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Objective: Severe acute respiratory syndrome coronavirus-2 uses angiotensin-converting enzyme-2 as a primary receptor for invasion. This study investigated angiotensin-converting enzyme-2 expression in the sinonasal mucosa of patients with chronic rhinosinusitis, as this could be linked to a susceptibility to severe acute respiratory syndrome coronavirus-2 infection., Methods: Ethmoid sinus specimens were obtained from 27 patients with eosinophilic chronic rhinosinusitis, 18 with non-eosinophilic chronic rhinosinusitis and 18 controls. The angiotensin-converting enzyme-2 and other inflammatory cytokine and chemokine messenger RNA levels were assessed by quantitative reverse transcription polymerase chain reaction. Angiotensin-converting enzyme-2 positive cells were examined immunohistologically., Results: The eosinophilic chronic rhinosinusitis patients showed a significant decrease in angiotensin-converting enzyme-2 messenger RNA expression. In the chronic rhinosinusitis patients, angiotensin-converting enzyme-2 messenger RNA levels were positively correlated with tumour necrosis factor-α and interleukin-1β (r = 0.4971 and r = 0.3082, respectively), and negatively correlated with eotaxin-3 (r = -0.2938). Angiotensin-converting enzyme-2 immunoreactivity was mainly localised in the ciliated epithelial cells., Conclusion: Eosinophilic chronic rhinosinusitis patients with type 2 inflammation showed decreased angiotensin-converting enzyme-2 expression in their sinus mucosa. Angiotensin-converting enzyme-2 regulation was positively related to pro-inflammatory cytokines, especially tumour necrosis factor-α production, in chronic rhinosinusitis patients.

The human paranasal sinuses are the major source of intrinsic nitric oxide (NO) production in the human airway. NO plays several roles in the maintenance of physiological homeostasis and the regulation of airway inflammation through the expression of three NO synthase (NOS) isoforms. Measuring NO levels can contribute to the diagnosis and assessment of allergic rhinitis (AR) and chronic rhinosinusitis (CRS). In symptomatic AR patients, pro-inflammatory cytokines upregulate the expression of inducible NOS (iNOS) in the inferior turbinate. Excessive amounts of NO cause oxidative damage to cellular components, leading to the deposition of cytotoxic substances. CRS phenotype and endotype classifications have provided insights into modern treatment strategies. Analyses of the production of sinus NO and its metabolites revealed pathobiological diversity that can be exploited for useful biomarkers. Measuring nasal NO based on different NOS activities is a potent tool for specific interventions targeting molecular pathways underlying CRS endotype-specific inflammation. We provide a comprehensive review of the functional diversity of NOS isoforms in the human sinonasal system in relation to these two major nasal disorders' pathologies. The regulatory mechanisms of NOS expression associated with the substrate bioavailability indicate the involvement of both type 1 and type 2 immune responses.

A potential mechanism underlying cigarette smoke-induced airway disease is insufficient tissue repair via altered production of matrix metalloproteinases (MMPs). Osteitis is a signature feature of recalcitrant chronic rhinosinusitis (CRS) and often results in revision surgery. The present study aimed to investigate MMP expression in the nasal tissues of asthmatic patients with CRS and any association with cigarette smoking and osteitis. Thirteen smokers with CRS and asthma, 16 non-smokers with CRS and asthma, and seven non-smoker asthmatic patients without CRS were prospectively recruited. The expression of MMPs and associated immunological factors in surgically-obtained nasal tissues was evaluated via real-time PCR and western blotting. Maximal bone thickness of the anterior ethmoid (AE) partition was measured in axial sinus computed tomography (CT) sections. MMP-1 and MMP-9 expression was increased in the nasal tissues of smokers with asthma and CRS via real-time PCR and western blot. Maximal AE partition bone thickness was greater in smokers with CRS and asthma than in non-smokers with CRS and asthma. MMP-1 and MMP-9 levels were correlated with maximal AE bone thickness. Cigarette smoking was associated with the up-regulation of MMP-1 and MMP-9 in the nasal tissues of patients with airway inflammatory diseases, and with AE osteitis, and with therapeutic resistence.

The nasal tissues have the main consecutive roles of moistening and heating the air entering the respiratory tract and detecting odor via the activation of olfactory receptors in the neuro-olfactory epithelium. Initially, nasal toxicology was investigated to better assess the risk of nasal injuries caused by environmental toxicants or their active metabolites. Later, the characterization of the nasal toxicological barrier was a research concern for the purposes of intranasal drug delivery. Both fields allowed for an increase in our knowledge of the nasal xenobiotic-metabolizing enzymes and transporters that are highly expressed in this tissue. In addition to airborne toxicants or drugs, the main substrates for these proteins are natural volatiles known as odorants that emanate from our daily environment (food, perfume, plants, materials, congeners, etc.). Accordingly, another emerging field of interest has been developed that aims to understand the function of odorant-metabolizing enzymes (OMEs) in olfaction. Early in this field of research, OMEs were suspected to participate in the clearance of odorants from the receptor environment to avoid their saturation and thus maintain the sensitivity of neuronal detection. Other roles of OMEs that could significantly modulate olfaction were also considered, such as the involvement of odorant primary metabolites in the olfactory response. By combining enzymatic, physiological and sensory experimental approaches, recent advances have markedly improved our understanding of the contributions of OMEs to the olfactory process. This review combines recent data from the literature regarding nasal OME identification, localization, and activity and highlights the function of OMEs in olfaction.

