Nasal Epithelial Cells Of Patients Research Articles

Bioinformatics analysis of nasal epithelial cell gene expression in seasonal and perennial allergic rhinitis

Objective: Transcriptome sequencing and bioinformatics analysis were performed on the gene expression of nasal epithelial cells in patients with seasonal allergic rhinitis (AR) and perennial AR, so as to obtain the differences in the gene expression of nasal epithelial cells between seasonal AR and perennial AR. Methods: The human nasal epithelial cell line(HNEpC) was cultured in vitro, treated with 100 μg/ml mugwort or house dust mite (HDM) extracts for 24 hours. Total cell RNA was extracted, and quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression of cytokines, including IL-6, IL-8, IL-33 and thymic stromal lymphopoietin (TSLP). From November 2019 to November 2020, 3 seasonal AR patients, 3 perennial AR patients, and 3 healthy controls who attended the Department of Otolaryngology Head and Neck Surgery, China-Japan Union Hospital of Jilin University were analyzed. The patients' primary nasal epithelial cells were cultured in vitro, treated with corresponding allergens for 24 hours. Total RNA was extracted for transcriptome sequencing, and the sequencing results were analyzed by bioinformatics. Results: The qPCR results showed that the cytokines IL-6, IL-8, IL-33 and TSLP of HNEpC treated with mugworts extracts and HDM extracts had the same trend of change. After the nasal epithelial cells from patients with seasonal AR and perennial AR were treated with corresponding allergens, there were differences in biological processes and signal pathways between those and control. Gene ontology (GO) enrichment analysis showed that the differentially expressed genes (DEG) in AR patients allergic to mugwort were mainly enriched in the oxidation-reduction process, the negative regulation of apoptosis process, and the cell adhesion; the DEG in AR patients allergic to HDM were mainly enriched in cell adhesion, the negative regulation of cell proliferation and the response to drug. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway showed that the DEG of AR patients allergic to mugwort were significantly enriched in arachidonic acid metabolism, p53 signaling pathway and transforming growth factor β (TGF-β) signaling pathway, while the DEG of AR patients allergic to HDM were mainly enriched in cells cycle, Fanconi anemia pathway and DNA replication. Gene Set Enrichment Analysis (GSEA) showed that the inflammatory response, TNF-α/NF-κB signaling pathway and IL-2/STAT5 signaling pathway were significantly up-regulated in AR patients allergic to mugwort, indicating the promotion of inflammatory response; and AR patients allergic to HDM had significant down-regulation of G2M, E2F, and MYC, indicating the inhibition of cell proliferation. The protein-protein interaction network showed that TNF and CDK1 were the most interacting proteins in mugwort and HDM allergic AR patients, respectively. Conclusion: Seasonal AR and perennial AR may affect the different biological processes and signal pathways of nasal epithelial cells, leading to differences in the occurrence and development of AR.

Increased Expression of TXNIP Facilitates Oxidative Stress in Nasal Epithelial Cells of Patients With Chronic Rhinosinusitis With Nasal Polyps.

Oxidative stress plays crucial roles in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Thioredoxin-interacting protein (TXNIP) is essential in the process of triggering oxidative stress. However, its role and mechanism in CRSwNP remain unclear. The present study sought to explore the role and mechanism of TXNIP in the pathogenesis of CRSwNP. Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess TXNIP, thioredoxin (TRX) expression in nasal tissue samples from patients with CRSwNP and control subjects. MDA level and SOD activity in nasal tissue homogenates were measured using MDA and SOD Assay Kit. To evaluate the role and mechanism of TXNIP in CRSwNP, human nasal epithelial cells (HNECs) were cultured and stimulated using TXNIP siRNA, with or without N-acetylcysteine (NAC, an ROS scavenger). Western blotting, real-time PCR, ROS detecting dye DCFH-DA, MDA and SOD Assay Kit were performed to assess the effects and mechanisms of stimulators on the cells. We found significantly increased levels of TXNIP and decreased levels of TRX protein, mRNA, positive cells, increased MDA level and decreased SOD activity in CRSwNP patients compared with control subjects. In vitro study, significantly altered levels of TXNIP, TRX, MDA, SOD and ROS in HNECs were found following treatment of TXNIP siRNA with or without NAC on HNECs. TXNIP expression was increased and TRX expression was decreased in CRSwNP at both protein and mRNA levels. MDA levels were increased and SOD activities were decreased in CRSwNP. TXNIP may have negative association with TRX, and then decrease SOD activities and increase MDA levels, resulting in the upregulation of ROS and oxidative stress in HNECs, which may play a pivotal role in the pathogenesis of CRSwNP. Future studies are expected to further explore the role and mechanism of TXNIP in CRSwNP.

