Search Results (76)

Primary ciliary dyskinesia (PCD) is a rare genetic disorder mainly characterized by impaired mucociliary clearance and chronic respiratory symptoms. From a consanguineous family, a male patient, although with respiratory complaints since birth, was diagnosed with PCD only in adulthood. Whole-exome sequencing disclosed a novel homozygous intronic single-nucleotide deletion, NM_001369.3(DNAH5):c.13723+4del, initially classified as of uncertain clinical significance. Digital highspeed videomicroscopy (HSVM) evidenced a null ciliary beating frequency; transmission electron microscopy showed absence of outer dynein arms (class-1); and immunofluorescence (IF) demonstrated markedly absent DNAH5 protein level in the apical cilia region with delocalization to the transition and basal-body regions. Bioinformatic analysis predicted altered splicing at the donor splice site of exon 78, whereas mRNA sequencing revealed two splicing defects: the mainly expressed transcript corresponding to exon 78 skipping and a minor transcript originated from a cryptic splice site in exon 78. The patient was infertile and showed severe oligoteratozoospermia. Sperm IF analysis revealed absence of DNAH5 from the flagellum with accumulation at the neck region. The family study confirmed homozygosity. The present results support a pathogenic role for the c.13723+4del variant and underscore the importance of integrating clinical, ultrastructural, DNA, mRNA and protein analyses to clarify and contribute to PCD diagnosis. Full article

Primary ciliary dyskinesia (PCD) is a rare ciliopathy resulting in chronic oto-sino-pulmonary disease. PCD diagnosis can be achieved by a combination of different diagnostic and adjuvant tools, including high-speed video-microscopy analysis (HSVA). A founder variant has been described in Puerto Rico as the most common cause of PCD in the island. Background/Objectives: In HSVA, objective parameters such as ciliary beat frequency (CBF) and subjective parameters such as ciliary beat pattern (CBP) shed light on the biophysical properties of cilia. However, the subjective nature of CBP creates a gap in knowledge; characteristics such as the length, angle, and bending index of cilia are poorly described. Our goal is to quantify cilia dynamics of the RSPH4A (c.921+3_921+6delAAGT (intronic)) founder variant in Puerto Rico through biophysical properties of cilia. This approach enhances longitudinal patient care by understanding treatment progress through biophysical ciliary function. Methods: We analyzed images from HSVA of six patients with PCD homozygous for the founder variant and six healthy controls (HC) (n = 12). Results: We found that ciliary length (PCD = 7.62 ± 0.95 μm, HC = 8.12 ± 1.36 μm, p = 0.204 ns), orientation vector (PCD = 7.20 ± 0.93 μm, HC = 7.25 ± 1.01 μm, p = 0.883 ns), straight angle (PCD = 1.67 ± 0.27 rad, HC = 1.76 ± 0.29 rad, p = 0.380 ns), and area (PCD = 2.35 ± 0.52 μm², HC = 2.10 ± 0.53 μm², p = 0.264 ns) did not have statistically significant differences between PCD and HC. In contrast, bending index (PCD = 1.06 ± 0.04, HC = 1.12 ± 0.09, p = 0.01), bent angle (PCD = 1.11 ± 0.30 rad, HC = 0.67 ± 0.21 rad, p < 0.0001), net angle (PCD = 0.56 ± 0.26 rad, HC = 1.09 ± 0.35 rad, p < 0.0001), amplitude (PCD = 5.77 ± 1.25 μm, HC = 7.99 ± 1.65 μm, p < 0.0001), and amplitude per second (PCD = 48.83 ± 13.23 A(s), HC = 91.66 ± 27.96 A(s), p < 0.0001) showed significant differences between both groups. Conclusions: Reduced angular excursion and amplitude in PCD demonstrate that the beating pattern of the RSPH4A founder variant is dysfunctional as compared with healthy controls. Our study provides an objective framework to understand the biophysical properties of the RSPH4A founder variant. Full article

