High-speed Video Microscopy Research Articles

Impact of local anesthesia on ciliary dyskinesia diagnosis by digital high-speed videomicroscopy.

It was hypothesized that local anesthesia administration would not significantly affect ciliary function in nasal epithelium. A prospective, simple-blind randomized study was conducted between 2020 and 2023. The study employed digital high-speed videomicroscopy to analyze ciliary beat frequency and pattern. A cohort of 38 participants was recruited, consisting of 25 healthy volunteers and 13 referred individuals (including seven diagnosed with PCD). Selection criteria ensured the absence of chronic respiratory diseases, recent respiratory tract infections, or regular use of nasal medications. Participants underwent nasal brushing with administration of lidocaine and naphazoline nasal spray in one nostril and saline in the contralateral nostril. Ciliary beat frequency and pattern were measured using digital high-speed video microscopy. Nasal spray administration did not significantly alter ciliary beat frequency or pattern compared to saline (p = 0.841 and p = 0.125, respectively). Subgroup analysis revealed consistent results across healthy volunteers, referred patients, and PCD patients. Local anesthesia with lidocaine and naphazoline spray did not affect ciliary function outcomes. These findings support the safe use of these agents in clinical practice for PCD diagnostic procedures. Further research with larger cohorts is warranted for validation.

Primary ciliary dyskinesia in children: clinical, laboratory-instrumental and genetic characteristics

Background. Primary ciliary dyskinesia (PCD) is an orphan disease, and diagnosis is difficult because there is no gold standard for diagnosis. Aim. Clinical, laboratory-instrumental, genetic characteristics of PCD in children. Materials and methods. From 2009 to 2024, 31 patients with a genetically confirmed diagnosis of PCD were observed as part of a multicenter, open-ended, descriptive pilot longitudinal study. Examination methods: clinical and anamnestic method; X-ray examination and computed tomography of the chest organs and paranasal sinuses, tracheobronchoscopy; sputum/aspirate cultures of the tracheobroncheal tree with determination of sensitivity to antibiotics; transmission electron microscopy, high-speed video microscopy of the ciliated epithelium, cytological examination of bronchoalveolar lavage; monitoring computer pulse oximetry, echocardiography, audiometry, spirometry with bronchodilator test. Results. Respiratory symptoms in the neonatal period have 80% of children with PCD, lateralization defects – 35%, congenital heart defects – 13%, bronchiectasis – 68%, purulent endobronchitis – 62%, year-round rhinitis – 84%, hearing loss, otitis – 65%. The average age of onset of symptoms was 1 [1; 1] weeks, and the verification of diagnosis was 6 [2,5; 8] years. The main pathogens of chronic respiratory infection with PCD are Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus. The most common cause of PCD was biallelic variants of the DNAH5 gene. Conclusion. The diagnosis of PCD should be based on the application of the maximum number of diagnostic tests.

Pathogenic variants in CFAP46, CFAP54, CFAP74, and CFAP221 cause Primary Ciliary Dyskinesia with a defective C1d projection of the central apparatus.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by insufficient mucociliary clearance leading to chronic airway infections. The diagnostic guideline of the European Respiratory Society (ERS) primarily recommends the evaluation of the clinical history (e. g. by the PICADAR prediction tool), nasal nitric oxide (nNO) production rate measurements, high-speed videomicroscopy analysis (HSVMA) of ciliary beating, and the assessment of ciliary axonemes via transmission electron microscopy (TEM). Genetic testing can be implemented as a last step. In this study, we aimed to characterise PCD with a defective C1d projection of the ciliary central apparatus (CA) and evaluated the applicability of the ERS diagnostic guideline to this PCD type. Using a high-throughput sequencing approach of genes encoding C1d components, we identified pathogenic variants in the novel PCD genes CFAP46 and CFAP54, and the known PCD gene CFAP221. To fully assess this PCD type, we also analysed individuals with pathogenic variants in CFAP74. Careful evaluation revealed that C1d-defective PCD is associated with normal situs composition, normal nNO-production rates, normal ciliary ultrastructure by TEM, and normal ciliary beating by HSVMA. Despite chronic respiratory disease, PICADAR does not reliably detect this PCD type. However, we could show by in-vitro ciliary transport assays that affected individuals exhibit insufficient ciliary clearance. Overall, this study extends the spectrum of PCD genes and highlights that C1d-defective PCD individuals elude diagnosis in the current diagnostic algorithm. To enable diagnosis, genetic testing should be prioritised in future diagnostic guidelines.