Reduced A20 expression in nasal epithelial cells correlates with disease severity in patients with chronic rhinosinusitis with nasal polyps

Abstract Objective Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial disorder characterized by heightened and sustained inflammation. A20 is a novel multifunctional protein with immunomodulatory effects. We sought to investigate the expression of A20 in sinonasal mucosa, and its association with local inflammation and disease severity in patients with CRSwNP. Methods The expression of A20 in sinonasal samples was assessed by quantitative RT-PCR, IHC, and Western blot. The numbers of local inflammatory cells were counted. CT scan findings and symptom severity were scored. Primary nasal epithelial cells were cultured to explore the regulation of A20 expression. Results Compared with control tissues and non-eosinophilic polyps, the mRNA and protein expression of A20 was significantly down-regulated in eosinophilic polyps. A20 was mainly expressed by epithelial cells. The expression of A20 in nasal epithelial cells was significantly reduced in eosinophilic polyps in comparison with those in non-eosinophilic polyps and control tissues. There was a further decrease in A20 expression in nasal epithelial cells in patients with eosinophilic polyps with asthma compared to those without asthma. A20 mRNA was induced by TNF-α, LPS, dsRNA, and dexamethasone, whereas was inhibited by IL-4 and IL-13 in human primary nasal epithelial cells. A20 mRNA levels in nasal epithelial cells negatively correlated with IL-4 and IL-13 levels and eosinophil numbers in sinonasal mucosa. Importantly, mRNA levels of A20 in nasal epithelial cells negatively correlated with disease duration and so on. Conclusions The decreased expression of A20 might contribute to eosinophilic inflammation and disease progression in patients with CRSwNP.

Hypomethylation of the IL8 promoter in nasal epithelial cells of patients with chronic rhinosinusitis with nasal polyps

IL-8 is an important chemokine implicated in the pathogenesis of chronic rhinosinusitis (CRS), but little is known about epigenetic regulation of IL8 in the pathogenesis of CRS. We sought to investigate the relationship between the DNA methylation level in the IL8 proximal promoter and CRS in Han Chinese subjects. Patients with chronic rhinosinusitis with nasal polyps (CRSwNP; n=187), patients with chronic rhinosinusitis without nasal polyps (CRSsNP; n=89), and control subjects (n=57) were enrolled in 2 independent cohorts. Purified human nasal epithelial cells from each participant were assessed for percentage DNA methylation of CpG sites in the IL8 proximal promoter by using bisulfite pyrosequencing and for functional aspects of methylation status by using invitro assays. DNA methylation of CpG sites 1, 2, and 3, respectively, in the IL8 proximal promoter was significantly decreased in human nasal epithelial cells of patients with CRSwNP compared with that in patients with CRSsNP (P<.001) and control subjects (P<.001). Percentage of DNA methylation of the CpG3 site was correlated negatively with both tissue eosinophilic cationic protein (P<.01) and myeloperoxidase (P<.05) levels. IL-1β (P<.001) and TNF-α (P<.01) significantly increased IL8 expression accompanied by a reduction in methylation at the CpG3 site (P<.001). Electrophoretic mobility shift assays demonstrated that methylation status of CpG3 changed the binding of octamer-binding transcription factor 1 and nuclear factor κB. Decreased DNA methylation of particularly CpG sites in the IL8 proximal promoter might play a role in the pathogenesis of CRSwNP.

