RESUMO
Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 µM (LE 0.35).
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Fibrose Cística/complicações , Fibrose Cística/mortalidade , Fibrose Cística/patologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêuticoRESUMO
Essential fatty acid deficiency has been observed in most patients with Cystic Fibrosis (CF); however, pancreatic supplementation does not restore the deficiency, suggesting a different pathology independent of the pancreas. At this time, the underlying pathological mechanisms are largely unknown. Essential fatty acids are obtained from the diet and processed by organs including the liver and intestine, two organs significantly impacted by mutations in the cystic fibrosis transmembrane conductance regulator gene (Cftr). There are several CF animal models in a variety of species that have been developed to investigate molecular mechanisms associated with the CF phenotype. Specifically, global and systemic mutations in Cftr which mimic genotypic changes identified in CF patients have been generated in mice, rats, sheep, pigs and ferrets. These mutations produce CFTR proteins with a gating defect, trafficking defect, or an absent or inactive CFTR channel. Essential fatty acids are critical to CFTR function, with a bidirectional relationship between CFTR and essential fatty acids proposed. Currently, there are limited analyses on the essential fatty acid status in most of these animal models. Of interest, in the mouse model, essential fatty acid status is dependent on the genotype and resultant phenotype of the mouse. Future investigations should identify an optimal animal model that has most of the phenotypic changes associated with CF including the essential fatty acid deficiencies, which can be used in the development of therapeutics.
Assuntos
Animais Geneticamente Modificados , Fibrose Cística/patologia , Modelos Animais de Doenças , Ácidos Graxos Essenciais/deficiência , Fenótipo , Animais , Fibrose Cística/etiologia , Fibrose Cística/metabolismo , Humanos , Transporte de ÍonsRESUMO
While approximately 2000 mutations have been discovered in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), only a small amount (about 10%) is associated with clinical cystic fibrosis (CF) disease. The discovery of the association between CFTR and the hyperactive epithelial sodium channel (ENaC) has raised the question of the influence of ENaC on the clinical CF phenotype. ENaC disturbance contributes to the pathological secretion, and overexpression of one ENaC subunit, the ß-unit, can give a CF-like phenotype in mice with normal acting CFTR. The development of ENaC channel modulators is now in progress. Both CFTR and ENaC are located in the cell membrane and are influenced by its lipid configuration. Recent studies have emphasized the importance of the interaction of lipids and these proteins in the membranes. Linoleic acid deficiency is the most prevailing lipid abnormality in CF, and linoleic acid is an important constituent of membranes. The influence on sodium excretion by linoleic acid supplementation indicates that lipid-protein interaction is of importance for the clinical pathophysiology in CF. Further studies of this association can imply a simple clinical adjuvant in CF therapy.
Assuntos
Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ácido Linoleico/deficiência , Animais , Membrana Celular/genética , Membrana Celular/patologia , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Humanos , Ácido Linoleico/metabolismo , CamundongosRESUMO
Numerous benefits are attributed to omega-3 fatty acids (OM3) especially in cardiovascular health. However, bioavailability and clinical efficacy depend on numerous factors, including OM3 form, food matrix effects (especially the lipid content of the diet), and metabolic capacity. Here, we show in humans that a "pre-digested" OM3-sn-1(3)-monoacylglycerol lipid structure (OM3-MAG) has a significantly greater absorption at high therapeutic doses (2.9 g/day) than the most commonly OM3-ethyl ester (3.1 g/day) form (used for the treatment of hypertriglyceridemia), and a comparable profile to other pre-digested OM3 free fatty acids (OM3-FFA) structure (3.2 g/day). Nutritional supplement doses of MAG resulted in similar increases in OM3 blood level, compared to OM3 triacylglycerols (OM3-TAG) supplements in obese subjects (1.2 g/day) under low fat diet, and in children with cystic fibrosis (1.0 g/day). These results suggest that both forms of pre-digested OM3-MAG and OM3-FFA are effectively absorbed and re-incorporated effectively into triacylglycerols inside the enterocytes, before being exported into the chylomicrons lipid transport system. The pre-digested OM3-MAG might provide a more effective therapy in severe cardiovascular conditions where high doses of OM3 are required and a low-fat diet is indicated, which limited digestive lipase activity.