CCL2 may contribute to the damage of fallopian tube epithelium in women with tubal endometriosis

Research QuestionHow does tubal endometriosis (TEM) impact the morphology and ultrastructure of the fallopian tube epithelium? What are the underlying pathophysiological mechanisms of TEM? DesignEpithelial samples of the fallopian tubes from patients with (TEM group) and without (non-TEM group) TEM were collected. The morphological characteristics, ultrastructure, ciliated cell percentage, and ciliary beat frequency (CBF) were assessed via hematoxylin-eosin staining, electron microscopy, and high-speed video microscopy. mRNA microarray analysis was conducted to identify differentially expressed genes (DEGs) in the fallopian tube epithelium, which were further validated using qPCR, immunohistochemistry, and Western blotting. The effects of recombinant CCL2 protein on primary human fallopian tube epithelial cells (FTEC) were examined in vitro. ResultsPatients with TEM exhibited significant abnormalities in the morphology and ultrastructure of the fallopian tube epithelium with reduced percentage of ciliated cells and CBF, compared to the non-TEM patients. Among the 765 DEGs identified in the fallopian tube epithelium, 512 genes were upregulated and 253 genes were downregulated in the TEM group. qPCR, immunohistochemistry, and Western blotting validation confirmed a significant upregulation of CCL2 expression in the TEM group. In vitro experiments revealed that recombinant CCL2 protein significantly reduced ciliated cell differentiation, length of cilia, and CBF in FTEC, suggesting a role for CCL2 in the development of the TEM-related pathological phenotype. ConclusionsThese findings indicate that CCL2 may contribute to the pathological phenotype associated with TEM and could be a crucial signaling molecule in the damage of fallopian tube epithelium in patients with TEM.

Evidence for secondary ciliary dyskinesia in patients with cystic fibrosis

BackgroundMucociliary clearance (MCC) impairment can be due to mucus abnormalities or to a ciliary dysfunction, which can be innate, or secondary to infection and/or inflammation. In cystic fibrosis (CF), it is well documented that MCC is impaired due to mucus abnormalities, but little is known concerning ciliary beating. This study aimed to confirm that ciliary dyskinesia is present in CF, and if this might be innate or secondary to the chronic infection and/or inflammation. MethodsCiliated epithelial samples were obtained by nasal brushing from 51 CF patients, and from 30 healthy subjects. Ciliary beating was evaluated using digital high-speed videomicroscopy at 37 °C, allowing to evaluate ciliary beat frequency (CBF) and the percentage of abnormal beat pattern (CBP); this was repeated after air-liquid interface (ALI) cell culture. ResultsCiliary dyskinesia was higher in CF patients than in healthy subjects, with a lower CBF and a higher percentage of abnormal CBP. Ciliary dyskinesia, already present in childhood, normalized after ALI cell culture. A chronic airway colonization did not worsen ciliary dyskinesia. ConclusionsWe showed that, in CF, a ciliary dyskinesia, present from childhood, might contribute to the impaired MCC. Our results also found that the abnormal ciliary beating was not associated with a chronic infection, and resolved after ALI cell culture, suggesting that ciliary dyskinesia in CF is not innate, and might be secondary to chronic inflammation.