Експресія HSP70 у епітеліальних клітинах слизової оболонки носа пацієнтів із хронічним поліпозним риносинуситом

Експресія HSP70 у епітеліальних клітинах слизової оболонки носа пацієнтів із хронічним поліпозним риносинуситом

Blocking histone deacetylase activity as a novel target for epithelial barrier defects in patients with allergic rhinitis

A defective epithelial barrier is found in patients with allergic rhinitis (AR) and asthma; however, the underlying mechanisms remain poorly understood. Histone deacetylase (HDAC) activity has been identified as a crucial driver of allergic inflammation and tight junction dysfunction. We investigated whether HDAC activity has been altered in patients with AR and in a mouse model of house dust mite (HDM)-induced allergic asthma and whether it contributed to epithelial barrier dysfunction. Primary nasal epithelial cells of control subjects and patients with AR were cultured at the air-liquid interface to study transepithelial electrical resistance and paracellular flux of fluorescein isothiocyanate-dextran (4kDa) together with mRNA expression and immunofluorescence staining of tight junctions. Air-liquid interface cultures were stimulated with different concentrations of JNJ-26481585, a broad-spectrum HDAC inhibitor. Invivo the effect of JNJ-26481585 on mucosal permeability and tight junction function was evaluated in a mouse model of HDM-induced allergic airway inflammation. General HDAC activity was greater in nasal epithelial cells of patients with AR and correlated inversely with epithelial integrity. Treatment of nasal epithelial cells with JNJ-26481585 restored epithelial integrity by promoting tight junction expression and protein reorganization. HDM-sensitized mice were treated with JNJ-26481585 to demonstrate the invivo role of HDACs. Treated mice did not have allergic airway inflammation and had no bronchial hyperreactivity. Moreover, JNJ-26481585 treatment restored nasal mucosal function by promoting tight junction expression. Our findings identify increased HDAC activity as a potential tissue-injury mechanism responsible for dysregulated epithelial cell repair, leading to defective epithelial barriers in AR. Blocking HDAC activity is a promising novel target for therapeutic intervention in patients with airway diseases.

&lt;i&gt;Staphylococcus aureus&lt;/i&gt; Enterotoxin B Disrupts Nasal Epithelial Barrier Integrity via TLR2 Activation

Background: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been shown to contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP), but the pathogenic mechanisms are largely unknown. Methods: We investigated the effect of SEB on nasal epithelial function in vitro on primary polyp and nasal epithelial cells of patients with CRSwNP and healthy controls respectively, and in vivo in a mouse model by measuring epithelial integrity and tight junction expression. The involvement of TLR2 signaling was studied in vitro by using TLR2 monoclonal antibodies and in vivo in tlr2-/- transgenic mice. Findings: We found that in vitro stimulation with SEB decreased polyp epithelial integrity by reducing occludin and ZO-1 protein expression. This impaired barrier function was associated with elevated expression of epithelial TLR2 and antagonizing TLR2 triggering prevented SEB-induced barrier disruption in vitro. In wild type mice, SEB applied in the nose increased mucosal permeability and decreased occludin and ZO-1 mRNA expression, whereas tlr2-/- mice had an intact mucosal barrier and normal tight junction expression after SEB exposure. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6, IL-8 and TNF, which was prevented when TLR2 triggering was antagonized. Interpretation: Our results indicate that SEB impairs epithelial barrier function in CRSwNP via TLR2 activation. Interfering with TLR2 activation may provide a way to avoid the pathophysiological consequences of S. aureus colonization in CRSwNP. Funding Statement: The author’s laboratories are supported by grants from the Belgian Federal Government (IUAP P7/30), IWT (TBM project 130260) and the research council of the KU Leuven (GOA 14/011). PWH, RS and DMB are recipients of a senior researcher fellowship from the Fund of Scientific Research (FWO), Flanders, Belgium. BS is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO). Declaration of Interests: The authors declare no competing interest related to this work. Ethics Approval Statement: All experiments were approved by the Medical Ethical Committee of the University Hospitals Leuven. Murine experiments were approved by the Ethical Committee for Animal Research at the KU Leuven.

