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1.
PLoS One ; 15(10): e0235803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031374

RESUMO

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.


Assuntos
Brônquios/imunologia , Fibrose Cística/imunologia , NF-kappa B/metabolismo , Prevotella/imunologia , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/imunologia , Receptores Toll-Like/metabolismo , Brônquios/metabolismo , Brônquios/microbiologia , Brônquios/patologia , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Interleucina-8/metabolismo , NF-kappa B/genética , Prevotella/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Transdução de Sinais , Receptores Toll-Like/imunologia
2.
APMIS ; 128(12): 647-653, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32794206

RESUMO

IL-2 is a pro-inflammatory and a Th1 inducing cytokine, which is important for activation of the cell-mediated immunity. IL-2 in serum and sputum has been observed to be reduced in cystic fibrosis (CF) patients. The present IL-2 treatment study of Pseudomonas aeruginosa (PA) lung infected mice was performed in order to evaluate the effect of IL-2 supplement. One hundred-and-twenty female BALB/c mice were divided into three groups: 1) IL-2 treatment/infection (TIG), 2) non-treatment/infection (NTIG), and 3) IL-2 treatment/non-infection (TNIG). The mice were challenged intra-tracheally with PA (PAO579) embedded in seaweed alginate to resemble the biofilm mode of growth. At day 0 and 1, the treatment groups received IL-2 s.c. Mice were killed on day 1 or 2, and cytokine production, lung pathology, and quantitative lung bacteriology were estimated. IL-2 treatment of infected mice reduced the number of mice with signs of macroscopic lung pathology at day 2 (p < 0.05). The reduced macroscopic pathology was paralleled by a reduced IL-1ß and TNF-α. In contrast, an increased PMN response at day 2 was observed in the IL-2 treated mice (p < 0.01). This was preceded by a significantly higher degree of microscopic inflammation at day 1 (p < 0.02). The IL-12 levels decreased in both groups of infected mice at day 2 (p < 0.01), however, significantly more in the IL-2 treated mice (p < 0.02). In accordance, but surprisingly, IFN-γ was significantly reduced in the IL-2 treated PA infected group at day 2 (p < 0.05). The present results indicate that early IL-2 treatment prolongs the PMN response but also reduces pro-inflammatory IL-1ß and TNF-α and macroscopic signs of pathology.


Assuntos
Interleucina-2/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética
4.
PLoS One ; 15(7): e0235638, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687499

RESUMO

IMPORTANCE: Sinonasal symptoms in patients suffering from cystic fibrosis can negatively influence the quality of life and sinuses can be a niche for pathogens causing infection and inflammation leading to a decrease of lung function. Ivacaftor, a potentiator of the Cystic Fibrosis Transmembrane Conductance Regulator protein, has shown improvement in pulmonary function in cystic fibrosis patients with different forms of class III gating mutations. However, the effects of ivacaftor on sinonasal pathology have hardly been studied. OBJECTIVE: To determine the impact of ivacaftor therapy on sinonasal pathology in patients with cystic fibrosis with an S1251N mutation. DESIGN: Prospective observational mono-center cohort study, between June 2015 and December 2016. SETTING: A tertiary referral center in Utrecht, The Netherlands. PARTICIPANTS: Eight patients with cystic fibrosis with an S1251N mutation, treated with the potentiator ivacaftor were investigated. EXPOSURES: Ivacaftor (Kalydeco, VX-770) therapy. Computed tomography imaging of paranasal sinuses. Nasal nitric oxide concentration measurements and nasal endoscopy. MAIN OUTCOMES AND MEASURES: Primary outcome is opacification of paranasal sinuses examined with computed tomography scan analysis and scaled by the modified Lund-Mackay score before and one year after treatment. Secondary outcomes are nasal nitric oxide concentration levels, sinonasal symptoms and nasal endoscopic findings before and approximately two months and in some cases one year after treatment. RESULTS: Computed tomography scan analysis showed a significant decrease in opacification of the majority of paranasal sinuses comparing the opacification score per paranasal sinus before and after one year of treatment with ivacaftor. Median nasal nitric oxide levels significantly improved from 220.00 (IQR:136.00-341.18) to 462.84 (IQR:233.17-636.25) (p = 0.017) parts per billion. Likewise, the majority of sinonasal symptoms and nasal endoscopic pathology decreased or resolved at two months after the use of ivacaftor. CONCLUSION AND RELEVANCE: Ivacaftor appears to improve sinonasal outcome parameters and thereby sinonasal health in patients with cystic fibrosis with an S1251N mutation.