Assuntos
Fibrose Cística/tratamento farmacológico , Suplementos Nutricionais , Ácidos Graxos Ômega-3 , Hipertrigliceridemia/tratamento farmacológico , Monoglicerídeos , Obesidade/tratamento farmacológico , Adulto , Disponibilidade Biológica , Quilomícrons/metabolismo , Fibrose Cística/sangue , Fibrose Cística/patologia , Enterócitos/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/farmacocinética , Feminino , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/patologia , Masculino , Pessoa de Meia-Idade , Monoglicerídeos/administração & dosagem , Monoglicerídeos/farmacocinética , Obesidade/sangue , Obesidade/patologia , Triglicerídeos/sangueRESUMO
Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared with immortalized cell lines and primary cell cultures, but implementation of iPSC-based high-throughput (HT) assays is still technically challenging. Here, we demonstrate the development and conduction of an organotypic HT Cl-/I- exchange assay using cystic fibrosis (CF) disease-specific iPSCs. The introduction of a halide-sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of â¼42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modeling can also be utilized for drug screening in a true HT format.
Assuntos
Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Células Epiteliais , Engenharia Genética , Células-Tronco Pluripotentes Induzidas , Quinolonas/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Deleção de SequênciaRESUMO
The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Microtúbulos/patologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Criança , Colesterol/metabolismo , Feminino , Furanos/farmacologia , Humanos , Ibuprofeno/farmacologia , Masculino , Camundongos Knockout , Células Sf9 , Spodoptera , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
The streptococci are increasingly recognized as a core component of the cystic fibrosis (CF) lung microbiome, yet the role that they play in CF lung disease is unclear. The presence of the Streptococcus milleri group (SMG; also known as the anginosus group streptococci [AGS]) correlates with exacerbation when these microbes are the predominant species in the lung. In contrast, microbiome studies have indicated that an increased relative abundance of streptococci in the lung, including members of the oral microflora, correlates with impacts on lung disease less severe than those caused by other CF-associated microflora, indicating a complex role for this genus in the context of CF. Recent findings suggest that streptococci in the CF lung microenvironment may influence the growth and/or virulence of other CF pathogens, as evidenced by increased virulence factor production by Pseudomonas aeruginosa when grown in coculture with oral streptococci. Conversely, the presence of P. aeruginosa can enhance the growth of streptococci, including members of the SMG, a phenomenon that could be exacerbated by the fact that streptococci are not susceptible to some of the frontline antibiotics used to treat P. aeruginosa infections. Collectively, these studies indicate the necessity for further investigation into the role of streptococci in the CF airway to determine how these microbes, alone or via interactions with other CF-associated pathogens, might influence CF lung disease, for better or for worse. We also propose that the interactions of streptococci with other CF pathogens is an ideal model to study clinically relevant microbial interactions.
Assuntos
Coinfecção/microbiologia , Fibrose Cística/microbiologia , Interações Microbianas/genética , Infecções Pneumocócicas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Streptococcus milleri (Grupo)/genética , Antibacterianos/uso terapêutico , Biofilmes/crescimento & desenvolvimento , Coinfecção/patologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Expressão Gênica , Humanos , Pulmão/microbiologia , Pulmão/patologia , Modelos Biológicos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/patologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Streptococcus milleri (Grupo)/efeitos dos fármacos , Streptococcus milleri (Grupo)/crescimento & desenvolvimento , Streptococcus milleri (Grupo)/patogenicidade , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat. METHODS: 10 adult male CF patients were recruited (11/2014â»1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84â»91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D9]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis. RESULTS: Supplementation increased plasma choline from 4.8 (4.1â»6.2) µmol/L to 10.5 (8.5â»15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D9-labeled PC was decreased (12.2 [10.5â»18.3] µmol/L vs. 17.7 [15.5â»22.4] µmol/L, p < 0.01), indicating D9-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9â»74.8)% to 78.3 (60.1â»83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37â»8.82)% to 0.84 (0.56â»1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. CONCLUSIONS: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials.
