scholarly journals Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells

2020 ◽  
Author(s):  
Wren E Michaels ◽  
Robert J Bridges ◽  
Michelle L Hastings
Keyword(s):  
Cystic Fibrosis ◽  
Membrane Conductance ◽  
Anion Channel ◽  
Autosomal Recessive Disorder ◽  
Premature Termination Codon ◽  
Chloride Secretion ◽  
Aberrant Splicing ◽  
Bronchial Epithelial ◽  
Cryptic Exon ◽  
Cf Transmembrane Conductance Regulator

Abstract Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5′ splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

scholarly journals Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells

2020 ◽  
Author(s):  
Wren E. Michaels ◽  
Robert J. Bridges ◽  
Michelle L. Hastings
Keyword(s):  
Cystic Fibrosis ◽  
Membrane Conductance ◽  
Anion Channel ◽  
Autosomal Recessive Disorder ◽  
Premature Termination Codon ◽  
Chloride Secretion ◽  
Aberrant Splicing ◽  
Bronchial Epithelial ◽  
Cryptic Exon ◽  
Cf Transmembrane Conductance Regulator

ABSTRACTCystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in more than 15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10kbC>T), which creates a new 5’ splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

scholarly journals Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis

2020 ◽  
Vol 21 (19) ◽  
pp. 7415
Author(s):  
Hicham Benabdelkamel ◽  
Hanadi Alamri ◽  
Meshail Okla ◽  
Afshan Masood ◽  
Mai Abdel Jabar ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Multiple Reaction Monitoring ◽  
Autosomal Recessive Disorder ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Serum Proteomics ◽  
Proteomic Approach ◽  
Significant Difference ◽  
Canonical Pathways ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

2005 ◽  
Vol 288 (5) ◽  
pp. L894-L902 ◽  
Author(s):  
James L. Kreindler ◽  
Alan D. Jackson ◽  
Philip A. Kemp ◽  
Robert J. Bridges ◽  
Henry Danahay
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Ion Transport ◽  
Cigarette Smoke ◽  
Apical Membrane ◽  
Chloride Secretion ◽  
Cigarette Smoke Extract ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.

scholarly journals Interleukin-17A induces bicarbonate secretion in normal human bronchial epithelial cells

2009 ◽  
Vol 296 (2) ◽  
pp. L257-L266 ◽  
Author(s):  
James L. Kreindler ◽  
Carol A. Bertrand ◽  
Robert J. Lee ◽  
Thomas Karasic ◽  
Shean Aujla ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Antimicrobial Peptide ◽  
Basolateral Membrane ◽  
Chloride Secretion ◽  
Bicarbonate Secretion ◽  
Immune Functions ◽  
Bronchial Epithelial ◽  
Interleukin 17A ◽  
Well Differentiated

The innate immune functions of human airways include mucociliary clearance and antimicrobial peptide activity. Both functions may be affected by changes in epithelial ion transport. Interleukin-17A (IL-17A), which has a receptor at the basolateral membrane of airway epithelia, is a T cell cytokine that has been shown to increase mucus secretion and antimicrobial peptide production by human bronchial epithelial (HBE) cells. Furthermore, IL-17A levels are increased in sputum from patients during pulmonary exacerbations of cystic fibrosis. Therefore, we investigated the effects of IL-17A on basal, amiloride-sensitive, and forskolin-stimulated ion transport in mature, well-differentiated HBE cells. Exposure of HBE monolayers to IL-17A for 48 h induced a novel forskolin-stimulated bicarbonate secretion in addition to forskolin-stimulated chloride secretion and resulted in alkalinization of liquid on the mucosal surface of polarized cells. IL-17A-induced bicarbonate secretion was cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, mucosal chloride-dependent, partially Na+-dependent, and sensitive to serosal, but not mucosal, stilbene inhibition. These data suggest that IL-17A modulates epithelial bicarbonate secretion and implicate a mechanism by which airway surface liquid pH changes may be abnormal in cystic fibrosis.

scholarly journals Cystic Fibrosis: Systems Biology Analysis from Homozygous p.Phe508del Variant Patients’ Samples Reveals Perturbations in Tissue-Specific Pathways