Apoptosis and programmed necrosis (necroptosis) determine cell fate, and antagonize infection. Execution of these complementary death pathways involves the formation of receptor-interacting protein kinase 1 (RIPK1) containing complexes. RIPK1 binds to adaptor proteins, such as TRIF (Toll-IL-1 receptor-domain-containing-adaptor-inducing interferon-beta factor), FADD (Fas-associated-protein with death domain), NEMO (NF-κB regulatory subunit IKKγ), SQSTM1 (sequestosome 1/p62), or RIPK3 (receptor-interacting protein kinase 3), which are involved in RNA sensing, NF-κB signaling, autophagosome formation, apoptosis, and necroptosis. We report that a range of rhinoviruses impair apoptosis and necroptosis in epithelial cells late in infection. Unlike the double-strand (ds) RNA mimetic poly I:C (polyinosinic:polycytidylic acid), the exposure of dsRNA to toll-like receptor 3 (TLR3) in rhinovirus-infected cells did not lead to apoptosis execution. Accordingly, necroptosis and the production of ROS (reactive oxygen species) were not observed late in infection, when RIPK3 was absent. Instead, a virus-induced alternative necrotic cell death pathway proceeded, which led to membrane rupture, indicated by propidium iodide staining. The impairment of dsRNA-induced apoptosis late in infection was controlled by the viral 3C-protease (3Cpro), which disrupted RIPK1-TRIF/FADD /SQSTM1 immune-complexes. 3Cpro and 3C precursors were found to coimmuno-precipitate with RIPK1, cleaving the RIPK1 death-domain, and generating N-terminal RIPK1 fragments. The depletion of RIPK1 or chemical inhibition of its kinase at the N-terminus did not interfere with virus progeny formation or cell fate. The data show that rhinoviruses suppress apoptosis and necroptosis, and release progeny by an alternative cell death pathway, which is controlled by viral proteases modifying innate immune complexes.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the paranasal sinuses, and its pathophysiology is not yet precisely known. It is suggested that oxygen free radicals play an important role in the pathogenesis of nasal polyposis. This study aimed to identify genetic polymorphisms of superoxide dismutase (SOD 2), catalase (CAT), and inducible nitric oxide synthase (iNOS) enzymes in eosinophilic CRSwNP and non-eosinophilic CRSwNP patients; the study also aimed to evaluate the effect of genetic polymorphism of antioxidant enzymes on CRSwNP etiopathogenesis. One hundred thirty patients, who received endoscopic sinus surgery due to CRSwNP, and 188 control individuals were included in this study. Nasal polyp tissues were divided into two groups histopathologically as eosinophilic CRSwNP and non-eosinophilic CRSwNP. Venous blood samples were taken from the patient and control groups. Polymorphisms in the Ala16Va1 gene, which is the most common variation of SOD-2 gene, and 21 A/T polymorphisms in catalase gene were evaluated with the restriction fragment length polymorphism method and -277 C/T polymorphism in the iNOS gene was evaluated with the DNA sequencing method. The GG genotype distribution for the (-277) A/G polymorphism in the iNOS gene was a statistically significant difference between eosinophilic CRSwNP and control groups (p < 0.05). The CC genotype distribution for the SOD2 A16V (C/T) polymorphism was not statistically significant in all groups (p > 0.05). The TT genotype distribution for the A/T polymorphism in catalase gene at position -21 was statistically significant differences in eosinophilic CRSwNP and control groups (p < 0.05). Increased free oxygen radical levels, which are considered effective factors in the pathogenesis of CRSwNP, can occur due to genetic polymorphism of enzymes in the antioxidant system and genetic polymorphism of antioxidant enzymes in eosinophilic CRSwNP patients might contribute to the pathophysiology.

Conclusion The HNE-TACE signalling pathway has an important role in the process of MUC5AC overexpression in chronic rhinosinusitis (CRS). Objectives To provide evidence of HNE-induced MUC5AC overexpression in CRS via TACE. Method HE and PAS staining were used to assess the pathological changes in sinus mucosa samples from CRS or normal control. HNE, TACE, and MUC5AC expression in the sinonasal mucosa was determined using immunohistochemistry (IHC) and real-time polymerase chain reaction (qRT-PCR). In addition, the MUC5AC and TACE expression was determined in a primary culture of human nasal mucosa epithelial cells in vitro. Results On HE staining, the main pathological feature in the sinus mucosa of CRS patients was hyperplasia of goblet cells, inflammatory cells, and submucosal glands. Mucosa from the two experimental groups also showed strong expression on PAS staining. IHC and qRT-PCR demonstrated that HNE, TACE, and MUC5AC expression was significantly higher in the CRS patients compared with control samples (p < 0.05). MUC5AC mRNA expression was higher in cells stimulated by HNE than in untreated cells (p < 0.05). MUC5AC mRNA expression was significantly reduced in cells pre-treated with the TACE inhibitor TAPI-1 prior to HNE stimulation, compared with untreated and HNE-stimulated cells (p < 0.01).

Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification (P <0.05), N classification (P <0.05) and total staging (P <0.01) in NPC, and patients with higher KLHDC4 expression had poorer overall (P <0.01) and metastasis-free survival (P <0.05) rates. Knockout (KO) of KLHDC4 via CRISPR/Cas9-mediated gene editing in NPC cell line dramatically inhibited cell proliferation, colony formation in soft agar and tumor formation in nude mice. In addition, cell migration and invasion were also impaired by KLHDC4 depletion as revealed by wound healing and Transwell assay. Mechanically, loss of KLHDC4 markedly induced spontaneous apoptosis in NPC cells, as evidenced by increased levels of cleaved caspase-3 and cleaved PARP. Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining. In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis. Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.

Background: The interest in administering drugs by intranasal route is currently increasing, particularly because it has shown to ensure drug therapeutic action by a rapid systemic absorption through the respiratory mucosa and/or a direct delivery of some molecules into the brain through the olfactory mucosa. Nevertheless, although intranasal administration avoids the first-pass hepatic biotransformation, several enzymes as well as efflux transporters can be found in the mammalian nasal epithelium., Methods: This work reviewed the expression and localization of the drug-metabolizing enzymes and efflux transporters in the mammalian nasal mucosa epithelium, as well as their influence on the pharmacokinetic and pharmacodynamic profiles of specific drugs, using one hundred and thirty five peer-reviewed papers undertaken from MEDLINE database, including review and research papers., Results: Most of the studies analyzed corroborate that oxidative and conjugative enzymes as well as multidrug resistance proteins and multidrug resistance-associated proteins efflux transporters are expressed in the nasal cavity, particularly in olfactory and respiratory mucosae, determining the access of drugs to central nervous system and bloodstream. Nonetheless, besides their well-documented anatomic location, either in laboratory animals or humans, there is still a lack of experimental information regarding the substrates of these enzymes and efflux transporters at the nasal cavity., Conclusion: The knowledge herein discussed important not only to select the drugs with better potential to be administered by intranasal route but also to optimize drug formulations, in which inhibitors of enzymes or efflux transporters may be included to increase drug systemic and central delivery.

