Search Results (55)

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

Background and Objective: Primary ciliary dyskinesia (PCD) is a rare genetic disorder that affects the mucociliary system, leading to progressive lung damage. This deterioration can result in bronchiectasis, atelectasis, and respiratory failure, necessitating lung transplantation in severe cases. This study aims to assess ciliary motility and ultrastructure in the bronchial epithelium of transplanted lungs in patients with PCD to determine whether mucociliary function is preserved post-transplantation. The findings seek to enhance scientific understanding and provide prognostic insights for these patients. Materials and Methods: A prospective observational study was conducted on two patients with PCD and advanced lung disease who underwent bilateral lung transplantation. Nasal and bronchial cilia samples were analyzed using high-speed videomicroscopy and transmission electron microscopy. Follow-up assessments included ciliary function analysis, lung rejection monitoring, and quality-of-life evaluations, with follow-up extending up to 30 months post-transplant. Results: Post-transplant evaluations demonstrated normal ciliary motility and ultrastructure in the transplanted lungs throughout the study period (up to 30 months), indicating the long-term preservation of mucociliary function. Conclusions: Transplanted lungs in patients with PCD maintain normal bronchial ciliary motility and structure in the long term, suggesting a favorable prognosis for both the graft and the recipient. These findings support the feasibility and long-term effectiveness of lung transplantation in patients with PCD. Full article

Background and clinical significance: Ciliary body medulloepithelioma (CBME) is a rare germinal tumor deriving from nonpigmented ciliary epithelium, usually occurring during the first decade of life. Typically, the diagnosis is delayed as a result of the tumor’s slow growth and late onset of symptoms. Primary enucleation is commonly required; nevertheless, globe-sparing means of therapy have been successful in selected cases. CBME is among the spectrum of neoplasms associated with DICER1 cancer predisposition syndrome. Case presentation: Herein, we report a case of a 6-year-old boy with a history of pleuropulmonary blastoma type II and DICER1 mutation who presented with leukocoria in his right eye. After a thorough ophthalmological evaluation, he was diagnosed with CBME. Because of the large size of the lesion and vitreous seeding, the eye was enucleated. Histopathology confirmed the diagnosis of a benign teratoid medulloepithelioma. After 2 years of regular ophthalmological examinations, a new lesion was detected in the left eye. Three cycles of transscleral cryotherapy allowed for tumor control and globe salvage. The patient remains recurrence-free 6 months after the last treatment. Conclusions: This report should raise awareness among clinicians about the possibility of bilateral CBME and the necessity of regular ophthalmological screening in patients with DICER1 syndrome, especially those previously treated for CBME. Full article

The nasal epithelium is the primary site for entry of respiratory viruses. In comparison to oral administration, nasal drug applications directed locally to the site of infection can serve as early interventional barriers against respiratory virus pathogenesis by limiting viral spread in the upper airway. Experiments on the diffusion of methylene blue and nanoparticles in both water and low pH conditions revealed that hydroxypropyl methylcellulose (HPMC) can act as an effective physical barrier. This study also evaluated the activity of HPMC as a barrier against common respiratory viruses, i.e., rhinovirus (RV) and influenza A virus (IAV) using the in vitro human nasal epithelial cell (hNEC) model. Utilizing the hNEC infection model, we assessed the protective effects of HPMC in pH 3.5 and pH 7 buffers against RV and IAV. Acidic and pH-neutral buffers and HPMC dissolved in acidic and pH-neutral buffers were administered for 4 h prior to virus infection and at 4 h post-infection (hpi). The apical supernatant was harvested at 24 hpi to determine the viral loads of RV and IAV (H1N1 and H3N2). HPMC was demonstrated to exert protective effects in the infected hNECs independent of acidic pH. Pre-treatment with HPMC in acidic buffer significantly diminished viral loads for both RV and IAV infections of hNECs. Similarly, direct treatment of HPMC in acidic buffer after infection (4 hpi) also effectively decreased viral loads of both RV and IAV. Moreover, treatment using HPMC in acidic buffer before or after infection did not affect the epithelial integrity and ciliary function of hNECs. This study demonstrates the protective effects of HPMC in acidic buffer against RV and IAV infections of the human nasal epithelium. Full article