Assuntos
Aminofenóis/uso terapêutico , Agonistas dos Canais de Cloreto/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Seios Paranasais/patologia , Quinolonas/uso terapêutico , Adolescente , Adulto , Estudos de Coortes , Fibrose Cística/genética , Fibrose Cística/patologia , Feminino , Genótipo , Humanos , Masculino , Óxido Nítrico/metabolismo , Seios Paranasais/diagnóstico por imagem , Seios Paranasais/metabolismo , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Centros de Atenção Terciária , Tomografia Computadorizada por Raios X , Adulto Jovem
5.
Cell ; 182(2): 429-446.e14, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32526206

RESUMO

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Genética Reversa/métodos , Idoso , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Fibrose Cística/patologia , DNA Recombinante , Feminino , Furina/metabolismo , Humanos , Imunização Passiva , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Sistema Respiratório/patologia , Serina Endopeptidases/metabolismo , Células Vero , Virulência , Replicação Viral
6.
Tunis Med ; 98(5): 420-422, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32548846

RESUMO

Cystic Fibrosis (CF) is a lethal autosomal recessive condition due to a defect at the level of the transmembrane conductance regulator gene which plays a role in cell homeostasis. Numerous mutations have been identified as the cause of this gene defect, with delF508 being one of the most common mutations in Tunisia. This is a case report describing, up to our knowledge, the second case of a patient with CF carrying a rare mutation: W19X. W19X is a nonsense mutation that has been previously identified in only one other Tunisian patient with CF. Since both incidence of this mutation have been described in Tunisia, it seems as if W19X is specific to Tunisian CF patient with significant morbidities. The information provided by this study contributes to defining the molecular spectrum of CF in Tunisia, in the aim of improving genetic testing and prenatal diagnosis.


Assuntos
Códon sem Sentido , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Fibrose Cística/diagnóstico , Fibrose Cística/patologia , Frequência do Gene , Testes Genéticos , Humanos , Triptofano/genética , Tunísia
7.
BMC Med Genet ; 21(1): 89, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32357917

RESUMO

BACKGROUND: The aim was to establish the true risk of having an affected child with Cystic Fibrosis (CF) in the Sicilian infertile population. METHODS: A longitudinal CFTR screening of 1279 Sicilian infertile patients for all CFTR mutations sequencing the entire gene by Next Generation Sequencing (NGS) was performed from patient's blood. RESULTS: One patient out of 16 was a carrier of a CFTR mutation. Twenty-four mutations were found. Theoretically one couple out of 256 was at risk of CF transmission. CONCLUSIONS: The risk of CF transmission is unexpectedly high in Sicily and with a high heterogeneity. Sequencing an entire and long gene such as CFTR makes accessible the true panel of mutations in a specific population and helps better to understand the true risk of having an affected child.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Alelos , Fibrose Cística/epidemiologia , Fibrose Cística/patologia , Feminino , Genótipo , Humanos , Masculino , Mutação/genética , Análise de Sequência de DNA , Sicília/epidemiologia
8.
Sci Rep ; 10(1): 8440, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439937

RESUMO

One of the most common mutations in Cystic Fibrosis (CF) patients is the deletion of the amino acid phenylalanine at position 508. This mutation causes both the protein trafficking defect and an early degradation. Over time, small molecules, called correctors, capable of increasing the amount of mutated channel in the plasma membrane and causing an increase in its transport activity have been developed. This study shows that incubating in vitro cells permanently transfected with the mutated channel with the correctors VX809, VX661 and Corr4a, and the combination of VX809 and Corr4a, a recovery of anion transport activity is observed. Interestingly, the permeability of bicarbonate increases in the cells containing corrected p.F508del CFTR channels is greater than the increase of the halide permeability. These different increases of the permeability of bicarbonate and halides are consistent with the concept that the structural conformation of the pore of the corrector-rescued p.F508del channels would be different than the normal wild type CFTR protein.


Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Bicarbonatos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Mutação , Animais , Membrana Celular , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Transporte Proteico , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(19): 10357-10367, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345720

RESUMO

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common symptoms include progressive lung disease and chronic digestive conditions. CF is the first human genetic disease to benefit from having five different species of animal models. Despite the phenotypic differences among the animal models and human CF, these models have provided invaluable insight into understanding disease mechanisms at the organ-system level. Here, we identify a member of the ABCC4 family, CG5789, that has the structural and functional properties expected for encoding the Drosophila equivalent of human CFTR, and thus refer to it as Drosophila CFTR (Dmel\CFTR). We show that knockdown of Dmel\CFTR in the adult intestine disrupts osmotic homeostasis and displays CF-like phenotypes that lead to intestinal stem cell hyperplasia. We also show that expression of wild-type human CFTR, but not mutant variants of CFTR that prevent plasma membrane expression, rescues the mutant phenotypes of Dmel\CFTR Furthermore, we performed RNA sequencing (RNA-Seq)-based transcriptomic analysis using Dmel\CFTR fly intestine and identified a mucin gene, Muc68D, which is required for proper intestinal barrier protection. Altogether, our findings suggest that Drosophila can be a powerful model organism for studying CF pathophysiology.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Intestinos/patologia , Mutação , Células-Tronco/patologia , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Humanos , Mucinas/genética , Mucinas/metabolismo , Fenótipo , Células-Tronco/metabolismo
10.
Nat Commun ; 11(1): 1979, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332735

RESUMO

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.


Assuntos
Adenina/química , Sistemas CRISPR-Cas , RNA Guia/química , RNA Mensageiro/química , Alelos , Animais , Linhagem Celular , Códon , Códon sem Sentido , Fibrose Cística/patologia , Edição de Genes , Células HEK293 , Humanos , Camundongos , Mutação , Nucleotídeos , Fenótipo , Plasmídeos , Transfecção , Uridina/análogos & derivados , Uridina/química
11.
Sci Rep ; 10(1): 5806, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242045

RESUMO

The clinical manifestation of cystic fibrosis (CF) is heterogeneous also in patients with the same cystic fibrosis transmembrane regulator (CFTR) genotype and in affected sibling pairs. Other genes, inherited independently of CFTR, may modulate the clinical manifestation and complications of patients with CF, including the severity of chronic sinonasal disease and the occurrence of chronic Pseudomonas aeruginosa colonization. The T2R38 gene encodes a taste receptor and recently its functionality was related to the occurrence of sinonasal diseases and upper respiratory infections. We assessed the T2R38 genotype in 210 patients with CF and in 95 controls, relating the genotype to the severity of sinonasal disease and to the occurrence of P. aeruginosa pulmonary colonization. The frequency of the PAV allele i.e., the allele associated with the high functionality of the T2R38 protein, was significantly lower in i) CF patients with nasal polyposis requiring surgery, especially in patients who developed the complication before 14 years of age; and ii) in CF patients with chronic pulmonary colonization by P. aeruginosa, especially in patients who were colonized before 14 years of age, than in control subjects. These data suggest a role for T2R38 as a novel modifier gene of sinonasal disease severity and of pulmonary P. aeruginosa colonization in patients with CF.


Assuntos
Fibrose Cística/genética , Genes Modificadores , Receptores Acoplados a Proteínas-G/genética , Adolescente , Adulto , Criança , Fibrose Cística/complicações , Fibrose Cística/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/etiologia , Pólipos Nasais/genética , Pneumonia/etiologia , Pneumonia/genética , Receptores Acoplados a Proteínas-G/metabolismo
12.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1145-L1157, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267731

RESUMO

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.


Assuntos
Fibrose Cística/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Espaço Intracelular/metabolismo , Resveratrol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Acetilação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carbazóis/farmacologia , Células Cultivadas , Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nariz/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Resorcinóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuínas/metabolismo , Estilbenos/farmacologia , Tubulina (Proteína)/metabolismo
13.
Rev Mal Respir ; 37(3): 218-221, 2020 Mar.
Artigo em Francês | MEDLINE | ID: mdl-32146055

RESUMO

Although cystic fibrosis is a monogenic disease, a considerable clinical phenotypic variability is observed in patients with the same CFTR mutations. Thanks to the development of new and powerful tools for carrying out genetic studies, several genes called "modifier genes" have been identified as being associated with the severity of the lung function disorder in cystic fibrosis patients. Among these genes, SLC6A14 may modulate the anti-infective response and epithelial integrity of the airways, thus providing a potential therapeutic target to improve the patient's lung function.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Fibrose Cística/genética , Genes Modificadores , Sistemas de Transporte de Aminoácidos/fisiologia , Animais , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Epistasia Genética/fisiologia , Genótipo , Humanos , Mutação
14.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L931-L942, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32130033

RESUMO

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.


