Nasal Epithelium Research Articles

Etiology of placental insufficiency in women in the second trimester of gestation during the epidemic of influenza A(H3N2) and pandemic influenza(H1N1)pdm

Aim. To study the etiology of placental insufficiency in women in the second trimester of gestation during the influenza A(H3N2) epidemic and the A(H1N1)pdm pandemic.Materials and methods. In 1865 patients in the second trimester of pregnancy, uncomplicated and complicated by exacerbation of chronic ENT pathology leading to the development of placental insufficiency, using laboratory methods in the blood, nasal epithelium and biological fluids, pathogens of monoand mixed infections (influenza A(H1N1)pdm), A(H3N2), influenza B, parainfluenza type 1-3, adenovirus, respiratory syncytial virus (RS), cytomegalovirus (CMV), herpes simplex virus type 1 and type 2 (HSV-1,2 type), candida infection (Candida albicans), chlamydia (Chlamydia trachomatis), mycoplasmosis (Mycoplasma hominis) and ureaplasmosis (Ureaplasma urealyticum) were detected. The first group consisted of 873 women with placental insufficiency diagnosed during the influenza A(H3N2) epidemic, the second group consisted of 890 patients with placental insufficiency diagnosed during circulation of the influenza A(H1N1)pdm in the population. The control group were 42 women without ENT diseases and placental insufficiency caused by monoand mixed viral infections during influenza A(H3N2) and 60 women with similar somatic, infectious and obstetric status, examined during pandemic influenza A(H1N1)pdm.Results. With the development of placental insufficiency in women of the second group, compared with the first one, mono-influenza B and parainfluenza types 1-3 are diagnosed less frequently, as well as a combination of influenza A(H1N1) pdm and influenza B; influenza A(H1N1)pdm and parainfluenza types 1-3; influenza A(H1N1)pdm and RS virus; CMV and Chlamydia trachomatis; Mycoplasma hominis and Ureaplasma urealyticum; HSV-1,2 type, Candida albicans and Chlamydia trachomatis; as well as HSV-1,2 type, Candida albicans and Ureaplasma urealyticum. At the same time, exacerbations of mono-CMV, mono-HSV-1,2 type and Candida albicans are more common, as well as combinations of CMV+HSV-1,2 type; CMV+Candida albicans; CMV+Ureaplasma urealyticum; and CMV+Mycoplasma hominis; CMV+HSV-1,2 type+Candida albicans.Conclusion. In the etiology of placental insufficiency in acute diseases of the ENT organs in women in the second trimester of gestation against the background of circulation of the A(H1N1)pdm influenza in the population, compared with placental dysfunction in pregnant women with a similar ENT pathology during the influenza A(H3N2) epidemic, there is an increase in the proportion of monocytomegalovirus, herpesvirus and candida infections, as well as mixed cytomegalovirus infection, the reactivation of which may be due to more pronounced virusinduced immunosuppression and hormonal status disorders, leading to local dysbiosis.

Metakrylan 2,3-epoksypropylu (metakrylan glicydylu) Dokumentacja proponowanych dopuszczalnych wielkości narażenia zawodowego

2,3-Epoxypropyl methacrylate (glycidyl methacrylate, GMA) is an organic chemical compound, an ester of methacrylic acid and 2,3-epoxypropan-1-ol. It is a colorless liquid with a fruity odor. It is used as a co-monomer for the production of epoxy polymers, as an adhesion promoter and cross-linking co-monomer in the production of vinyl and acrylic resins. This substance is on the ACSH (The Advisory Committee on Safety and Health at Work) priority list for developing a proposal for an EU limit value. So far, no normative values in the air of the working environment have been defined in Poland in regard of 2,3-epoxypropyl methacrylate. The MAC value of 0,3 mg/m3 and STEL value of 0,6 mg/m3 have been proposed. The basis for calculating the MAC value was the LOAEC value of 3,55 mg/m3 for the irritation of the nasal epithelium in mice (olfactory epithelium metaplasia). Taking into account the carcinogenicity of GMA and its effect on the skin, the following notations have been proposed: “Carc. 1B” – carcinogenic substance of hazard category 1B, “C” – corrosive substance, “A” – skin sensitizing substance, “Ft” – fetotoxic substance, and “Skin” – skin absorption of the substance may be just as important as for inhalation exposure. There is no basis for setting BEI value.

