Nasal Epithelium Research Articles

Rare presence and function of neuroendocrine cells in the nasal mucosa.

There is growing evidence that neurogenic inflammation contributes to the pathophysiology of upper airway diseases, with nasal hyperreactivity (NHR) being a key symptom. The rare neuroendocrine cells (NECs) in the epithelium have been linked to the pathophysiology of bronchial and intestinal hyperreactivity, however their presence in the nasal mucosa and their potential role in NHR remains unclear. Therefore, we studied the presence of NECs in the nasal epithelium of controls, allergic rhinitis patients and chronic rhinosinusitis with nasal polyps patients, and their link to NHR. The expression of typical NECs markers, CHGA, ASCL1 and CGRP, were evaluated on gene and protein level in human samples using real-time quantitative PCR (RT-qPCR), western blot, immunohistochemistry fluorescence staining, RNA scope assay, flow cytometry and single cell RNA-sequencing. Furthermore, the change in peak nasal inspiratory flow after cold dry air provocation and visual analogue scale scores were used to evaluate NHR or disease severity, respectively. Limited gene expression of the NECs markers CHGA and ASCL1 was measured in patients with upper airway diseases and controls. Gene expression of these markers did not correlate with NHR severity nor disease severity. In vitro, CHGA and ASCL1 expression was also evaluated in primary nasal epithelial cell cultures from patients with upper airway disease and controls using RT-qPCR and western blot. Both on gene and protein level only limited CHGA and ASCL1 expression was found. Additionally, NECs were studied in nasal biopsies of patients with upper airway diseases and controls using immunohistochemistry fluorescence staining, RNA scope and flow cytometry. Unlike in ileum samples, CHGA could not be detected in nasal biopsies of patients with upper airway diseases and control subjects. Lastly, single cell RNA-sequencing of upper airway tissue could not identify a NEC cluster. In summary, in contrast to the bronchi and gut, there is only limited evidence for the presence of NECs in the nasal mucosa, and without correlation with NHR, thereby questioning the relevance of NECs in upper airway pathology.

THE 72-KDA PROTEIN OF NAEGLERIA FOWLERI PLAYS AN IMPORTANT ROLE IN THE ADHESION OF TROPHOZOITES TO BALB/C MICE NASAL EPITHELIUM.

Naegleria fowleri is a protozoan that causes primary amebic meningoencephalitis (PAM). The infection occurs when the trophozoites enter the nasal cavity, adhere to the nasal mucosa, invade the epithelium, and migrate until they reach the olfactory bulb. Like other pathogens, there is evidence that the adhesion of N. fowleri to host cells is an important factor in the process of cytopathogenicity and disease progression. However, the factors involved in the adhesion of the pathogen to the cells of the nasal epithelium have not been characterized. The objective of this study was to identify a protein on the surface of N. fowleri, which could act as adhesin to the mouse nasal epithelium in the PAM model. The interaction between proteins of extracts of N. fowleri and cells of the nasal epithelium of BALB/c mice was analyzed using overlay and Western blot assays. A 72-kDa band of N. fowleri interacted directly with epithelial cell proteins, this polypeptide band was purified and analyzed by mass spectrometry. Analysis revealed that polypeptide bands of 72 kDa contained peptides that matched the membrane protein, actin 1 and 2, and Hsp70. Moreover, the N. fowleri extracts resolved in 2D-SDS-PAGE showed that 72-kDa spot interacted with proteins of mouse epithelial cells, which include characteristics of the theoretical data of molecular weight and pH obtained in the analysis by mass spectrometry. Immunofluorescence tests showed that this protein is located on the surface of trophozoites and plays an important role in the adhesion of amoeba either in vitro or in vivo assays, suggesting that this protein contributes during the N. fowleri invasion and migration to the brain, causing primary amoebic meningoencephalitis.

Transcriptomic analysis of primary nasal epithelial cells reveals altered interferon signalling in preterm birth survivors at one year of age.

Introduction: Many survivors of preterm birth (<37weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-β [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.

Nasal Epithelium Transcriptomics Predict Clinical Response to Elexacaftor/Tezacaftor/Ivacaftor.

Elexacaftor/tezacaftor/ivacaftor (ETI) has made a substantial positive impact for people living with CF (pwCF). However, there can be substantial variability in efficacy, and we lack adequate biomarkers to predict individual response. We thus aimed to identify transcriptomic profiles in nasal respiratory epithelium that predict clinical response to ETI treatment. We obtained nasal epithelial samples from pwCF prior to ETI initiation and performed a transcriptome-wide analysis of baseline gene expression to predict changes in FEV1 (∆FEV1), year's best FEV1 (∆ybFEV1), and body mass index (∆BMI). Using the top differentially expressed genes (DEGs), we generated transcriptomic risk scores (TRS) and evaluated their predictive performance. The study included 40 pwCF aged ≥6 years (mean 27.7 [SD=15.1] years; 40% female). After ETI initiation, FEV1 improved ≥5% in 22 (61.1%) participants and ybFEV1 improved ≥5% in 19 (50%). TRS were constructed using top over-expressed and under-expressed genes for each. Adding the ∆FEV1 TRS for to a model with age, sex, and baseline FEV1 increased the AUC from 0.41 to 0.88; the ∆ybFEV1 TRS increased the AUC from 0.51 to 0.88; and the ∆BMI TRS increased the AUC from 0.46 to 0.92. Average accuracy was thus ~85% in predicting the response to the three outcomes. Results were similar in models further adjusted for F508del zygosity and previous CFTR modulator use. In conclusion, we identified nasal epithelial transcriptomic profiles that help accurately predict changes in FEV1 and BMI with ETI treatment. These novel TRS could serve as predictive biomarkers for clinical response to modulator treatment in pwCF.

Sanfeng Tongqiao Dripping Pills alleviate House Dust Mite-induced allergic rhinitis in mice by inhibiting Th2 differentiation and repairing the nasal epithelial barrier

BackgroundSanfeng Tongqiao Dripping Pills (SFTQ) has clinically demonstrated a promising therapeutic effect on allergic rhinitis (AR). However, the active ingredients and underlying mechanisms of SFTQ remain unclear. PurposeExploring the effects, mechanisms, and active ingredients of SFTQ in the treatment of AR is valuable. Study designThe mechanisms of SFTQ and its active ingredients in treating AR were investigated through in vivo and in vitro studies. MethodsA HDM-induced AR model was established in BALB/c mice. The effects of SFTQ in treating AR were evaluated by AR-like symptoms, EOS count, and pathological changes in the nasal tissue in vivo. The effects of SFTQ active components on epithelial cells (ECs) were evaluated in Poly(I:C) and TNF-α co-stimulated human nasal ECs (RPMI-2650). Additionally, the effects of SFTQ active components on splenocytes proliferation and Th cell differentiation were assessed. A co-culture system of ECs and T lymphocytes was established to investigate the impact of Th2 cells on the structure and function of ECs. The effects of SFTQ ingredients on ECs, T lymphocytes, and the HDM-induced AR model were further confirmed through in vivo and in vivo studies, respectively. ResultsSFTQ significantly alleviated AR-like symptoms and pathological changes in the nasal tissue of AR mice. The treatment elevated the expression of Occludin and E-cadherin in the nasal epithelium and reduced the percentage of Th2 cells in cervical lymph nodes (CLN). Among the active compounds of SFTQ, L-Menthone and Pulegone notably downregulated IL-33 levels in activated ECs, while Hesperetin significantly decreased TSLP and IL-33 levels. In the co-culture system of ECs and Th2 cells, exposure to Baicalin, Wogonin, and Pulegone increased the TEER value of ECs, while notably inhibiting the production of TSLP and IL-33. Furthermore, in HDM-induced AR mice, treatments with Baicalin, Luteolin, and Hesperetin effectively inhibited AR-like symptoms. Additionally, Luteolin and Hesperetin significantly reduced the inflammatory cells infiltration and the population of Th2 cells in AR mice. ConclusionSFTQ and its active ingredients effectively alleviated HDM-induced AR in mice by inhibiting Th2 cell differentiation and repairing the nasal epithelial barrier. Our study can provide a scientific basis for SFTQ to be used in clinical treatment of AR.

Mutagenicity evaluation of methyl tertiary- butyl ether in multiple tissues of transgenic rats following whole body inhalation exposure.

Methyl tertiary-butyl ether (MTBE) is used as a component of motor vehicle fuel to enhance combustion efficiency and to reduce emissions of carbon monoxide and nitrogen oxides. Although MTBE was largely negative in the in vitro and in vivo genotoxicity studies, isolated reports of positive findings along with the observation of tumors in the rat cancer bioassays raised concern for its in vivo mutagenic potential. To investigate this, transgenic male Big Blue Fischer 344 rats were exposed to 0 (negative control), 400, 1000, and 3000 ppm MTBE via whole body inhalation for 28 consecutive days, 6 h/day. Mutant frequencies (MF) at the cII locus of the transgene in the nasal epithelium (portal of entry tissue), liver (site of primary metabolism), bone marrow (rapidly proliferating tissue), and kidney (tumor target) were analyzed (5 rats/exposure group) following a 3-day post-exposure manifestation period. MTBE did not induce a mutagenic response in any of the tissues investigated. The adequacy of the experimental conditions to detect induced mutations was confirmed by utilizing tissue samples from animals treated with the known mutagen ethyl nitrosourea. These data provide support to the conclusion that MTBE is not an in vivo mutagen and male rat kidney tumors are not likely the result of a mutagenic mode of action.

PLUNC inhibits invasion and metastasis in nasopharyngeal carcinoma by inhibiting NLRP3 inflammasome activation

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx. Palate, lung, and nasal epithelium clone (PLUNC) has been identified as an early secreted protein that is specifically expressed in the nasopharynx. The aim of this study was to determine the role and mechanism of PLUNC in NPC. We used mRNA sequencing (seq) combined with ribosome-nascent chain complex (RNC)-seq to determine the biological role of PLUNC. The expression of epithelial-to-mesenchymal transition (EMT)-related molecules was detected by western blotting. Then, cell migration and invasion were detected by wound healing and Transwell chamber assays. NPC cells were injected into the tail vein of nude mice to explore the biological role of PLUNC in vivo. The sequencing results showed that PLUNC inhibited the progression of NPC and its expression was correlated with that of NOD-like receptors. Experiments confirmed that PLUNC inhibited the invasion and metastasis of NPC cells by promoting the ubiquitination degradation of NLRP3. PLUNC overexpression in combination with the treatment by MCC950, an inhibitor of NLRP3 inflammasome activation, was most effective in inhibiting NPC invasion and metastasis. In vivo experiments also confirmed that the combination of PLUNC overexpression and MCC950 treatment effectively inhibited the lung metastasis of NPC cells. In summary, our research suggested that PLUNC inhibited the invasion and metastasis of NPC by inhibiting NLRP3 inflammasome activation, and targeting the PLUNC-NLRP3 inflammasome axis could provide a new strategy for the diagnosis and treatment of NPC patients.

Long-Term PM2.5 Exposure and Upregulation of CLCA1 Expression in Nasal Epithelium from Youth with Asthma.

Little is known about long-term PM2.5 exposure and airway epithelial gene expression. To test for association between long-term PM2.5 exposure and nasal epithelial gene expression in youth with asthma. Transcriptome-wide association study (TWAS) of long-term PM2.5 in nasal epithelium from youth aged 6-20 years in the: 1) Epigenetic Variation and Childhood Asthma in Puerto Ricans study (EVA-PR, n=182); 2) Vitamin D Kids Asthma Study (VDKA, n=58); and 3) Stress and Treatment Response in Puerto Rican and African American Children with Asthma study (STAR, n=81). Satellite hybrid models were used to estimate PM2.5 exposure in the prior year at each participant's residence. Multivariable negative binomial regression was used for each TWAS, adjusting for age, sex, and other covariates. A meta-analysis of all TWAS results was then conducted using an inverse variance-weighted average approach. Most participants (~95%) in the meta-analysis of TWAS for PM2.5 exposure identified as Puerto Rican or Black. Long-term PM2.5 was associated with: 1) upregulated expression of CLCA1 (calcium-activated chloride channel regulator 1, false discovery rate-adjusted P [FDR-P]=0.008), SYCP2 (synaptonemal complex protein 2, FDR-P=0.01), and CYP2A6 (cytochrome p450 family 2 subfamily A member 6, FDR-P=0.02), and 2) downregulated expression of EDAR (ectodysplasin A receptor, FDR-P=0.01). In a meta-analysis, CLCA1 upregulation was associated with ≥1 positive allergen-specific IgE (FDR-P <0.001) and increased blood eosinophils (FDR-P <0.001) and total IgE (FDR-P <0.001). In a meta-analysis of TWASs in predominantly Puerto Rican and Black youth with asthma, long-term PM2.5 exposure was associated with upregulated airway epithelial CLCA1 expression, in turn linked to biomarkers of T2-high immunity.

Cyclodextrin inclusion complex and amorphous solid dispersions as formulation approaches for enhancement of curcumin’s solubility and nasal epithelial membrane permeation

BackgroundCurcumin is a compound that occurs in the rhizomes of the turmeric plant (Curcuma longa) and has shown potential for the treatment of illnesses including certain neurodegenerative diseases. The bioavailability of curcumin is hindered by its extremely poor aqueous solubility.ResultsThis study aimed to apply formulation strategies such as inclusion complex formation with hydroxypropyl-β-cyclodextrin (HPβCD), as well as amorphous solid dispersion (ASD) formation with poly(vinylpyrrolidone-co-vinyl acetate) (PVP VA64) and hydroxypropyl methylcellulose (HPMC) to increase curcumin’s solubility and thereby its nasal epithelial membrane permeation. The curcumin formulations were evaluated by means of DSC, TGA, FT-IR, XRPD, microscopic imaging, aqueous solubility and membrane permeation across nasal respiratory and olfactory epithelial membranes. The solubility of curcumin was substantially increased by the formulations from 8.4 µg/ml for the curcumin raw material to 79.0 µg/ml for the HPβCD inclusion complex, 256.4 µg/ml for the HPMC ASD and 314.9 µg/ml for the PVP VA64 ASD. The HPMC ASD only slightly changed the membrane permeation of curcumin, while the PVP VA64 ASD decreased the membrane permeation of curcumin. The HPβCD inclusion complex enhanced the nasal epithelial membrane permeation of curcumin statistically significantly across the olfactory epithelial tissue and extensively across the respiratory epithelial tissue.ConclusionComplexation of curcumin with HPβCD enhanced the solubility of curcumin and thereby also increased its permeation across excised nasal respiratory and olfactory epithelial tissue. This indicated high potential of the curcumin-HPβCD complex for nose-to-brain delivery of curcumin for treatment of neurodegenerative diseases by means of intranasal administration.Graphical abstract

An in vitro study of the impact of IL-17A and IL-22 on ciliogenesis in nasal polyps epithelium via the Hippo-YAP pathway

Cilia loss and impaired motile ciliary functions are one of the typical pathological features of chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin-17A (IL-17A) and interleukin-22 (IL-22) are the canonical cytokines of type 3 inflammation, exhibiting similar functional effects on epithelial cells. In this study, we sought to examine the effects of IL-17A and IL-22 on ciliated cells and investigate the potential involvement of Hippo-Yes-associated protein (YAP) signaling in their influence on ciliogenesis. We assessed both the mRNA and protein expression levels of IL-17A and IL-22 in nasal tissues obtained from patients with CRSwNP and compared them to those from healthy controls. To further explore the impact of IL-17A and IL-22, we established a primary human nasal epithelial cell (hNEC) model using different concentrations (2 ng/mL, 10 ng/mL, 50 ng/mL) for a duration of 28 days in an air-liquid interface (ALI) culture. Additionally, we employed the inhibitor verteporfin (VP) to investigate whether IL-17A andIL-22 exert their effects on ciliated cells via Hippo-YAP pathway. The mRNA and protein levels of IL-17A and IL-22 in CRSwNP were significantly higher than those in healthy controls, revealing a robust correlation between IL-17A and IL-22. YAP was highly expressed in the nucleus of ciliated cells in CRSwNP and displayed a positive correlation with clinical symptoms. Both IL-17A and IL-22 were found to reduce the number of ciliated cells. IL-17A, but not IL-22, suppressed ciliogenesis by disrupting the proper development and docking of the basal body of ciliated cells, resulting in motile ciliary dysfunctions. Furthermore, the expression of YAP within the nucleus of ciliated cells gradually declined as these cells reached the final stage of differentiation. However, this process was obstructed by IL-17A only. YAP inhibitors, such as Verteporfin, markedly reversed the effects of IL-17A by increasing the proportion of ciliated cells, suppressing nuclear YAP expression in these cells, and enhancing ciliary beating frequency. Both IL-17A and IL-22 are overexpressed in nasal epithelium of CRSwNP, which is associated with the impairment of epithelial cell differentiation. Furthermore, IL-17A has been shown to exert a disruptive effect on morphogenesis of motile cilia via activation of YAP.

Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

Establishment of a human nasal epithelium model of histamine-induced inflammation to assess the activity of fexofenadine as an inverse agonist and its link to clinical benefit.

Fexofenadine (FEX) is an antihistamine that acts as an inverse agonist against histamine (HIS) receptor 1 (H1R), which mediates the allergic reaction. Inverse agonists may be more potent than neutral antagonists, as they bind the same receptor as the agonist (HIS) but stabilize the inactive form and induce an opposite pharmacological response, suppressing the basal activity of H1R and preventing HIS from binding. This study aims to establish and validate a model of HIS-induced inflammation based on fully reconstituted human nasal epithelial tissue to assess the activity of FEX as an inverse agonist in this model and explore its link to clinical benefit. The model was developed using nasal MucilAir™ (Epithelix) in vitro epithelium challenged by HIS. Two conditions were assessed in a side-by-side comparison: tissue was exposed to HIS + FEX with or without FEX pre-treatment (one-hour prior to HIS challenge). Tissue functionality, cytotoxicity, H1R gene expression, and inflammatory cytokines were assessed. HIS at 100µM induced significant 3.1-fold and 2.2-fold increases for inflammatory biomarkers interleukin (IL)-8 and IL-6, respectively (p < 0.0001), as well as rapid upregulation of H1R mRNA. Inflammatory biomarkers were inhibited by FEX and H1R expression was significantly reduced (p < 0.0001). FEX alone decreased H1R expression at all doses tested. With one-hour FEX pre-treatment, there was significantly higher downregulation of IL-8 (p < 0.05) and further downregulation of H1R expression and IL-6 versus without FEX pre-treatment; the effects of FEX were improved from 22% to 40%. A model of HIS-induced airway inflammation was established based on IL-8, IL-6 and H1R gene expression and was validated with FEX. FEX works as an inverse agonist, with a higher effect when used before+during versus only during the HIS challenge. Taking FEX before+during allergen exposure, or when symptoms first occur, may reduce basal activity and H1R gene expression, providing stronger protection against the worsening of symptoms upon allergen exposure.

Welfare Assessment in Pigs Using the Salivary Proteome.

Identifying the potential presence of stress at the pig farm is fundamental since it affects pig welfare. As a result, a reliable and straightforward tool to monitor stress could record the welfare status of the animals. Although numerous methods to assess the welfare of pigs have been developed in the past, no gold standard has been established yet. Recently, the value of saliva as a tool to identify chronic stress in piglets was explored, as it can be collected fast and non-invasively. Since the protein composition, i.e., the proteome of porcine saliva, responds to stress, the affected proteins could be used as salivary stress biomarkers. The present review first defines stress and its relationship with welfare. Next, the porcine gland-specific salivary proteome is characterized. Finally, six potential salivary biomarkers for stress are proposed, i.e., odorant-binding protein, vomeromodulin-like protein, chitinase, lipocalin-1, long palate lung and nasal epithelium protein, and alpha-2-HS-glycoprotein.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Primary Amoebic Meningoencephalitis with Progression to Brain Death Following Naegleria Fowleri Infection in a Teenage Female

Abstract Naegleria fowleri is a high-temperature freshwater-living amoeba that causes primary amoebic meningoencephalitis (PAM) by invading nasal epithelium. N. fowleri lives ubiquitously in high-temperature freshwater, but only around 400 cases of PAM have been published worldwide. The mortality rate is 98%. No treatment is 100% effective. A previously healthy 17-year-old female presented to an urgent care center with fever, headache, sore throat, ear pain, and dizziness. She had swum in freshwater 5 days prior. Her symptoms progressed to altered mental status, photo- and phonophobia, and neck stiffness. Within 4 days, she developed increased intracranial pressure (ICP) and eventually brain death. Two unsuccessful lumbar punctures were attempted before the third provided cerebrospinal fluid (CSF) for polymerase chain reaction (PCR) analysis. Magnetic resonance imaging showed diffuse cerebral edema, effacement of basal cisterns, tonsillar herniation with diffuse loss of gray–white matter differentiation, leptomeningitis, bifrontal encephalitis with evolving frontal lobe cortical infarcts, and ventriculitis. She was treated with metronidazole, vancomycin, ceftriaxone, acyclovir, and doxycycline. Her increased ICP progressed to brain death, and she died 11 days after lake exposure. CSF PCR was reported positive for N. fowleri the day after her death. Despite advances in diagnostic testing for N. fowleri with PCR, mortality rate is high and current treatments are highly ineffective. This case highlights the importance of epidemiological exposure and considering PAM on the differential diagnosis. Although headache and fever are benign symptoms, they could also represent the first stages of a deadly disease and their progression should be addressed promptly.

Multi-omics in nasal epithelium reveals three axes of dysregulation for asthma risk in the African Diaspora populations

Asthma has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omic signatures of asthma focusing on populations of African ancestry. RNASeq and DNA methylation data are generated from nasal epithelium including cases (current asthma, N = 253) and controls (never-asthma, N = 283) from 7 different geographic sites to identify differentially expressed genes (DEGs) and gene networks. We identify 389 DEGs; the top DEG, FN1, was downregulated in cases (q = 3.26 × 10−9) and encodes fibronectin which plays a role in wound healing. The top three gene expression modules implicate networks related to immune response (CEACAM5; p = 9.62 × 10−16 and CPA3; p = 2.39 × 10−14) and wound healing (FN1; p = 7.63 × 10−9). Multi-omic analysis identifies FKBP5, a co-chaperone of glucocorticoid receptor signaling known to be involved in drug response in asthma, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. This work reveals molecular dysregulation on three axes – increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response – that may play a critical role in asthma within the African Diaspora.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Uncovering strain- and age-dependent innate immune responses to SARS-CoV-2 infection in air-liquid-interface cultured nasal epithelia

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Gold Nanoparticles: Tunable Characteristics and Potential for Nasal Drug Delivery.

A general procedure to prepare gold nanourchins (GNUs) via a seed-mediated method was followed using dopamine hydrochloride as a reducing agent and silver nitrate salt (AgNO3) as a shape-directing agent. The novelty of this study comes from the successful incorporation of the prepared gold urchins as an aqueous suspension in a nasal pressurized metered dose inhaler (pMDI) formulation and the investigation of their potential for olfactory targeting for direct nose-to-brain drug delivery (NTBDD). The developed pMDI formulation was composed of 0.025% w/w GNUs, 2% w/w Milli-Q water, and 2% w/w EtOH, with the balance of the formulation being HFA134a propellant. Particle integrity and aerosolization performance were examined using an aerosol exposure system, whereas the nasal deposition profile was tested in a sectioned anatomical replica of human nasal airways. The compatibility of the gold dispersion with the nasal epithelial cell line RPMI 2650 was also investigated in this study. Colloidal gold was found to be stable following six-month storage at 4 °C and during the lyophilization process utilizing a pectin matrix for complete re-dispersibility in water. The GNUs were intact and discrete following atomization via a pMDI, and 13% of the delivered particles were detected beyond the nasal valve, the narrowest region in the nasal cavity, out of which 5.6% was recovered from the olfactory region. Moreover, the formulation was found to be compatible with the human nasal epithelium cell line RPMI 2650 and excellent cell viability was observed. The formulated GNU-HFA-based pMDI is a promising approach for intranasal drug delivery, including deposition in the olfactory region, which could be employed for NTBDD applications.

SARS-CoV-2-Induced Type I Interferon Signaling Dysregulation in Olfactory Networks Implications for Alzheimer's Disease.

Type I interferon signaling (IFN-I) perturbations are major drivers of COVID-19. Dysregulated IFN-I in the brain, however, has been linked to both reduced cognitive resilience and neurodegenerative diseases such as Alzheimer's. Previous works from our group have proposed a model where peripheral induction of IFN-I may be relayed to the CNS, even in the absence of fulminant infection. The aim of our study was to identify significantly enriched IFN-I signatures and genes along the transolfactory route, utilizing published datasets of the nasal mucosa and olfactory bulb amygdala transcriptomes of COVID-19 patients. We furthermore sought to identify these IFN-I signature gene networks associated with Alzheimer's disease pathology and risk. Gene expression data involving the nasal epithelium, olfactory bulb, and amygdala of COVID-19 patients and transcriptomic data from Alzheimer's disease patients were scrutinized for enriched Type I interferon pathways. Gene set enrichment analyses and gene-Venn approaches were used to determine genes in IFN-I enriched signatures. The Agora web resource was used to identify genes in IFN-I signatures associated with Alzheimer's disease risk based on its aggregated multi-omic data. For all analyses, false discovery rates (FDR) <0.05 were considered statistically significant. Pathways associated with type I interferon signaling were found in all samples tested. Each type I interferon signature was enriched by IFITM and OAS family genes. A 14-gene signature was associated with COVID-19 CNS and the response to Alzheimer's disease pathology, whereas nine genes were associated with increased risk for Alzheimer's disease based on Agora. Our study provides further support to a type I interferon signaling dysregulation along the extended olfactory network as reconstructed herein, ranging from the nasal epithelium and extending to the amygdala. We furthermore identify the 14 genes implicated in this dysregulated pathway with Alzheimer's disease pathology, among which HLA-C, HLA-B, HLA-A, PSMB8, IFITM3, HLA-E, IFITM1, OAS2, and MX1 as genes with associated conferring increased risk for the latter. Further research into its druggability by IFNb therapeutics may be warranted.