Background and Purpose: The anti-inflammatory and immunomodulatory effects of macrolides include the ability to decrease mucus secretion and inhibit inflammatory mediators in chronic rhinosinusitis. Nevertheless, their mechanisms of action remain to be determined. Here we have investigated the effects of macrolide antibiotics (clarithromycin, azithromycin and josamycin; representating the 14-, 15- and 16-membered macrolides) on endogenous steroids in human sinonasal epithelial cells and mouse nasal mucosa., Experimental Approach: The effects of macrolides on the expression of steroid-converting enzymes [11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2)], steroid-synthesizing enzymes (3β-HSD, CYP21, CYP11B1 and CYP11A1) and cortisol levels were assessed in cultured human epithelial cells. In control and adrenalectomized mice , these enzymes and corticosterone levels were evaluated in nasal mucosa and serum after administration of macrolides., Key Results: The expression levels of 3β-HSD, CYP21, 11β-HSD1 and CYP11B1 increased in human epithelial cells treated with clarithromycin and azithromycin, whereas the expression levels of 11β-HSD2 and CYP11A1 were not affected. Josamycin had no effects on the expression of these enzymes. Cortisol levels increased in epithelial cells treated with clarithromycin or azithromycin. The expression of 3β-HSD, CYP11A1, CYP21, CYP11B1 and 11β-HSD1 was upregulated in nasal mucosa of mice treated with clarithromycin or azithromycin, but not in adrenalectomized mice., Conclusions and Implications: This study provides evidence that 14- and 15-membered macrolide antibiotics may affect the expression of steroid-synthesizing and steroid-converting enzymes in human sinonasal epithelial cells and mouse nasal mucosa, increasing the endogenous cortisol levels in sinonasal mucosa., (© 2015 The British Pharmacological Society.)

Background: Nuclear factor κB (NF-κB) is a vital transcription factor that is activated by numerous inflammatory stimuli. Its activity is tightly regulated by a family of deubiquitinating enzymes (A20, Cezanne, cylindromatosis [CYLD]) that function in a negative-feedback loop, a process that prevents chronic and systemic inflammation. This study seeks to characterize the expression and functional role of NF-κB-regulating deubiquitinases in the sinonasal epithelium., Methods: Expression of A20, Cezanne, and CYLD was assessed in normal sinonasal tissue using immunohistochemistry. Cultured sinonasal epithelial cells (SNECs) were stimulated with proinflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin 4 [IL]-4, IL-13) or lipopolysaccharide (LPS) and changes in NF-κB activation and deubiquitinase expression were assessed using Western blots and quantitative real-time polymerase chain reaction (qRT-PCR), respectively., Results: NF-κB was activated in response to LPS and TNF-α, but not IL-4 or IL-13. A20, Cezanne, and CYLD were all expressed in sinonasal tissue, primarily along the apical surface of the epithelium. Proinflammatory mediators primarily affected expression of A20, with upregulation by LPS and TNF-α and downregulation by IL-4 and IL-13., Conclusion: The NF-κB-regulating deubiquitinases A20, Cezanne, and CYLD are expressed in sinonasal tissue and are differentially induced by proinflammatory cytokines and the microbial antigen, LPS. These results suggest an important role for NF-κB-regulating deubiquitinases in mucosal immunity and homeostasis., (© 2015 ARS-AAOA, LLC.)

Objectives/hypothesis: To investigate the effect of oral plus intranasal corticosteroid (CS) treatment on nasal polyp (NP) mucosa remodeling from patients with severe chronic rhinosinusitis with nasal polyps (CRSwNP)., Study Design: Case series, retrospective study., Methods: Patients (n = 18) with severe CRSwNP were treated with oral prednisone for 2 weeks and intranasal budesonide for 12 weeks. NP biopsies were obtained from patients biopsies before (w0) and after 2 weeks (w2) and 12 weeks (w12) of CS treatment. Matrix metalloprotease 1 (MMP-1), MMP-2, MMP-7, MMP-9, and tissue inhibitor of metalloprotease type 1 (TIMP-1) expression was evaluated by immunohistochemistry in cell and tissue structures. Epithelial damage, eosinophil infiltration, and collagen content were also examined in NP tissues before and after CS treatment., Results: Compared to w0: 1) oral plus intranasal CS significantly (P < .01) increased presence of submucosal glands at w2, decreased epithelial cell hyperplasia at w12, and decreased tissue eosinophilia at w2 and w12; 2) CS treatment significantly (P < .05) increased immunoreactivity for MMP-1 and MMP-2 in the epithelium at w2, but decreased immunoreactivity for MMP-9 in the epithelium at w2 and w12; 3) at w12, CS significantly (P < .05) reduced MMP-9 immunoreactive positivity and intensity in the extracellular matrix, while increasing total collagen amount in the extracellular matrix; and 4) CS treatment significantly (P < .01) reduced the number of eosinophils and their MMP and TIMP-1 immunoreactive expression., Conclusions: CS treatment modulates NP mucosa remodeling, particularly by promoting epithelial repair, regulating tissue remodeling markers, increasing total collagen content, and reducing tissue eosinophil infiltration., Level of Evidence: 4, (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: Chronic rhinosinusitis with nasal polyposis (CRSwNP) and asthma coexist frequently and share similar features of inflammation and remodeling. Remodeling has become an important concept in the pathophysiology of asthma and CRSwNP. It happens early in the development of these diseases and is relatively resistant to treatments. The key enzymes responsible for remodeling are matrix metalloproteinases (MMPs). In this study we examined whether asthma and CRSwNP share similar MMP profiles., Methods: Nasal secretion and serum specimens of controls (19 subjects) and patients with asthma (12), CRSwNP (39), or both (16) were collected between December 2007 and May 2009. Groups were divided into 2 subgroups according to atopy. MMP-7, MMP-9, MMP-13, tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 and TIMP-2, myeloperoxidase (MPO), and human neutrophil elastase (HNE) were measured using enzyme-linked immunosorbent assay (ELISA), and MMP-8 was determined using immunofluorometric assay. High-sensitivity C-reactive protein (hs-CRP) was measured to estimate systemic involvement., Results: Patients with asthma, CRSwNP, or both exhibited lower MMP-9, MMP-9/TIMP-1, MMP-9/TIMP-2, and MPO in nasal secretions (p < 0.05 in CRSwNP) and higher MMP-9, MMP-9/TIMP-1, MMP-9/TIMP-2, and HNE in serum (p < 0.05 in all groups) compared to controls, whereas no difference in MMP-7, MMP-13, TIMP-1, and TIMP-2 were detected. Atopy increased nasal MMP-9 and MPO expression. hs-CRP was higher in patients with CRSwNP and asthma compared to controls., Conclusion: Our findings suggest shared pathomechanisms behind asthma and CRSwNP. Contrasting local vs systemic results reflect a different ability of healthy mucosa to react to exogenous stimuli, possibly indicating a protective function of MMP-9 and possibly also MMP-8 in the airways., (© 2015 ARS-AAOA, LLC.)

