scholarly journals β2-Adrenergic receptor agonists activate CFTR in intestinal organoids and subjects with cystic fibrosis

2016 ◽  
Vol 48 (3) ◽  
pp. 768-779 ◽  
Author(s):  
Lodewijk A.W. Vijftigschild ◽  
Gitte Berkers ◽  
Johanna F. Dekkers ◽  
Domenique D. Zomer-van Ommen ◽  
Elizabeth Matthes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Adrenergic Receptor ◽  
Ex Vivo ◽  
Gene Mutations ◽  
Airway Epithelial Cells ◽  
Potential Difference ◽  
Response To Treatment ◽  
Receptor Agonists ◽  
Intestinal Organoids

We hypothesized that people with cystic fibrosis (CF) who expressCFTR(cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptor agonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with theCFTRgenotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. Thein vivoresponse to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effectivein vivo. Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administeredex vivoto organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR functionin vivoand to select route of administration.

Uncertainty in the diagnosis of cystic fibrosis: Possible role of in vivo nasal potential difference measurements

1998 ◽  
Vol 132 (4) ◽  
pp. 596-599 ◽  
Author(s):  
David C. Wilson ◽  
Lynda Ellis ◽  
Julian Zielenski ◽  
Mary Corey ◽  
Wan F. Ip ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Potential Difference ◽  
Nasal Potential Difference

Examining basal chloride transport using the nasal potential difference response in a murine model

2001 ◽  
Vol 281 (5) ◽  
pp. L1173-L1179 ◽  
Author(s):  
Kristine G. Brady ◽  
Thomas J. Kelley ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Inbred Mice ◽  
Inbred Strains ◽  
Potential Difference ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Inbred Strains Of Mice ◽  
Cystic Fibrosis Transmembrane

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate ( Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.

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 Peroxisome proliferator-activated receptor-γ in cystic fibrosis lung epithelium

2008 ◽  
Vol 295 (2) ◽  
pp. L303-L313 ◽  
Author(s):  
Aura Perez ◽  
Anna M. van Heeckeren ◽  
David Nichols ◽  
Sanhita Gupta ◽  
Jean F. Eastman ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Acute Infection ◽  
Airway Epithelial Cells ◽  
Peroxisome Proliferator ◽  
Airway Epithelial ◽  
Peroxisome Proliferator Activated Receptor ◽  
Pparγ Agonists

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-κB activation. Peroxisome proliferator-activated receptor-γ (PPARγ) inhibits NF-κB activity and is reported to be reduced in CF. If PPARγ participates in regulatory dysfunction in the CF lung, perhaps PPARγ ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARγ expression and binding to NF-κB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFα/IL-1β. An animal model of CF was used to evaluate the potential of PPARγ agonists as therapeutic agents in vivo. In vitro, PPARγ agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFα/IL-1β stimulation. Less NF-κB bound to PPARγ in CF than normal cells, in two different assays; PPARγ agonists abrogated this reduction. PPARγ bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARγ inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARγ agonists in reducing IL-8 secretion. In vivo, administration of PPARγ agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARγ inhibits the inflammatory response in CF, at least in part by interaction with NF-κB in airway epithelial cells. PPARγ agonists may be therapeutic in CF.

scholarly journals Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alane Blythe C. Dy ◽  
Paul R. Langlais ◽  
Natalie K. Barker ◽  
Kenneth J. Addison ◽  
Sasipa Tanyaratsrisakul ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Transmembrane Protein ◽  
Protein A ◽  
Asthma Severity ◽  
Surfactant Protein ◽  
Protective Role ◽  
Airway Epithelial Cells ◽  
Mucus Production ◽  
Differentiation Marker

AbstractSurfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.

scholarly journals Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

2021 ◽  
Author(s):  
Maja C Funk ◽  
Jan G Gleixner ◽  
Florian Heigwer ◽  
Erica Valentini ◽  
Zeynep Aydin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mhc Class Ii ◽  
Ex Vivo ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Extrinsic Factors ◽  
Intestinal Organoids ◽  
Persistent Inflammation ◽  
A Cell

During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.

scholarly journals Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing

2021 ◽  
Author(s):  
Mira Tafa ◽  
Sevim Naz Karışık ◽  
Ece Begüm Aksoy ◽  
Rüya Aslan
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Animal Model ◽  
Ex Vivo ◽  
Low Cost ◽  
Cationic Liposome ◽  
Chloride Ions ◽  
Common Mutation ◽  
Cftr Protein