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, includingthe cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

Red ginseng aqueous extract improves mucociliary transport dysfunction and histopathology in CF rat airways

BackgroundWe previously discovered that Korean red ginseng aqueous extract (RGAE) potentiates the TMEM16A channel, improved mucociliary transport (MCT) parameters in CF nasal epithelia in vitro, and thus could serve as a therapeutic strategy to rescue the MCT defect in cystic fibrosis (CF) airways. The hypothesis of this study is that RGAE can improve epithelial Cl− secretion, MCT, and histopathology in an in-vivo CF rat model. MethodsSeventeen 4-month old CFTR–/– rats were randomly assigned to receive daily oral control (saline, n = 9) or RGAE (Ginsenosides 0.4mg/kg/daily, n = 8) for 4 weeks. Outcomes included nasal Cl− secretion measured with the nasal potential difference (NPD), functional microanatomy of the trachea using micro-optical coherence tomography, histopathology, and immunohistochemical staining for TMEM16a. ResultsRGAE-treated CF rats had greater mean NPD polarization with UTP (control = -5.48 +/- 2.87 mV, RGAE = -9.49 +/- 2.99 mV, p < 0.05), indicating, at least in part, potentiation of UTP-mediated Cl− secretion through TMEM16A. All measured tracheal MCT parameters (airway surface liquid, periciliary liquid, ciliary beat frequency, MCT) were significantly increased in RGAE-treated CF rats with MCT exhibiting a 3-fold increase (control, 0.45+/-0.31 vs. RGAE, 1.45+/-0.66 mm/min, p < 0.01). Maxillary mucosa histopathology was markedly improved in RGAE-treated cohort (reduced intracellular mucus, goblet cells with no distention, and shorter epithelial height). TMEM16A expression was similar between groups. ConclusionRGAE improves TMEM16A-mediated transepithelial Cl− secretion, functional microanatomy, and histopathology in CF rats. Therapeutic strategies utilizing TMEM16A potentiators to treat CF airway disease are appropriate and provide a new avenue for mutation-independent therapies.

Age-differential CD13 and interferon expression in airway epithelia affect SARS-CoV-2 infection - Effects of vitamin D

Young age and high vitamin D plasma levels have been associated with lower SARS-CoV-2 infection risk and favourable disease outcomes. This study investigated mechanisms associated with differential responses to SARS-CoV-2 across age groups and effects of vitamin D. Nasal epithelia were collected from healthy children and adults and cultured for four weeks at the air-liquid interface with and without vitamin D. Gene expression and DNA methylation were investigated. Surface protein expression was confirmed by immunofluorescence while vitamin D receptor recruitment to the DNA was analysed through chromatin immunoprecipitation. HEp-2 cells were used for protein co-immunoprecipitation and luciferase reporter assays. Compared to children, airway epithelia from adults show higher viral RNA recovery following infection. This was associated with higher ANPEP/CD13, reduced type I interferon expression, and differential DNA methylation. In cells from adults, exposure to vitamin D reduced TTLL-12 expression, a negative regulator of the interferon response. This was mediated by vitamin D receptor recruitment to TTLL12, where it instructs DNA methylation through DNA methyltransferase 1. This study links age-dependent differential expression of CD13 and type I interferon to variable infection of upper airway epithelia. Furthermore, it provides molecular evidence for vitamin D reducing viral replication by inhibiting TTLL-12.