Assuntos
Deficiência de Colina/tratamento farmacológico , Colina/uso terapêutico , Fibrose Cística/tratamento farmacológico , Volume Expiratório Forçado/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Adolescente , Adulto , Colina/sangue , Colina/farmacologia , Deficiência de Colina/sangue , Deficiência de Colina/complicações , Fibrose Cística/sangue , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Suplementos Nutricionais , Insuficiência Pancreática Exócrina/sangue , Insuficiência Pancreática Exócrina/complicações , Insuficiência Pancreática Exócrina/tratamento farmacológico , Fígado Gorduroso/sangue , Fígado Gorduroso/etiologia , Fígado Gorduroso/prevenção & controle , Humanos , Fígado/metabolismo , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fosfatidilcolinas/sangue , Triglicerídeos/sangue , Adulto JovemRESUMO
Cystic fibrosis (CF), one of the most common genetic disorders, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In spite of significant improvement in patient life expectancy, the disease remains lethal and incurable. Clinically, CF lung disease claims the most morbidity and mortality, characterized by chronic bacterial infection, persistent neutrophilic inflammation, and purulent small airway obstruction. Although all these manifestations are highly associated with neutrophils, the actual role of this phagocyte in the disease pathogenesis has not been fully appreciated. One of the major obstacles impeding such progress is the lack of CF neutrophil cell lines. Taking advantage of the new CRISPR/Cas9 gene-editing technology, we have generated a homozygous ΔF508-CF promyelocytic cell line from HL-60 cells, from which unlimited CF neutrophil cells can be differentiated. The derived cells showed defective CFTR presentation, deficient phagosomal hypochlorous acid (HOCl) production, and compromised microbial killing. Such a phenotype recapitulates that of primary neutrophils from CF patients. Thus, the established human CF promyelocytic cell line should be a useful tool for future CF basic research and drug screening.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , DNA/genética , Células Precursoras de Granulócitos/patologia , Terapia de Alvo Molecular/métodos , Mutação , Apoptose , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Análise Mutacional de DNA , Avaliação Pré-Clínica de Medicamentos/métodos , Células Precursoras de Granulócitos/metabolismo , Humanos , FenótipoRESUMO
Genistein is a naturally occurring isoflavone found in soy. Genistein has been shown to increase the open probability of the most common cystic fibrosis (CF) disease-associated mutation, ∆F508-CFTR. Mice homozygous for the ∆F508 mutation are characterized with severe intestinal disease and require constant laxative treatment for survival. This pathology mimics the intestinal obstruction (meconium ileus) seen in some cystic fibrosis patients. This study tested whether dietary supplementation with genistein would reduce the dependence of the ∆F508 CF mouse model on laxatives for survival, thereby improving mortality rates. At weaning (21 days), homozygous ∆F508 mice were maintained on one of three diet regimens for a period of up to 65 days: normal diet, normal diet plus colyte, or genistein diet. Survival rates for males were as follows: standard diet (38%, n = 21), standard diet plus colyte (83%, n = 42) and genistein diet (60%, n = 15). Survival rates for females were as follows: standard diet (47%, n = 19), standard diet plus colyte (71%, n = 38), and genistein diet (87%, n = 15). Average weight of male mice fed genistein diet increased by ~2.5 g more (p = 0.006) compared to those with colyte treatment. Genistein diet did not change final body weight of females. Expression of intestinal SGLT-1 increased 2-fold (p = 0.0005) with genistein diet in females (no change in males, p = 0.722). Expression of GLUT2 and GLUT5 was comparable between all diet groups. Genistein diet reduced the number of goblet cells per micrometer of crypt depth in female (p = 0.0483), yet was without effect in males (p = 0.7267). The results from this study demonstrate that supplementation of diet with genistein for ~45 days increases the survival rate of female ∆F508-CF mice (precluding the requirement for laxatives), and genistein only improves weight gain in males.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Genisteína/administração & dosagem , Laxantes/administração & dosagem , Mutação , Animais , Fibrose Cística/mortalidade , Fibrose Cística/patologia , Suplementos Nutricionais , Eletrólitos/administração & dosagem , Feminino , Células Caliciformes/patologia , Homozigoto , Enteropatias/tratamento farmacológico , Enteropatias/patologia , Jejuno/patologia , Masculino , Camundongos , Polietilenoglicóis/administração & dosagem , Taxa de SobrevidaRESUMO