Primary ciliary dyskinesia: a case report of double DNAH11 mutant alleles

Primary ciliary dyskinesia (PCD), a rare disorder, is genetically varied. Mutations in proteins involved in the structure, function, or assembly of cilia are known to determine situs inversus, male infertility, and chronic destructive airway disease. PCD is inherited by an autosomal recessive pattern of inheritance in most cases. Nonetheless, patterns of autosomal dominant and X-linked inheritance have been mentioned. A history of recurrent upper and lower respiratory tract infections raised clinical suspicion of primary ciliary dyskinesia in a 10-year-old patient. Genetic tests were performed using next-generation sequencing technology (Illumina NextGen) with the multiplex ligation-dependent probe amplification technique for primary ciliopathies and syndromes subject to differential diagnosis. Genetic testing identified two pathogenic variants, not previously associated with a case report in the literature, c.7727A>G (p.Asp2576Gly) and c.8578G>A (p.Gly2860Ser), within the DNAH11 gene, which is associated with autosomal recessive PCD. The result also reported mutations in other genes involved in autosomal recessive PCD (DNAH8, DNAH9 and ZMYND10), which were classified as variants with uncertain clinical significance. Transmission electron microscopy of respiratory cilia and nasal nitric oxide measurement cannot be used to diagnose PCD in patients with DNAH11 mutations because the structure of cilia is normal, and the levels of NO are not constantly low. High-speed video microscopy analysis can be helpful because DNAH11 mutations cause a distinct phenotype of PCD. Nevertheless, the mutation analysis of various PCD-causing genes remains the easiest to conduct and with good results. Genetic research on PCD has identified a number of significant ciliary genes in recent years, offering fresh perspectives on the molecular processes underlying cilia assembly and function. This facilitates the development of new methods for the diagnosis, prevention, and treatment of PCD. However, because it is a highly complex and heterogeneous disease, the field of gene diagnosis and therapy in PCD is still in its infancy.

Heterozygous cis HYDIN mutations cause primary ciliary dyskinesia.

The product of ciliary gene HYDIN is an integral component for c2b projection within the motile cilia central pair (CP) apparatus. Biallelic mutations of this gene cause primary ciliary dyskinesia (PCD), an uncommon heterogeneous recessive disorder affecting motile cilia, resulting in defective mucociliary clearance that leads to chronic suppurative lung disease. Nasal brushing samples were collected from two siblings attending the Victorian Diagnostic service for PCD. Nasal airway epithelial cells (NAECs) were cultured before cilia structure and function studies using high-speed video microscopy (HSVM), transmission electron microscopy, and immunofluorescence. Cultured NAECs from both siblings showed defective cilia beating patterns under HSVM. A confirmatory PCD diagnosis was achieved through immunofluorescence, which showed the loss of HYDIN and the associated protein SPEF2 from the cilia axoneme. This case report details the diagnosis of two siblings who displayed similar defective cilia beating phenotypes seen in patients with PCD bearing recessive HYDIN mutations. Uniquely, both siblings carry two previously unreported HYDIN mutations, which are in the cis position, demonstrating the possibility for disease manifestation without biallelic mutations of ciliary genes. The authors declare no funding support for this study.

Stanovenie mukociliárneho klírensu u pacientov so symptómami laryngofaryngeálneho refl uxu – preliminárne výsledky

