Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans

1995 ◽  
Vol 268 (2) ◽  
pp. C297-C307 ◽  
Author(s):  
S. N. Smith ◽  
D. M. Steel ◽  
P. G. Middleton ◽  
F. M. Munkonge ◽  
D. M. Geddes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Intestinal Tract ◽  
Chloride Secretion ◽  
Sodium Absorption ◽  
Wild Type ◽  
Nasal Potential Difference ◽  
New Treatments ◽  
Exon 10

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.

Effect of Oral Digoxin, Topical Ouabain and Salbutamol on Transepithelial Nasal Potential Difference in Patients with Cystic Fibrosis

1995 ◽  
Vol 89 (3) ◽  
pp. 277-284 ◽  
Author(s):  
D. G. Peckham ◽  
A. Conn ◽  
C. Chotai ◽  
S. Lewis ◽  
A. J. Knox
Keyword(s):  
Cystic Fibrosis ◽  
Potential Difference ◽  
Sodium Absorption ◽  
Double Blind ◽  
Double Blind Placebo ◽  
Control Subjects ◽  
Healthy Control ◽  
Nasal Potential Difference

1. Airway epithelium in cystic fibrosis is characterized by a defect in chloride secretion across the apical membrane and an increase in sodium absorption. The increased rate of sodium absorption can be inhibited in vitro by ouabain, a Na+-K+-ATPase inhibitor, and in cystic fibrosis patients the number and activity of nasal epithelial Na+-K+-ATPase pumps is increased. 2. We have performed a series of studies to determine whether drugs which modify airway epithelial Na+-K+-ATPase activity in vitro can modify nasal potential in cystic fibrosis patients in vivo. As transepithelial nasal potential difference measurements were used to study the effect of drug modulation of airway epithelial ion transport in vivo, the repeatability of the technique was first evaluated. In order to assess the effectiveness of the technique used for measuring nasal potential difference, a pilot study was carried out using topical amiloride, a drug which has previously been shown to inhibit airway epithelium sodium transport in vivo. We then studied the effects of ouabain and digoxin, two inhibitors of Na+-K+-ATPase, and salbutamol, a drug which activates Na+-K+-ATPase, on nasal potential difference. 3. In study 1, nasal potential difference measurements were repeated on non-consecutive days in 20 patients with cystic fibrosis and 20 healthy individuals. Healthy subjects had a mean (SEM) potential difference value of −19.5 (0.9) mV, with a 95% range for a single estimate of 75–133%. In patients with cystic fibrosis, the mean (SEM) potential difference was −40.4 (2.1) mV, with a 95% range for a single estimate of 74–136%. 4. In an initial pilot study, the effect of topical amiloride on nasal potential difference was investigated on two consecutive days in four cystic fibrosis patients and four healthy control subjects, in a double-blind, placebo-controlled, randomized cross-over study. Nasal transepithelial potential was measured before and at 5, 15, 30, 45 and 60 min after the intranasal administration of 0.4 ml of a fine spray of 1 mmol/l amiloride or 0.9% saline placebo to both nostrils. Amiloride was associated with a maximal reduction in nasal potential difference at 15 min of 49% and 41% in cystic fibrosis patients and control subjects, respectively. Compared with saline, the amiloride response was significant in both groups (P < 0.025). 5. In study 2, the effect of topical ouabain and salbutamol on nasal potential difference was investigated in ten cystic fibrosis patients and ten healthy control subjects, in a double-blind, placebo-controlled, randomized cross-over study. Nasal transepithelial potential was measured before and at 5, 15, 30, 45 and 60 min after the intranasal administration of either 0.4 ml of a fine spray of 5 mg/ml salbutamol, 0.25 mg/ml ouabain or 0.9% saline placebo to both nostrils. There was no significant change in nasal potential difference with either ouabain, salbutamol or placebo in either healthy control subjects or patients with cystic fibrosis. 6. In study 3, we performed a randomized, double-blind, placebo-controlled cross-over study of oral digoxin on nasal potential difference, spirometry and sweat electrolytes for 2 weeks in 11 patients with cystic fibrosis. During the treatment period, patients had a mean (range) serum digoxin level after the first and second week of therapy of 0.9 (0.3–1.4) μg/l and 1.1 (0.4–2.2) μg/l, respectively. There was no significant difference in the change in nasal potential difference measurements, forced expiratory volume in 1 s and sweat Na/Cl concentrations between the digoxin and placebo trial periods. 7. In conclusion, neither topical ouabain nor systemic digoxin had any effect on nasal potential difference in cystic fibrosis. Inhibitors of Na+-K+-ATPase are therefore unlikely to find a role in the treatment of cystic fibrosis. The lack of a detrimental effect of salbutamol on nasal potential difference is reassuring, as β-agonists are widely used in patients with cystic fibrosis.

