scholarly journals Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heidi J. Nick ◽  
Pamela L. Zeitlin ◽  
Sangya Yadav ◽  
Preston E. Bratcher
Keyword(s):  
Ion Transport ◽  
Ussing Chamber ◽  
Treatment Method ◽  
Accurate Estimate ◽  
Airway Epithelial ◽  
Epithelial Cultures ◽  
Transepithelial Voltage ◽  
Induced Changes

AbstractQuantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.

scholarly journals Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Patrick R. Secor ◽  
Lia A. Michaels ◽  
Kate S. Smigiel ◽  
Maryam G. Rohani ◽  
Laura K. Jennings ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Chronic Infection ◽  
Bacterial Invasion ◽  
Airway Epithelial ◽  
Content Type ◽  
Epithelial Cultures ◽  
Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

scholarly journals SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

2020 ◽  
Author(s):  
Bin Zhou ◽  
Tran Thi Nhu Thao ◽  
Donata Hoffmann ◽  
Adriano Taddeo ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Mouse Model ◽  
Competitive Advantage ◽  
Nasal Airway ◽  
Airway Epithelial ◽  
Founder Effects ◽  
Fitness Advantage ◽  
Epithelial Cultures ◽  
Bottle Neck

AbstractDuring the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

scholarly journals Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice

2020 ◽  
Author(s):  
Andrea J. Pruijssers ◽  
Amelia S. George ◽  
Alexandra Schäfer ◽  
Sarah R. Leist ◽  
Lisa E. Gralinksi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Human Lung ◽  
Viral Disease ◽  
Lung Cells ◽  
Airway Epithelial ◽  
Human Lung Cells ◽  
Epithelial Cultures ◽  
Viral Target

SUMMARYSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 μM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 μM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.

Decreased ion transport by tracheal epithelium of the basenji-greyhound dog

1994 ◽  
Vol 76 (4) ◽  
pp. 1489-1493 ◽  
Author(s):  
T. L. Croxton ◽  
M. Takahashi ◽  
C. A. Hirshman
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Airway Epithelial Cells ◽  
Tracheal Epithelium ◽  
Airway Epithelial ◽  
Beta Adrenergic ◽  
Electrical Potentials ◽  
Short Circuit Currents

The Basenji-Greyhound (BG) dog model shows altered beta-adrenergic function in both airway smooth muscle and leukocytes. To investigate a possible beta-adrenergic pathway defect in airway epithelial cells of BG dogs, we studied the electrophysiological behavior of tracheal epithelia in vitro and measured tracheal electrical potentials in vivo. Baseline short-circuit currents of isolated tracheal epithelia from BG (n = 6) and mongrel control dogs (n = 7) were 18.7 +/- 3.4 and 43.7 +/- 4.2 microA/cm2, respectively (P = 0.001). Significant differences between short-circuit currents of BG and control epithelia persisted after inhibition of Cl- secretion by indomethacin or stimulation by isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate. In vivo tracheal potentials were also significantly less (P = 0.01) in BG dogs (-22.3 +/- 2.5 mV; n = 12) than in control dogs (-32.5 +/- 2.6 mV; n = 10), and intravenous indomethacin reduced the tracheal potential of BG dogs but had no effect in control animals. There was no correlation in BG dogs between tracheal potential and the dose of methacholine required to double total lung resistance. These data suggest that ion transport by tracheal epithelium is decreased in BG dogs, that this difference is not due to diminished beta-adrenergic activity, and that cyclooxygenase products are important in maintaining tracheal potential in vivo in this model.

scholarly journals Drug Disposition in the Lower Gastrointestinal Tract: Targeting and Monitoring

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 161
Author(s):  
Glenn Lemmens ◽  
Arno Van Camp ◽  
Stephanie Kourula ◽  
Tim Vanuytsel ◽  
Patrick Augustijns
Keyword(s):  
Drug Absorption ◽  
Drug Disposition ◽  
Drug Stability ◽  
Ussing Chamber ◽  
Dissolution Kinetics ◽  
Comprehensive Overview ◽  
Drug Molecules ◽  
Epithelial Physiology