A brainstem circuit for the expression of defensive facial reactions in rat

The brainstem houses neuronal circuits that control homeostasis of vital functions. These include the depth and rate of breathing1,2 and, critically, apnea, a transient cessation of breathing that prevents noxious vapors from entering further into the respiratory tract. Current thinking is that this reflex is mediated by two sensory pathways. One known pathway involves vagal and glossopharyngeal afferents that project to the nucleus of the solitary tract.3,4,5 Yet, apnea induced by electrical stimulation of the nasal epithelium or delivery of ammonia vapors to the nose persists after brainstem transection at the pontomedullary junction, indicating that the circuitry that mediates this reflex is intrinsic to the medulla.6 A second potential pathway, consistent with this observation, involves trigeminal afferents from the nasal cavity that project to the muralis subnucleus of the spinal trigeminal complex.7,8 Notably, the apneic reflex is not dependent on olfaction as it can be initiated even after disruption of olfactory pathways.9 We investigated how subnucleus muralis cells mediate apnea in rat. By means of electrophysiological recordings and lesions in anesthetized rats, we identified a pathway from chemosensors in the nostrils through the muralis subnucleus and onto both the preBötzinger and facial motor nuclei. We then monitored breathing and orofacial reactions upon ammonia delivery near the nostril of alert, head-restrained rats. The apneic reaction was associated with a grimace, characterized by vibrissa protraction, wrinkling of the nose, and squinting of the eyes. Our results show that a brainstem circuit can control facial expressions for nocifensive and potentially pain-inducing stimuli.

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways

BackgroundAcute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects.MethodsPrimary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air–liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and l-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, l-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge.ResultsCS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and l-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline.ConclusionAltogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.

Nose to Brain Delivery of Transferrin conjugated PLGA nanoparticles for clonidine

The present work focuses on the development of surface modified transferrin PLGA nanoparticles loaded with clonidine for nose to brain delivery. The CLD-Tf-PLGA-NPs were developed using double emulsification, followed by solvent evaporation and characterization. Particle size, PDI and Zeta potential of the nanoparticles was 199.5 ± 1.36 nm, 0.291, −17.4 ± 6.29 mV respectively with EE% 86.2 ± 2.12 %, and DL%, 7.8 ± 0.48 %. TEM, SEM and FTIR analysis were carried out to confirm the size and transferrin coating over the surface of nanoparticles. In-vitro drug release profile were studied in PBS (pH 7.4) and SNF (pH 5.5) for 72 h and highest release was observed in PBS 89.54 ± 3.17 %. Cellular assays were conducted on Neuro-2a cells to check the cytotoxicity and uptake of Tf-modified PLGA nanoparticles and the cell viability% was obtained to be 61.85 ± 4.48 % even at maximum concentration (40Cmax) with uptake of approximately 97 %. Histopathological studies were also performed to identify the cytotoxicity on nasal epithelium along with in-vivo biodistribution and pharmacodynamics studies to assess the concentration of drug in the mice brain and behavioural responses after intranasal delivery of surface modified nanoparticles. The results showed significant increase in concentration of drug in brain and behavioural improvements in mice (p < 0.05).

Upper Respiratory Tract OC43 Infection Model for Investigating Airway Immune-Modifying Therapies.