scholarly journals Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

1999 ◽  
Vol 277 (4) ◽  
pp. C833-C839 ◽  
Author(s):  
Beate Illek ◽  
Lei Zhang ◽  
Nancy C. Lewis ◽  
Richard B. Moss ◽  
Jian-Yun Dong ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Adrenergic Stimulation ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Nasal Potential Difference ◽  
Cystic Fibrosis Transmembrane

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ΔF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P o = 0.047 ± 0.019. Addition of genistein activated Cl currents ∼10-fold, and the P o of G551D-CFTR increased to 0.49 ± 0.12, which is a P o similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired ΔF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, −2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to β-adrenergic stimulation) found in healthy subjects.

scholarly journals Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis–based cell and mice models

2020 ◽  
Vol 6 (47) ◽  
pp. eabc5911
Author(s):  
Anindit Mukherjee ◽  
Kelvin D. MacDonald ◽  
Jeonghwan Kim ◽  
Michael I. Henderson ◽  
Yulia Eygeris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Therapeutic Potential ◽  
Lipid Nanoparticles ◽  
Sodium Absorption ◽  
Airway Surface Liquid ◽  
Surface Liquid ◽  
Cf Transmembrane Conductance Regulator ◽  
Airway Cells

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

scholarly journals Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway

2009 ◽  
Vol 296 (1) ◽  
pp. C131-C141 ◽  
Author(s):  
Lihua Liang ◽  
Kelvin MacDonald ◽  
Erik M. Schwiebert ◽  
Pamela L. Zeitlin ◽  
William B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Protein Tyrosine Kinase ◽  
Secretory Pathway ◽  
Chloride Secretion ◽  
Airway Epithelial ◽  
Screening Assay ◽  
Intracellular Ca ◽  
Compound Screening

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl− channel. Its dysfunction limits Cl− secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl− channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca2+ activate the epithelial CaCCs, which provides an alternative Cl− secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl− secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl− secretion in CF via a pathway independent of CFTR.

scholarly journals Osteal Macrophages and Megakaryocytes Increase Adipogenesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nikhil Tewari ◽  
Deepa Kanagasabapathy ◽  
Rachel J. Blosser ◽  
Edward F. Srour ◽  
Angela Bruzzaniti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Sorting ◽  
Fetal Liver ◽  
Fold Increase ◽  
Wild Type ◽  
Bone Marrow Adipose Tissue ◽  
New Treatments

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified.   We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

scholarly journals Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo

2018 ◽  
Vol 76 (7) ◽  
Author(s):  
Evida A Dennis ◽  
Mamie T Coats ◽  
Sarah Griffin ◽  
Bing Pang ◽  
David E Briles ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Continuous Flow ◽  
Biofilm Formation ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Pneumococcal Colonization ◽  
Phase Variants

AbstractMucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

scholarly journals Lack of Sphingosine Causes Susceptibility to Pulmonary Staphylococcus Aureus Infections in Cystic Fibrosis

2016 ◽  
Vol 38 (6) ◽  
pp. 2094-2102 ◽  
Author(s):  
Shaghayegh Tavakoli Tabazavareh ◽  
Aaron Seitz ◽  
Peter Jernigan ◽  
Carolin Sehl ◽  
Simone Keitsch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pulmonary Infection ◽  
Bacterial Survival ◽  
Pulmonary Infections ◽  
Wild Type ◽  
High Susceptibility ◽  
Bronchial Epithelial

Background: Pulmonary Staphylococcus aureus (S. aureus) infections occur early in a high percentage of cystic fibrosis (CF) patients and it is believed that these infections facilitate further colonization of CF lungs with Pseudomonas aeruginosa (P. aeruginosa). Previous studies demonstrated a marked reduction of sphingosine in tracheal and bronchial epithelial cells in CF compared to wild type mice, while ceramide is massively increased in CF mice. Methods: We investigated the effect of C18-sphingosine and C16-ceramide on S. aureus in vitro. Based on our results we performed pulmonary infections with S. aureus and tested the influence of sphingosine inhalation. Results: In vitro incubation of S. aureus with C18-sphingosine rapidly killed S. aureus, while C16-ceramide did not affect bacterial survival, but abrogated the effect of C18-sphingosine when applied together. The in vivo infection experiments revealed a high susceptibility of CF mice to pulmonary infection with S. aureus. Inhalation of C18-sphingosine rescued CF mice from pulmonary infections with different clinical S. aureus isolates, including a methicillin-resistant S. aureus (MRSA) strain. Conclusions: Our data indicate that the imbalance between ceramide and sphingosine in the CF respiratory tract prevents killing of S. aureus and causes the high susceptibility of CF mice to pulmonary S. aureus infections.