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Joice de Faria Poloni ◽  
Thaiane Rispoli ◽  
Maria Lucia Rossetti ◽  
Cristiano Trindade ◽  
José Eduardo Vargas
Keyword(s):  
Cystic Fibrosis ◽  
Enrichment Analysis ◽  
Autosomal Recessive Disorder ◽  
Bronchial Epithelium ◽  
Interaction Networks ◽  
Homozygous State ◽  
Functional Interaction ◽  
Tissue Specific ◽  
Ppi Networks ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by diverse genetic variants for the CF transmembrane conductance regulator (CFTR) protein. Among these, p.Phe508del is the most prevalent variant. The effects of this variant on the physiology of each tissue remains unknown. This study is aimed at predicting cell signaling pathways present in different tissues of fibrocystic patients, homozygous for p.Phe508del. The study involved analysis of two microarray datasets, E-GEOD-15568 and E-MTAB-360 corresponding to the rectal and bronchial epithelium, respectively, obtained from the ArrayExpress repository. Particularly, differentially expressed genes (DEGs) were predicted, protein-protein interaction (PPI) networks were designed, and centrality and functional interaction networks were analyzed. The study reported that p.Phe508del-mutated CFTR-allele in homozygous state influenced the whole gene expression in each tissue differently. Interestingly, gene ontology (GO) term enrichment analysis revealed that only “neutrophil activation” was shared between both tissues; however, nonshared DEGs were grouped into the same GO term. For further verification, functional interaction networks were generated, wherein no shared nodes were reported between these tissues. These results suggested that the p.Phe508del-mutated CFTR-allele in homozygous state promoted tissue-specific pathways in fibrocystic patients. The generated data might further assist in prediction diagnosis to define biomarkers or devising therapeutic strategies.

scholarly journals Ionocytes and CFTR Chloride Channel Expression in Normal and Cystic Fibrosis Nasal and Bronchial Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2090
Author(s):  
Paolo Scudieri ◽  
Ilaria Musante ◽  
Arianna Venturini ◽  
Daniela Guidone ◽  
Michele Genovese ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Chloride Channel ◽  
Ex Vivo ◽  
Chloride Secretion ◽  
Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Functional Studies ◽  
The Difference

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

scholarly journals Postnatal airway growth in cystic fibrosis piglets

2017 ◽  
Vol 123 (3) ◽  
pp. 526-533 ◽  
Author(s):  
Ryan J. Adam ◽  
Mahmoud H. Abou Alaiwa ◽  
Drake C. Bouzek ◽  
Daniel P. Cook ◽  
Nicholas D. Gansemer ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Lung Volume ◽  
Growth And Development ◽  
Anion Channel ◽  
Postnatal Period ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Disease Pathogenesis ◽  
Cf Transmembrane Conductance Regulator

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis. NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis.

scholarly journals Clinical development of triple-combination CFTR modulators for cystic fibrosis patients with one or two F508del alleles

2019 ◽  
Vol 5 (2) ◽  
pp. 00082-2019 ◽  
Author(s):  
Jennifer L. Taylor-Cousar ◽  
Marcus A. Mall ◽  
Bonnie W. Ramsey ◽  
Edward F. McKone ◽  
Elizabeth Tullis ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Anion Channel ◽  
Triple Combination ◽  
Open Label ◽  
Cftr Protein ◽  
Trafficking Defects ◽  
Cf Transmembrane Conductance Regulator ◽  
And Function ◽  
Cftr Modulators

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR) that result in diminished quantity and/or function of the CFTR anion channel. F508del-CFTR, the most common CF-causing mutation (found in ∼90% of patients), causes severe processing and trafficking defects, resulting in decreased CFTR quantity and function. CFTR modulators are medications that increase the amount of mature CFTR protein (correctors) or enhance channel function (potentiators) at the cell surface.Combinations of CFTR correctors and potentiators (i.e. lumacaftor/ivacaftor, tezacaftor/ivacaftor) have demonstrated clinical benefit in subsets of patients. However, none are approved for patients with CF heterozygous for F508del-CFTR and a minimal function mutation, i.e. a mutation that produces either no protein or protein that is unresponsive to currently approved CFTR modulators. Next-generation CFTR correctors VX-659 and VX-445, each in triple combination with tezacaftor and ivacaftor, improve CFTR processing, trafficking and function in vitro and have demonstrated clinical improvements in phase 2 studies in patients with CF with one or two F508del-CFTR alleles.Here, we present the rationale and design of four randomised phase 3 studies, and their open-label extensions, evaluating VX-659 (ECLIPSE) or VX-445 (AURORA) plus tezacaftor and ivacaftor in patients with one or two F508del-CFTR alleles.

