Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface

Wong, Sharon L.; Pandžić, Elvis; Kardia, Egi; Allan, Katelin M.; Whan, Renée; Waters, Shafagh A.

doi:10.3390/jpm12101668

Cited by 2 publications

(2 citation statements)

References 46 publications

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“…Mature ALI cultures were stained, as previously described [ 75 ]. Briefly, cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton-X in PBS on ice for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

Wong

Kardia

Vijayan

et al. 2023

IJMS

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Localized and chronic hypoxia of airway mucosa is a common feature of progressive respiratory diseases, including cystic fibrosis (CF). However, the impact of prolonged hypoxia on airway stem cell function and differentiated epithelium is not well elucidated. Acute hypoxia alters the transcription and translation of many genes, including the CF transmembrane conductance regulator (CFTR). CFTR-targeted therapies (modulators) have not been investigated in vitro under chronic hypoxic conditions found in CF airways in vivo. Nasal epithelial cells (hNECs) derived from eight CF and three non-CF participants were expanded and differentiated at the air–liquid interface (26–30 days) at ambient and 2% oxygen tension (hypoxia). Morphology, global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility and ion transport) of basal stem cells and differentiated cultures were assessed. hNECs expanded at chronic hypoxia, demonstrating epithelial cobblestone morphology and a similar proliferation rate to hNECs expanded at normoxia. Hypoxia-inducible proteins and pathways in stem cells and differentiated cultures were identified. Despite the stem cells’ plasticity and adaptation to chronic hypoxia, the differentiated epithelium was significantly thinner with reduced barrier integrity. Stem cell lineage commitment shifted to a more secretory epithelial phenotype. Motile cilia abundance, length, beat frequency and coordination were significantly negatively modulated. Chronic hypoxia reduces the activity of epithelial sodium and CFTR ion channels. CFTR modulator drug response was diminished. Our findings shed light on the molecular pathophysiology of hypoxia and its implications in CF. Targeting hypoxia can be a strategy to augment mucosal function and may provide a means to enhance the efficacy of CFTR modulators.

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Section: Methodsmentioning

confidence: 99%

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

Wong

Kardia

Vijayan

et al. 2023

IJMS

Self Cite

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“…Differentiated hNECs were processed as previously described ( Wong et al, 2022c ). Briefly, samples were fixed in 4% paraformaldehyde for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient

Allan

Astore

Kardia

et al. 2023

Front. Mol. Biosci.

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Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56 years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown.Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1 s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot.Results: The participant ceased ETI treatment after 3 months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25 μA/cm2) and not enhanced with ETI (5.73 ± 0.48 μA/cm2), aligning with the individual’s clinical evaluation as a non-responder to ETI.Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

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Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface

Cited by 2 publications

References 46 publications

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient

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