Neurohumoral control of esophageal epithelial electrolyte transport

1980 ◽  
Vol 239 (1) ◽  
pp. G5-G11 ◽  
Author(s):  
D. D. Boyd ◽  
C. N. Carney ◽  
D. W. Powell
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Electrolyte Transport ◽  
Sodium Absorption ◽  
Metabolic Substrates ◽  
Submucosal Glands ◽  
Neurohumoral Control

The neurohumoral control of epithelial esophageal electrolyte transport was investigated by studying the effect of various hormones and neuroeffector agents on the potential difference (PD) in vivo or on the electrical parameters of electrolyte transport in vitro. The rabbit esophagus, which has no submucosal esophageal glands, demonstrated no effect of pentagastrin, cholecystokinin octapeptide, or synthetic secretin in vivo, and no effect of these hormones or of vasopressin, aldosterone, carbachol, epinephrine, or cAMP in vitro. The rabbit esophagus did respond to metabolic substrates (glucose) in vitro by increasing sodium absorption. In contrast, the opossum esophagus, which contains extensive submucosal glands, had a lower electrical resistance, PD, short-circuit current, and sodium absorption with higher chloride secretion. This esophagus responded to carbachol and epinephrine by sodium and chloride secretion. We believe that only the submucosal glands of the esophagus are under significant neurohumoral control while the sodium transporting function of the stratified squamous epithelium of this organ is important in maintaining its barrier function.

scholarly journals The novel mutation c.1210-3C>G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis

2020 ◽  
Author(s):  
Xinyue Zhao ◽  
Keqiang Liu ◽  
Wenshuai Xu ◽  
Meng Xiao ◽  
Qianli Zhang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Chinese Patient ◽  
Wild Type ◽  
Mutant Type ◽  
Pathogenic Mutations ◽  
In Cis ◽  
Exon 10

Abstract Purpose To identify potential pathogenic mutations in a Chinese patient with cystic fibrosis (CF) and subsequently study its splicing effect on cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in vitro. Methods Genomic DNA was extracted from peripheral blood leukocytes of the patient and his parents. To detect the possible pathogenic mutations in this patient, Sanger sequencing was conducted on all 27 coding exons of CFTR and their flanking intronic regions. Minigene constructs of the wild type and the identified mutant type were produced and transfected into HEK293T cells. Total RNA was extracted and reverse-transcribed into cDNA, with which as the template polymerase chain reaction (PCR) was performed to amplify the corresponding region. Original TA cloning and Sanger sequencing of the resultant PCR products were performed to analyze their splicing patterns. Results The patient is a compound heterozygote of c.2909G>A, p.Gly970Asp in exon 18 and c.1210-3C>G in cis with a poly-T of 5T (T5) sequence, 3 bp upstream in intron 9. As reported, c.2909G>A, p.Gly970Asp is considered to be the most frequent CFTR mutation among Chinese CF patients. c.1210-3C>G, a variant adjacent to the 3’ splice site, may affect splicing and reduce the levels of normal mRNA. We validated this hypothesis by a minigene assay in vitro, which showed that the wild-type plasmid containing c.1210-3C together with the T7 sequence produced a normal transcript as well as a partial exon 10-skipping transcript, whereas the mutant plasmid containing c.1210-3G in cis with the T5 sequence caused almost all mRNA to skip exon 10. Conclusion c.1210-3C>G, newly identified in our patient, in combination with the T5 sequence in cis affects CFTR gene splicing and produces nearly no normal transcripts in vitro, which makes it a pathogenic mutation in this patient. Moreover, this patient carries a p.Gly970Asp mutation, which reinforces the high frequency of this mutation in Chinese CF patients.

scholarly journals Current and Emerging Therapies to Combat Cystic Fibrosis Lung Infections

2021 ◽  
Vol 9 (9) ◽  
pp. 1874
Author(s):  
Jim Manos
Keyword(s):  
Cystic Fibrosis ◽  
Bacterial Infections ◽  
Current Method ◽  
Antimicrobial Treatment ◽  
Bacterial Survival ◽  
In Vivo Testing ◽  
Lung Transplants ◽  
New Treatments

The ultimate aim of any antimicrobial treatment is a better infection outcome for the patient. Here, we review the current state of treatment for bacterial infections in cystic fibrosis (CF) lung while also investigating potential new treatments being developed to see how they may change the dynamics of antimicrobial therapy. Treatment with antibiotics coupled with regular physical therapy has been shown to reduce exacerbations and may eradicate some strains. Therapies such as hypertonic saline and inhaled PulmozymeTM (DNase-I) improve mucus clearance, while modifier drugs, singly and more successfully in combination, re-open certain mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR) to enable ion passage. No current method, however, completely eradicates infection, mainly due to bacterial survival within biofilm aggregates. Lung transplants increase lifespan, but reinfection is a continuing problem. CFTR modifiers normalise ion transport for the affected mutations, but there is conflicting evidence on bacterial clearance. Emerging treatments combine antibiotics with novel compounds including quorum-sensing inhibitors, antioxidants, and enzymes, or with bacteriophages, aiming to disrupt the biofilm matrix and improve antibiotic access. Other treatments involve bacteriophages that target, infect and kill bacteria. These novel therapeutic approaches are showing good promise in vitro, and a few have made the leap to in vivo testing.

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