Respiratory virus infections initiate and transmit from the upper respiratory tract (URT). Coronaviruses, including OC43, are a major cause of respiratory infection and disease. Failure to mount an effective antiviral immune response in the nasal mucosa increases the risk of severe disease and person-to-person transmission, highlighting the need for URT infection models to support the development of nasal treatments that improve coronavirus antiviral immunity. We aimed to determine if OC43 productively infected the mouse URT and would therefore be a suitable model to assess the efficacy and mechanism of action of nasal-targeting immune-modifying treatments. We administered OC43 via intranasal inoculation to wild-type Balb/c mice and assessed virus airway tropism (by comparing total respiratory tract vs. URT-only virus exposure) and characterized infection-induced immunity by quantifying specific antiviral cytokines and performing gene array assessment of immune genes. We then assessed the effect of immune-modulating therapies, including an immune-stimulating TLR2/6 agonist (INNA-X) and the immune-suppressing corticosteroid fluticasone propionate (FP). OC43 replicated in nasal respiratory epithelial cells, with peak viral RNA observed 2 days after infection. Prophylactic treatment with INNA-X accelerated expression of virus-induced IFN-λ and IFN-stimulated genes. In contrast, intranasal FP treatment increased nasal viral load by 2.4 fold and inhibited virus-induced IFN and IFN-stimulated gene expression. Prior INNA-X treatment reduced the immune-suppressive effect of FP. We demonstrate that the mouse nasal epithelium is permissive to OC43 infection and strengthen the evidence that TLR2 activation is a β-coronavirus innate immune determinant and therapeutic target.

Transfersome, an ultra-deformable lipid-based drug nanocarrier: an updated review with therapeutic applications.

The application of nanotechnology with integration of chemical sciences is increasing continuously in the management of diseases. The drug's physicochemical and pharmacological characteristics are enhanced by application of nanotechnological principles. Several nanotechnology-based formulations are being investigated to improve patient compliance. One such novel nanocarrier system is transfersome (TFS) and is composed of natural biocompatible phospholipids and edge activators. Morphologically, TFS are similar to liposomes but functionally, these are ultra-deformable vesicles which can travel through pores smaller than their size. Because of their amphipathic nature, TFS have the potential to deliver the drugs through sensitive biological membranes, especially the blood-brain barrier, skin layers, and nasal epithelium. Different molecular weight drugs can be transferred inside the cell by encapsulation into the TFS. Knowing the tremendous potentiality of TFS, the present work provides an in-depth and detailed account (pharmaceutical and preclinical characteristics) of TFS incorporating different categories of therapeutic moieties (anti-diabetic, anti-inflammatory, anti-cancer, anti-viral, anti-fungal, anti-oxidant, cardiovascular drugs, CNS acting drugs, vaccine delivery, and miscellaneous applications). It also includes information about the methods of preparation employed, significance of excipients used in the preparation, summary of clinical investigations performed, patent details, latest investigations, routes of administration, challenges, and future progresses related to TFS.

High-throughput bioprinting of the nasal epithelium using patient-derived nasal epithelial cells

Progenitor human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models through biofabrication. However, this approach has limitations in terms of achieving the intricate three-dimensional (3D) structure of the natural nasal epithelium. 3D bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of progenitor hNECs ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4 weeks air–liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes, such as disease modeling, immunological studies, and drug screening.

The viral fitness and intrinsic pathogenicity of dominant SARS-CoV-2 Omicron sublineages BA.1, BA.2, and BA.5

Among the Omicron sublineages that have emerged, BA.1, BA.2, BA.5, and their related sublineages have resulted in the largest number of infections. While recent studies demonstrated that all Omicron sublineages robustly escape neutralizing antibody response, it remains unclear on whether these Omicron sublineages share any pattern of evolutionary trajectory on their replication efficiency and intrinsic pathogenicity along the respiratory tract. We compared the virological features, replication capacity of dominant Omicron sublineages BA.1, BA.2 and BA.5 in the human nasal epithelium, and characterized their pathogenicity in K18-hACE2, A129, young C57BL/6, and aged C57BL/6 mice. We found that BA.5 replicated most robustly, followed by BA.2 and BA.1, in the differentiated human nasal epithelium. Consistently, BA.5 infection resulted in higher viral gene copies, infectious viral titres and more abundant viral antigen expression in the nasal turbinates of the infected K18-hACE2 transgenic mice. In contrast, the Omicron sublineages are continuously attenuated in lungs of infected K18-hACE2 and C57BL/6 mice, leading to decreased pathogenicity. Nevertheless, lung manifestations remain severe in Omicron sublineages-infected A129 and aged C57BL/6 mice. Our results suggested that the Omicron sublineages might be gaining intrinsic replication fitness in the upper respiratory tract, therefore highlighting the importance of global surveillance of the emergence of hyper-transmissive Omicron sublineages. On the contrary, replication and intrinsic pathogenicity of Omicron is suggested to be further attenuated in the lower respiratory tract. Effective vaccination and other precautions should be in place to prevent severe infections in the immunocompromised populations at risk. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Effect of TFF3 on tight junction protein in eosinophilic chronic sinusitis and its related mechanism

Objective: To study the effect of trefoil factor family (TFF) 3 on the expression of tight junctions (TJs) in the nasal mucosa epithelium of eosinophilic chronic rhinosinusitis (eCRS) and its mechanism. Methods: From September to December 2020, eligible patients from the Department of Otorhinolaryngology of the First Affiliated Hospital of Nanchang University were recruited, including 11 control patients and 37 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), from whom nasal mucosa and nasal polyp tissue samples were collected. Immunohistochemistry (IHC) was used to detect the localization and expression intensity of TFFs (TFF1, TFF2 and TFF3) and TJs (occudin, claudin-1 and ZO-1) in nasal mucosa. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the mRNA and protein expression. A cell model of tight junction injury in human nasal epithelial cells (HNECs) through stimulation with interleukin (IL)-13 was also established. The optimal modeling concentration and time for HNECs were determined, which were subsequently treated with TFF3 and/or a phosphoinositide 3-kinase (PI3K)-specific inhibitor (LY294002). Finally, RT-qPCR and WB were used to assess the effects of TFF3 on tight junctions and the PI3K/serine/threonine kinase (Akt) signaling pathway. Data were analyzed statistically using GraphPad Prism 7 software. Results: IHC results showed that the expression of TFF1 and TFF3 in nasal mucosa of eCRS group was significantly higher than that of control group (t=4.62, P=0.002; t=5.89, P<0.001), respectively, mainly expressed in goblet cell. The expression of occludin, claudin-1 and ZO-1 in the nasal mucosa of the eCRS group was lower than that of the control group (occludin t=3.98, P=0.019; claudin-1 t=5.15, P=0.002; ZO-1 t=5.42, P=0.001), respectively. WB results showed that the expression of TFF3 in non-eosinophilic chronic sinusitis (Non-eCRS) group and eCRS group was higher than that in the control group (t=3.62, P=0.036; t=5.93, P<0.001). The expression of occludin, claudin-1 and ZO-1 in eCRS group was lower than that in the control group (occludin t=5.14, P=0.002; claudin-1 t=6.35, P<0.001; ZO-1 t=6.64, P<0.001), respectively. The RT-qPCR results showed that compared with the control group, the levels of TFF1 and TFF3 mRNA were increased in the nasal mucosal epithelium of the Non-eCRS and eCRS groups (TFF1 t=3.98, P=0.046, t=4.89, P=0.002; TFF3 t=3.50, P=0.044, t=6.78, P<0.001). There was no statistically significant difference in TFF2 mRNA levels between the Non-eCRS and eCRS groups (t=1.34, P=0.061; t=3.37, P=0.055). Compared with the control group, Non-eCRS and eCRS groups showed a decrease in the mRNA levels of occludin, claudin-1 and ZO-1 (occludin t=4.27, P=0.011, t=5.61, P=0.007; claudin-1 t=3.62, P=0.036, t=6.80, P<0.001; ZO-1 t=3.47, P=0.047, t=7.86, P<0.001). The mRNA levels of TFF3 and TJs in eCRS nasal mucosa tissue showed a moderate positive correlation (occludin r=0.661, claudin-1 r=0.614, ZO-1 r=0.548, all P<0.001); TFF1 showed a low degree of positive correlation with the expression of occludin, claudin-1 and ZO-1 (occludin r=0.467, P=0.040; claudin-1 r=0.362, P=0.012; ZO-1 r=0.425, P=0.025). The establishment of cell models showed that compared with normal HNECs, the mRNA expression of TFF3 was most significantly increased at a concentration of 50 ng/ml stimulated by IL-13 (t=3.72, P=0.013); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.18, P=0.031; claudin-1 t=3.86, P=0.010; ZO-1 t=5.16, P=0.002). The expression of TFF3 mRNA increased most significantly after 15 hours of IL-13 stimulation (t=3.14, P=0.034); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.97, P=0.010; claudin-1 t=4.78, P=0.004; ZO-1 t=5.16, P=0.004). TJs damage model could be established by treating HNECs with 50 ng/ml IL-13 for 15 hours. Intervention experiments showed that compared with the IL-13 group, the IL-13+TFF3 group showed an increase in TJs mRNA expression (occludin t=6.10, P=0.009; claudin-1 t=5.90, P=0.013; ZO-1 t=9.44, P=0.007). Compared with the IL-13 group, the expression of TJs protein in the IL-13+TFF3 group increased (occludin t=3.23, P=0.013; claudin-1 t=9.40, P=0.017; ZO-1 t=2.23, P=0.032); The expression of TJs protein decreased in the IL-13+TFF3+LY294002 group (occludin t=4.73, claudin-1 t=8.77, ZO-1 t=3.51, all P<0.001). Compared with the IL-13+TFF3 group, the IL-3+TFF3+LY294002 group showed a decrease in PI3K and p-Akt/Akt protein expression (PI3K t=13.29, p-Akt/Akt t=10.30, all P<0.001). The increased mRNA and protein expression of occludin, claudin-1 and ZO-1 induced by TFF3 were also inhibited by LY294002. Conclusion: TFF3 can up-regulate the expression of occludin, claudin-1, and ZO-1 through PI3K/Akt pathway, and has a certain protective effect on the nasal mucosal epithelial barrier, providing a new idea for treating eCRS.

Novel DNA Promoter Hypermethylation in Nasal Epithelium of Asthma.

Abnormal epigenetic alterations influenced by external factors and affecting DNA expression contribute to the development of asthma. However, the role of the nasal epithelium in airway inflammation remains unknown. The objective of this study is to identify novel DNA promoter hypermethylation, which suppresses mRNA expression in nasal epithelial of asthma. Microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Gene expression and DNA promoter methylation sites in key correlated modules between asthma and normal were identified by weighted gene co-expression network analysis. Gene Oncology and Kyoto Encyclopedia of Genes and Genomes were conducted to analyse the function of genes. Further validation was performed in human BEAS-2B cells challenged by IL-4 or IL-13. Lightcyan, lightgreen, midnightblue, cyan and tan modules in the mRNA expression dataset showed a close relationship with asthma, in which genes were enriched in TNF, IL-17, ErbB, MAPK and Estrogen signalling pathways. Blue and turquoise modules in the methylation profiling dataset were associated with asthma. Forty nine lowly expressed genes were identified to be correlated with aberrant DNA hypermethylation of promoters. Among these genes, the mRNA levels of <i>BCL10, GADD45B</i>, <i>LSR</i> and </i>SQSTM1</i> were downregulated in BEAS-2B cells challenged with IL-4 or IL-13. Four potential genes in the nasal epithelium, by hypermethylating their own DNA promoter, might mediate the inflammatory response in the pathogenesis of asthma. Analyzing epigenomic data by integrated bioinformatics helps to understand the role of DNA methylation in asthma, with the goal of providing new perspectives for diagnosis and therapy.