Autoantibodies (AAbs) play an important role in the development of autoimmune diseases. While many AAbs induce apoptosis of target cells, a distinct subgroup, termed functional autoantibodies (fAAbs) against G protein–coupled receptors (GPCRs), can modulate physiological receptor signaling without inducing cell death. The functional activity of GPCR-fAAbs may be influenced by various cofactors, including inflammation (e.g., inflammatory cytokine, ciliary neurotrophic factor (CNTF)) and ischemia. As ischemia triggers a substantial release of arachidonic acid (AA) from membrane phospholipids, the present study aimed to examine exploratively the influence of AA, eicosapentaenoic acid (EPA), and CNTF on the responses of spontaneously beating neonatal rat cardiomyocytes to GPCR agonists and GPCR-fAAbs. AA and EPA differentially influenced responses in cardiomyocytes induced by GPCR-fAAbs: AA altered the functional responses associated with adrenergic β₂-fAAb, adrenergic α₁-fAAb, angiotensin II (AT1)-fAAb, endothelin A (ETA)-fAAb and angiotensin 1–7 MAS-fAAbs. However, muscarinergic M₂-fAAb responses remained largely unaffected. In contrast, EPA attenuated the responses to β₂-fAAb, α₁-fAAb, AT1-fAAb, and ETA-fAAb, while MAS-fAAb and M₂-fAAb responses were not markedly altered. CNTF acted as a time-dependent modulator of cardiomyocyte chronotropic responses and influenced the magnitude of GPCR-mediated signaling on a cardiomyocyte bioassay. Together, these findings might suggest that lipid mediators such as AA and EPA or CNTF may modulate functional responses of cardiomyocytes associated with GPCR-fAAbs. Full article

Background/Objectives: Primary ciliary dyskinesia (PCD) is a rare inherited disorder caused by dysfunction of motile cilia, leading to chronic respiratory disease. Diagnosis is challenging due to heterogeneous and non-specific clinical manifestations and the absence of a single definitive diagnostic test. Current diagnostic strategies rely on a combination of functional, ultrastructural, and genetic analyses. The objective of this study was to evaluate whether ciliary beat frequency (CBF), combined with ciliary beat pattern (CBP) assessment using digital high-speed video microscopy (DHSV), could serve as an effective first-line screening tool to identify patients requiring further diagnostic investigations. Methods: This single-center retrospective study included 65 patients (52 children and 13 adults) with clinical suspicion of PCD. Ciliary beat analysis was performed on nasal or bronchial samples using DHSV and Sisson–Ammons Video Analysis software. CBF and CBP were assessed and compared between patients with confirmed PCD and those in whom PCD was excluded based on transmission electron microscopy (TEM) and/or molecular genetic analysis. Results: Fifteen patients were diagnosed with PCD. Mean CBF was significantly lower in the PCD group compared with the non-PCD group (3.3 Hz vs. 8.1 Hz; p < 0.001). A CBF cut-off value of 5.25 Hz yielded a sensitivity of 78.6% and a specificity of 95.7%. Three patients with PCD had CBF values above this threshold; however, two of them exhibited abnormal CBP. Sample type, patient age, and the presence of airway pathogens did not significantly influence CBF measurements. Conclusions: CBF and CBP analysis using DHSV represents a useful first-line screening tool within a multifaceted diagnostic approach for PCD, allowing rapid identification of patients who should undergo further confirmatory testing. Full article

In chronic obstructive pulmonary disease (COPD), dysregulated calcium homeostasis, oxidative stress, and mucus hypersecretion converge to suppress ciliary beat frequency (CBF), thereby compromising mucociliary clearance (MCC). These mechanisms are subject to pharmacological modulation. Long-acting muscarinic antagonists (LAMAs) exert direct cilia-stimulatory effects and may counteract pathogen-induced mucin overproduction without impairing clearance. Long-acting β₂-agonists (LABAs) enhance ciliary activity through the cAMP–PKA–dynein (cyclic adenosine monophosphate–protein kinase A–dynein) signalling pathway. Inhaled corticosteroids (ICSs), although largely neutral on CBF, provide indirect protection by suppressing IL-13–driven inflammation. Phosphodiesterase (PDE)-4 inhibitors sustain intracellular cAMP and promote ciliary motility, though their clinical use remains limited by adverse effects. Emerging evidence suggests that dual and triple therapies may provide additive or synergistic benefits for preserving mucociliary function. Clinically, ex vivo CBF interpretation may be influenced by ongoing pharmacotherapy and tissue sampling site. Nasal brush samples may predominantly reflect systemic rather than inhaled therapy. Moreover, differences in PDE isoform expression between nasal and bronchial epithelium further complicate direct extrapolation of results. Rigorous patient stratification by treatment regimen is therefore essential to reconcile inconsistencies reported across studies. Ultimately, preservation of MCC in COPD depends on a delicate balance between oxidative stress and pharmacological modulation of ciliary function. Full article

