scholarly journals Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 603
Author(s):  
Zhongyu Liu ◽  
Justin D. Anderson ◽  
Lily Deng ◽  
Stephen Mackay ◽  
Johnathan Bailey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ex Vivo ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Genotypic Diversity ◽  
Mutant Gene ◽  
Airway Epithelial ◽  
Predictive Capacity ◽  
Apical Side

We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.

Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells

2000 ◽  
Vol 279 (6) ◽  
pp. C1925-C1937 ◽  
Author(s):  
Laurence Bulteau ◽  
Renaud Dérand ◽  
Yvette Mettey ◽  
Thierry Métayé ◽  
M. Rachel Morris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Cystic Fibrosis Gene ◽  
Intracellular Camp ◽  
Airway Epithelial ◽  
Whole Cell ◽  
Xanthine Derivative ◽  
Human Airway ◽  
High Level

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl− current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

Cystic fibrosis and beta-adrenergic response of airway epithelial cell cultures

1986 ◽  
Vol 251 (4) ◽  
pp. R818-R822 ◽  
Author(s):  
J. H. Widdicombe
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Ionophore A23187 ◽  
Airway Epithelial ◽  
Cell Sheets ◽  
Maximal Increase ◽  
Cl Secretion ◽  
Camp Levels

Confluent cell sheets were cultured from the tracheal epithelium of normal humans or from tracheal and nasal epithelia of patients with cystic fibrosis (CF). Changes in short-circuit current (Isc) or cyclic AMP (cAMP) levels in response to 10(-5) M isoproterenol were measured. In CF tracheal cells the response to isoproterenol was transient, and the maximal increase in Isc was one-tenth normal. In CF nasal cells, isoproterenol or epinephrine caused only small transient increases in Isc. However, in both CF nasal and tracheal cells, the Ca ionophore, A23187, caused relatively large increases in Isc that were inhibited by the Cl transport blocker, bumetanide, suggesting that Cl secretion can be induced by raising intracellular levels of Ca. In normal tracheal cell sheets, cAMP levels increased within 15 s of isoproterenol addition and continued to increase for up to 20 min. Resting levels of cAMP in CF tracheal cells were not statistically different from those of normal cells and showed linear increases for up to 4 min after addition of isoproterenol. Changes in cAMP in CF nasal cells were similar to the changes in CF tracheal cells. After 2 min, all three cell types showed cAMP levels elevated approximately equal to 10-fold. These results suggest that receptor-activated stimulation of adenylate cyclase is normal in CF. However, though raised cAMP levels stimulate Cl secretion in normal, they are unable to do so in CF airway epithelial cells.

scholarly journals Assessment of Distinct Electrophysiological Parameters in Rectal Biopsies for the Choice of the Best Diagnosis/Prognosis Biomarkers for Cystic Fibrosis

2020 ◽  
Vol 11 ◽  
Author(s):  
Iris A. L. Silva ◽  
Aires Duarte ◽  
Fernando A. L. Marson ◽  
Raquel Centeio ◽  
Tereza Doušová ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Features ◽  
Ex Vivo ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Predictive Biomarker ◽  
Sweat Chloride ◽  
Best Parameter ◽  
Rectal Biopsies

Most cases of Cystic Fibrosis (CF) are diagnosed early in life. However, people with atypical CF forms pose diagnosis dilemmas, requiring laboratory support for diagnosis confirmation/exclusion. Ex vivo analysis of fresh rectal biopsies by Ussing chamber has been the best discriminant biomarker for CF diagnosis/prognosis so far. Here we aimed to evaluate different electrophysiological parameters from Ussing chamber analysis of rectal biopsies from people with CF (PwCF) to establish the one with highest correlations with clinical features as the best CF diagnosis/prognosis biomarker. We analyzed measurements of CFTR-mediated Cl– secretion in rectal biopsies from 143 individuals (∼592 biopsies), the largest cohort so far analyzed by this approach. New parameters were analyzed and compared with the previous biomarker, i.e., the IBMX (I)/Forskolin (F)/Carbachol (C)-stimulated short-circuit current (I’sc–I/F/C). Correlations with clinical features showed that the best parameter corresponded to voltage measurements of the I/F + (I/F/CCH) response (VI/F+I/F/C), with higher correlations vs. I’sc–I/F/C for: sweat chloride (59 vs. 52%), fecal elastase (69 vs. 55%) and lung function, measured by FEV1 (27 vs. 20%). Altogether data show that VI/F+I/F/C is the most sensitive, reproducible, and robust predictive biomarker for CF diagnosis/prognosis effectively discriminating classical, atypical CF and non-CF groups.