Background: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated., Results: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues., Conclusions: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.

Rhinitis and asthma are the most common respiratory diseases in children. We assessed whether airway inflammation markers were associated with nasal allergies and self-reported symptoms of wheeze and rhinitis in 130 children 6-12 year old in an epidemiological context. Independent of sex and age, the fraction of exhaled nitric oxide (FeNO) and nasal mast cell (MC) activation (tryptase ≥ 5 ng/mL) were positively associated with wheeze, rhinitis and with nasal allergy. Nasal eosinophil cationic protein (ECP) and exhaled breath condensate (EBC) markers (pH, 8-isoprostane, interleukin-1β) were not associated with symptoms or with nasal allergy. In conclusion, FeNO and nasal tryptase reflect allergic inflammation in the respiratory system.

Background: Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown., Methods: To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function., Results: PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs., Conclusions: PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

Naphthalene (NA), a ubiquitous environmental pollutant that can cause pulmonary and nasal toxicity in laboratory animals, requires cytochrome P450 (P450)-mediated metabolic activation to cause toxicity. Our recent study using a Cyp2f2-null mouse showed that CYP2F2 plays an essential role in NA-induced lung toxicity, but not in NA-induced nasal toxicity. The aim of this study was to determine whether mouse CYP2A5, abundantly expressed in nasal olfactory mucosa (OM) and the liver, but less in the lung, plays a major role in the bioactivation and toxicity of NA in the OM. We found, by comparing Cyp2a5-null and wild-type (WT) mice, that the loss of CYP2A5 expression led to substantial decreases in rates of NA metabolic activation by OM microsomes. The loss of CYP2A5 did not cause changes in systemic clearance of NA (at 200 mg/kg, i.p.). However, the Cyp2a5-null mice were much more resistant than were WT mice to NA-induced nasal toxicity (although not lung toxicity), when examined at 24 hours after NA dosing (at 200 mg/kg, i.p.), or to NA-induced depletion of total nonprotein sulfhydryl in the OM (although not in the lung), examined at 2 hours after dosing. Thus, mouse CYP2A5 plays an essential role in the bioactivation and toxicity of NA in the OM, but not in the lung. Our findings further illustrate the tissue-specific nature of the role of individual P450 enzymes in xenobiotic toxicity, and provide the basis for a more reliable assessment of the potential risks of NA nasal toxicity in humans.

Respiratory epithelial adenomatoid hamartoma (REAH) is regarded as a rare tumor of the nasal cavity. The mechanisms driving the development of REAH are unknown, and its nature as a benign tumor, hamartoma, or reactive inflammatory process is still open to discussion. A total of 150 consecutive patients operated on for nasal polyposis (NP) were extensively checked for the diagnosis of REAH. The profile of REAH occurring in association with NP was compared with solitary REAH in a series of 19 cases. The possible role of tryptase-producing mast cells (MC) and of metalloproteinases MMP2 and MMP9 in REAH development was investigated by immunohistochemistry. REAH lesions were identified in 35% of patients who had surgery for NP (53/150). The distribution of the lesions suggested that REAH originated in the olfactory cleft. Solitary REAH occurred about 20 times less frequently than those observed in an NP context but shared the same microscopic characteristics. Tryptase-producing MCs were recruited at high density in REAH (135/10 hpf), compared with inflammatory polyps (45/10 hpf; P<0.00005) and hypertrophied turbinates (51/10 hpf; P<0.0005). REAH also showed constant MMP9 expression and to a lesser degree MMP2 expression in epithelial cells. If solitary REAH is a relatively rare lesion, we demonstrated that an exhaustive sampling allows the detection of a high proportion of NP-associated REAH, sharing the same clinical and histologic characteristics with solitary REAH. Tryptase-producing MCs, possibly in association with MMP expression, may play a central role in REAH formation.

Introduction: Nasal polyps are smooth outgrowths assuming a shape of grapes, formed from the nasal mucosa, limiting air flow by projecting into a lumen of a nasal cavity. Up to now the surgical resection is the best method of their treatment, but etiology and pathogenesis of the nasal polyps is not yet fully established., Aim of the Study: The aim of the study was the assessment of the selected lysosomal exoglycosidases activity in the nasal polyps. In this study the activity of β-galactosidase, α-mannosidase and α-fucosidase was determined in the tissue of the nasal polyps obtained from 40 patients (10F, 30M) and control tissues derived from mucosa of lower nasal conchas obtained during mucotomy from 20 patients (10F, 10M)., Results: We observed significant lower values of GAL, FUC and tendency to decrease of MAN and GLU concentration in nasal polyps (P) in comparison to control healthy nasal mucosa (C). In nasal polyp tissue (P) no differences of GAL, MAN and FUC specific activity in comparison to control mucosa (C) were found., Conclusions: Our research supports bioelectrical theory of the nasal polyps pathogenesis and directs attention at research on glycoconjugates and glycosidases of the nasal mucosa extracellular matrix., (Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.)

Objectives: We investigated the role of inducible nitric oxide synthase (iNOS) in the pathogenesis of sinonasal polyps., Methods: Adult patients (21 men, 3 women) with nasal polyposis underwent functional endoscopic sinus surgery. Nine adults without polyps (6 men) who underwent septoplasty and/or rhinoplasty served as controls. Polyp specimens came from three regions: the maxillary sinus (10), ethmoid sinus (14), and nasal cavity (10). Control group samples (9) came from the inferior turbinate. Specimens were evaluated in eight mucosal layers for count and distribution of inflammatory cells and iNOS expression. An iNOS positivity index (PI) was determined for the epithelium (E), subepithelial layer of the lamina propria (SE), and deep paraglandular layer of the mucosa (D)., Results: Polymorphonuclear cell (PMNC) % values of the ethmoid and maxillary sinus and overall ethmoid sinus PI were significantly higher in the polyp group. Patients with longer polyp duration, D-perivascular (D-pv), and a higher Brinkmann index had decreased ethmoid sinus D PIs. However, in older patients and patients with longer polyp duration, perivascular PIs increased in maxillary sinus SE and D, respectively. Furthermore, as PMNC % and iNOS-PMNC PI increased, SE&#95;glandular and epithelial&#95;apical iNOS values decreased. In the ethmoid and maxillary sinuses, iNOS&#95;D&#95;. endothelial values increased but decreased in the nasal cavity., Conclusions: iNOS may play a role in sinonasal polyp pathogenesis, especially in mucosal SE and D layers. Increased vascular permeability, stromal edema, inflammatory cell migration into the stroma of the mucosa, and increased mucosal gland secretion may result in polyp formation.