Assuntos
Células Acinares/metabolismo , Cílios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Pulmão/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Animais , Carbacol/farmacologia , Cátions Monovalentes , Cloretos/metabolismo , Cílios/efeitos dos fármacos , Cílios/patologia , Colforsina/farmacologia , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Modelos Animais de Doenças , Técnicas Eletroquímicas , Eletrodos , Deleção de Genes , Expressão Gênica , Humanos , Transporte de Íons , Pulmão/efeitos dos fármacos , Pulmão/patologia , Potássio/metabolismo , Sódio/metabolismo , Suínos
15.
Cell Mol Life Sci ; 77(17): 3311-3323, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32166393

RESUMO

The solute carrier family 6 member 14 (SLC6A14) protein imports and concentrates all neutral amino acids as well as the two cationic acids lysine and arginine into the cytoplasm of different cell types. Primarily described as involved in several cancer and colonic diseases physiopathological mechanisms, the SLC6A14 gene has been more recently identified as a genetic modifier of cystic fibrosis (CF) disease severity. It was indeed shown to have a pleiotropic effect, modulating meconium ileus occurrence, lung disease severity, and precocity of P. aeruginosa airway infection. The biological mechanisms explaining the impact of SLC6A14 on intestinal and lung phenotypes of CF patients are starting to be elucidated. This review focuses on SLC6A14 in lung and gastrointestinal physiology and physiopathology, especially its involvement in the pathophysiology of CF disease.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Fibrose Cística/patologia , Trato Gastrointestinal/metabolismo , Pulmão/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Doenças do Colo/genética , Doenças do Colo/metabolismo , Doenças do Colo/patologia , Fibrose Cística/genética , Fibrose Cística/metabolismo , Variação Genética , Humanos , Desequilíbrio de Ligação , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Índice de Gravidade de Doença
16.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L873-L887, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32160007

RESUMO

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.


Assuntos
Ácido Acético/administração & dosagem , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/genética , Obstrução das Vias Respiratórias/induzido quimicamente , Fibrose Cística/induzido quimicamente , Diminazena/análogos & derivados , Canais Iônicos Sensíveis a Ácido/metabolismo , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/patologia , Animais , Animais Recém-Nascidos , Bicarbonatos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Diminazena/farmacologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Suínos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia
17.
Int J Mol Sci ; 21(4)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098269

RESUMO

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Ionóforos , Mucosa Respiratória/metabolismo , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Células Epiteliais/patologia , Humanos , Transporte de Íons/efeitos dos fármacos , Ionóforos/síntese química , Ionóforos/química , Ionóforos/farmacologia , Muco/metabolismo , Mucosa Respiratória/patologia
18.
Am J Pathol ; 190(5): 977-993, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084371

RESUMO

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística , Modelos Animais de Doenças , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Camundongos Knockout , Mutação , Fenótipo , Ratos , Ratos Sprague-Dawley
19.
Molecules ; 25(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046275

RESUMO

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of ß-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.


Assuntos
Fibrose Cística/terapia , DNA/genética , Edição de Genes/métodos , Terapia Genética/métodos , Ácidos Nucleicos Peptídicos/genética , Talassemia beta/terapia , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , DNA/metabolismo , Modelos Animais de Doenças , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/metabolismo , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/metabolismo , Reparo de DNA por Recombinação , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
20.
J Immunol ; 204(6): 1650-1660, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32060135

RESUMO

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.


Assuntos
Fibrose Cística/imunologia , Interleucina-33/metabolismo , Pulmão/patologia , Mucina-5AC/metabolismo , Células Th2/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Fibrose Cística/patologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/metabolismo , Canais Epiteliais de Sódio/genética , Humanos , Interleucina-33/genética , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Muco/imunologia , Muco/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Células Th2/metabolismo
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