Partial correction of defective Cl− secretion in cystic fibrosis epithelial cells by an analog of squalamine

2001 ◽  
Vol 281 (5) ◽  
pp. L1164-L1172 ◽  
Author(s):  
Canwen Jiang ◽  
Edward R. Lee ◽  
Mathieu B. Lane ◽  
Yong-Fu Xiao ◽  
David J. Harris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Synthetic Analog ◽  
Airway Epithelial ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Partial Correction

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl− transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl− transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl− gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ∼30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl− currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl− solution, indicating restoration of Cl− secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl− secretion.

Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice

1998 ◽  
Vol 274 (4) ◽  
pp. C875-C882 ◽  
Author(s):  
Rabindra Tirouvanziam ◽  
Mama Desternes ◽  
Anouar Saari ◽  
Edith Puchelle ◽  
Bruno Péault ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Severe Combined Immunodeficiency ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Scid Mice ◽  
Tracheal Mucosa ◽  
Induced Changes

We measured the bioelectric properties of 14 cystic fibrosis (CF) and 33 non-CF human fetal tracheal xenografts in severe combined immunodeficiency (SCID) mice. All xenografts exhibited a mature airway-type epithelium irrespective of their gestational age, duration of engraftment, and genotype. The in vivo potential difference and the in vitro baseline short-circuit current ( I sc) were significantly higher in non-CF than in CF xenografts. In non-CF xenografts, sequential addition of amiloride, forskolin, and ATP resulted in a 39.4% decrease, a 24.1% increase, and a 43.6% increase in I sc, respectively. In CF xenografts, forskolin had no significant effect on I sc, whereas amiloride- and ATP-induced changes in I sc were proportionally higher than in non-CF xenografts (−60.0 and +68.8%, respectively). These results indicate that the bioelectric properties of non-CF xenografts are similar to those of postnatal airways and that CF xenografts exhibit lower baseline electrogenic activity than non-CF xenografts but similar regulation of ion transport processes to postnatal CF airways. This model of mature human fetal tracheal mucosa may help gain insight into early CF airway pathogenesis.

scholarly journals Chlorzoxazone or 1-EBIO increases Na+absorption across cystic fibrosis airway epithelial cells

2001 ◽  
Vol 281 (5) ◽  
pp. L1123-L1129 ◽  
Author(s):  
Lin Gao ◽  
James R. Yankaskas ◽  
Catherine M. Fuller ◽  
Eric J. Sorscher ◽  
Sadis Matalon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Cystic Fibrosis Airway ◽  
Cf Transmembrane Conductance Regulator

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl− secretion by increasing basolateral K+ conductance ( G K) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778–787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across ΔF508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na+ absorption. The addition of chlorzoxazone (400 μM) or 1-EBIO (2 mM) increased the amiloride-sensitive I sc ∼2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G K with a K+ gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an ∼10-fold increase in clotrimazole-sensitive G K. In contrast, chlorzoxazone did not alter epithelial Na+ channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G K alone can modulate epithelial Na+ transport.

scholarly journals Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Thorax ◽  
2018 ◽  
Vol 73 (9) ◽  
pp. 847-856 ◽  
Author(s):  
Aristides D Tagalakis ◽  
Mustafa M Munye ◽  
Rositsa Ivanova ◽  
Hanpeng Chen ◽  
Claire M Smith ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Fluid Transport ◽  
Transfection Efficiency ◽  
Short Circuit ◽  
Beat Frequency ◽  
Short Circuit Current ◽  
Mouse Lung

IntroductionLoss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.MethodsWe investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.ResultsNanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.ConclusionNanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Hyperabsorption of Na+ and raised Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice

1994 ◽  
Vol 266 (5) ◽  
pp. C1478-C1483 ◽  
Author(s):  
B. R. Grubb ◽  
R. N. Vick ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Na Channel ◽  
Cl Secretion ◽  
Regulatory Link

We investigated the effect of homozygous genetic disruption of the murine cystic fibrosis transmembrane regulator (CFTR) gene on regulation of the rates of Na+ absorption and Cl- secretion by nasal epithelia in cystic fibrosis (CF) mice. The basal in vivo nasal potential difference (PD; -28.8 +/- 1.8 mV, n = 10) and amiloride-sensitive PD (delta 13.8 +/- 1.0 mV, n = 10) were raised in CF mice compared with controls [-7.8 +/- 0.8 mV, n = 14 (basal); delta 4.5 +/- 0.7 mV, n = 14 (amiloride)], consistent with raised Na+ transport. In vitro studies of freshly excised nasal epithelia confirmed that CF epithelia exhibited a greater basal equivalent short-circuit current (Ieq; 63.5 +/- 12 microA/cm2, n = 15) vs. control (30.2 +/- 7.2 microA/cm2, n = 16) and amiloride-sensitive Ieq (delta 46.2 +/- 12.5 microA/cm2) vs. control (delta 11.3 +/- 4.5 microA/cm2). Tissue from normal mice failed to secrete Cl- in response to ionomycin (delta Ieq: -1.2 +/- 1.9 microA/cm2, n = 18), whereas CF murine tissue responded with a large rise in Ieq (delta 55.1 +/- 19.1 microA/cm2, n = 13). We conclude that CF murine nasal epithelia exhibit Na+ hyperabsorption, providing strong evidence for a regulatory link between CFTR and Na+ channel activity in airway epithelia. We speculate that upregulation of the Ca(2+)-mediated Cl- secretory pathway buffers the severity of airway disease in the CF mouse.

Ion transport, ciliary activity, and mechanosensitivity of sinusal mucosa: an in vitro study

1996 ◽  
Vol 271 (3) ◽  
pp. L349-L358 ◽  
Author(s):  
D. Leuba ◽  
Y. De Ribaupierre ◽  
P. Kucera
Keyword(s):  
Fluid Transport ◽  
Short Circuit ◽  
Fluid Velocity ◽  
In Vitro Study ◽  
Short Circuit Current ◽  
Transepithelial Transport ◽  
Ciliary Activity ◽  
Apical Side ◽  
K Secretion

The hypothesis that relative movement between respiratory epithelium and surrounding fluid modulates both ciliary activity and mucosal secretion-absorption properties was tested. Fresh human (HM) and bovine sinusal mucosae (BM) were mounted in transparent chambers perfused with defined media at 36 degrees C. The fluid advanced along the ciliated surface at 15-30 microns/s. Transepithelial transport of electrolytes was studied by using voltage-clamp technique. Ciliary beating frequency (CBF) was recorded by using laser reflectometry. Average transmucosal potential difference (apical side negative) and short-circuit current (Isc) were -0.5 mV and 17 microA.cm-2 for HM and -1.4 mV and 28 microA.cm-2 for BM. Average CBF was 900 beats/min. Ionic pathways included basolateral Na,K-ATPase, K+ channels and symports for Na-Cl and Na-glucose, and apical channels for Na+ (absorption) and for Cl- and K+ (secretion). Increase of fluid velocity up to 300 microns/s induced significant increase of both Isc (63%) and CBF (29%). Such adaptations of transepithelial fluid transport and ciliary activity to hydrodynamic conditions might reflect a mechanism of coordination between the secretion of mucus electrolytes and mucociliary clearance.

scholarly journals Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

2014 ◽  
Vol 306 (3) ◽  
pp. L284-L291 ◽  
Author(s):  
A. R. Moran ◽  
Y. Norimatsu ◽  
D. C. Dawson ◽  
K. D. MacDonald
Keyword(s):  
Cystic Fibrosis ◽  
Cigarette Smoke ◽  
Xenopus Oocytes ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chronic Obstructive ◽  
Voltage Dependent ◽  
Dependent Inhibition

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

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