Intraocularly, fatty acid-binding protein 4 (FABP4) and 5 (FABP5) mainly originate from human ocular choroidal fibroblasts (HOCF), and human nonpigmented ciliary epithelium (HNPCE) cells have been suggested to be pivotally involved in intraocular pathophysiology. To elucidate the unidentified regulatory mechanisms of the gene expression and protein secretion of FABPs, the effects of glucose levels, fatty acids (FAs), and peroxisome proliferator-activated receptor (PPAR) modulators were studied. To elucidate the additional biological role of FABPs, laser choroidal neovascularization (CNV) in Fabp4^−/− and Fabp4/5^−/− mice was analyzed by fluorescein angiography. By changing glucose levels, the secretion and expression of FABP4 in HOCF were significantly upregulated, whereas the secretion and expression of FABP5 in HNPCE decreased. The administration of various FAs, particularly docosahexaenoic acid (DHA), markedly increased the expression and secretion of both FABPs. PPAR modulators also influenced the secretion and expression of FABPs. In vivo, wild-type retina exhibited evident CNV with high fluorescein intensity, while Fabp4^−/− retina showed reduced CNV formation and Fabp4/5^−/− retina displayed evident CNV along with vitreous leakage. These findings suggest that (1) the production and secretion of intraocular FABP4 and FABP5 are distinctly regulated by glucose levels, FAs, and PPARs; and (2) intraocular FABP4 and FABP5 are critical for inducing retinal neovascularization and maintaining the blood-aqueous barrier. Full article

The ability of a cell to keep its volume constant irrespective of intra- and extracellular conditions is essential for cellular homeostasis and survival. The purpose of this study is to elaborate a theoretical model of cell volume homeostasis and to apply it to a simulation of human aqueous humor (AH) production. The model assumes a cell with a spherical shape and only radial deformation satisfying the property that the cell volume in rest conditions equals that of the cell couplets constituting the ciliary epithelium of the human eye. The cytoplasm is described as a homogeneous mixture containing fluid, ions, and neutral solutes whose evolution is determined by net production mechanisms occurring in the intracellular volume and by water and solute exchange across the membrane. Averaging the balance equations over the cell volume leads to a coupled system of nonlinear ordinary differential equations (ODEs) which are solved using the $\theta$-method and the Matlab function ode15s. Simulation tests are conducted to characterize the set of parameters corresponding to baseline conditions in AH production. The model is subsequently used to investigate the relative importance of (a) impermeant charged proteins; (b) sodium–potassium (Na+/K+) pumps; (c) carbonic anhydrase (CA) in the AH production process; and (d) intraocular pressure. Results suggest that (a) and (b) play a role; (c) lacks significant weight, at least for low carbon dioxide values; and (d) plays a role for the elevated values of intraocular pressure. Model results describe a higher impact from charged proteins and Na+/K+ ATPase than CA on AH production and cellular volume. The computational virtual laboratory provides a method to further test in vivo experiments and machine learning-based data analysis toward the prevention and cure of ocular diseases such as glaucoma. 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

Polycystic ovarian syndrome (PCOS) is a heterogeneous condition characterized by hyperandrogenism (HA), polycystic ovaries, and dysfunctional ovulation, and it is associated with metabolic problems such as insulin resistance (IR) and obesity. After having investigated the morphological and antioxidant/antiglycative alterations on mouse ovaries and uteri, we here focus on PCOS oviducts, a tract of the reproductive system essential for the nourishment and transport of gametes and embryos. The modulating effects of L-carnitine (LC) and acetyl-L-carnitine (ALC) were also assessed. CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg of L-carnitine (LC) and 0.20 mg of acetyl-L-carnitine (ALC). Oviducts were then subjected to histology and immunohistochemistry to evaluate their morphology and collagen deposition, and steroidogenesis. Oxidative, mitochondrial, and methylglyoxal (MG)-dependent damage was also investigated. Transmission electron microscopy was used to detect ultrastructural alterations. The PCOS oviducts were affected by hyperfibrosis, hyperplasia, hypertrophy, and altered steroidogenesis, with oxidative alterations associated with MethylGlyoxal-Advanced Glycation End product (MG-AGE) accumulation. A reduced ciliary coverage and numerous dilated intercellular spaces were found in the epithelium. LC-ALC administration mitigated PCOS oviductal alterations. These results provide evidence for the detrimental action of oxidative and glycative stress in PCOS oviducts, confirming a protective role of carnitines on the PCOS phenotype. Full article