Summary: Introduction: Mucociliary clearance represents an important protective mechanism of the respiratory tract. Results of multiple studies indicate that gastroesophageal reflux can participate in worsening mucociliary clearance leading to laryngopharyngeal reflux symptoms including recurrent upper respiratory tract infections. Thereby, we decided to provide ciliary beat frequency of the upper respiratory tract epithelium in patients with laryngopharyngeal reflux. Materials and methods: We enrolled 23 patients with laryngopharyngeal reflux symptoms. Control group consisted of 9 healthy volunteers. Patients were enrolled upon a positive questionnaire reflux symptom index (RSI >13 points), with no proton pump inhibitor treatment. We obtained a nasopharyngeal swab and evaluated ciliary beat frequency of the respiratory epithelium using highspeed videomicroscopy and special sofware Ciliary Analysis. We compared the values of ciliary beat frequency with values from healthy volunteers. Results: We provided median, minimal and maximum values of ciliary beat frequency. Median ciliary beat frequency in healthy volunteers was significantly higher than in patients with laryngopharyngeal reflux symptomatology (10.86 ± 1.67 vs. 6.6 ± 2.2; P <0.001). Maximum ciliary beat frequency was also significantly higher in healthy volunteers than in patients with LPR (17.9 ± 0.95 vs. 15.3 ± 2.39; P <0.001). Minimal value had no significant difference. Correlation analysis showed an inverse correlation between median ciliary beat frequency of the respiratory epithelium and LPR symptoms (RSI value) (P <0.04). Conclusion: Patients with laryngopharyngeal reflux symptoms had lower ciliary beat frequency compared to healthy volunteers. The degree of frequency defect correlates with the degree of their laryngopharyngeal symptoms. More studies are needed to confirm direct evidence of gastroesophageal reflux etiology in mucociliary clearance worsening. Key words: mucociliary clearance – laryngopharyngeal reflux – cilia

Characterization of pathogenic genetic variants in Russian patients with primary ciliary dyskinesia using gene panel sequencing and transcript analysis

BackgroundPrimary ciliary dyskinesia (PCD) is a group of rare genetically heterogeneous disorders caused by defective cilia and flagella motility. The clinical phenotype of PCD patients commonly includes chronic oto-sino-pulmonary disease, infertility, and, in about half of cases, laterality defects due to randomization of left–right body asymmetry. To date, pathogenic variants in more than 50 genes responsible for motile cilia structure and assembly have been reported in such patients. While multiple population-specific mutations have been described in PCD cohorts from different countries, the data on genetic spectrum of PCD in Russian population are still extremely limited.ResultsThe present study provides a comprehensive clinical and genetic characterization of 21 Russian families with PCD living in various country regions. Anomalies of ciliary beating in patients` respiratory epithelial cells were confirmed by high-speed video microscopy. In the most cases, custom-designed panel sequencing allowed to uncover causative variants in well-known or rarely mentioned PCD-related genes, including DNAH5, DNAH11, CFAP300, LRRC6, ZMYND10, CCDC103, HYDIN, ODAD4, DNAL1, and OFD1. The variations comprised common mutations, as well as novel genetic variants, some of which probably specific for Russian patients. Additional targeted analysis of mRNA transcripts from ciliated cells enabled us to specify functional effects of newly identified genetic variants in DNAH5 (c.2052+3G>T, c.3599-2A>G), HYDIN (c.10949-2A>G, c.1797C>G), and ZMYND10 (c.510+1G>C) on splicing process. In particular, the splice site variant c.2052+3G>T, detected in four unrelated families, resulted in skipping of exon 14 in DNAH5 transcripts and, according to haplotype analysis of affected probands, was proposed as an ancestral founder mutation in Udmurt population.ConclusionsThe reported data provide a vital insight into genetic background of primary ciliary dyskinesia in the Russian population. The findings clearly illustrate the utility of gene panel sequencing coupled with transcriptional analysis in identification and clinical interpretation of novel genetic variants.

Evaluation of Open-Source Ciliary Analysis Software in Primary Ciliary Dyskinesia: A Comparative Assessment.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder characterized by alterations in motile cilia function. The diagnosis of PCD is challenging due to the lack of standardized methods in clinical practice. High-speed video microscopy analysis (HSVA) directly evaluates ciliary beat frequency (CBF) in PCD. Recently, open-source ciliary analysis software applications have shown promise in measuring CBF accurately. However, there is limited knowledge about the performance of different software applications, creating a gap in understanding their comparative effectiveness in measuring CBF in PCD. We compared two open-source software applications, CiliarMove (v219) and Cilialyzer (v1.2.1-b3098cb), against the manual count method. We used high-speed videos of nasal ciliary brush samples from PCD RSPH4A-positive (PCD (RSPH4A)) patients and healthy controls. All three methods showed lower median CBF values for patients with PCD (RSPH4A) than in healthy controls. CiliarMove and Cilialyzer identified lower CBF in patients with PCD (RSPH4A), similarly to the manual count. Cilialyzer, CiliarMove, and manual count methods demonstrated statistical significance (p-value < 0.0001) in the difference of median CBF values between patients with PCD (RSPH4A) and healthy controls. Correlation coefficients between the manual count values against both software methods demonstrated positive linear relationships. These findings support the utility of open-source software-based analysis tools. Further studies are needed to validate these findings with other genetic variants and identify the optimal software for accurate CBF measurement in patients with PCD.

