveland, Robert Harris; Pier, Gerald B.
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
EN_US
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Introduction: Mutations in both alleles of the cystic fibrosis transmembrane conductance regulator gene result in the disease cystic fibrosis, which usually manifests as chronic sinopulmonary disease, pancreatic insufficiency, elevated sodium chloride loss in sweat, infertility among men due to agenesis of the vas deferens and other symptoms including liver disease. Case presentation: We describe a pair of African-American brothers, aged 21 and 27, with cystic fibrosis. They were homozygous for a rare frameshift mutation in the cystic fibrosis transmembrane conductance regulator 3791delC, which would be expected to cause significant morbidity. Although 80% of cystic fibrosis patients are colonized with Pseudomonas aeruginosa by eight years of age, the older brother had no serum opsonic antibody titer to P. aeruginosa by age 13 and therefore would have failed to mount an effective antibody response to the alginate (mucoid polysaccharide) capsule of P. aeruginosa. He was not colonized with P. aeruginosa until 24 years of age. Similarly, the younger brother was not colonized with P. aeruginosa until age 20 and had no significant lung disease. Conclusion: Despite a prevailing idea in cystic fibrosis research that the amount of functional cystic fibrosis transmembrane conductance regulator predicts clinical status...

Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Tuggle, Katherine L.; Birket, Susan E.; Cui, Xiaoxia; Hong, Jeong; Warren, Joe; Reid, Lara; Chambers, Andre; Ji, Diana; Gamber, Kevin; Chu, Kengyeh K.; Tearney, Guillermo; Tang, Li Ping; Fortenberry, James A.; Du, Ming; Cadillac, Joan M.; Bedwell, David M
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes...

Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer

Limberis, M.; Anson, D.; Fuller, M.; Parsons, D.
Fonte: Mary Ann Liebert Inc Publ Publicador: Mary Ann Liebert Inc Publ
Tipo: Artigo de Revista Científica
Publicado em //2002 EN
Relevância na Pesquisa
156.39%
The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.

Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase

Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, Peter
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 ENG
Relevância na Pesquisa
126.23%
Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.; Fundação para a Ciência e Tecnologia

Diagnóstico clínico e laboratorial da fibrose cística : métodos clássicos e novas perspectivas = Clinical and laboratorial diagnosis of cystic fibrosis: classical methods and new perspectives; Clinical and laboratorial diagnosis of cystic fibrosis : classical methods and new perspectives

Maria de Fátima Corrêa Pimenta Servidoni
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 21/07/2014 PT
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126.43%
A Fibrose Cística (FC) é uma doença genética autossômica recessiva, comum em caucasianos. Tem incidência de 1: 2.500 a 1: 6.000 nascidos vivos e 1: 25 em portadores sãos na Europa e EUA e no Brasil a incidência estimada é de 1:10.000 nascidos vivos. É causada pela presença de dois genes CFTR (do inglês Cystic Fibrosis Transmembrane Conductance Regulator) mutados, que codificam uma proteína também denominada CFTR. A CFTR é o principal canal de Cloro (Cl-), é expressa na membrana apical das células epiteliais dos tratos respiratório e digestório (pâncreas, fígado e intestino), nas glândulas sudoríparas e salivares, e no aparelho reprodutor masculino. Regula o transporte de iôns e de água.O comprometimento ou a ausência da função da CFTR promove a desidratação das mucosas com produção de um muco viscoso com consequente obstrução das vias respiratórias e ductos das glândulas exócrinas determinando o fenótipo da FC. O grau de função da CFTR será determinante da gravidade da doença. Até à data, já foram descritas cerca de 2000 mutações no gene CFTR. A F508del é a mutação mais prevalente, está presente em 85% dos pacientes a nível mundial e em 65% no Brasil. As mutações podem ser classificadas em 6 grupos de acordo com o defeito molecular e celular e determina o fenótipo da FC. Pode ser classificado em: clássico e não-clássico. O clássico é o mais conhecido e frequente e apresenta sintomas graves. O não-clássico ocorre em cerca de 15% dos doentes e apresenta sintomas mais brandos...

Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.

Mohamed, A; Ferguson, D; Seibert, F S; Cai, H M; Kartner, N; Grinstein, S; Riordan, J R; Lukacs, G L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/1997 EN
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The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide...