Search Results (24)

COVID-19 has triggered the rapid adoption of human organoid-based tissue culture models to overcome the limitations of the commonly used Vero cell line that did not fully recapitulate SARS-CoV-2 infection of human tissues. As the primary site of SARS-CoV-2 infection, the human nasal epithelium (HNE) cultivated in vitro and differentiated at air–liquid interface (ALI) is an ideal model to study infection processes and for testing anti-viral antibodies and drugs. However, the need for primary basal cells to establish the ALI-HNE limits the scalability of this model system. To try and bypass this bottleneck, we devised an ALI-differentiated form of the human adenocarcinoma cell line Calu-3, reported to model most aspects of authentic SARS-CoV-2 infection, including viral entry. The ALI-Calu-3 were tested for infection by a panel of SARS-CoV-2 variants, including ancestral (VIC01) and early pandemic lineages (VIC2089, Beta, Delta), and Omicron subvariants (BA2.75, BA4, BA5, XBB1.5). All tested lineages infected the ALI-HNE. In stark contrast, infection of the ALI-Calu-3 by Omicron subvariants BA4 and XBB1.5 was reduced. These data support the use of ALI-Calu-3 as a complementary, intermediary model for most but not all SARS-CoV-2 lineages, and places the ALI-HNE as the benchmark culture model for SARS-CoV-2 infection. 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

Background: Cholesteatoma, characterized by the abnormal growth of keratinizing squamous epithelium in ectopic locations, most commonly arises in the middle ear. Its occurrence in the sinonasal tract is rare and presents significant diagnostic and management challenges. These lesions can lead to severe complications like bone erosion, intracranial involvement, and orbital spread. This narrative review aims to summarize the current knowledge on cholesteatomas in these regions, focusing on epidemiology, pathophysiology, diagnosis, and treatment. Methods: A comprehensive review of the English literature was conducted, focusing on reported cases of cholesteatomas in the nasal cavity and paranasal sinuses. This review examines key aspects, including epidemiological data, imaging findings, surgical strategies, and postoperative outcomes. The role of diagnostic tools, particularly computed tomography and diffusion-weighted magnetic resonance imaging, in distinguishing cholesteatomas from other sinonasal lesions is also discussed. Results: As of March 2025, 51 cases of paranasal sinus cholesteatoma were reported. The frontal sinus is the most commonly affected site, followed by the maxillary, ethmoid, and sphenoid sinuses. Diagnosis is often delayed due to nonspecific symptoms, such as nasal congestion and recurrent infections. Surgical excision is the primary treatment, with endoscopic techniques being favored for their minimally invasive nature. Recurrence remains a major concern, and although very rare, cases of squamous cell carcinoma have also been observed in association with cholesteatoma. Conclusions: Nasal and paranasal sinus cholesteatomas require early recognition and intervention to prevent complications. Advances in imaging and surgery have improved outcomes; however, further research is needed to refine therapies and understand disease mechanisms. 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

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

Alterations of the BRAF gene and the resulting changes in the BRAF protein are one example of molecular cancer profiling in humans and dogs. We tested 227 samples of canine carcinomas from different anatomical sites (anal sac (n = 23), intestine (n = 21), liver (n = 21), lungs (n = 19), mammary gland (n = 20), nasal cavity (n = 21), oral epithelium (n = 18), ovary (n = 20), prostate (n = 21), thyroid gland (n = 21), urinary bladder (n = 22)) with two commercially available primary anti-BRAF^V600E antibodies (VE1 Ventana, VE1 Abcam). The immunohistochemical results were confirmed with droplet digital PCR (ddPCR). BRAF^V595E-mutated cases were found in canine prostatic (16/21), urothelial (17/22), and oral squamous cell carcinomas (4/18), while other carcinoma types tested negative. Both antibodies showed consistent results, with intracytoplasmic immunolabeling of tumour cells, making them reliable tools for detecting the BRAF^V595E mutation in canine carcinomas. In conclusion, identifying BRAF mutations from biopsy material offers a valuable opportunity to enhance cancer treatment strategies (BRAF inhibitors) in canine urothelial carcinomas, prostatic carcinomas, and oral squamous cell carcinomas. Full article

A recent estimate indicates that up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to associated symptoms, leading to a USD 50 billion annual loss of salary. Post-COVID (Long COVID) neurologic symptoms are due to the initial robust replication of SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation of the olfactory epithelium (OE) and the central nervous system (CNS), and the OE becoming a persistent infection site. Previously, our group showed that Epigallocatechin-3-gallate-palmitate (EC16) nanoformulations possess strong antiviral activity against human coronavirus, suggesting this green tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug to eliminate the persistent SARS-CoV-2 infection, leading to restored olfactory function and reduced inflammation in the CNS. The objective of the current study was to determine the compatibility of the nanoformulations with human nasal primary epithelial cells (HNpECs). Methods: Nanoparticle size was measured using the ZetaView Nanoparticle Tracking Analysis (NTA) system; contact antiviral activity was determined by TCID50 assay for cytopathic effect on MRC-5 cells; post-infection inhibition activity was determined in HNpECs; and cytotoxicity for these cells was determined using an MTT assay. The rapid inactivation of OC43 (a β-coronavirus) and 229E (α-coronavirus) viruses was further characterized by transmission electron microscopy. Results: A saline-based nanoformulation containing 0.1% w/v EC16 was able to inactivate 99.9999% β-coronavirus OC43 on direct contact within 1 min. After a 10-min incubation of infected HNpECs with a formulation containing drug-grade EC16 (EGCG-4′ mono-palmitate or EC16m), OC43 viral replication was inhibited by 99%. In addition, all nanoformulations tested for their effect on cell viability were comparable to normal saline, a regularly used nasal irrigation solution. A 1-min incubation of an EC16 nanoformulation with either OC43 or 229E showed an altered viral structure. Conclusion: Nanoformulations containing EC16 showed properties compatible with nasal application to rapidly inactivate SARS-CoV-2 residing in the olfactory mucosa and to reduce inflammation in the CNS, pending additional formulation and safety studies. Full article

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1. Full article

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations. Full article

Background and Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) presently remains a difficult disease to manage. Antimicrobial and defense proteins are important factors that could help characterize the role of microorganisms in CRSwNP pathogenesis, as the concept of microbial dysbiosis in CRS is still being considered. Our aim is to investigate the complex appearance, relative distribution and interlinks of human β defensin 2 (HBD-2), human β defensin 3 (HBD-3), human β defensin 4 (HBD-4), and cathelicidin LL 37 (LL 37) in chronic rhinosinusitis with nasal polyps (CRSwNP)-affected human nasal mucosa. Materials and Methods: The study group consisted of 48 samples from patients with CRSwNP. Samples were collected during functional endoscopic sinus surgery. The control group consisted of 17 normal healthy nasal mucosa samples gathered during routine septoplasty. β-defensin-2, β-defensin-3, β-defensin-4 and cathelicidin LL 37 in tissue were detected via immunohistochemical analysis. Results: HBD-2, HBD-3 and LL 37 were significantly decreased in epithelial cells in both primary and recurrent nasal polyp samples (p < 0.001) in comparison to control samples. HBD-2 was decreased in the subepithelial connective tissue of primary nasal polyp samples when compared to both recurrent polyp (p = 0.050) and control (p = 0.033) samples. In subepithelial connective tissue, significantly more HBD-3-positive structures were observed in primary nasal polyp samples (p = 0.049) than in control samples. In primary polyp samples, moderate correlations between connective tissue HBD-3 and connective (R = 0.584, p = 0.001) and epithelial tissue LL 37 (R = 0.556, p = 0.002) were observed. Conclusions: Decreased HBD-2, HBD-3 and LL 37 concentrations in the epithelium suggest a dysfunction of the epithelial barrier in patients with nasal polyps. Decreased subepithelial connective tissue HBD-2 suggests different responses to nasal microbiota in patients with primary nasal polyps compared to recurrent nasal polyps. Increased HBD-3 in subepithelial connective tissue suggests a possible role of this antimicrobial peptide in the pathogenesis of primary nasal polyps. Full article