Polysaccharide-peptide complexes stabilized around nanoemulsion droplets: A new approach for nasal delivering of pramlintide and insulin

Insulin (HIns) and pramlintide (Pram) are peptide drugs currently used to modulate blood glucose concentrations in diabetic patients. Glucose metabolism impairment has also been described to occur in the brain of diabetic and non-diabetic Alzheimer's patients. In this context, restoring the brain glucose balance by HIns and Pram administration has been proposed as a therapeutic approach for Alzheimer's disease treatment. However, despite their potential application in therapeutics, clinical use of these peptides has some drawbacks as their metabolic instability via protease degradation and undesirable pharmacokinetic properties, besides being delivered by invasive and inconvenient administration routes. Considering the above mentioned, in this study, we describe the development of Pram and HIns polyelectrolyte complexes stabilized around nanoemulsion droplets as a new approach to delivering these peptides by nasal route. Firstly, dextran sulfate/Pram (PECDexS/Pram) and chitosan/HIns (PECChi/HIns) polyelectrolyte complexes were prepared and characterized according to their physicochemical properties. Polyelectrolyte complexes-coated nanoemulsions (NE-PECDexS/Pram and NE-PECChi/HIns) were then obtained and characterized according to size, zeta potential, morphology, drug recovery, and association efficiency. Both NE-PECDexS/Pram and NE-PECChi/HIns showed desirable nanotechnological properties before and after incubation in a simulated nasal fluid pH 5.5 at 37 °C and during 30-day storage at 8 °C. Negatively charged NE-PECDexS/Pram showed improved mucodiffusion properties and led to a more sustained permeation profile of Pram across the nasal epithelium. On the contrary, the positively charged NE-PECChi/HIns led to a faster mucosal permeation of HInS compared to the free peptide. These results could support future in vivo studies concerning the potential use of these new drug delivery systems for treating metabolic brain disorders by the nasal route.

In vitro fitness of SARS-CoV-2 variants as assessed by competition experiments followed by ddRT-PCR and whole genome sequencing.

To develop a new method for reliable and rapid determination of the fitness of SARS-CoV-2 variants of concern. Competition experiments between two SARS-CoV-2 variants were performed in cells of the upper (nasal human airway epithelium) and lower (Calu-3 cells) respiratory tracts followed by quantification of variant ratios by droplet digital reverse transcription (ddRT)-PCR. In competition experiments, the delta variant outcompeted the alpha variant in both cells of the upper and lower respiratory tracts. A 50/50% mixture of delta and omicron variants indicated a predominance of omicron in the upper respiratory tract whereas delta predominated in the lower respiratory tract. There was no evidence of recombination events between variants in competition as assessed by whole gene sequencing. Differential replication kinetics were shown between variants of concern which may explain, at least partly, the emergence and disease severity associated with new SARS-CoV-2 variants.

H1N1 influenza virus infection through NRF2-KEAP1-GCLC pathway induces ferroptosis in nasal mucosal epithelial cells.

Influenza A virus can induce nasal inflammation by stimulating the death of nasal mucosa epithelium, however, the mechanism is not clear. In this study, to study the causes and mechanisms of nasal mucosa epithelial cell death caused by Influenza A virus H1N1, we isolated and cultured human nasal epithelial progenitor cells (hNEPCs) and exposed them to H1N1 virus after leading differentiation. Then we performed high-resolution untargeted metabolomics and RNAseq analysis of human nasal epithelial cells (hNECs) infected with H1N1 virus. Surprisingly, H1N1 virus infection caused the differential expression of a large number of ferroptosis related genes and metabolites in hNECs. Furthermore, we have observed a significant reduction in Nrf2/KEAP1 expression, GCLC expression, and abnormal glutaminolysis. By constructing overexpression vector of GCLC and the shRNAs of GCLC and Keap1, we determined the role of NRF2-KEAP1-GCLC signaling pathway in H1N1 virus-induced ferroptosis. In addition, A glutaminase antagonist, JHU-083, also demonstrated that glutaminolysis can regulate the NRF2-KEAP1-GCLC signal pathway and ferroptosis. According to this study, H1N1 virus can induce the ferroptosis of hNECs via the NRF2-KEAP1-GCLC signal pathway and glutaminolysis, leading to nasal mucosal epithelial inflammation. This discovery is expected to provide an attractive therapeutic target for viral-induced nasal inflammation.

