LSC Abstract – Oxidative stress in nasal epithelial cells from patients with primary ciliary dyskinesia

2016 ◽  
Author(s):  
Ana Reula ◽  
Shirley Camacho ◽  
Amparo Escribano ◽  
Sara Pastor ◽  
Silvia Castillo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Primary Ciliary Dyskinesia ◽  
Nasal Epithelial Cells ◽  
Ciliary Dyskinesia

Oxidative stress in ciliated nasal epithelial cells from patients with primary ciliary dyskinesia

2016 ◽  
Author(s):  
Ana Reula ◽  
Amparo Escribano ◽  
Silvia Castillo ◽  
Sara Pastor ◽  
Silvia Vicente ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Primary Ciliary Dyskinesia ◽  
Nasal Epithelial Cells ◽  
Ciliary Dyskinesia

scholarly journals A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia

2020 ◽  
Vol 9 (11) ◽  
pp. 3753
Author(s):  
Janice L. Coles ◽  
James Thompson ◽  
Katie L. Horton ◽  
Robert A. Hirst ◽  
Paul Griffin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Ciliary Dyskinesia ◽  
High Speed ◽  
Ex Vivo ◽  
Cell Damage ◽  
Airway Epithelial Cells ◽  
University Hospital ◽  
Mitigation Measures ◽  
Diagnostic Certainty ◽  
Ciliary Dyskinesia

Air–liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.

scholarly journals Higher throughput drug screening for rare respiratory diseases: Readthrough therapy in primary ciliary dyskinesia

2021 ◽  
pp. 2000455
Author(s):  
Dani Do Hyang Lee ◽  
Daniela Cardinale ◽  
Ersilia Nigro ◽  
Colin R. Butler ◽  
Andrew Rutman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
High Throughput Screening ◽  
Primary Ciliary Dyskinesia ◽  
Respiratory Diseases ◽  
Beat Frequency ◽  
Screening Method ◽  
Respiratory Epithelium ◽  
Basal Cells ◽  
Translational Readthrough ◽  
Ciliary Dyskinesia

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that it is possible to expand from such biopsies. Here, we describe an immunofluorescence screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary ultrastructure, beat pattern and beat frequency indicate that a range of different PCD defects can be retained in these cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia, RGMC), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. The screening system allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. Restoration of basal body formation in the patient's nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD.

scholarly journals Effect of High, Medium, and Low Molecular Weight Hyaluronan on Inflammation and Oxidative Stress in anIn VitroModel of Human Nasal Epithelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Giusy Daniela Albano ◽  
Anna Bonanno ◽  
Luca Cavalieri ◽  
Eleonora Ingrassia ◽  
Caterina Di Sano ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Weight ◽  
Epithelial Cells ◽  
Signal Pathway ◽  
Mrna Levels ◽  
Ros Production ◽  
Nasal Epithelial Cells ◽  
Pathway Activation ◽  
Nasal Inflammation ◽  
Anti Inflammatory

IL-17A is involved in the activation of oxidative stress and inflammation in nasal epithelial cells. Hyaluronan (HA) in its high molecular weight form (HMW-HA) shows anti-inflammatory responses in contrast to low and medium molecular weight HA (LMW-HA and MMW-HA). The aim of this study was to investigate the pro- or anti-inflammatory biologic function of HA at different molecular weight in anin vitromodel of nasal inflammation IL-17A mediated. We evaluated the ERK1/2 and IκBαphosphorylation, NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 protein, and mRNA levels, in nasal epithelial cells RPMI 2650 stimulated with recombinant human (rh) IL-17A. Furthermore, the cells were treated with HMW-HA, MMW-HA, LMW-HA, and U0126. Our results showed that rhIL-17A increased the ERK1/2, IκBαphosphorylation and NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 proteins, and mRNA levels. The addiction of HMW-HA or U0126 showed a significant downregulatory effect on inflammation due to the rhIL-17A stimulation in nasal epithelial cells. IL-17A is able to generate oxidative stress and inflammation via the activation of ERK1/2/NF-κB pathway in nasal epithelial cells. The HMW-HA might represent a coadjuvant of the classic anti-inflammatory/antioxidative treatment of nasal epithelial cells during IL-17A nasal inflammation.

scholarly journals Primary Ciliary Dyskinesia in Mice Lacking the Novel Ciliary Protein Pcdp1

2007 ◽  
Vol 28 (3) ◽  
pp. 949-957 ◽  
Author(s):  
Lance Lee ◽  
Dean R. Campagna ◽  
Jack L. Pinkus ◽  
Howard Mulhern ◽  
Todd A. Wyatt ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Male Infertility ◽  
Primary Ciliary Dyskinesia ◽  
Single Gene ◽  
Ependymal Cells ◽  
The Novel ◽  
Gene Encoding ◽  
Respiratory Epithelial ◽  
Ciliated Epithelial Cells ◽  
Ciliary Dyskinesia

ABSTRACT Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.

scholarly journals Mutation of CFAP57 causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms

2019 ◽  
Author(s):  
Ximena M. Bustamante-Marin ◽  
Amjad Horani ◽  
Mihaela Stoyanova ◽  
Wu-Lin Charng ◽  
Mathieu Bottier ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Ciliary Dyskinesia ◽  
Large Population ◽  
Exome Capture ◽  
Swimming Velocity ◽  
Population Sampling ◽  
Dynein Arms ◽  
Motile Cilia ◽  
Ciliary Dyskinesia ◽  
Sampling Probability

AbstractPrimary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process.SignificanceMotile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.

scholarly journals High-content screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia

2020 ◽  
Author(s):  
Dani Do Hyang Lee ◽  
Daniela Cardinale ◽  
Ersilia Nigro ◽  
Colin R. Butler ◽  
Andrew Rutman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Primary Ciliary Dyskinesia ◽  
Respiratory Diseases ◽  
Primary Cell Culture ◽  
Respiratory Epithelium ◽  
High Content Screening ◽  
Basal Cells ◽  
Air Liquid Interface ◽  
Ciliary Dyskinesia

AbstractDevelopment of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique’s broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in the MCIDAS gene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patient’s nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD.SummaryWe describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures.

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