A straightforward ex vivo approach has been developed and refined to enable high-resolution imaging and quantitative assessment of motile cilia function in mouse airway epithelial tissue, allowing critical insights into cilia motility and cilia generated flow using different mouse models or following different sample treatments. In this method, freshly excised mouse trachea is cut longitudinally through the trachealis muscle which is then sandwiched between glass coverslips within a thin silicon gasket. By orienting the tissue along its longitudinal axis, the natural curling of the trachealis muscle helps maintain the sample in a configuration optimal for imaging along the full tracheal length. High-speed video microscopy, utilizing differential interference contrast (DIC) optics and a fast digital camera capturing at >200 frames per second is then used to record ciliary motion. This enables detailed measurement of both cilia beat frequency (CBF) and waveform characteristics. The application of 1 µm microspheres to the bathing media during imaging allows for additional analysis of fluid flow generated by ciliary activity. The entire procedure typically takes around 40 min to complete per animal: ~30 min for tissue harvest and sample mounting, then ~10 min for imaging samples and acquiring data. Full article

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows. Full article

Cultures of primary human nasal epithelial cells (hNECs) differentiated at the air–liquid interface (ALI) represent a sophisticated and widely used model of the human upper respiratory epithelium. Despite the availability of various cell culture insert types and the well-established understanding that different culture media influence the cell culture characteristics, the possible impact of the insert brand remains rather underexplored. We cultured hNECs from nineteen healthy adult donors on three distinct brands of commercially available inserts—Corning^® Transwell^®, CELLTREAT^®, and ThinCert^®—and compared the ciliary activity and cellular composition of the cultures using high-speed video microscopy and flow cytometry, respectively. Additionally, we employed an alternative method of hNEC culture setup—the inverted condition—wherein the hNECs were seeded on the basal side of the insert with the idea to avoid mucus accumulation. Our results show that ciliary activity and cell type composition did not differ between insert types for both culture conditions. However, we found a higher ciliary beat frequency and a lower active (ciliated) area in the inverted setup compared to the conventional setup across all three insert brands. These findings indicate that all three mentioned insert types yield comparable cell cultures. Full article

Multiciliated cells generate fluid flow along epithelial surfaces, and defects in their development or function cause primary ciliary dyskinesia. The fluid flow is generated by the coordinated beating of motile cilia, which are microtubule-based organelles. The base of each cilium, the basal body, is anchored to the apical cell membrane and surrounded by a dense apical cytoskeleton of actin, microtubules, and intermediate filaments. Several cell adhesion proteins play a role in the connection of the basal body to the apical cytoskeleton. Here, we show that the desmosomal protein plakophilin3, a member of the armadillo family of proteins, localizes to the striated rootlet in Xenopus laevis multiciliated cells. Knockdown of plakophilin 3 leads to significant defects in cilia-generated fluid flow and basal body docking. These defects are cell-autonomous and independent of cell intercalation and gross changes in the actin cytoskeleton. These findings suggest a crucial role for PKP3 in basal body apical migration and docking in multiciliated cells, highlighting a novel connection between desmosomal proteins and ciliary function. Full article

Mucociliary clearance, the ability of the respiratory tract to protect the integrity of the airways through the mechanical removal of potentially harmful substances, is of enormous importance during intensive care treatment. The present study aimed to evaluate the influence of clinically relevant inotropic agents on mucociliary clearance. The particle transport velocity (PTV) of isolated murine tracheae was measured as a surrogate for mucociliary clearance in the presence of dobutamine, epinephrine, and milrinone. Inhibitory substances were applied to elucidate the signal transduction cascades and the value and origin of calcium ions which provoke alterations in mucociliary clearance function. Dobutamine, epinephrine, and milrinone increased the PTV in a dose-dependent manner with half maximal effective concentrations of 75.7 nM, 87.0 nM, and 13.7 µM, respectively. After the depletion of intracellular calcium stores, no increase in PTV was observed after administering any of the three inotropic agents. While dobutamine and epinephrine activated β-adrenergic receptors, epinephrine used both the phospholipase C (PLC) and protein kinase A (PKA) pathway to promote the release of intracellular Ca²⁺. However, dobutamine primarily acted on the PKA pathway, having only a minor influence on the PLC pathway. The induced changes in PTV following milrinone administration required both the PKA and PLC pathway, although the PKA pathway was responsible for most of the induced changes. In conclusion, the common inotropic agents dobutamine, epinephrine, and milrinone increase murine PTV in a concentration-dependent manner and ultimately release Ca²⁺ from intracellular calcium stores, suggesting the function of changes in mucociliary clearance in the respiratory tract. Full article

