RESUMO
BACKGROUND: Information on the level of knowledge about cystic fibrosis (CF) among affected people and their families is still scarce. OBJECTIVE: This study aimed to translate, cross-culturally adapt and analyze the psychometric properties of the Brazilian version of Cystic Fibrosis Knowledge Scale (CFKS). MATERIALS AND METHODS: The translation and cross-cultural adaptation involved the stages of translation, synthesis of translations, reverse translation, synthesis of reverse translations, review by a multi-professional committee of experts and pre-testing. The reliability, viability, construct, predictive, concurrent and discriminant validity were investigated. RESULTS: The sample consisted of 40 individuals with cystic CF, 47 individuals with asthma, 242 healthcare workers and 81 students from the health area. The Brazilian version of the CFKS presented high internal consistency (α = 0.91), moderate floor and ceiling effects, without differences in the test-retest scores. An analysis of factorial exploration identified three dimensions. Confirmatory factor analysis led to an acceptable data-model fit. There was good predictive validity, with a difference in the scores among all the evaluated groups (p <0.001), as well as good discriminant validity since individuals with asthma had greater knowledge of asthma compared to CF (r = 0.401, p = 0.005; r2 = 0.162). However, there was no difference between the diagnosis time and knowledge about CF (r = -0.25, p = 0.11; r2 = 0.06), either between treatment adherence and knowledge about CF (r = -0.04, p = 0.77; r2 = 0.002). CONCLUSION: The Brazilian version of the CFKS indicated that the scale is able to provide valid, reliable and reproducible measures for evaluating the knowledge about CF.
Assuntos
Fibrose Cística/patologia , Conhecimento , Psicometria/métodos , Adolescente , Adulto , Asma/patologia , Asma/psicologia , Brasil , Comparação Transcultural , Fibrose Cística/psicologia , Feminino , Pessoal de Saúde/psicologia , Humanos , Masculino , Estudantes/psicologia , Inquéritos e Questionários , Tradução , Adulto JovemRESUMO
The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl-) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3-1 CF cells, and S9 (IB3-1 expressing wt-CFTR), and C38 (IB3-1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3-1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3-1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/patologia , Mitocôndrias/patologia , Dinâmica Mitocondrial , Mutação , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Células Epiteliais/metabolismo , Humanos , Transporte de Íons , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Cystic fibrosis (CF), an autosomal recessive genetic disease, is recognized as one of the most prevalent diseases in Caucasian populations. Epidemiological data show that the incidence of CF varies between countries and ethnic groups in the same region. CF occurs due to pathogenic variants in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), located on chromosome 7q31.2. To date, more than 2,000 variants have been registered in the CFTR database. The study of these variants leads to the diagnosis and the possibility of a specific treatment for each patient through precision medicine. In this study, complete screening of CFTR was performed through next-generation sequencing (NGS) to gain insight into the variants circulating in the population of Rio de Janeiro and to provide patient access to treatment through genotype-specific therapies. Samples from 93 patients with an inconclusive molecular diagnosis were subjected to full-length screening of CFTR using an Illumina NGS HiSeq platform. Among these patients, 46 had two pathogenic variants, whereas 12 had only one CFTR variant. Twenty-four variants were not part of our routine screening. Of these 24 variants, V938Gfs∗37 had not been described in the CF databases previously. This research achieved a molecular diagnosis of the patients with CF and identification of possible molecular candidates for genotype-specific treatments.