The increasing prevalence of colonic diseases calls for a better understanding of the various colonic drug absorption barriers of colon-targeted formulations, and for reliable in vitro tools that accurately predict local drug disposition. In vivo relevant incubation conditions have been shown to better capture the composition of the limited colonic fluid and have resulted in relevant degradation and dissolution kinetics of drugs and formulations. Furthermore, drug hurdles such as efflux transporters and metabolising enzymes, and the presence of mucus and microbiome are slowly integrated into drug stability- and permeation assays. Traditionally, the well characterized Caco-2 cell line and the Ussing chamber technique are used to assess the absorption characteristics of small drug molecules. Recently, various stem cell-derived intestinal systems have emerged, closely mimicking epithelial physiology. Models that can assess microbiome-mediated drug metabolism or enable coculturing of gut microbiome with epithelial cells are also increasingly explored. Here we provide a comprehensive overview of the colonic physiology in relation to drug absorption, and review colon-targeting formulation strategies and in vitro tools to characterize colonic drug disposition.

scholarly journals Insulin induces rapid and specific rearrangement of the cytoskeleton of rat mesangial cells in vitro.

1996 ◽  
Vol 44 (2) ◽  
pp. 91-101 ◽  
Author(s):  
A K Berfield ◽  
G J Raugi ◽  
C K Abrass
Keyword(s):  
Mesangial Cells ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Direct Effects ◽  
Ligand Effects ◽  
Igf I ◽  
Induced Changes ◽  
Secondary Ligand

Mesangial cells (MCs) grown without supplemental insulin (SI-MCs) express a quiescent phenotype and extracellular matrix (ECM) composition similar to MCs in vivo. In contrast, MCs routinely propagated in insulin (SI+MCs) are stimulated to proliferate, change their phenotype, and produce large amounts of collagens I and III. These effects of insulin may in part be mediated through cytoskeletal rearrangement. Differences in cytoskeletal arrangement were compared between SI-MCs and SI+MCs and 1 hr after addition of insulin (1 nM) or IGF-1 (100 nM) to SI-MCs. Cells were examined by light microscopy, electron microscopy, and immunostaining for specific cytoskeletal proteins and fibronectin. Insulin induced rapid rearrangement of stress fibers. Surface ruffling, actin aggregation, vimentin retraction, rearrangement of vinculin in focal adhesions, and fibronectin extraction were apparent. These direct effects of insulin on the SI-MC cytoskeleton occurred before insulin-induced changes in ECM composition. IGF-I induced cytoskeletal reorganization distinct from insulin. These observations demonstrate that insulin and IGF-I have unique effects on the MC cytoskeleton, which is turn may mediate secondary ligand effects on MCs.

scholarly journals A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 631
Author(s):  
Luis Soriano ◽  
Tehreem Khalid ◽  
Fergal J. O'Brien ◽  
Cian O'Leary ◽  
Sally-Ann Cryan
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Responses ◽  
Bronchial Epithelial Cells ◽  
Lung Fibroblasts ◽  
Drug Induced ◽  
Bronchial Epithelial ◽  
Culture Model ◽  
Epithelial Cultures

Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

The anterior and posterior ‘stomach’ regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine

1999 ◽  
Vol 202 (3) ◽  
pp. 247-252 ◽  
Author(s):  
T.M. Clark ◽  
A. Koch ◽  
D.F. Moffett
Keyword(s):  
Steady State ◽  
Aedes Aegypti ◽  
Ion Transport ◽  
Malpighian Tubules ◽  
Transport Mechanisms ◽  
Regional Specialization ◽  
Negative Deflection ◽  
Electrical Polarity

The ‘stomach’ region of the larval mosquito midgut is divided into histologically distinct anterior and posterior regions. Anterior stomach perfused symmetrically with saline in vitro had an initial transepithelial potential (TEP) of −66 mV (lumen negative) that decayed within 10–15 min to a steady-state TEP near −10 mV that was maintained for at least 1 h. Lumen-positive TEPs were never observed in the anterior stomach. The initial TEP of the perfused posterior stomach was opposite in polarity, but similar in magnitude, to that of the anterior stomach, measuring +75 mV (lumen positive). This initial TEP of the posterior stomach decayed rapidly at first, then more slowly, eventually reversing the electrical polarity of the epithelium as lumen-negative TEPs were recorded in all preparations within 70 min. Nanomolar concentrations of the biogenic amine 5-hydroxytryptamine (5-HT, serotonin) stimulated both regions, causing a negative deflection of the TEP of the anterior stomach and a positive deflection of the TEP of the posterior stomach. Phorbol 12,13-diacetate also caused a negative deflection of the TEP of the anterior stomach, but had no effect on the TEP of the posterior stomach. These data demonstrate that 5-HT stimulates region-specific ion-transport mechanisms in the stomach of Aedes aegypti and suggest that 5-HT coordinates the actions of the Malpighian tubules and midgut in the maintenance of an appropriate hemolymph composition in vivo.

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