Cystic Fibrosis is a rare genetic disease that affects the transmission of chloride ions due to mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Even though there are nearly 2000 mutations identified to be related to the condition, the most common mutation is F508del; deletion of a phenylalanine residue at 508. On the other hand, G542X which is a Class I mutation is also found very commonly and there are no modulator treatments available for it. Furthermore, it was investigated that R553X mutation can as well be corrected simultaneously with G542X mutation. Therefore, the main focus is on designing a gene therapy project that can correct all these three mutations at once by utilizing the prime editing technique via lipid-based delivery. In this way, the mutations can be edited through plasmids that were designed containing 2 pegRNAs and the Cas enzyme. To implement such an approach efficiently, both ex vivo, an animal model, and in vivo steps are to be designed. For the cell line, fibroblasts are selected due to their simplicity and low cost. The animal model of the experiment is determined to be a ferret concerning the high similarity to the human's CFTR protein and finally, the procedure will follow on a direct application in human Cystic Fibrosis patients. The plasmids are thought to be delivered through a cationic liposome that will reach the lungs with the aid of a nebulizer. At the last stage of the experimental procedure, Sanger Sequencing will be done to see if the desired edit within the CFTR has been performed successfully, and Next Generation Sequencing will be executed to see if there has been an off-target mutation in the remainder of the genome. Whereas for detecting the presence and expression of CFTR protein in humans, immunodetection with flow cytometry will be conducted.

Targeted Treatments: Pre-Clinical Evaluation Of Efficacy By Tracking Clonal Diversity In CLL Xenograft Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 875-875
Author(s):  
Nicholas J Davies ◽  
Ceri E Oldreive ◽  
Angelo Agathanggelou ◽  
Clive Gould ◽  
Guy Pratt ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Clonal Evolution ◽  
Xenograft Model ◽  
Response To Therapy ◽  
Response To Treatment ◽  
Proliferating Cells ◽  
Targeted Treatments ◽  
Xenograft Models

Abstract Progressive CLL with DNA damage response defects caused by ATM or TP53 inactivation is still a therapeutic problem. There is currently a growing appreciation of the subclonal complexity in this type of leukaemia, mostly supported by whole genome analyses of CLL samples such as SNP arrays and next generation sequencing, both highlighting the role of subclonal variations in progression and therapeutic response. Although revealing, these analyses do not inform about the clonal make-up of individual proliferating cells and such information might be necessary to determine effectiveness of novel targeted treatments. In order to determine and follow the clonal evolutionary millieu in individual CLL cases we developed multi-colour fluorescence in situ hybridization (MCF) to analyse CLL samples at the single cell level. We screened 134 samples for the presence of 11q and 17p deletions of which 25 were identified with one of these cytogenetic defects and these were screened alongside 28 samples with normal 17p and 11q loci for 13q and 6q deletion as well as trisomy 12. We found that all but four of the samples with 11q or 17p deletions had at least one other genomic abnormality, whilst only two of the 28 samples with normal 17p and 11q loci harboured two genomic abnormalities. We subsequently performed MCF on the 27 samples with multiple genetic abnormalities and generated evolutionary trees for each of the samples. Two types of clonal evolution were identified: linear and branched, with the latter being the more common. We were able to analyse eleven of these samples post-treatment and found that whilst the clonality of some samples was largely unaffected by treatment, others showed treatment-induced differences in the subclonal make-up. Furthermore, some samples also exhibited signs of evolution with the generation of novel subclones upon treatment. Pre-clinical testing of novel therapeutic agents in xenograft models requires that the subclonal architecture of engrafted samples is representative of the donor sample. To this end, modifications were made to two primary CLL xenograft models. Firstly, samples from three different CLLs with a complex karyotype and multiple subclones were engrafted with autologous ex-vivo stimulated T-cells into NOG mice. All CLL subclones displayed a capacity to engraft and proliferate in this xenograft model. Furthermore, the MCF protocol was implemented to assess subclonality in vivo. Mice were randomised to Rituximab or control saline treatment three times over a period of five days. Analysis of engrafted cells in the spleen a week later displayed a good response to Rituximab with a significantly lower number of hCD45+ CD19+ CD5+cells. However, upon isolation of CLL cells by FACS and assessment of clonal architecture using MCF it was shown that Rituximab treatment affected all subclones with a differential subclonal response. This resulted in a greater proportion of the CLL subclones with the greatest genetic complexity harbouring both 11q and 6q deletions still remaining. Finally, to recapitulate patient response to therapy, T-cell depleted, pre-treatment PBMC from a patient poorly responsive to bendamustine + Rituximab were engrafted in humanised mice. As in the patient, bi-weekly therapy for 3 weeks resulted in a limited, poor response to therapy with a corresponding small reduction of CFSE-labelled CLL PBMC and hCD45+ CD19+ cells. We conclude that the majority of CLLs display a branched pattern of evolution and that the subclone dynamic is an important determinant of CLL proliferation and response to treatment. We suggest that novel targeted therapies should be tested in the context of their ability to eradicate CLL subclones with the highest proliferative capacity, as these subclones are most likely to evolve. The development of an MCF protocol, in combination with the xenograft model, provides a powerful tool to help predict overall and subclonal responses to therapy in the patient. Disclosures: No relevant conflicts of interest to declare.

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