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a typical type-2 inflammation involving several cytokines and is associated with epithelial cell dysfunction. Oncostatin M (OSM) (belonging to the interleukin(IL)-6 family) could be a key driver of epithelial barrier dysfunction. Therefore, we investigated the presence of OSM and IL-6 and the expression pattern of tight junctions (TJs) in the nasal tissue of CRSwNP patients and controls using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Then, their potential role in the epithelial barrier was evaluated in vitro in 27 different primary cultures of human nasal epithelial cells (HNECs) by measuring TJ expression and transepithelial electric resistance (TEER) with or without OSM or IL-6 (1, 10, and 100 ng/mL). The effect on ciliary beating efficiency was evaluated by high-speed videomicroscopy and on repair mechanisms with a wound healing model with or without OSM. OSM and IL-6 were both overexpressed, and TJ (ZO-1 and occludin) expression was decreased in the nasal polyps compared to the control mucosa. OSM (100 ng/mL) but not IL-6 induced a significant decrease in TJ expression, TEER, and ciliary beating efficiency in HNECs. After 24 h, the wound repair rate was significantly higher in OSM-stimulated HNECs at 100 ng/mL. These results suggest that OSM could become a new target for monoclonal antibodies. Full article

SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies. Full article

Allergic rhinitis (AR) is a common upper-airway inflammatory disease of the nasal mucosa caused by immunoglobulin (IgE)-mediated inflammation. AR causes various painful clinical symptoms of the nasal mucosa that worsen the quality of daily life, necessitating the urgent development of therapeutic agents. Herein, we investigated the effects of Caesalpinia sappan Linn. heartwood water extract (CSLW), which has anti-inflammatory and antioxidant properties, on AR-related inflammatory responses. We examined the anti-inflammatory and anti-allergic effects of CSLW in ovalbumin (OVA)-induced AR mice and in primary human nasal epithelial cells (HNEpCs). Administration of CSLW mitigated allergic nasal symptoms in AR mice, decreased total immune cell and eosinophil counts in nasal lavage fluid, and significantly reduced serum levels of OVA-specific IgE, histamine, and Th2 inflammation-related cytokines. CSLW also inhibited the infiltration of several inflammatory and goblet cells, thereby ameliorating OVA-induced thickening of the nasal mucosa tissue. We found that CSLW treatment significantly reduced infiltration of eosinophils and production of periostin, MUC5AC, and intracellular reactive oxygen species through the Keap1/Nrf2/HO-1 pathway in HNEpCs. Thus, our findings strongly indicate that CSLW is a potent therapeutic agent for AR and can improve the daily life of patients by controlling the allergic inflammatory reaction of the nasal epithelium. Full article

The nasal epithelium is a key portal for infection by respiratory viruses such as SARS-CoV-2 and represents an important target for prophylactic and therapeutic interventions. In the present study, we test the safety and efficacy of a newly developed nasal spray (AM-301, marketed as Bentrio) against infection by SARS-CoV-2 and its Delta variant on an in vitro 3D-model of the primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in pre-viral load and post-viral load application on airway epithelium. No toxic effects of AM-301 on the nasal epithelium were found. Prophylactic treatment with AM-301 significantly reduced viral titer vs. controls over 4 days, reaching a maximum reduction of 99% in case of infection from the wild-type SARS-CoV-2 variant and more than 83% in case of the Delta variant. When AM-301 administration was started 24 h after infection, viral titer was reduced by about 12-folds and 3-folds on Day 4. The results suggest that AM-301 is safe and significantly decelerates SARS-CoV-2 replication in cell culture inhibition assays of prophylaxis (pre-viral load application) and mitigation (post-viral load application). Its physical (non-pharmaceutical) mechanism of action, safety and efficacy warrant additional investigations both in vitro and in vivo for safety and efficacy against a broad spectrum of airborne viruses and allergens. Full article

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium. Full article