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
Acute pulmonary exacerbations (APE) involving Pseudomonas aeruginosa are associated with increased morbidity and mortality in cystic fibrosis (CF) patients. Drug resistance is a significant challenge to treatment. Ceftazidime-avibactam (CZA) demonstrates excellent in vitro activity against isolates recovered from CF patients, including drug-resistant strains. Altered pharmacokinetics (PK) of several beta-lactam antibiotics have been reported in CF patients. Therefore, this study sought to characterize the PK of CZA and perform target attainment analyses to determine the optimal treatment regimen. The PK of CZA in 12 adult CF patients administered 3 intravenous doses of 2.5 g every 8 h infused over 2 h were determined. Population modeling utilized the maximum likelihood expectation method. Monte Carlo simulations determined the probability of target attainment (PTA). An exposure target consisting of the cumulative percentage of a 24-h period that the free drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (fT>MIC) was evaluated for ceftazidime (CAZ), and an exposure target consisting of the cumulative percentage of a 24-h period that the free drug concentration exceeds a 1-mg/liter threshold concentration (fT>1 mg/liter) was evaluated for avibactam (AVI). Published CAZ and CZA MIC distributions were incorporated to evaluate cumulative response probabilities. CAZ and AVI were best described by one-compartment models. The values of total body clearance (CL; CAZ CL, 7.53 ± 1.28 liters/h; AVI CL, 12.30 ± 1.96 liters/h) and volume of distribution (V; CAZ V, 18.80 ± 6.54 liters; AVI V, 25.30 ± 4.43 liters) were broadly similar to published values for healthy adults. CZA achieved a PTA (fT>MIC, 50%) of >0.9 for MICs of ≤16 mg/liter. The overall likelihood of a treatment response was 0.82 for CZA, whereas it was 0.42 for CAZ. These data demonstrate improved pharmacodynamics of CZA in comparison with those of CAZ and provide guidance on the optimal dosing of CZA for future studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT02504827.).
Assuntos
Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacocinética , Compostos Azabicíclicos/uso terapêutico , Ceftazidima/farmacocinética , Ceftazidima/uso terapêutico , Fibrose Cística/patologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Inibidores de beta-Lactamases/uso terapêutico , Adulto , Idoso , Antibacterianos/farmacocinética , Combinação de Medicamentos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Método de Monte Carlo , Estudos Prospectivos , Pseudomonas aeruginosa/isolamento & purificação , Infecções Respiratórias/microbiologia , Adulto Jovem , Inibidores de beta-Lactamases/farmacocinéticaRESUMO
Chronic inflammation that progressively disrupts the lung tissue is a hallmark of cystic fibrosis (CF). In mice, vardenafil, an inhibitor of phosphodiesterase type 5 (PDE5), restores transepithelial ion transport and corrects mislocalization of the most common CF mutation, F508del-CFTR. It also reduces lung pro-inflammatory responses in mice and in patients with CF. To test the hypothesis that macrophages are target effector cells of the immunomo-dulatory effect of vardenafil, we isolated lung macrophages from mice homozygous for the F508del mutation or invalidated for the cftr gene and from their corresponding wild-type (WT) littermates. We then evaluated the effect of vardenafil on the classical M1 polarization, mirroring release of pro-inflammatory cytokines. We confirmed that macrophages from different body compartments express CF transmembrane conductance regulator (CFTR) and showed that vardenafil targets the cells through PDE5- and CFTR-dependent mechanisms. In the presence of the F508del mutation, vardenafil down-regulated overresponses of the M1 markers, tumour necrosis factor (TNF)-α and inducible nitric oxide synthase (NOS)-2. Our study identifies lung macrophages as target cells of the anti-inflammatory effect of vardenafil in CF and supports the view that the drug is potentially beneficial for treating CF as it combines rescue of CFTR protein and anti-inflammatory properties.
