scholarly journals Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia

Cilia ◽  
2017 ◽  
Vol 6 (1) ◽  
Author(s):  
June Kehlet Marthin ◽  
Elizabeth Munkebjerg Stevens ◽  
Lars Allan Larsen ◽  
Søren Tvorup Christensen ◽  
Kim Gjerum Nielsen
Keyword(s):  
Airway Epithelium ◽  
Primary Ciliary Dyskinesia ◽  
Ex Vivo ◽  
Methodological Approach ◽  
Three Dimensional ◽  
Patient Specific ◽  
Nasal Airway ◽  
Ciliary Dyskinesia

scholarly journals Primary ciliary dyskinesia with normal ultrastructure: three-dimensional tomography detects absence of DNAH11

2018 ◽  
Vol 51 (2) ◽  
pp. 1701809 ◽  
Author(s):  
Amelia Shoemark ◽  
Thomas Burgoyne ◽  
Robert Kwan ◽  
Mellisa Dixon ◽  
Mitali P. Patel ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Structural Abnormality ◽  
Healthy Controls ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Causal Variants ◽  
Diagnostic Samples ◽  
Ciliary Dyskinesia

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.

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.

A Methodology to Analyze Changes in Lipid Core and Calcification Onto Fibrous Cap Vulnerability: The Human Atherosclerotic Carotid Bifurcation as an Illustratory Example

2009 ◽  
Vol 131 (12) ◽  
Author(s):  
Dimitrios E. Kiousis ◽  
Stephan F. Rubinigg ◽  
Martin Auer ◽  
Gerhard A. Holzapfel
Keyword(s):  
Ex Vivo ◽  
Geometric Model ◽  
Three Dimensional ◽  
Carotid Bifurcation ◽  
Vulnerability Index ◽  
Patient Specific ◽  
Lipid Core ◽  
Fibrous Cap ◽  
Treatment And Prevention ◽  
Morphological Criteria

A lipid core that occupies a high proportion of the plaque volume in addition to a thin fibrous cap is a predominant indicator of plaque vulnerability. Nowadays, noninvasive imaging modalities can identify such structural components, however, morphological criteria alone cannot reliably identify high-risk plaques. Information, such as stresses in the lesion’s components, seems to be essential. This work presents a methodology able to analyze the effect of changes in the lipid core and calcification on the wall stresses, in particular, on the fibrous cap vulnerability. Using high-resolution magnetic resonance imaging and histology of an ex vivo human atherosclerotic carotid bifurcation, a patient-specific three-dimensional geometric model, consisting of four tissue components, is generated. The adopted constitutive model accounts for the nonlinear and anisotropic tissue behavior incorporating the collagen fiber orientation by means of a novel and robust algorithm. The material parameters are identified from experimental data. A novel stress-based computational cap vulnerability index is proposed to assess quantitatively the rupture-risk of fibrous caps. Nonlinear finite element analyses identify that the highest stress regions are located at the vicinity of the shoulders of the fibrous cap and in the stiff calcified tissue. A parametric analysis reveals a positive correlation between the increase in lipid core portion and the mechanical stress in the fibrous cap and, hence, the risk for cap rupture. The highest values of the vulnerability index, which correlate to more vulnerable caps, are obtained for morphologies for which the lipid cores were severe; heavily loaded fibrous caps were thus detected. The proposed multidisciplinary methodology is able to investigate quantitatively the mechanical behavior of atherosclerotic plaques in patient-specific stenoses. The introduced vulnerability index may serve as a more quantitative tool for diagnosis, treatment and prevention.

scholarly journals Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

2021 ◽  
pp. mbc.E20-12-0806
Author(s):  
Yanhe Zhao ◽  
Justine Pinskey ◽  
Jianfeng Lin ◽  
Weining Yin ◽  
Patrick R. Sears ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Tract Infections ◽  
Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

scholarly journals Organoids in Lung Cancer Management

2021 ◽  
Vol 8 ◽  
Author(s):  
Yushi Li ◽  
Joyce W. Y. Chan ◽  
Rainbow W. H. Lau ◽  
Winnie W. Y. Cheung ◽  
Alissa Michelle Wong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Tumor ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Patient Specific ◽  
Preclinical Model ◽  
Cancer Tissue ◽  
Cancer Management

Lung cancer is a complex milieu of genomically altered cancer cells, a diverse collection of differentiated cells and nonneoplastic stroma. Lung cancer organoids is a three-dimensional structure grown from patient cancer tissue that could mimic in vivo complex behavior and cellular architecture of the cancer. Furthermore, the genomic alterations of the primary lung tumor is captured ex vivo. Lung cancer organoids have become an important preclinical model for oncology studies in recent years. It could be used to model the development of lung cancer, investigate the process of tumorigenesis, and also study the signaling pathways. The organoids could also be a platform to perform drug screening and biomarker validation of lung cancer, providing a promising prediction of patient-specific drug response. In this review, we described how lung cancer organoids have opened new avenues for translating basic cancer research into clinical therapy and discussed the latest and future developments in organoid technology, which could be further applied in lung cancer organoids research.

scholarly journals CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia

2020 ◽  
Vol 117 (24) ◽  
pp. 13571-13579 ◽  
Author(s):  
Alan M. Robinson ◽  
Satoe Takahashi ◽  
Eva J. Brotslaw ◽  
Aisha Ahmad ◽  
Emma Ferrer ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Basal Bodies ◽  
Nasal Airway ◽  
Directional Flow ◽  
Multiple Phenotypes ◽  
Multiciliated Cells ◽  
Motile Cilia ◽  
Ciliary Dyskinesia ◽  
Different Tissues ◽  
Proper Orientation

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a “9 + 2” microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3),Camsap3tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination ofCamsap3tm1a/tm1amice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, inCamsap3tm1a/tm1a, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs ofXenopusembryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

scholarly journals Airway–artery quantitative assessment on chest computed tomography in paediatric primary ciliary dyskinesia

2020 ◽  
Vol 6 (3) ◽  
pp. 00210-2019
Author(s):  
Valentina Ferraro ◽  
Eleni-Rosalina Andrinopoulou ◽  
Anna Marthe Margaretha Sijbring ◽  
Eric G. Haarman ◽  
Harm A.W.M. Tiddens ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Primary Ciliary Dyskinesia ◽  
Quantitative Assessment ◽  
Three Dimensional ◽  
Bronchial Tree ◽  
Wall Thickening ◽  
Bronchial Wall ◽  
Chest Computed Tomography ◽  
Structural Abnormalities ◽  
Ciliary Dyskinesia

Chest computed tomography (CT) is the gold standard for detecting structural abnormalities in patients with primary ciliary dyskinesia (PCD) such as bronchiectasis, bronchial wall thickening and mucus plugging. There are no studies on quantitative assessment of airway and artery abnormalities in children with PCD. The objectives of the present study were to quantify airway and artery dimensions on chest CT in a cohort of children with PCD and compare these with control children to analyse the influence of covariates on airway and artery dimensions.Chest CTs of 13 children with PCD (14 CT scans) and 12 control children were collected retrospectively. The bronchial tree was segmented semi-automatically and reconstructed in a three-dimensional view. All visible airway–artery (AA) pairs were measured perpendicular to the airway centre line, annotating per branch inner and outer airway and adjacent artery diameter and computing inner airway diameter/artery ratio (AinA ratio), outer airway diameter/artery ratio (AoutA ratio), wall thickness (WT), WT/outer airway diameter ratio (Awt ratio) and WT/artery ratio.In the children with PCD (38.5% male, mean age 13.5 years, range 9.8–15.3) 1526 AA pairs were measured versus 1516 in controls (58.3% male, mean age 13.5 years, range 8–14.8). AinA ratio and AoutA ratio were significantly higher in children with PCD than in control children (both p<0.001). Awt ratio was significantly higher in control children than in children with PCD (p<0.001).Our study showed that in children with PCD airways are more dilated than in controls and do not show airway wall thickening.

scholarly journals Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

2019 ◽  
Vol 21 (1) ◽  
pp. 69 ◽  
Author(s):  
Giuseppantonio Maisetta ◽  
Lucia Grassi ◽  
Semih Esin ◽  
Esingül Kaya ◽  
Andrea Morelli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Primary Ciliary Dyskinesia ◽  
Ethylenediaminetetraacetic Acid ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Clinical Prognosis ◽  
Airway Infections ◽  
Ciliary Dyskinesia

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Sign in / Sign up

Export Citation Format

Share Document