Human immunodeficiency virus type-1 (HIV-1) associated comorbidities account for the majority of poor health outcomes in people living with HIV (PLWH) in the era of antiretroviral therapy. Lung-related comorbidities such as chronic obstructive pulmonary disease (COPD) and bacterial pneumonia are primarily responsible for increased morbidity and mortality in PLWH, even when compensated for smoking. Smokers and COPD patients demonstrate cilia shortening, attenuated ciliary beat frequency (CBF), dysfunctional ciliated cells along with goblet cell hyperplasia, and mucus hypersecretion. This is exacerbated by the fact that almost 60% of PLWH smoke tobacco, which can exacerbate inflammation and mucociliary clearance (MCC) dysfunction. This study shows that HIV Tat alters the microRNAome in airway epithelial cells and upregulates miR-34a-5p with consequent suppression of its target, Sirtuin 1 (SIRT1). SIRT1 is known to suppress Metalloproteinase 17 (ADAM17), a protease activating Notch signaling. HIV and cigarette smoke (CS) upregulate ADAM17. ADAM17 upregulation followed by SIRT1 suppression can lead to decreased ciliation, mucus hypersecretion, and attenuated MCC, a hallmark of chronic bronchitis in smokers and COPD. It is, therefore, essential to understand the pathophysiological mechanism resulting in acquired Notch dysregulation and its downstream impact on HIV-infected smokers. Full article

Organ-on-a-chip (OOC) devices mimic human organs, which can be used for many different applications, including drug development, environmental toxicology, disease models, and physiological assessment. Image data acquisition and analysis from these chips are crucial for advancing research in the field. In this study, we propose a label-free morphology imaging platform compatible with the small airway-on-a-chip system. By integrating deep learning and image recognition techniques, we aim to analyze the differentiability of human small airway epithelial cells (HSAECs). Utilizing cell imaging on day 3 of culture, our approach accurately predicts the differentiability of HSAECs after 4 weeks of incubation. This breakthrough significantly enhances the efficiency and stability of establishing small airway-on-a-chip models. To further enhance our analysis capabilities, we have developed a customized MATLAB program capable of automatically processing ciliated cell beating images and calculating the beating frequency. This program enables continuous monitoring of ciliary beating activity. Additionally, we have introduced an automated fluorescent particle tracking system to evaluate the integrity of mucociliary clearance and validate the accuracy of our deep learning predictions. The integration of deep learning, label-free imaging, and advanced image analysis techniques represents a significant advancement in the fields of drug testing and physiological assessment. This innovative approach offers unprecedented insights into the functioning of the small airway epithelium, empowering researchers with a powerful tool to study respiratory physiology and develop targeted interventions. Full article

Digital high-speed videomicroscopy (DHSV) is a crucial tool for evaluating ciliary function in children suspected of primary ciliary dyskinesia (PCD). However, until now, samples are taken without anesthesia due to uncertainty about its effect on ciliary function and DHSV interpretation. This study aimed to investigate the impact of general anesthesia on ciliary functional analysis by DHSV in a series of three patients listed for ENT surgeries, which could improve diagnostic procedures for pediatric patients. Patient 1 (7-year-old girl) underwent adenotonsillectomy and tympanostomy placement tube, while patients 2 (17-month-old boy) and 3 (15-month-old girl) underwent adenoidectomy and tympanostomy placement tube. All patients underwent nasal brushing before general anesthesia (control sample). Experimental samples were taken in the contralateral nostril at the time of equilibration of the anesthetic agents (sevoflurane, propofol, sufentanil). Ciliary beat frequency and pattern were measured using digital high-speed videomicroscopy. Our findings highlighted the variability of respiratory ciliary function under general anesthesia among individuals. Our results emphasize the need for caution when interpreting ciliary function data obtained during general anesthesia. Further research with larger cohorts is warranted for validation. Full article

An application of CO₂/HCO₃⁻-free solution (Zero-CO₂) did not increase intracellular pH (pH_i) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pH_i of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pH_i of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pH_i of c-hNECs caused pH_i, CBF and CBA to decrease upon Zero-CO₂ application, while a low pH_i of c-hBECs caused them to increase. An extremely high pH_i was generated by a high rate of HCO₃⁻ influx via interactions between CAIV and Na⁺/HCO₃⁻ cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pH_i, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO₂ application. In conclusion, the interactions of CAIV and NBC maximize HCO₃⁻ influx to increase pH_i in c-hNECs. This novel mechanism causes pH_i to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO₂ application, and appears to play a crucial role in maintaining pH_i, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO₂) with respiration. Full article

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. Full article