Tc17/IL-17A Up-Regulated the Expression of MMP-9 via NF-κB Pathway in Nasal Epithelial Cells of Patients With Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease of the upper airways involving nasal cavity and sinus. Deriving both from its clinical complexity with protean clinical manifestations as well its pathogenetic heterogeneity, the molecular mechanisms contributing to the pathogenesis of CRS remain unclear, and attract a wide interest in the field. Current evidences indicate that IL-17A is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNP). However, its pathogenetic role in regulation of tissue remodeling of CRSwNP remains unknown. The present study aimed to investigate the cellular origins and functions of IL-17A cytokine in CRSwNP, and further determined whether IL-17A could affect the expression of metalloproteinases (MMPs), the remodeling factors of CRSwNP. The results showed that the expression of IL-17A was upregulated in nasal tissues of patients with CRSwNP compared to those with chronic rhinosinusitis without nasal polyps (CRSsNP) and controls. CD8+ cytotoxic T lymphocytes (Tc) were major IL-17A producers in nasal tissues of CRSwNP. Interleukin (IL)-17-producing CD8+ T cells (Tc17) was significantly higher in nasal tissues of CRSwNP than CRSsNP and controls. Nonetheless, no difference was observed among the IL-17A in peripheral blood lymphocytes of these three groups. Moreover, in the same patients, IL-17A expression was negligible in lymphocytes of peripheral blood when compared with nasal tissues. Increased gene and protein expression of MMP-7 and MMP-9 in patients with CRSwNP compared with controls were observed. In CRSwNP samples, IL-17A receptor (IL-17AR) co-localized with MMP-9 and they were mainly expressed in the epithelial cells. MMP-9 expression was up-regulated both in Primary human nasal epithelial cells (PHNECs) and a nasal epithelial cell line (RPMI 2650) by IL-17A treatment, and diminished by anti-IL-17AR treatment. Furthermore, IL-17A promoted the expression of MMP-9 by activating the NF-κB signal pathway. Thus, our results have revealed a crucial role of IL-17A and Tc cells on pathogenesis and tissue remodeling of CRSwNP.

Can Sevoflurane Induce Micronuclei Formation in Nasal Epithelial Cells of Adult Patients?

Volatile anaesthetics can inhibit the bronchociliary clearence in a dose- and time-dependend way. Moreover, they can have potential mutagenic/carcinogenic effects under chronic exposure. A genotoxicity test -micronuclei assay- was carried out in nasal epithelial cells to analyze the genotoxic effect of sevoflurane in adult patients undergoing general anesthesia. In this study, micronucleus (MN) assay was conducted using nasal epithelial cells of 37 adult patients (age, 18-65 years) who underwent elective, minor, short surgical procedures under general anaesthesia with sevoflurane. Anaesthesia was induced and maintained using 8% sevoflurane (in 6 L min-1 of oxygen) and an inspired concentration of 2% in O2-air mixture, respectively. Nasal epithelial samples were collected at three time points: before anaesthesia induction (T1), after recovery from anaesthesia in the postanaesthesia care unit (T2) and on postoperative day 21 (T3). Sevoflurane significantly increased mean MN (‰) frequencies in nasal epithelial cells at T2 (6.97±2.33) and T3 (6.22±2.47) compared with those at T1 (3.84±1.89) (p<0.001). Similar result were observed for MN frequencies if the patients were analysed with regard to age (>40 or <40 years) or sex. Short-term administration of sevoflurane anaesthesia induces MN formation in nasal epithelial cells of this patient population. Further studies are required for evaluation of the results. The prolonged administration of volatile anaesthetics in various risk groups and surgical protocols should be conducted for evaluating their safety.

Digitoxin for Airway Inflammation in Cystic Fibrosis: Preliminary Assessment of Safety, Pharmacokinetics, and Dose Finding

Cystic fibrosis (CF) lung disease progresses by a combination of airway inflammation, bacterial colonization, and infection. Airway inflammation is predominantly neutrophilic and complicates airway clearance therapies through cellular debris; excessive DNA; excessive and viscous mucus; and high concentrations of neutrophils, IL-8, and related cytokines liberated along the nuclear factor-κB signaling pathway. We conducted a preliminary, single-site, randomized, double-blind, placebo-controlled study to evaluate the effects over 28 days of two dose levels (0.05 mg and 0.1 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on safety, pharmacokinetics, and inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease. Patients with CF 18-45 years old with any genotype combination were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure (1) the pharmacokinetics of digitoxin in sera of patients with stable CF; (2) safety indices, including ECG changes and sputum microbiology; (3) the effect of digitoxin on gene expression in nasal epithelial cells of patients with stable CF; and (4) quality-of-life scores using the Cystic Fibrosis Questionnaire-Revised. It took several weeks to achieve a therapeutic serum level of digitoxin in subjects with CF. No safety concerns emerged during the study. Digitoxin treatment showed a trend toward reduction in sputum free neutrophil elastase and neutrophil counts, but not a reduction in sputum IL-8. Digitoxin treatment did not reach statistical significance for the primary or secondary outcome measures over the 28-day study period. However, the nasal mRNA from the group receiving 0.1 mg of digitoxin daily had a distinct distribution of global gene expression levels as compared with either the 0.05-mg dose or placebo treatment. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment, and mucus hypersecretion. At a dose of 0.1 mg daily for 28 days, digitoxin was safe for adults with CF lung disease, but it did not achieve a significant decrease in sputum inflammatory markers. Clinical trial registered with www.clinicaltrials.gov (NCT00782288).