Assuntos
Fibrose Cística/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Dicloridrato de Vardenafila/farmacologia , Animais , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Macrófagos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos Endogâmicos CFTR , Terapia de Alvo Molecular/métodos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Magnetic nanoparticles (MNPs) have gained much attention due to their unique properties such as biocompatibility and biodegradability as well as magnetic and heat-medicated characteristics. Due to these inherent properties, MNPs have been widely used in various biomedical applications including targeted drug delivery and hyperthermia-based therapy. Hyperthermia is a promising approach for the thermal activation therapy of several diseases, including pulmonary diseases. Additionally, due to their large loading capacity and controlled release ability, several MNP-based drug delivery systems have been emerged for treatment of cystic fibrosis and lung cancer. This review provides an overview on the unique properties of MNPs and magnetic-mediated hyperthermia with emphasis on the recent biomedical applications of MNPs in treatment of both lung cancer and cystic fibrosis.
Assuntos
Fibrose Cística/terapia , Sistemas de Liberação de Medicamentos/métodos , Hipertermia Induzida/métodos , Neoplasias Pulmonares/terapia , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Animais , Fibrose Cística/patologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/uso terapêutico , Técnicas de Transferência de Genes , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologiaRESUMO
La fibrosis quística (FQ) es una enfermedad genética hereditaria, causada por mutaciones en el gen CFTR, por lo que se requiere una prueba molecular de ADN (ácido desoxirribonucleico) para la confirmación del diagnóstico clínico, que oriente el pronóstico del paciente y permita dirigir el tratamiento más acertado así como la correcta asesoría familiar. La intención del siguiente trabajo es proveer una herramienta sencilla y precisa que permita la comprensión de la importancia de un examen de diagnóstico molecular en pacientes confirmados clínicamente o sospechosos de poseer fibrosis quística y su interpretación por parte del personal médico o del paciente. También se describen los últimos avances en el diagnóstico molecular de la FQ en la población venezolana, se reportan las mutaciones más frecuentes, el panel de mutaciones sugerido para esta población y también se reseña el estatus del servicio para diagnóstico molecular de FQ más robusto establecido en el país(AU)
Cystic fibrosis (CF) is an inherited genetic disorder caused by mutations in the CFTR gene, which necessarily requires a molecular DNA test to confirm the clinical diagnosis, orient the patient's prognosis and to direct the most successful patient's treatment and proper family counseling. The objective of the following paper is to provide a simple and accurate tool that would allow the understanding of the importance of a molecular diagnostic test in patients with cystic fibrosis and their interpretation by the patient or medical personnel. Also we highlighted recent advances in the molecular diagnosis of CF in the Venezuelan population, reported the most frequent mutations, the mutations panel suggested and reported the status of the CF molecular diagnosis service more robust established the country(AU)
Assuntos
Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/patologia , Doenças Genéticas Inatas , DNA , Diagnóstico Clínico , Medicina InternaRESUMO
In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Éxons/genética , Edição de Genes/métodos , Loci Gênicos/genética , Sequência de Bases , Linhagem Celular , Fibrose Cística/genética , DNA Complementar/genética , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Células Epiteliais/metabolismo , Genótipo , Humanos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Dedos de ZincoRESUMO
High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Fibrose Cística/tratamento farmacológico , Células Epiteliais/metabolismo , Ibuprofeno/farmacologia , Microtúbulos/metabolismo , Acetilação , Adenilato Quinase , Linhagem Celular , Fibrose Cística/patologia , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cultura Primária de Células , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Complexo Shelterina , Proteínas de Ligação a Telômeros/metabolismoRESUMO
