scholarly journals Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells

2019 ◽  
Author(s):  
Bianca Maria Rotoli ◽  
Rossana Visigalli ◽  
Amelia Barilli ◽  
Francesca Ferrari ◽  
Massimiliano G. Bianchi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Delivery ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Apical Side ◽  
Basolateral Side ◽  
Subsequent Degradation ◽  
Carnitine Transport ◽  
Air Liquid Interface

ABSTRACTIn human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study is to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI were used as comparison. In EpiAirway™, ATB0,+ was highly expressed and functional on the apical side while OCTN2 transporter was active on the basolateral side. Calu-3 cells showed a different pattern of expression and activity for ATB0,+: indeed, L-carnitine uptake on apical side was evident in Calu-3 at 8 days of culture but not in fully differentiated 21d ALI culture. As both ATB0,+ and OCTN2, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the studies of drug inhalation and pulmonary delivery.

scholarly journals A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

2021 ◽  
Author(s):  
Thuc Nguyen Dan Do ◽  
Arnab K. Chatterjee ◽  
Philippe A. Gallay ◽  
Michael D. Bobardt ◽  
Laura Vangeel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Antiviral Agents ◽  
Airway Epithelial Cells ◽  
Liquid Interface ◽  
Airway Epithelial ◽  
Apical Side ◽  
Basolateral Side ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Air Liquid Interface

There are, besides Remdesivir (RDV), no approved antivirals for the treatment and/or prophylaxis of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI) and infected at the apical side with either one of two different SARS-CoV-2 isolates. The virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35°C than at 37°C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. GS-441524 (the parent nucleoside of Remdesivir), EIDD-1931 (the active metabolite of Molnupiravir) and IFN (β1 and λ1) all resulted in a dose-dependent inhibition of viral RNA and infectious virus titers at the apical side. However, AT-511 (a guanosine nucleotide previously reported to inhibit SARS-CoV-2) failed to inhibit viral replication. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

scholarly journals Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell® culture inserts

2020 ◽  
Author(s):  
Abiram Chandiramohan ◽  
Mohammedhossein Dabaghi ◽  
Jennifer A. Aguiar ◽  
Nicholas Tiessen ◽  
Mary Stewart ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Exposure System ◽  
Bronchial Epithelial ◽  
Human Airway ◽  
3D Printed ◽  
Air Liquid Interface ◽  
Whole Smoke

AbstractAccessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy and recreational cannabis use. In the present study, we developed and validated a novel 3D printed In Vitro Exposure System (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells.Using commercially available design software and a 3D printer, we designed a four-chamber Transwell® insert holder for exposures to whole smoke. Software was used to model gas distribution, concentration gradients, velocity profile and shear stress within IVES. Following simulations, primary human bronchial epithelial cells cultured at air-liquid interface on Transwell® inserts were exposed to whole cannabis smoke. Following 24 hours, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine and gene expression.Whole smoke delivered through IVES possesses velocity profiles consistent with uniform gas distribution across the four chambers and complete mixing. Airflow velocity ranged between 1.0-1.5 μm s−1 and generated low shear stresses (<< 1 Pa). Human airway epithelial cells exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated IL-1 family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control.IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with human airway epithelial cells grown under air-liquid interface culture conditions.

scholarly journals Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell® culture inserts

2020 ◽  
pp. 00705-2020
Author(s):  
Abiram Chandiramohan ◽  
Mohammedhossein Dabaghi ◽  
Jennifer A. Aguiar ◽  
Nicholas Tiessen ◽  
Mary Stewart ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Smoke Exposure ◽  
Airway Epithelial ◽  
Exposure System ◽  
Human Airway ◽  
3D Printed ◽  
Air Liquid Interface ◽  
Human Airway Epithelial Cells

Accessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy and recreational cannabis use. In the present study, we developed and validated a novel 3D printed In Vitro Exposure System (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells.Using commercially available design software and a 3D printer, we designed a four-chamber Transwell® insert holder for exposures to whole smoke. COMSOL® Multiphysics software was used to model gas distribution, concentration gradients, velocity profile and shear stress within IVES. Following simulations, primary human bronchial epithelial cells cultured at air-liquid interface on Transwell® inserts were exposed to whole cannabis smoke using a modified version of the Foltin Puff procedure. Following 24 h, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine and gene expression.Whole smoke delivered through IVES possesses velocity profiles consistent with uniform gas distribution across the four chambers and complete mixing. Airflow velocity ranged between 1.0–1.5 µm s−1 and generated low shear stresses (≪ 1 Pa). Human airway epithelial cells exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated IL-1 family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control, validating IVES smoke exposure impacts in human airway epithelial cells at a molecular level.The growing legalisation of cannabis on a global scale must be paired with research related to potential health impacts of lung exposures. IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with human airway epithelial cells grown under air-liquid interface culture conditions.