Characterization of the ciliary beating efficiency in primary diffuse chronic rhinosinusitis.

Ciliary dysfunction may result in chronic airway inflammation and infection causing injury and structural changes to the airway epithelium, leading to a variety of diseases, like bronchiectasis and primary diffuse chronic rhinosinusitis (CRS). Currently, ciliary beating analysis has mainly been studied through the measure of the ciliary beating frequency (CBF) by high-speed digital video microscopy (HSDV). However, a normal CBF has been described in different forms of primary and acquired ciliary dyskinesia.

Does dispersed phase inertia affect the shape of sheared emulsion droplets?

Inertial effects on sheared emulsion droplets are a topic of scientific and industrial interest for several applications from processing to microfluidics. Most of the literature have addressed so far the role of inertia of the continuous phase, which is known to affect shear-induced droplet deformation and migration at values of the Reynolds number of the external fluid Rec &amp;gt; 1. However, less attention has been paid to the case of inertial effects inside the droplets, corresponding to values of the Reynolds number of the droplet fluid Red &amp;gt; 1. Such a case is especially relevant when the viscosity ratio λ between the droplet and the external fluid is ≪ 1, which is typical of water-in-oil emulsions where the low values of droplet viscosity can result in Red &amp;gt; 1, while Rec &amp;lt; 1 due to the larger oil viscosity. Here, we focus on the effect of droplet inertia under shear flow at λ ≪ 1 by high-speed video microscopy experiments in a microcapillary and by numerical simulations based on a front-tracking finite-difference method. The results unveil the droplet's three-dimensional shape under shear flow at low viscosity ratios and show that droplet inertia tends to increase droplet deformation and orientation along the flow direction and to form two vortices inside the droplets even at small Rec. The latter findings are at variance with the case of external fluid inertia, where droplets become more aligned with the velocity gradient direction.

Primary Ciliary Dyskinesia: A Clinical Review.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed videomicroscopy analysis (HSVMA), immunofluorescent staining, axonemal ultrastructure analysis via transmission electron microscopy (TEM), and genetic testing. Notably, there is no single gold standard confirmatory or exclusionary test. Currently, 54 causative genes involved in cilia assembly, structure, and function have been linked to PCD; this rare disease has a spectrum of clinical manifestations and emerging genotype-phenotype relationships. In this review, we provide an overview of the structure and function of motile cilia, the emerging genetics and pathophysiology of this rare disease, as well as clinical features associated with motile ciliopathies, novel diagnostic tools, and updates on genotype-phenotype relationships in PCD.

Secondary ice production – no evidence of efficient rime-splintering mechanism

Abstract. Mixed-phase clouds are essential for Earth’s weather and climate system. Ice multiplication via secondary ice production (SIP) is thought to be responsible for the observed strong increase in ice particle number concentration in mixed-phase clouds. In this study, we focus on the rime splintering also known as the Hallett–Mossop (HM) process, which still lacks physical and quantitative understanding. We report on an experimental study of rime splintering conducted in a newly developed setup under conditions representing convective mixed-phase clouds in the temperature range of −4 to −10 °C. The riming process was observed with high-speed video microscopy and infrared thermography, while potential secondary ice (SI) particles in the super-micron size range were detected by a custom-built ice counter. Contrary to earlier HM experiments, where up to several hundreds of SI particles per milligram of rime were found at −5 °C, we found no evidence of productive SIP, which fundamentally questions the importance of rime splintering. Further, we could exclude two potential mechanisms suggested to be the explanation for rime splintering: the freezing of droplets upon glancing contact with the rimer and the fragmentation of spherically freezing droplets on the rimer surface. The break-off of sublimating fragile rime spires was observed to produce very few SI particles, which is insufficient to explain the large numbers of ice particles reported in earlier studies. In the transition regime between wet and dry growth, in analogy to phenomena of the deformation of drizzle droplets upon freezing, we also observed the formation of spikes on the rimer surface, which might be a source of SIP.