When herbivores come in contact with volatile plant secondary compounds (PSC) that enter the nasal passages the only barrier between the nasal cavity and the brain is the nasal epithelium and the biotransformation enzymes present there. The expression of two biotransformation enzymes Cytochrome P450 2B (CYP2B) and glutathione-S-transferase (GST) was investigated in the nasal epithelia and livers of three populations of woodrats. One population of Neotoma albigula was fed juniper that contains volatile terpenes. Juniper caused upregulation of CYP2B and GST in the nasal epithelium and the expression of CYP2B and GST in the nasal epithelium was correlated to liver expression, showing that the nasal epithelia responds to PSC and the response is similar to the liver. Two populations of Neotoma bryanti were fed creosote that contains less volatile phenolics. The creosote naive animals upregulated CYP2B in their nasal epithelia while the creosote experienced animals upregulated GST. There was no correlation between CYP2B and GST expression in the nasal epithelia and livers of either population. The response of the nasal epithelium to PSC seems to be an evolved response that is PSC and experience dependent., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microbial products play a role in the pathogenesis of allergic diseases; ubiquitin E3 ligase A20 (A20) is an important molecule in regulating inflammation in the body. The present study aims to elucidate the role of A20 in processing the absorbed microbial products in nasal epithelial cells. Human nasal mucosal specimens were collected from patients with or without chronic rhinitis and analyzed by immunohistochemistry. Human nasal epithelial cell line, RPMI2650 cell, was employed to assess the role of A20 in processing the absorbed staphylococcal enterotoxin B (SEB). The RPMI2650 cells absorbed SEB in the culture. The increase in A20 was observed in RPMI2650 cells in parallel to the absorption of SEB. A20 is a critical molecule in the degradation of SEB in the nasal epithelial cells by promoting the tethering of endosomes and lysosomes. A20 plays a critical role in processing of the absorbed SEB in nasal epithelial cells.

Matrix metalloproteinases (MMPs) play an important role in respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. It was hypothesized that MMP-8 and MMP-9 may function as biological markers to assess disease severity in viral lower respiratory tract infections in children. MMP-8 and MMP-9 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) and granulocytes obtained in both the acute and recovery phase from 153 children with mild, moderate, and severe viral lower respiratory tract infections were determined using real-time PCR. In addition, MMP-8 and MMP-9 concentrations in blood and nasopharyngeal specimens were determined during acute mild, moderate, and severe infection, and after recovery using ELISA. Furthermore, PBMCs and neutrophils obtained from healthy volunteers were stimulated with RSV, LPS (TLR4 agonist), and Pam3Cys (TLR2 agonist) in vitro. Disease severity of viral lower respiratory tract infections in children is associated with increased expression levels of the MMP-8 and MMP-9 genes in both PBMCs and granulocytes. On the contrary, in vitro experiments showed that MMP-8 and MMP-9 mRNA and protein expression in PBMCs and granulocytes is not induced by stimulation with RSV, the most frequent detected virus in young children with viral lower respiratory tract infections. These data indicate that expression levels of the MMP-8 and MMP-9 genes in both PBMCs and neutrophils are associated with viral lower respiratory tract infections disease severity. These observations justify future validation in independent prospective study cohorts of the usefulness of MMP-8 and MMP-9 as potential markers for disease severity in viral respiratory infections., (Copyright © 2012 Wiley Periodicals, Inc.)

CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (∼100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (∼0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (∼0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

Objectives/hypothesis: The existence of nasal mucosa remodeling in allergic rhinitis is controversial. Few data are available on the dynamics of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in nasal fluid after an allergen challenge. We examined whether an immediate allergic reaction that induces nasal congestion and inflammation is able to also induce changes in remodeling parameters in nasal fluid., Study Design: Controlled experimental study., Methods: Ten patients with allergic occupational rhinitis due to flour underwent a control and active inhalation challenge with serial monitoring of nasal congestion and nasal symptoms with acoustic rhinometry and a visual analogue scale. Levels of remodeling markers (MMP-2, MMP-7, MMP-9, MMP-13, TIMP-1, TIMP-2) and inflammatory cells in nasal fluid were measured before the challenge and at 30 minutes, 6 hours, and 24 hours following the challenge., Results: In contrast to the control challenge, the flour challenge induced nasal symptoms and significant decreases in nasal volume in all subjects. After the flour challenge, a significant increase in nasal levels of TIMP-2 and a nonsignificant increase in TIMP-1 levels were observed, whereas no significant changes in nasal levels of MMPs were documented., Conclusions: This study showed that after an inhalation challenge with an occupational allergen, the nasal mucosa displayed an imbalance in favor of TIMPs enzymes activity as compared to MMPs enzymes activity, represented in an increase in nasal levels of TIMP-2 during the course of the early reaction following the allergen challenge., (Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.)

Background: Nitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood., Objective: To characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP)., Methods: Normal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies., Results: The proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP., Conclusion: All isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues., (Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

In this study, we evaluated effect of glycyrrhizin on immunity function in allergic rhinitis (AR) mice. The AR mice model were induced by dripping ovalbumin in physiological saline (2 mg mL⁻¹, 10 μL) into the bilateral nasal cavities using a micropipette. After the AR model was induced, mice were randomly divided into six groups: the normal control, model, lycopene 20 mg kg⁻¹ (as positive control drug) group, and glycyrrhizin 10, 20, 30 mg kg⁻¹ groups. After the sensitization day 14, lycopene (20 mg/kg BW) and glycyrrhizin (10, 20 and 30 mg/kg BW) were given orally for 20 days once a day. Mice in the normal control and model groups were given saline orally once a day for 20 days. Results showed that glycyrrhizin treatment could dose-dependently significantly reduce blood immunoglobulin E (IgE), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), nitrous oxide (NO), tumor necrosis factor-alpha (TNF-α) levels and nitrous oxide synthase (NOS) activity and enhance blood immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interleukin-2 (IL-2) and interleukin-12 (IL-12) levels in AR mice. Furthermore, glycyrrhizin treatment could dose-dependently significantly enhance acetylcholinesterase (AchE) activity and reduce substance P (SP) level in peripheral blood and nasal mucosa of AR mice. We conclude that glycyrrhizin can improve immunity function in AR mice, suggesting a potential drug for the prevention and therapy of AR.

Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

Background: Matrix metalloproteinase (MMP) is involved in the remodeling process of inflammatory airway diseases and is correlated with the severity of asthma. We hypothesized that MMP was associated with the severity of chronic rhinosinusitis with nasal polyps (CRSwNPs). We also investigated the effect of allergy on the expression of MMP in the polyp., Methods: The expression of MMP-2 and -9 was investigated in recurrent nasal polyps of 30 patients and in nonrecurrent nasal polyps of 31 patients undergoing endoscopic sinus surgery. These expressions were then compared with those in control nasal mucosal samples obtained from 32 patients with chronic hypertrophic rhinitis. Demographic data, Lund-Mackay (LM) score, polyp grade, and allergy status were obtained for all patients. Tissue samples were assessed via immunohistochemistry., Results: MMP-2 and -9 were constantly expressed in recurrent NPs, primary NPs, and control nasal mucosa. The expression of MMP-9 was significantly enhanced in glands and MMP-2 positivity was significantly increased in surface epithelium for patients with NPs when compared with control nasal mucosa. The expression of MMP-9 and -2 was not correlated with polyp grade and LM score. Allergic status is an independent factor in the expression of MMP-2 and -9., Conclusion: These results suggested up-regulation of MMP-9, and MMP-2 in gland and surface epithelium, respectively, were characteristic of NPs. Therefore, patients with allergy will exhibit greater MMP-2 and -9 positivity. However, the MMP-2 and -9 expression intensity was not correlated with the severity of CRS with nasal polyposis.

Dexamethasone cipecilate (DX-CP, 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel synthetic corticosteroid used to treat allergic rhinitis. The pharmacological effect of DX-CP is considered to be mainly due to its active de-esterified metabolite (DX-17-CPC). To investigate the in vitro metabolism of DX-CP in human liver, DX-CP was incubated with human liver microsomes and S9. In addition, a metabolism study of DX-CP with human nasal mucosa was carried out in order to elucidate whether DX-17-CPC is formed in nasal mucosa, the site of action of DX-CP. DX-17-CPC was the major metabolite in both liver microsomes and S9. Two new epoxide metabolites, UK1 and UK2, were detected in liver S9, while only UK1 was detected in liver microsomes. This suggests that cytosol enzymes are responsible for the formation of UK2. In human nasal mucosa, DX-CP was mainly transformed into DX-17-CPC. By using recombinant human carboxylesterases (CESs), the reaction was shown to be catalyzed by CES2. These results provide the evidence that the active metabolite DX-17-CPC is the main contributor to the pharmacological action after the intranasal administration of DX-CP to humans.

Background: Matrix metalloproteinases (MMPs) are key enzymes responsible for extracellular matrix degradation contributing to the progressive histological changes seen in lower airway disease, including asthma. MMP-9 and TIMP-1 have also shown some role in the pathogenesis of chronic rhinosinusitis (CRS) and nasal polyposis (NP)., Objective: We aim to determine variability in expression of MMP-9 and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in sinus tissue from distinct patient populations presenting with nasal polyposis., Methods: The expression of MMP-9 and TIMP-1 was investigated in nasal polyp tissue from 6 aspirin-sensitive (AS) and 6 aspirin-tolerant (AT) patients undergoing endoscopic sinus surgery for chronic rhinosinusitis with nasal polyposis (CRSwNP). Sinus mucosa from 6 patients with chronic rhinosinusitis without nasal polyposis (CRSsNP) was used as control. The MMP-9 and TIMP-1 expression was measured using immunofluorescence technique and graded using manual and computerized methods., Results: Expression of TIMP-1 was significantly reduced in the AS group when compared with both the AT and CRSsNP (control) groups (P < .001). The MMP-9/TIMP-1 ratio was significantly increased in the AS group when compared with other patient groups (P < .001). The MMP- 9 expression was similar between study and control groups., Conclusion: These results support the importance of MMP-9 and TIMP-1 expression in nasal polyp formation. The decreased expression of TIMP-1 in AS patients may promote the effects of MMP-9 expression and thus contribute to tissue remodeling and inflammatory changes. This finding may lead to further understanding of disease severity and resistance to treatment in this group of patients, as well as the pathogenesis of nasal polyps., (Copyright © 2011 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

Background: Collecting mucosal biopsies is invasive and creates additional inflammation, hampering a better understanding of nasal and sinus disease evolution and response to treatment. We examine whether sinus secretion collection can replace tissue biopsy for protein determination., Methods: Prior to surgical intervention for chronic rhinosinusitis (CRS), a piece of gelatin foam was used to collect secretions from the ethmoid mucosa. A tissue biopsy was then taken from the same location. Matrix metalloproteinase-9 (MMP-9) protein levels were measured in each sample., Results: MMP-9 protein levels in secretions and tissues were significantly correlated (p = 0.0033, r = 0.52, by Pearson correlation)., Conclusion: Secretion collection can replace tissue biopsy for MMP-9 determinations, reducing morbidity. Furthermore, secretion collection allows sequential sampling from the same location., (Copyright © 2011 American Rhinologic Society-American Academy of Otolaryngic Allergy, LLC.)