Endogenous Protease Inhibitors in Airway Epithelial Cells Contribute to Eosinophilic Chronic Rhinosinusitis

Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen-induced production of IL-25, IL-33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA-induced pathology. Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.

WS06.2 Altered epithelial Cl− secretion in primary human nasal epithelial cells of patients with chronic rhinosinusitis

WS06.2 Altered epithelial Cl− secretion in primary human nasal epithelial cells of patients with chronic rhinosinusitis

Epithelial cell-derived micro RNA-146a generates interleukin-10-producing monocytes to inhibit nasal allergy.

The aberrant immunity plays an important role in the pathogenesis of allergic diseases. Micro RNAs (miR) are involved in regulating the immunity in the body. This study aims to test a hypothesis that miR-146a induces the expression of interleukin (IL)-10 in monocytes (Mos). In this study, the levels of miR-146a were determined by real time RT-PCR. The IL-10+ Mos were evaluated by flow cytometry. The miR-146a-laden exosomes were generated with RPMI2650 cells (an airway epithelial cell line). An allergic rhinitis mouse model was developed. The results showed that nasal epithelial cells expressed miR-146a, which was markedly lower in the nasal epithelial cells of patients with nasal allergy than that in healthy controls. Exposure to T helper (Th)2 cytokines suppressed the levels of miR-146a in the nasal epithelial cells. The nasal epithelial cell-derived miR-146a up regulated the expression of IL-10 in Mos. The inducible IL-10+ Mos showed an immune suppressor effect on the activities of CD4+ effector T cells and the Th2 polarization in a mouse model of allergic rhinitis. In summary, nasal epithelial cells express miR-146a, the latter is capable of inducing IL-10 expression in Mos, which suppress allergic reactions in the mouse nasal mucosa.

The effect of Epigallocatechin-3-gallate in allergic airway inflammation

Background: The variety of beneficial effects of green tea has been reported. Epigallocatechin-3-gallate (EGCG) is a major component of green tea extract. The biological activity of EGCG includes anti-allergic and anti-inflammatory activities. However, the precise effect of EGCG on the allergic airway inflammation has not been fully defined. Methodology: In lung tissues of an asthma model-mouse, and nasal epithelial cells of patients with allergic rhinitis, the effect and brief signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction, immunohistochemical, and immunoblot analysis. Results: In the asthma model-mouse, mucus production, MUC5B expression, p38 mitogen-activated protein kinase (MAPK) and matrix metalloprotease (MMP)-9 expression of the asthma group were significantly higher than in the control group. Extracellular signal related kinase (ERK)1/2 MAPK expression did not change. Mucus production, MUC5B expression, p38 MAPK expression, and MMP-9 expression of the asthma + EGCG group were significantly lower than in the asthma group. In the nasal epithelial cells of patients with allergic rhinitis, EGCG significantly decreased phorbol 12-myristate 13-acetate (PMA)-induced MUC5B and MMP-9 expression. Conclusion: These results suggest that EGCG reduces mucin expression in the asthma model-mouse and nasal epithelial cells of patients with allergic inflammation.

The effect of Epigallocatechin-3-Gallate in allergic airway inflammation

The variety of beneficial effects of green tea has been reported. Epigallocatechin-3-gallate (EGCG) is a major component of green tea extract. The biological activity of EGCG includes anti-allergic and anti-inflammatory activities. However, the precise effect of EGCG on the allergic airway inflammation has not been fully defined. In lung tissues of an asthma model-mouse, and nasal epithelial cells of patients with allergic rhinitis, the effect and brief signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction, immunohistochemical, and immunoblot analysis. In the asthma model-mouse, mucus production, MUC5B expression, p38 mitogen-activated protein kinase (MAPK) and matrix metalloprotease (MMP)-9 expression of the asthma group were significantly higher than in the control group. Extracellular signal related kinase (ERK)1/2 MAPK expression did not change. Mucus production, MUC5B expression, p38 MAPK expression, and MMP-9 expression of the asthma + EGCG group were significantly lower than in the asthma group. In the nasal epithelial cells of patients with allergic rhinitis, EGCG significantly decreased phorbol 12-myristate 13-acetate (PMA)-induced MUC5B and MMP-9 expression. These results suggest that EGCG reduces mucin expression in the asthma model-mouse and nasal epithelial cells of patients with allergic inflammation.

11 A new multiplex PCR method for the quantification of aberrant transcripts from nasal epithelial cells of patients

11 A new multiplex PCR method for the quantification of aberrant transcripts from nasal epithelial cells of patients

A critical role of IL-33 in experimental allergic rhinitis

We reported previously that serum levels of IL-33 are significantly increased in patients with allergic rhinitis (AR). However, very little is known about the role of IL-33 for the development of AR. We thought to develop a novel murine model of ragweed pollen-specific AR and examined the pathologic role for ragweed-induced IL-33 in the development of AR manifestation using IL-33-deficient (il33(-/-)) mice. Ragweed-immunized and ragweed-challenged mice were examined for early- and late-phase nasal responses. IL-33 protein expression in the nasal epithelial cells of the AR murine model and patients with AR were assessed by using confocal microscopy. After nasal challenge with ragweed pollen, ragweed-immunized wild-type mice manifested early-phase (sneezing) and late-phase (eosinophilic and basophilic accumulation) responses. In contrast, il33(-/-) and FcεRI(-/-) mice did not have both early- and late-phase AR responses. IL-33 protein was constitutively expressed in the nucleus of nasal epithelial cells and was promptly released into nasal fluids in response to nasal exposure to ragweed pollen. In human subjects we revealed constitutive expression of IL-33 protein in the nasal epithelial cells of healthy control subjects and downregulated expression of IL-33 protein in inflamed nasal epithelial cells of patients with AR. IL-33-stimulated mast cells and basophils contributed to the early- and late-phase AR manifestation through increasing histamine release and production of chemoattractants for eosinophils/basophils, respectively. Ragweed pollen-driven endogenous IL-33 contributed to the development of AR responses. IL-33 might present an important therapeutic target for the prevention of AR.

TGF-β Signaling May Play a Role in the Development of Goblet Cell Hyperplasia in a Mouse Model of Allergic Rhinitis

Transforming growth factor-beta (TGF-beta) levels are elevated in the nasal mucosa in allergic rhinitis. However, because TGF-beta is secreted extracellulary in latent complexes, it remains unclear whether the local TGF-beta expression actually drives active signaling and affects the pathophysiology of allergic rhinitis. The objective of this study is to investigate whether TGF-beta signaling is activated in allergic rhinitis and plays a role in the pathophysiology of allergic rhinitis. An ovabumin (OVA)-sensitized and -nasally challenged mouse model of allergic rhinitis was established and phosphorylation of Smad2 in the nasal mucosa was examined by immunohistochemistry. In addition, the effects of the pharmacological inhibition of endogenous TGF-beta signaling on the allergic rhinitis model were histologically examined. Furthermore, phosphorylation of Smad2 in the nasal mucosa samples obtained from patients with allergic rhinitis was also evaluated. In the mouse model of allergic rhinitis, OVA challenge induced phosphorylation of Smad2 predominantly in epithelial cells in the nasal mucosa. In addition, the administration of an inhibitor of TGF-beta type I receptor kinase activity during OVA challenge suppressed goblet cell hyperplasia in the nasal mucosa. Furthermore, phosphorylated Smad2 expression increased in nasal epithelial cells in patients with allergic rhinitis. These results suggest that TGF-beta signaling is activated in epithelial cells in the nasal mucosa in allergic rhinitis and may contribute to the development of goblet cell hyperplasia.

