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.

Defective cholinergic Cl− secretion and detection of K+ secretion in rectal biopsies from cystic fibrosis patients

2000 ◽  
Vol 278 (4) ◽  
pp. G617-G624 ◽  
Author(s):  
M. Mall ◽  
A. Wissner ◽  
H. H. Seydewitz ◽  
J. Kuehr ◽  
M. Brandis ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Channel Blockers ◽  
K Secretion ◽  
Transepithelial Voltage ◽  
Human Airways ◽  
Rectal Biopsies

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl− secretion and an inverse response of the short-circuit current ( I sc) toward stimulation with carbachol (CCh). Alternative Cl− channels are found in airway epithelia and have been attributed to residual Cl− secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I sc upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca2+-dependent Cl− conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I sc were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I sc in CF rectal biopsies but caused a negative I sc in non-CF subjects. The CCh-induced negative I sc in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I sc was significantly enhanced in CF and was caused by activation of a luminal K+ conductance, as shown by the use of the K+ channel blockers Ba2+ and tetraethylammonium. Moreover, a cAMP-dependent luminal K+conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl− channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl−conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca2+- and cAMP-dependent K+ secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

2000 ◽  
Vol 279 (1) ◽  
pp. G132-G138 ◽  
Author(s):  
Lane L. Clarke ◽  
Matthew C. Harline ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
John T. Turner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Receptor Activation ◽  
Biliary Disease ◽  
Anion Conductance ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Chamber Studies

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO3 − secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y2 nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca2+-activated Cl− secretion. However, the value of this treatment in facilitating transepithelial HCO3 − secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca2+-dependent anion conductance during activation of luminal P2Y2receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ( I sc) that declined to a stable plateau phase lasting 30–60 min. The plateau I sc after UTP was Cl− independent, HCO3 − dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I sc and serosal-to-mucosal HCO3 − flux ( J s→m) during a 30-min period. Substitution of Cl− with gluconate in the luminal bath to inhibit Cl−/HCO3 −exchange did not prevent the increase in J s→mand I sc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J s→m and I sc. We conclude that P2Y2 receptor activation results in a sustained (30–60 min) increase in electrogenic HCO3 − secretion that is mediated via an intracellular Ca2+-dependent anion conductance in CF gallbladder.

scholarly journals Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

2011 ◽  
Vol 301 (4) ◽  
pp. L587-L597 ◽  
Author(s):  
Louise C. Pyle ◽  
Annette Ehrhardt ◽  
Lisa High Mitchell ◽  
LiJuan Fan ◽  
Aixia Ren ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
High Throughput Screening ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Regulatory Domain ◽  
Thyroid Cells ◽  
Binding Domains ◽  
Mechanistic Basis ◽  
Rat Thyroid

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

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.

Coconut milk regulates the allergic response in Balb/c mice sensitized with whey proteins

2018 ◽  
Vol 8 (1) ◽  
pp. 19-25
Author(s):  
Yamina Benaissa ◽  
Samia Addou ◽  
Wafaa Dib ◽  
Hadria Grar ◽  
Omar Kheroua ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Ex Vivo ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Short Circuit Current ◽  
Coconut Milk ◽  
Intestinal Damage

In this work the effect of coconut milk on mice sensitized to cow's milk proteins was evaluated. Balb/c mice treated orally for twenty eight days with coconut milk were sensitized intraperitoneally with β-lactoglobulin (β-Lg) or α-Lactoglobulin (α-Lac). We used Ussing chamber to analyze ex vivo electrical parameters characterizing the intestinal tissue of mice by measuring the variations of the short current circuit Isc (μA/cm2) as well as the epithelial conductance (G). Jejunal fragments of sensitized and treated mice were mounted in Ussing chamber and stimulated by the deposit of β-Lg or α-Lac. Symptom scores were determined after in vivo challenge to β-Lg or α-Lac. Intestinal damage was assessed by histological analysis. Coconut milk influ-enced the electrophysiological parameters by significantly decreasing the short-circuit current (Isc) (p < 0.001) and the epithelial conductance ((p< 0.01 and (p < 0.001, respectively). Moreover, in coconut milk-treated mice, no significant clinical symptoms were observed. Analysis of histological sections revealed that coconut milk reduced the microscopic lesions induced by β-Lg or α-Lac sensitization. We speculate that the administration of coconut milk could prevent the systemic and anaphylactic responses in sensitized mice.

Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. C332-C341 ◽  
Author(s):  
N. J. Willumsen ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Na Transport ◽  
Airway Epithelia ◽  
Human Nasal Epithelium ◽  
Epithelial Cultures

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

Tissue- and Salinity-specific Na–Cl Cotransporter (NCC) Orthologues Involved in the Sea lamprey (Petromyzon Marinus) Adaptive Osmoregulation

2021 ◽  
Author(s):  
A. Barany ◽  
C. A. Shaughnessy ◽  
R. M. Pelis ◽  
J. Fuentes ◽  
J. M. Mancera ◽  
...  
Keyword(s):  
Water Absorption ◽  
Ex Vivo ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Petromyzon Marinus ◽  
Short Circuit Current ◽  
Sea Lamprey ◽  
Intestinal Tissue ◽  
Distal Intestine ◽  
Phylogenetic Comparison

Abstract Two ncc orthologues (termed ncca and nccb) were found in the sea lamprey genome, whereas nkcc2 was not. In a phylogenetic comparison among other vertebrate amino acids, NCC and NKCC deduced sequences, the sea lamprey NCC’s occupied basal positions within the NCC clade. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Acclimation to seawater increased nccb mRNA in the intestine and kidney. Intestinal nccb mRNA also increased during late metamorphosis. The electrophysiological approach in the Ussing chamber of intestinal tissue ex vivo showed significant differences between freshwater and seawater-acclimated juveniles. Luminal application of indapamide (NCC inhibitor) resulted in 73 and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. The luminal application of bumetanide (NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited ex vivo intestinal water absorption. Our results indicate that NCCb is likely the key passive ion cotransporter protein for ion uptake by the lamprey intestine to facilitate water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA expression during metamorphosis are likely critical to the development of whole animal salinity tolerance.

scholarly journals Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

2006 ◽  
Vol 74 (5) ◽  
pp. 2937-2946 ◽  
Author(s):  
A. Ghosh ◽  
D. R. Saha ◽  
K. M. Hoque ◽  
M. Asakuna ◽  
S. Yamasaki ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Hela Cells ◽  
Histopathological Examination ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Toxin Gene ◽  
Dependent Manner ◽  
A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

scholarly journals Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 351 ◽  
Author(s):  
C. Tse ◽  
Julie In ◽  
Jianyi Yin ◽  
Mark Donowitz ◽  
Michele Doucet ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Adult Stem Cell ◽  
Short Circuit Current ◽  
Watery Diarrhea ◽  
E Coli ◽  
Electrogenic Ion Transport

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Sign in / Sign up

Export Citation Format

Share Document