Advancing Primary Ciliary Dyskinesia Diagnosis through High-Speed Video Microscopy Analysis.

Primary ciliary dyskinesia (PCD) is an inherited disorder that impairs motile cilia, essential for respiratory health, with a reported prevalence of 1 in 16,309 within Hispanic populations. Despite 70% of Puerto Rican patients having the RSPH4A [c.921+3_921+6del (intronic)] founder mutation, the characterization of the ciliary dysfunction remains unidentified due to the unavailability of advanced diagnostic modalities like High-Speed Video Microscopy Analysis (HSVA). Our study implemented HSVA for the first time on the island as a tool to better diagnose and characterize the RSPH4A [c.921+3_921+6del (intronic)] founder mutation in Puerto Rican patients. By applying HSVA, we analyzed the ciliary beat frequency (CBF) and pattern (CBP) in native Puerto Rican patients with PCD. Our results showed decreased CBF and a rotational CBP linked to the RSPH4A founder mutation in Puerto Ricans, presenting a novel diagnostic marker that could be implemented as an axillary test into the PCD diagnosis algorithm in Puerto Rico. The integration of HSVA technology in Puerto Rico substantially enhances the PCD evaluation and diagnosis framework, facilitating prompt detection and early intervention for improved disease management. This initiative, demonstrating the potential of HSVA as an adjunctive test within the PCD diagnostic algorithm, could serve as a blueprint for analogous developments throughout Latin America.

A new software for automated analysis of respiratory tract ciliary epithelium movement for the diagnosis of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare hereditary disease. In this ciliopathy, the disturbed structure and motility of the ciliary epithelium negatively affects the ciliary function and leads to prominent decrease or absence of mucociliary clearance. The European guidelines recommend analyzing the cilia beat frequency (СBF) in a native preparation or in ALI culture using light microscopy as one of the methods to confirm the diagnosis of PCD.The aim of this project was to create software for automated analysis of the movement/beating of the ciliary epithelium of the respiratory tract for the diagnosis of primary ciliary dyskinesia using digital high-speed video microscopy in vivo and in vitro.Methods. Five healthy donors and 10 patients with suspected PCD underwent nasal epithelial brush biopsy. The preparations were examined with a transmission electron microscope in vivo. Epithelial cells were also isolated from the nasal biopsy specimen, and ciliogenesis of these cells was performed by ALI-culturing, followed by digital high-speed video microscopy and assessment of the number of active cells and cilia beating frequency. The resulting video images were used to create the software.Results. Software for determination of ciliary epithelium beat frequency in primary ciliary dyskinesia (PCD HighSpeed Video Microscopy Analysis – PCD HSVMA) was created to optimize the diagnosis of PCD by light microscopy (software registration number 2023687245). The software is designed to count the number of active cells of ciliary epithelium and CBF (Hz) by digital high-speed video microscopy in vivo and in vitro in ALI-culture. PCD HSVMA software features storage of patient data, display of heat map, formation of a large server database of patients and video files, building of color and static histograms, processing of several areas in one video. Our software has a number of advantages over CiliarMove and Cilialyzer and has high correlation of CFB (Hz) estimation with these products.Conclusion. Our software can be used for improvement of PCD diagnostics in laboratories of healthcare institutions, in scientific institutions and can be included in specialist educational programs for laboratory doctors, pediatricians, general practitioners, pulmonologists, diagnosticians (endoscopists).

Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the...

Next-Generation Sequencing-Based Copy Number Variation Analysis in Chinese Patients with Primary Ciliary Dyskinesia Revealed Novel DNAH5 Copy Number Variations.

The online version contains supplementary material available at 10.1007/s43657-023-00130-0.

Identification of a novel RPGR mutation associated with retinitis pigmentosa and primary ciliary dyskinesia in a Slovak family: a case report

BackgroundThe mutations in the RPGR (retinitis pigmentosa GTPase regulator) gene are the most common cause of X-linked retinitis pigmentosa (XLRP), a rare genetic disorder affecting the photoreceptor cells in the retina. Several reported cases identified this gene as a genetic link between retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), characterised by impaired ciliary function predominantly in the respiratory tract. Since different mutations in the same gene can result in various clinical manifestations, it is important to describe a correlation between the gene variant and the observed phenotype.MethodsTwo young brothers from a non-consanguineous Slovak family with diagnosed retinal dystrophy and recurrent respiratory infections were examined. Suspected PCD was diagnosed based on a PICADAR questionnaire, nasal nitric oxide analysis, transmission electron microscopy, high-speed video microscopy analysis, and genetic testing.ResultsWe identified a novel frameshift RPGR mutation NM_001034853: c.309_310insA, p.Glu104Argfs*12, resulting in a complex X-linked phenotype combining PCD and RP. In our patients, this mutation was associated with normal ultrastructure of respiratory cilia, reduced ciliary epithelium, more aciliary respiratory epithelium, shorter cilia, and uncoordinated beating with a frequency at a lower limit of normal beating, explaining the clinical manifestation of PCD in our patients.ConclusionThe identified novel pathogenic mutation in the RPGR gene expands the spectrum of genetic variants associated with the X-linked PCD phenotype overlapping with RP, highlighting the diversity of mutations contributing to the disorder. The described genotype–phenotype correlation can be useful in clinical practice to recognise a broader spectrum of PCD phenotypes as well as for future research focused on the genetic basis of PCD, gene interactions, the pathways implicated in PCD pathogenesis, and the role of RPGR protein for the proper functioning of cilia in various tissues throughout the body.

Specialized proresolving mediator resolvin E1 corrects the altered cystic fibrosis nasal epithelium cilia beating dynamics

In cystic fibrosis (CF), impaired mucociliary clearance leads to chronic infection and inflammation. However, cilia beating features in a CF altered environment, consisting of dehydrated airway surface liquid layer and abnormal mucus, have not been fully characterized. Furthermore, acute inflammation is normally followed by an active resolution phase requiring specialized proresolving lipid mediators (SPMs) and allowing return to homeostasis. However, altered SPMs biosynthesis has been reported in CF. Here, we explored cilia beating dynamics in CF airways primary cultures and its response to the SPMs, resolvin E1 (RvE1) and lipoxin B4 (LXB4). Human nasal epithelial cells (hNECs) from CF and non-CF donors were grown at air-liquid interface. The ciliary beat frequency, synchronization, orientation, and density were analyzed from high-speed video microscopy using a multiscale Differential Dynamic Microscopy algorithm and an in-house developed method. Mucins and ASL layer height were studied by qRT-PCR and confocal microscopy. Principal component analysis showed that CF and non-CF hNEC had distinct cilia beating phenotypes, which was mostly explained by differences in cilia beat organization rather than frequency. Exposure to RvE1 (10 nM) and to LXB4 (10 nM) restored a non-CF-like cilia beating phenotype. Furthermore, RvE1 increased the airway surface liquid (ASL) layer height and reduced the mucin MUC5AC thickness. The calcium-activated chloride channel, TMEM16A, was involved in the RvE1 effect on cilia beating, hydration, and mucus. Altogether, our results provide evidence for defective cilia beating in CF airway epithelium and a role of RvE1 and LXB4 to restore the main epithelial functions involved in the mucociliary clearance.