Increased expression of HLA‐DR and CD86 in nasal epithelial cells in allergic rhinitics: antigen presentation to T cells and up‐regulation by diesel exhaust particles

Summary Background A proportion of nasal epithelial cells (NEC) in patients with allergic rhinitis (AR) are known to express the major histocompatibility complex Class II molecule (HLA‐DR). Objective We hypothesized that NEC may play a role in antigen presentation to T cells. To elucidate the possible role of NEC in antigen presentation, we examined the expression of HLA‐DR, CD80 and CD86 in NEC, their regulation by cytokines and the capacity of NEC to induce antigen‐specific proliferation of T cells. Methods We examined the expression of HLA‐DR, CD80 and CD86 in nasal epithelial scrapings of patients with seasonal allergic rhinitis (SAR) to Japanese cedar pollen pre‐season and in‐season, by immunohistochemistry. Next, we examined the effect of IL‐1β, TNF‐α, (IFN‐γ), IL‐4 α, IL‐13 and diesel exhaust particles (DEP) on the HLA‐DR, CD80 and CD86 expression in cultured nasal epithelial cells (CNEC), by flow cytometry. Further, we analysed the capacity of mite antigen (Der f II)‐pulsed mitomycin‐C‐treated CNEC to induce proliferation of autologous T cells from patients with perennial allergic rhinitis. Results NEC constitutively expressed HLA‐DR and CD86, but not CD80. The expression of HLA‐DR and CD86 in NEC was significantly increased in‐season, in patients with SAR as compared with that of pre‐season. While IFN‐γ up‐regulated the expression of HLA‐DR, IL‐1β and TNF‐α up‐regulated the expression of CD86 in CNEC. Furthermore, in the presence of mite antigen, CNEC induced the proliferation of autologous peripheral blood T lymphocytes. Anti‐CD86 and anti‐HLA‐DR monoclonal antibody but not anti‐CD80 inhibited the epithelial cell‐induced T cell proliferation. Stimulation with a combination of DEP and mite antigen significantly up‐regulated HLA‐DR and CD86 expression in CNEC. Conclusions These studies suggest that NEC in patients with AR may play a role in antigen presentation through the enhanced expression of HLA‐DR and CD86. Furthermore, these results suggest the possibility that DEP may enhance the antigen‐presenting function of CNEC.

Pharmacologic therapy for stop mutations: how much CFTR activity is enough?

The purpose of this review is to summarize the recent approaches using mutation-specific therapy to correct the genetic defect according to the molecular mechanism by which the mutation causes the defects in cystic fibrosis transmembrane conductance regulator (CFTR). Premature stop mutations (class I mutations) account for 5 to 10% of the total mutant alleles in cystic fibrosis patients, and in certain subpopulations the incidence is much higher. The aminoglycoside antibiotics can suppress premature termination codons by permitting translation to continue to the normal termination of the transcript. The susceptibility to suppression by aminoglycosides depends on the stop codon itself and on the sequence context surrounding it. In vitro studies in cell lines expressing stop mutations and in mice have shown that aminoglycosides caused a dose-dependent increase in CFTR expression and restored functional CFTR to the apical membrane. Clinical studies also provided evidence that the aminoglycoside gentamicin can suppress these CFTR premature stop mutations in affected patients. A recent double-blind, placebo-controlled, crossover study has demonstrated restoration of CFTR function by topical application of gentamicin to the nasal epithelium of cystic fibrosis patients carrying stop mutations. In 21% of the patients there was a complete normalization of all the electrophysiologic abnormalities caused by the CFTR defect, and in 68% there was restoration of either chloride or sodium transport. Furthermore, immunohistochemical staining to the C-terminal part of the CFTR was demonstrated via peripheral staining for CFTR in scraped nasal epithelial cells of patients carrying stop mutations. Inconsistent results were reported regarding the required level of corrected CFTR that has to be reached to achieve normal function. Achieving CFTR activity of 10 to 35% might be needed to prevent significant pulmonary morbidity. It is as yet unknown how much corrected mutant CFTR must reach the apical membrane to induce a clinically relevant beneficial effect. The future goal is to maximize the effect of stop-codon supressors on CFTR while minimizing side effects, but further studies must be performed to find a safer compound that may be administered in small children from the time of diagnosis.