scholarly journals 22: MITOTIC ASYNCHRONY INDUCES A PRO-INFLAMMATORY STATE IN AIRWAY EPITHELIUM

2016 ◽  
Vol 64 (3) ◽  
pp. 816.1-816
Author(s):  
S Alcala ◽  
A Benton ◽  
A Watson ◽  
E Reeves ◽  
J Damsker ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Immune Cell ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Immune Mediated ◽  
Inflammatory State ◽  
Novel Method ◽  
Air Liquid Interface ◽  
Tgf Β1

Purpose of StudyMitotic behaviors are likely important for maintaining and restoring homeostasis in lung diseases with epithelial injury. We recently proposed that regenerative asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to pro-inflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that regenerative asynchrony is associated with pro-inflammatory/fibrotic cytokine secretion, here we provide proof of cause-and-effect.Methods UsedIn vitro experiments were performed wherein airway epithelial cells were mitotically asynchronous due to disease state and then resynchronized via capture of the G1/S checkpoint via pulse exposure to dexamethasone, simvastatin, or aphidicolin. Experiments utilized a novel method we developed for inducing mitotic asynchrony in normal progenitors. Induced populations were used to elucidate if TGF- β1 plays a role in the resynchronization process.Summary of ResultsHuman asthmatic fully-differentiated air–liquid interface airway epithelial mitosis was asynchronous relative to normal epithelia. Mitotic capture increased the percentage of progenitors in G1. This resynchronization in the asthmatic epithelia reduced basolateral TGF-β1 secretion. We next examined whether inducing mitotic asynchrony in normal epithelial cells would result in TGF-β1 secretion. Mitotic asynchrony was induced and samples showed moderate asynchrony at 6 and 12 hours that resolved spontaneously by 48 hours. These cells show elevated TGF-β1 secretion at 12 hours compared to either cell population in isolation. Regulation of TGF-β1 is being investigated as a possible mechanism for synchronization through contact and non-contact dependent experiments. Additionally, blocking TGF-β1 delays resynchronization.ConclusionsCumulative analysis shows mitotic synchrony is the homeostatic state in airway epithelial progenitor populations and poorly-synchronized mitosis (as in asthma) induces TGF-β1 secretion and a pro-inflammatory/pro-fibrotic airway. This finding establishes rationale for targeting progenitor cell mitotic behavior rather than immune-mediated inflammation in fibrotic disease.

A Toolbox for Studying Respiratory Viral Infections Using Air-Liquid Interface Cultures of Human Airway Epithelial Cells

2021 ◽  
Author(s):  
Aubrey Nicole Michi ◽  
David Proud
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Viral Infections ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Respiratory Viral Infections ◽  
Air Liquid Interface ◽  
Human Airway Epithelial Cells

Submerged cultures of primary human airway epithelial cells, or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

scholarly journals Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza a Virus Infection in C57BL/6 Mice

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 509 ◽  
Author(s):  
Meenakshi Tiwary ◽  
Robert J. Rooney ◽  
Swantje Liedmann ◽  
Kim S. LeMessurier ◽  
Amali E. Samarasinghe
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Early Phase ◽  
Influenza A ◽  
Airway Epithelial Cells ◽  
Epithelial Barrier ◽  
Virus Infectivity ◽  
Airway Epithelial ◽  
In Vivo Models

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

The wound healing capacity of undifferentiated and differentiated airway epithelial cells in vitro

2018 ◽  
Vol 112 ◽  
pp. 163-168 ◽  
Author(s):  
Cynthia M. Schwartz ◽  
Braedyn A. Dorn ◽  
Selam Habtemariam ◽  
Cynthia L. Hill ◽  
Tendy Chiang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

scholarly journals In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary-Based Alpha-1-Antitrypsin Deficiency Treatment

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1281
Author(s):  
Shan Guan ◽  
Max Darmstädter ◽  
Chuanfei Xu ◽  
Joseph Rosenecker
Keyword(s):  
Epithelial Cells ◽  
Transfection Efficiency ◽  
Genetic Diseases ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Significant Toxicity ◽  
Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin

In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial number of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT, while bringing along the unique advantages of IVT-mRNA.

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