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE. Full article

Coronaviruses (CoVs) are a class of respiratory viruses with the potential to cause severe respiratory diseases by infecting cells of the upper respiratory tract, bronchial epithelium, and lung. The airway cilia are distributed on the surface of respiratory epithelial cells, forming the first point of contact between the host and the inhaled coronaviruses. The function of the airway cilia is to oscillate and sense, thereby defending against and removing pathogens to maintain the cleanliness and patency of the respiratory tract. Following infection of the respiratory tract, coronaviruses exploit the cilia to invade and replicate in epithelial cells while also damaging the cilia to facilitate the spread and exacerbation of respiratory diseases. It is therefore imperative to investigate the interactions between coronaviruses and respiratory cilia, as well as to elucidate the functional mechanism of respiratory cilia following coronavirus invasion, in order to develop effective strategies for the prevention and treatment of respiratory viral infections. This review commences with an overview of the fundamental characteristics of airway cilia, and then, based on the interplay between airway cilia and coronavirus infection, we propose that ciliary protection and restoration may represent potential therapeutic approaches in emerging and re-emerging coronavirus pandemics. Full article

Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air–liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses. Full article

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells. Full article

The impact of visual impairment, such as blindness, on quality of life is immeasurable. However, effective ocular drug delivery into the eyes has not yet been established, primarily due to the impermeability imposed by the blood–retinal barrier (BRB) based on the tight junctions and efflux transporters at the endothelium or the epithelium in oral or intravenous administration, as well as the dilution with tear fluid and excretion through the nasolacrimal duct in eye drop administration. Furthermore, intravitreous injections induce pain and fear in patients. Unmet medical needs persist in ocular diseases such as age-related macular degeneration and diabetic retinopathy. Therefore, innovative non-invasive administration methods should be developed. Drug-releasing soft contact lenses (DR-SCLs) affixed to the eye’s surface can continuously and locally deliver their loaded drugs to the eyes. The use of DR-SCLs is expected to greatly enhance the bioavailability and patient adherence to the drug regimen. It is known that several solute carrier (SLC) transporters are expressed in various parts of the eyes, including the cornea, the ciliary body, and the bulbar conjunctiva. Carrier-mediated transport through SLC transporters may occur in addition to passive diffusion. Moreover, nanoparticles can be loaded into DR-SCLs, offering various intelligent approaches based on modifications to induce receptor-mediated endocytosis/transcytosis or to control the loaded drug release within this delivery system. In this perspective review, I discuss the implementation and potential of DR-SCL-mediated ocular drug delivery, particularly focusing on low-molecular-weight compounds because of their fine distribution in living body, ease of handling, and ease of manufacturing. Full article

Nontuberculous mycobacteria (NTM) are environmental and ubiquitous, but only a few species are associated with disease, often presented as nodular/bronchiectatic or cavitary pulmonary forms. Bronchiectasis, airways dilatations characterized by chronic productive cough, is the main presentation of NTM pulmonary disease. The current Cole’s vicious circle model for bronchiectasis proposes that it progresses from a damaging insult, such as pneumonia, that affects the respiratory epithelium and compromises mucociliary clearance mechanisms, allowing microorganisms to colonize the airways. An important bronchiectasis risk factor is primary ciliary dyskinesia, but other ciliopathies, such as those associated with connective tissue diseases, also seem to facilitate bronchiectasis, as may occur in Lady Windermere syndrome, caused by M. avium infection. Inhaled NTM may become part of the lung microbiome. If the dose is too large, they may grow excessively as a biofilm and lead to disease. The incidence of NTM pulmonary disease has increased in the last two decades, which may have influenced the parallel increase in bronchiectasis incidence. We propose that ciliary dyskinesia is the main promoter of bronchiectasis, and that the bacteria most frequently involved are NTM. Restoration of ciliary function and impairment of mycobacterial biofilm formation may provide effective therapeutic alternatives to antibiotics. Full article

The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors’ cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance. Full article