Objectives/hypothesis: It is known that arginase may be a regulator of diverse pathways, including production of nitric oxide (NO). Increased expression of arginase has been reported in several inflammatory lung diseases, including allergic asthma, suggesting that this may be a common feature underlying the pathophysiology of airway hyperreactivity. Thus, arginase I and II may play a role in the pathogenesis of allergic rhinitis. The distribution pattern and level of expression of arginase I and II were therefore determined in normal and allergic nasal mucosa., Study Design: Controlled, prospective study., Methods: The distribution pattern and level of expression of arginase I and II in normal and allergic nasal mucosa were evaluated using RT-PCR, immunohistochemistry, and Western blotting., Results: The level of expression of arginase I and II mRNA was increased in allergic nasal mucosa in comparison with normal nasal mucosa. In normal nasal mucosa, arginase I and II were expressed in the surface epithelium, submucosal glands, vascular endothelium, and fibroblasts. In allergic nasal mucosa, both enzymes were also localized to similar sites, in addition to inflammatory cells, and the level of expression were greatly increased compared with normal nasal mucosa. These findings were verified by Western blotting., Conclusions: These results indicate that arginase I and II may play a role in the pathophysiology of allergic rhinitis, and suggest the possible role of the L-arginine metabolic pathway through modulation of L-arginine availability as a substrate for nitric oxide synthase (NOS) and arginase in the pathogenesis of allergic rhinitis., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Genotoxicity experiments with exfoliated nasal mucosa cells are a promising minimally invasive approach for the detection of DNA-damaging compounds in ambient air. Results of single cell gel electrophoresis (SCGE) assays with individual cells and organ cultures from bioptic material show that DNA damage caused by compounds such as nitrosamines, polycyclic aromatic hydrocarbons and pesticides can be detected. Biochemical studies indicate that enzymes involved in the metabolism of environmental mutagens are represented in nasal cells. Several protocols for experiments with nasal cells have been developed and it was shown that formaldehyde, metals, styrene and crystalline silica induce DNA damage in SCGE and/or in micronucleus studies; furthermore, it was also found that polluted urban air causes DNA instability in nasal epithelial cells. Comparisons of these data with results obtained in lymphocytes and buccal cells indicate that nasal cells are in general equally sensitive. Broad variations in the baseline levels, differences of results obtained in various studies as well as the lack of information concerning the impact of confounding factors on the outcome of experiments with these cells indicate the need for further standardisation of the experimental protocols.

Chelidonic acid (CA) is known as an inhibitor of the rat brain glutamate decarboxylase. However, the pharmacological effects of CA in allergic reactions have not yet been defined. Here, we show the effects and the mechanism of CA in the ovalbumin (OVA)-sensitized allergic rhinitis (AR) model. CA significantly decreased the number of nasal/ear rubs and increment of IgE levels in the AR mice. The level of interferon-γ was enhanced while the level of IL-4 was reduced on the spleen tissue of the CA-administered AR mice. Expressions of IL-1β and cyclooxygenase-2 were inhibited by CA administration in the nasal mucosa tissues. Infiltration of eosinophils and mast cells was decreased in the CA-administered AR mice. Furthermore, CA decreased the caspase-1 activity in the same nasal mucosa tissue and human mast cell line, HMC-1. Our results indicate that CA may attenuate allergic reaction by inhibition of caspase-1 activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Odor information is decoded by a combination of odorant receptors, and thus transformed into discrete spatial patterns of olfactory glomerular activity. It has been found, however, that for some odorants, there are differences between the ligand specificity of an odorant receptor in vitro and its corresponding glomerulus in vivo. These observations led us to hypothesize that there exist prereceptor events that affect the local concentration of a given odorant in the nasal mucus, thus causing the apparent specificity differences. Here we show that odorants with functional groups such as aldehydes and esters are targets of metabolic enzymes secreted in the mouse mucus, resulting in their conversion to the corresponding acids and alcohols. The glomerular activation patterns elicited by an enzyme-targeted odorant in the olfactory bulb was different in the presence of an enzyme inhibitor in the mucosa, suggesting that the enzymatic conversion occurs fast enough to affect recognition of the odorant at the levels of olfactory sensory neurons. Importantly, olfactory discrimination tests revealed that mice behaviorally trained to associate an enzyme-targeted odorant to sugar rewards could not discriminate the odorant after treatment with the enzyme inhibitor. These results reveal that the enzymatic conversion of odorants in the nasal mucus appears be fast enough to affect olfactory perception, which sheds light on the previously unappreciated role of nasal mucosal enzymes in odor sensation.

Unlabelled: Nasal polyposis affects about 1 to 4% of the population. Polyps develop in oedematous and inflammated mucous membrane. In spite of the intensive research the pathomechanism of their development is not fully understood. The majority of the theories concerning the development of nasal polyps emphasize the role of the inflammatory process causing the rupture of the epithelium and the basal membrane. Cathepsin D is one of important mediators of inflammatory processes, that may be involved in the pathogenesis of nasal polyposis., The Aim of the Study: was to establish the role of the cathepsin D in the pathogenesis of nasal polyps. MATHERIAL AND METHOD: Tissues were taken from 39 patients treated with endoscopic sinus surgery due to chronic rhinosinusitis with polyps. The activity of the cathepsin D was assesed with spectrofotometric method using the specific inhibitor (pepstatin) in tissue of nasal polyps, in oedematous and the inflammated mucous membrane of the nasal conchae and the samples of mucous membrane taken from the nasal septum., Results: Statistically significant difference in cathepsin D activity between polypoid tissue, inflammated mucosa and the mucous membrane of the nasal septum was detected (t-student test, p < 0.05). No difference in the activity of this enzyme was observed between the polypoid tissue and the inflammated mucosa., Conclusion: Increased activity of the cathepsin D in nasal polyps and inflammatory changed mucosa confirm the important role of the cathepsin D in inflammatory processes leading to damage and subsequent remodeling of mucous membrane. We believe that further research on the activity of other proteolytic enzymes is necessary to demonstrate the differences between the inflammable changed mucous membrane and nasal polyps.

In this study, the expression and inducibility of CYP2C33, CYP2C42, CYP2C49, CYP2B22, CYP3A22, CYP3A29, and CYP3A46 were investigated at activity and/or transcriptional level in liver, kidney, small intestine, respiratory, and olfactory nasal mucosa of control and phenobarbital (PB)-treated pigs. PB treatment resulted in an up-regulation of mRNA levels of all analyzed CYPs in liver, of CYP2C42 and CYP2C49 in kidney, of CYP2C42, CYP2C49, CYP2B22, and CYP3As in small intestine. In liver microsomes from PB-treated pigs, these transcriptional activations were accompanied by an increase of various marker activities of human CYP2B6, CYP3As, CYP2C9, CYP2C19. Among the extrahepatic tissues, a significant induction by PB was observed only in kidney for the marker activities of CYP2C9. Taken together, our results demonstrated that the PB administration in pigs induced at least in liver, in addition to CYP2B22 and CYP3As, the expression of CYP2C33, CYP2C42, and CYP2C49 at transcriptional and activity levels. Furthermore our findings showed that the catalytic activities of porcine CYP2Cs are different amongst those observed and with respect to the human counterparts. Thus, the use of pigs as a model for humans in studies using drugs as substrates and/or inducers of CYP2Cs should be considered carefully.