Protein‐Based Face Mask with High SARS‐CoV‐2 Neutralization Ability and Breathability

AbstractCurrent intramuscular vaccines cannot effectively prevent severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) transmission and infection in nasal epithelium. Although face masks represent an additional preventive measure against SARS‐CoV‐2, their low breathability hinders wearing compliance. Herein, the development of SuperNeut, a protein‐based breathable high‐efficiency face mask, is reported. The mask contains KP‐LCB1, a fusion protein that can capture SARS‐CoV‐2 spike proteins on one end and attach to keratin fibres on the other. The meltblown fabric is replaced by a thin electrospun keratin layer that allows uniform adhesion of KP‐LCB1, which serves as an effective barrier to filter incoming viruses, while substantially improving breathability. The studies demonstrate that the fusion protein is stable and can efficiently neutralise multiple SARS‐CoV‐2 wildtype (WT), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529). The breathability of SuperNeut mask compared to commercial surgical masks is also quantified. Importantly, it is shown that SuperNeut Mask is able to effectively prevent SARS‐CoV‐2 Delta transmission in Syrian hamster model. Overall, the highly breathable SuperNeut mask can efficiently prevent SARS‐CoV‐2 transmission. This design serves as a proof‐of‐concept that face mask designs can be improved with biological components. The neutralising proteins in this mask can be altered to combat future respiratory disease outbreaks.

The immune mechanism of the nasal epithelium in COVID-19-related olfactory dysfunction.

During the first waves of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, olfactory dysfunction (OD) was reported as a frequent clinical sign. The nasal epithelium is one of the front-line protections against viral infections, and the immune responses of the nasal mucosa may be associated with OD. Two mechanisms underlying OD occurrence in COVID-19 have been proposed: the infection of sustentacular cells and the inflammatory reaction of the nasal epithelium. The former triggers OD and the latter likely prolongs OD. These two alternative mechanisms may act in parallel; the infection of sustentacular cells is more important for OD occurrence because sustentacular cells are more likely to be the entry point of SARS-CoV-2 than olfactory neurons and more susceptible to early injury. Furthermore, sustentacular cells abundantly express transmembrane protease, serine 2 (TMPRSS2) and play a major role in the olfactory epithelium. OD occurrence in COVID-19 has revealed crucial roles of sustentacular cells. This review aims to elucidate how immune responses of the nasal epithelium contribute to COVID-19-related OD. Understanding the underlying immune mechanisms of the nasal epithelium in OD may aid in the development of improved medical treatments for COVID-19-related OD.

Mechanisms of SARS-CoV-2-associated anosmia.

Anosmia, the loss of the sense of smell, is one of the main neurological manifestations of COVID-19. Although the SARS-CoV-2 virus targets the nasal olfactory epithelium, current evidence suggests that neuronal infection is extremely rare in both the olfactory periphery and the brain, prompting the need for mechanistic models that can explain the widespread anosmia in COVID-19 patients. Starting from work identifying the non-neuronal cell types that are infected by SARS-CoV-2 in the olfactory system, we review the effects of infection of these supportive cells in the olfactory epithelium and in the brain and posit the downstream mechanisms through which sense of smell is impaired in COVID-19 patients. We propose that indirect mechanisms contribute to altered olfactory system function in COVID-19-associated anosmia, as opposed to neuronal infection or neuroinvasion into the brain. Such indirect mechanisms include tissue damage, inflammatory responses through immune cell infiltration or systemic circulation of cytokines, and downregulation of odorant receptor genes in olfactory sensory neurons in response to local and systemic signals. We also highlight key unresolved questions raised by recent findings.