Assuntos
Cromossomos Humanos Par 7/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Mutação , Adolescente , Adulto , Brasil , Criança , Pré-Escolar , Estudos de Coortes , Fibrose Cística/diagnóstico , Fibrose Cística/etnologia , Fibrose Cística/patologia , Feminino , Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Medicina de Precisão , População BrancaRESUMO
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3- transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1ß loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1ß autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (â¼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Lactato Desidrogenase 5/metabolismo , Ácido Láctico/metabolismo , Pulmão/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/patologia , Receptores ErbB/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Pulmão/patologiaRESUMO
BACKGROUND: Elevated extracellular DNA levels are found in the sputum of patients with cystic fibrosis (CF). However, studies investigating the association of extracellular DNA with CF severity are scarce. OBJECTIVE: To evaluate the association of extracellular DNA levels with pulmonary function, antibiotic use, and hospitalization in CF patients. METHODS: This cross-sectional study included CF patients aged ≥5 years who were clinically stable and produced spontaneously expectorated sputum. Extracellular DNA in sputum was quantified, and extracellular DNA networks were seen with immunofluorescence microscopy. Also, cell death profile was assessed. Data on pulmonary function, airway colonization, antibiotic use, and hospitalization in the previous year were collected. Patients were divided into two groups based on median DNA level. RESULTS: Thirty-three patients were included. Their mean age was 16.3 ± 6.2 years, mean forced expiratory volume in the first second (FEV1) was 67.0 ± 26.7 (% of the predicted), and mean DNA level was 241.9 ± 147.2 µg/mL. There were significant correlations of DNA level with FEV1 (r = -0.60; p < 0.001) and forced vital capacity (r = -0.59; p < 0.001). Moreover, patients with higher DNA level (>243.0 µg/mL) had lower FEV1 (52.1 ± 27.8% vs. 81.1 ± 16.2%; p = 0.001) and required more hospitalizations (68.8% vs. 35.3%; p = 0.05). Additional findings were the presence of extracellular DNA networks and low rates of necrosis and apoptosis. CONCLUSION: Elevated extracellular DNA levels in CF sputum are associated with reduced pulmonary function and increased hospitalizations.
Assuntos
Fibrose Cística/diagnóstico , Fibrose Cística/fisiopatologia , Armadilhas Extracelulares/metabolismo , Volume Expiratório Forçado , Hospitalização/estatística & dados numéricos , Pulmão/fisiopatologia , Escarro/metabolismo , Capacidade Vital , Adolescente , Adulto , Apoptose , Biomarcadores/metabolismo , Criança , Estudos Transversais , Fibrose Cística/patologia , Feminino , Humanos , Pulmão/patologia , Masculino , Necrose , Testes de Função Respiratória , Índice de Gravidade de Doença , Adulto JovemRESUMO
Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the ß-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying ß-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.
Assuntos
Antibacterianos/farmacologia , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Fibrose Cística/patologia , Humanos , Mutação/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/isolamento & purificação , Sistema Respiratório/microbiologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genéticaRESUMO
BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). There are over 2000 different pathogenic and non-pathogenic variants described in association with a broad clinical heterogeneity. The most common types of mutations in this gene are single nucleotide substitutions or small deletions and insertions. However, large rearrangements, such as large duplications or deletions, are also a possible cause of CF; these variations are rarely tested in routine screenings, and much of them remain unidentified in some populations, especially those with high ethnic heterogeneity. METHODS: The present study utilized the Multiplex Ligation-dependent Probe Amplification (MLPA) technique for the detection of duplications and deletions in 165 CF patients from the Rio de Janeiro State (Brazil), which after extensive mutational screening, still exhibited one or two unidentified CF alleles. RESULTS: Five patients with alterations in MLPA signals were detected. After validation, we identified three copy number variations, one large duplication (CFTRdup2-3) and two large deletions (CFTRdel25-26 and CFTRdel25-27-CTTNBP2). Two detected deletions were not validated. They were false positives caused by a small deletion of 18 base pairs (232del18) and a point mutation (S168L) in the probe binding site. CONCLUSION: Our results highlight the importance of screening for large rearrangements in CF cases with no or only one CFTR mutation defined.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/patologia , Pré-Escolar , Fibrose Cística/etnologia , Fibrose Cística/genética , DNA/química , DNA/genética , DNA/metabolismo , Variações do Número de Cópias de DNA , Feminino , Deleção de Genes , Duplicação Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Técnicas de Amplificação de Ácido Nucleico/métodos , Mutação PuntualRESUMO
