scholarly journals The CULTEX RFS: A Comprehensive Technical Approach for theIn VitroExposure of Airway Epithelial Cells to the Particulate Matter at the Air-Liquid Interface

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Michaela Aufderheide ◽  
Beat Halter ◽  
Niklas Möhle ◽  
Dieter Hochrainer
Keyword(s):  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Liquid Interface ◽  
Alternative Methods ◽  
Airway Epithelial ◽  
Nanosized Particles ◽  
Dose Dependent Decrease ◽  
Air Liquid Interface

The EU Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) demands the implementation of alternative methods for analyzing the hazardous effects of chemicals including particulate formulations. In the field of inhalation toxicology, a variety ofin vitromodels have been developed for such studies. To simulate thein vivosituation, an adequate exposure device is necessary for the direct exposure of cultivated lung cells at the air-liquid interface (ALI). The CULTEX RFS fulfills these requirements and has been optimized for the exposure of cells to atomized suspensions, gases, and volatile compounds as well as micro- and nanosized particles. This study provides information on the construction and functional aspects of the exposure device. By using the Computational Fluid Dynamics (CFD) analysis, the technical design was optimized to realize a stable, reproducible, and homogeneous deposition of particles. The efficiency of the exposure procedure is demonstrated by exposing A549 cells dose dependently to lactose monohydrate, copper(II) sulfate, copper(II) oxide, and micro- and nanoparticles. All copper compounds induced cytotoxic effects, most pronounced for soluble copper(II) sulfate. Micro- and nanosized copper(II) oxide also showed a dose-dependent decrease in the cell viability, whereby the nanosized particles decreased the metabolic activity of the cells more severely.

scholarly journals Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Haiyan Wang ◽  
Zhenzhen Zhang ◽  
Xing Xie ◽  
Beibei Liu ◽  
Yanna Wei ◽  
...  
Keyword(s):  
Mycoplasma Hyopneumoniae ◽  
Liquid Interface ◽  
Respiratory Pathogen ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Migration Ability ◽  
Content Type ◽  
Air Liquid Interface

ABSTRACT Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo. We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniae in vitro. Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo

2006 ◽  
Vol 290 (6) ◽  
pp. L1202-L1209 ◽  
Author(s):  
Theodore A. Sarafian ◽  
Nancy Habib ◽  
Michael Oldham ◽  
Navindra Seeram ◽  
Ru-Po Lee ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Function ◽  
A549 Cells ◽  
Cross Flow ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Red Fluorescence ◽  
Tracheal Epithelial Cells

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8–10 times higher than those measured in blood (75 ± 38 ng/g wet wt tissue vs. 9.2 ± 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.

scholarly journals Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ivy Aneas ◽  
Donna C. Decker ◽  
Chanie L. Howard ◽  
Débora R. Sobreira ◽  
Noboru J. Sakabe ◽  
...  
Keyword(s):  
Association Studies ◽  
Regulatory Region ◽  
Airway Epithelial Cells ◽  
Genome Wide Association Studies ◽  
Airway Epithelial ◽  
Allele Specific ◽  
Enhancer Blocking ◽  
Underlying Mechanisms

AbstractGenome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

2021 ◽  
Author(s):  
Colleen M Bartman ◽  
Kimberly E Stelzig ◽  
David R Linden ◽  
Y. S. Prakash ◽  
Sergio E Chiarella
Keyword(s):  
Epithelial Cell ◽  
Liquid Interface ◽  
Airway Epithelial ◽  
Loss Of Function ◽  
Sirna Transfection ◽  
Simple Method ◽  
Primary Epithelial Cell ◽  
Transfection Protocol ◽  
Air Liquid Interface

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and utility of a simple method for siRNA transfection of HBEs in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology of HBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

scholarly journals Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

2020 ◽  
Vol 318 (6) ◽  
pp. L1158-L1164
Author(s):  
Emily Mavin ◽  
Bernard Verdon ◽  
Sean Carrie ◽  
Vinciane Saint-Criq ◽  
Jason Powell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Real Time ◽  
Aerobic Glycolysis ◽  
Airway Epithelial Cells ◽  
Respiratory Epithelium ◽  
Liquid Interface ◽  
Human Nasal Epithelial Cells ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

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