Cystic fibrosis-related diabetes: an update on pathophysiology, diagnosis, and treatment

2020 ◽  
Vol 33 (7) ◽  
pp. 835-843 ◽  
Author(s):  
Crésio Alves ◽  
Thais Della-Manna ◽  
Cristiano Tulio Maciel Albuquerque
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Glucose ◽  
Microvascular Complications ◽  
Pubertal Development ◽  
Anion Channel ◽  
Autosomal Recessive Disorder ◽  
Tolerance Test ◽  
Oral Glucose ◽  
Postprandial Plasma Glucose ◽  
Glucose Levels

AbstractCystic fibrosis (CF) is a highly prevalent autosomal recessive disorder that is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene (7q31.2), which encodes the CFTR chloride-anion channel that is expressed in several tissues. Life expectancy has increased significantly over the past few decades due to therapeutic advances and early diagnosis through neonatal screening. However, new complications have been identified, including CF-related diabetes (CFRD). The earliest detectable glycemic abnormality is postprandial hyperglycemia that progresses into fasting hyperglycemia. CFRD is associated with a decline in lung function, impairments in weight gain and growth, pubertal development, and increased morbidity and mortality. Annual screening with oral glucose tolerance test is recommended beginning at the age of 10, and screenings are recommended for any age group during the first 48 h of hospital admission. Fasting plasma glucose levels ≥126 mg/dL (7.0 mmol/L) or 2-h postprandial plasma glucose levels ≥200 mg/dL (11.1 mmol/L) that persist for more than 48 h are diagnostic criteria for CFRD. Under stable health condition, the diagnosis is made when laboratory abnormalities in accordance with the American Diabetes Association criteria are detected for the first time; however, levels of HbA1c <6.5% do not rule out the diagnosis. Treatment for CFRD includes insulin replacement and a hypercaloric and hyperproteic diet that does not restrict carbohydrates, fats or salt, and diabetes self-management education. The most important CFRD complications are nutritional and pulmonary disease deterioration, though the microvascular complications of diabetes have already been described.

scholarly journals Impact of the different mutations in the cystic fibrosis gene in children with chronic rhinosinusitis

2019 ◽  
Vol 11 (5) ◽  
pp. 211-214
Author(s):  
Mafalda da Silva Ferreira ◽  
João Elói Moura ◽  
Ana Margarida Amorim ◽  
Ana Bernardo Ferreira ◽  
Luís Filipe Silva
Keyword(s):  
Cystic Fibrosis ◽  
Chronic Rhinosinusitis ◽  
Upper Airway ◽  
Autosomal Recessive Disorder ◽  
The Body ◽  
University Hospital ◽  
Cystic Fibrosis Gene ◽  
Lower Airway ◽  
Persistent Symptoms ◽  
Cf Transmembrane Conductance Regulator

Background: Cystic fibrosis (CF) is a worldwide disease occurring mostly in caucasians. It is an autosomal recessive disorder that leads to a malfunction of CF transmembrane conductance regulator (CFTR). These mutations cause an ionic disorder on the body fluids and a modification on its consistency. Affects multiple parts of the body and rhinosinusitis is a common manifestation of the upper airway affection. Material and methods: This retrospective study performed a statistical analysis of the prevelence of chronic rhinosinusitis with polyposis, genotipe and mortality in 30 children under 18 years with cystic fibrosis followed in the CF unit of Coimbra University Hospital. Results: The mean age of this study was 12,9 years. Phenylalanine deletion at position 508 (F508delF508del) was the most prevalent genotype (66,7%). Females patients had an higher prevalence of morbidities, however male patients had an higher mortality rate 20% comparing to 6,7%. Nasal polyposis was present only in the living ones with F508delF508del genotype. ENT (ear, nouse and throut) symptons and an abnormal ENT examination were mostly observed in F508delF508del genotype. Conclusions: CF is a lifelong disease that requires long-term surveillance and compliance. The involvement of the lower airway is prevalent in young chlidren. The uper airway symptoms becomes more important with disease progression. Nasal poliposis is prevalent on the older ones with F508delF508del genotype. In this kind of patients with persistent symptoms, who have failed medical management, are often considered appropriate candidates for functional endoscopic surgery.

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