Recombinant adeno-associated virus (rAAV) vector platforms have shown considerable therapeutic success in gene therapy for inherited disorders. In cystic fibrosis (CF), administration of first-generation rAAV2 was safe, but clinical benefits were not clearly demonstrated. Therefore, next-generation vectors that overcome rate-limiting steps in rAAV transduction are needed to obtain successful gene therapy for this devastating disease. In this study, we evaluated the effects of single-strand or self-complementary (sc) rAAV vectors containing single or multiple tyrosine-to-phenylalanine (Y-F) mutations in capsid surface-exposed residues on serotypes 2, 8 or 9. For this purpose, CF bronchial epithelial (CFBE) cells were transduced with rAAV vectors, and the transgene expression of enhanced green fluorescence protein (eGFP) was analyzed at different time points. The effects of vectors on the cell viability, host cell cycle and in association with co-adjuvant drugs that modulate intracellular vector trafficking were also investigated. Six rAAV vectors demonstrated greater percentage of eGFP+ cells compared to their counterparts at days 4, 7 and 10 post-transduction: rAAV2 Y(272,444,500,730)F, with 1.95-, 3.5- and 3.06-fold increases; rAAV2 Y(252,272,444,500,704,730)F, with 1.65-, 2.12-, and 2-fold increases; scrAAV2 WT, with 1.69-, 2.68-, and 2.32-fold increases; scrAAV8 Y773F, with 57-, 6.06-, and 7-fold increases; scrAAV9 WT, with 7.47-, 4.64-, and 3.66-fold increases; and scrAAV9 Y446F, with 8.39-, 4.62-, and 4.4-fold increases. At days 15, 20, and 30 post-transduction, these vectors still demonstrated higher transgene expression than transfected cells. Although the percentage of eGFP+ cells reduced during the time-course analysis, the delta mean fluorescence intensity increased. These vectors also led to increased percentage of cells in G1-phase without eliciting any cytotoxicity. Prior administration of bortezomib or genistein did not increase eGFP expression in cells transduced with either rAAV2 Y(272,444,500,730)F or rAAV2 Y(252,272,444,500,704,730)F. In conclusion, self-complementary and tyrosine capsid mutations on rAAV serotypes 2, 8, and 9 led to more efficient transduction than their counterparts in CFBE cells by overcoming the intracellular trafficking and second-strand DNA synthesis limitations.
Assuntos
Fibrose Cística/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Substituição de Aminoácidos/genética , Brônquios/metabolismo , Brônquios/patologia , Brônquios/virologia , Fibrose Cística/patologia , Fibrose Cística/terapia , Fibrose Cística/virologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Mutação , Fenilalanina/genética , Sorogrupo , Transdução Genética/métodos , Tirosina/genéticaRESUMO
Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na+/K+ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.
Assuntos
Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Espaço Extracelular/química , Pulmão/metabolismo , Pulmão/microbiologia , Animais , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Pulmão/patologiaRESUMO
BACKGROUND: The variation in cystic fibrosis (CF) lung disease not always is explained by the CFTR genotype, so it has become apparent that modifier genes must play a considerable role in the phenotypic heterogeneity of CF, so we investigated the association of allelic variants in modifier genes that modulate the severity of lung function in a group of Mexican patients diagnosed with CF. METHODS: We included 140 CF patients classified according to lung phenotype and analyzed 17 single nucleotide polymorphisms (SNPs) by TaqMan® allelic discrimination. RESULTS: We demonstrated that patients with GG or GC genotype of the allelic variant rs11003125 (MBL2-550) of the MBL2 gene exhibit most of the lung manifestations at an earlier age; and the rs1042713 allelic variant of ADRB2 gene, showed statistical difference only with the age of first spirometry. When we used the dominant model, the MBL2 allele rs11003125 (MBL2-550; p = 0.022, Odds Ratio (OR) 2.87, 95% CI 1.14-7.27) was significantly associated with CF patients as risk factor, and the ADRB2 allele rs1042713 (p.Arg16Gly; p = 0.005, Odds Ratio (OR) 0.37, 95% CI 0.19-0.75) was significantly associated with CF patients as protect factor. CONCLUSIONS: Our findings suggest that the MBL2 and ADRB2 genes exerts an important genetic influence on the lung disease in our patients. Taking into account our results, we insist on not leaving aside this type of studies, since having techniques such as GWAS or WES will be able to advance in achieving a better quality of life for CF patients with severe lung disease.