The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fibrose Cística/tratamento farmacológico , Lipoxinas/farmacologia , Infecções por Pseudomonas/microbiologia , Junções Íntimas/metabolismo , Linhagem Celular , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Humanos , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/fisiologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Mucosa Respiratória/microbiologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/microbiologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Cystic fibrosis (CF) patients are at high risk for vitamin K deficiency. The effects of vitamin K supplementation are very ambiguous. Therefore, we aimed to define the determinants of vitamin K deficiency in a large cohort of supplemented - 146 (86.9%) and non-supplemented - 22 (13.1%) CF patients. Vitamin K status was assessed using prothrombin inducted by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (u-OC). The pathological PIVKA-II concentration (≥ 2 ng/ml) and abnormal percentage of osteocalcin (≥ 20%) were found in 72 (42.8%) and 60 (35.7%) subjects, respectively. We found that liver involvement, diabetes, and glucocorticoid therapy were potential risk factors for vitamin K deficiency. Pathological concentrations of PIVKA-II occurred more frequently in patients with pancreatic insufficiency and those who have two severe mutations in both alleles of the CFTR gene. Pathological percentage of u-OC was found more frequently in adult CF patients and those not receiving vitamin K. However, it seems that there are no good predictive factors of vitamin K deficiency in CF patients in everyday clinical care. Early vitamin K supplementation in CF patients seems to be warranted. It is impossible to clearly determine the supplementation dose. Therefore, constant monitoring of vitamin K status seems to be justified.
Assuntos
Fibrose Cística/patologia , Vitamina K/análise , Adolescente , Adulto , Antibacterianos/uso terapêutico , Biomarcadores/análise , Criança , Pré-Escolar , Estudos de Coortes , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Suplementos Nutricionais , Feminino , Genótipo , Glucocorticoides/uso terapêutico , Humanos , Imunoensaio , Lactente , Coeficiente Internacional Normatizado , Masculino , Estado Nutricional , Osteocalcina/análise , Osteocalcina/química , Polimorfismo de Nucleotídeo Único , Precursores de Proteínas/análise , Protrombina/análise , Infecções por Pseudomonas/tratamento farmacológico , Análise de Regressão , Fatores de Risco , Deficiência de Vitamina K/etiologia , Adulto JovemRESUMO
BACKGROUND: Cystic fibrosis (CF) leads to advanced lung disease despite aggressive care. Persistent inflammation and oxidative stress contribute to exacerbations and disease progression. Flaxseed (FS), a dietary botanical supplement with high fiber, lignan phenolics, and omega-3 fatty acids has anti-inflammatory and antioxidant properties in murine models of acute and chronic lung injury. This pilot study was designed to determine whether CF patients could tolerate FS, evaluate circulating FS metabolites, and study biomarkers of lung damage, as a prelude to studying clinical outcomes. METHODS: 10 CF patients and 5 healthy volunteers consumed 40 g of FS daily for 4 weeks with safety and tolerability being assessed. Urine was evaluated for systemic oxidative stress and plasma for FS metabolites (enterolignans) and cytokine levels. Buccal swabs were analyzed for gene expression of Nrf2-regulated antioxidant enzymes including Heme Oxygenase-1 (HO-1) and NAD(P)H Quinone Oxidoreductase 1 (NQO1). RESULTS: All subjects completed the study without serious adverse events. Plasma levels of enterolignans were detectable in both healthy controls and CF volunteers. CF patients were stratified based on plasma enterolignan levels after 2 weeks of FS administration into high- (174 to 535 nM ED and 232 to 1841 nM EL) and low- (0 to 32 nM ED and 0 to 40 nM EL) plasma lignan cohorts. The low enterolignan level cohort experienced a statistically significant drop in urinary inflammatory IsoP and plasma TNFα levels, while demonstrating higher average NQO1 mRNA levels in buccal epithelium compared to high-lignan patients. CONCLUSIONS: This pilot study demonstrated that FS is tolerated by CF patients. FS metabolites could be detected in the plasma. Future studies will assess appropriate dosing and target populations for FS, while exploring clinical outcomes. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02014181 .