Objective: To evaluate the lipopolysaccharide induction of cyclooxygenase-2 (COX-2) in long-term epithelial cultures from nasal polyp tissue of patients with cystic fibrosis (CF) and severe nasal polyposis., Design: Experimental and histologic study., Setting: Department of Otorhinolaryngology, University of Ulm, Ulm, Germany., Participants: Nasal polyp tissue was evaluated from 9 patients with CF and obstructing nasal polyps undergoing elective sinus surgery. Nasal mucosa from the hypertrophic inferior turbinate of 9 patients without a history of CF or aspirin intolerance undergoing nasal corrective surgery served as control specimens., Interventions: Tissue culturing, Western blotting, and tissue staining with hematoxylin-eosin., Main Outcome Measures: The expression and lipopolysaccharide induction of COX-2 was detected and compared with those of the control group. Tissue was analyzed for the presence of inflammatory cells such as neutrophils, eosinophils, mast cells, plasma cells, lymphocytes, and monocytes., Results: COX-2 was detectable in tissue specimens from all of the patients with CF and control subjects. In patients with CF, however, COX-2 expression was significantly lower in lipopolysaccharide-stimulated, long-term cultured epithelial cells compared with control tissue. Polyps from patients with CF contained markedly more neutrophils, macrophages, and plasma cells than did nasal mucosa from hypertrophic inferior turbinates of controls., Conclusions: COX-2 expression in lipopolysaccharide-stimulated, long-term cultured epithelial cells in patients with CF is decreased compared with that in hypertrophic turbinate mucosa. The estimated defect in lipopolysaccharide responsiveness and the reduced induction of COX-2 needs further investigation.

Objective: To study the feasibility of ginsenoside Rg1 administrated from nasal route., Method: The safty of ginsenoside Rg1 was evaluated by testing cilia movement of the in situ toad palate model. The stability of ginsenoside Rg1 to nasal enzymes and pH was evaluated by HPLC method. Nasal circulatory perfusion test in vivo had been empolyed to investigated absorption effect of ginsenoside Rg1 from nasal mucosa., Result: Ginsenoside Rg1 was safe to the cilia movement. Meanwhile, it's stable to nasal enzymes and pH. Ginsenoside Rg1 can be absorbed from nasal route., Conclusion: It's feasible to administrate ginsenoside Rg1 from nasal route.

Objectives: Characterize the expression and glucuronidation activities of the human uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) 2A2., Method: UGT2A1 was cloned from nasal mucosa mRNA. Synthetic cDNA for UGT2A2 was constructed assuming exon sharing between UGT2A1 and UGT2A2 (Mackenzie et al., Pharmacogenetics and Genomics 2005, 15:677-685). Exon 1 of UGT2A2 was amplified from genomic DNA and combined with exons 2-6 of UGT2A1. UGT2A3 was cloned from liver mRNA. Quantitative reverse-transcribed-PCR (RT-PCR) was used to evaluate the expression of all the three UGTs of subfamily 2A in different tissues. Recombinant UGT2A1, UGT2A2 and UGT2A3 were expressed in baculovirus-infected insect cells and analyzed for glucuronidation activity towards different substrates., Results: DNA sequencing of RT-PCR products from human nasal mucosa mRNA, confirmed exon sharing between UGT2A1 and UGT2A2. In addition, it indicated that the N-terminal signal peptide sequence of UGT2A2 is the longest among the human UGTs. Quantitative RT-PCR revealed that both UGT2A1 and UGT2A2 are mainly expressed in the nasal mucosa, and that their expression level in fetal samples was much higher than in adults. Activity assays with recombinant UGTs 2A1-2A3 showed broad substrate selectivity for UGT2A1 and UGT2A2. Although glucuronidation rates and substrate affinities were mostly higher in UGT2A1, the Km values for UDP-glucuronic acid were similar in both UGTs. In addition, there were regioselectivity differences between the two UGTs and, with a few substrates, particularly ethinylestradiol, the activity of UGT2A2 was higher., Conclusion: UGT2A2 is mainly expressed in the nasal mucosa and it has glucuronidation activity towards several different endobiotic and xenobiotic substrates.

Unlabelled: The airways are made up of ciliated epithelium which secretes mucous, protecting the respiratory tract from particles inhaled during breathing. Its is paramount to understand the physiology and the mechanisms involved in mucociliary activity. Literature suggests that Nitric oxide (NO), especially the one produced by iNOS expression, maintains the mucociliary function and the immune defense of the nasal cavity., Aim: to assess NO participation and the enzymatic pathways in the production of NO and mucociliary transport, using constructive and inductive NO synthetase inhibitors, L-NAME and aminoguanidine, respectively., Materials and Methods: frog palates were prepared and immersed in ringer (control), L-NAME or aminoguanidine solutions. The palates were immersed in these solutions for four periods of 15 minutes. Mucociliary transport measures were carried out before and after each exposure., Results: control palates maintained stable their transportation speed. L-NAME increased, while aminoguanidine reduced mucous transportation velocity., Conclusion: unspecific cNOS block with L-NAME and relatively specific iNOS block with aminoguanidine results leads us to propose that depending on the pathway, the NO can increase or reduce mucociliary transport in frog palates.

Objective: Chitin is a recognition element for tissue infiltration by innate cells implicated in allergy and helminth immunity, and this process can be negatively regulated by vertebrate chitinases. Acidic mammalian chitinase (AMCase) and chitotriosidase (ChT) have chitinolytic activity, but little is known about their roles in nasal polyps., Study Design: A prospective controlled study., Setting: A tertiary referral center., Subjects and Methods: Nineteen subjects with nasal polyps and 12 subjects with deviated nasal septums were recruited to obtain inferior turbinate mucosa samples. The expression levels of AMCase and ChT were compared in nasal polyp and inferior turbinate tissue samples. The tissue samples were analyzed by reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemical staining., Results: AMCase and ChT were detected in all nasal polyps and inferior turbinate tissues. AMCase and ChT messenger RNA and protein expression were significantly higher in nasal polyps than in inferior turbinate tissues. In nasal polyps, AMCase-positive and ChT-positive cells were detected in the epithelium, inflammatory cells, and submucosal gland., Conclusions: AMCase and ChT may be important mediators in the pathogenesis of nasal polyps. Nasal polyps appear to have elevated levels of chitinases, and the presence or growth of chitin-containing pathogens might enhance chitinase expression, resulting in nasal polyp formation and growth in susceptible individuals.

Objective: To determine the influence of allergic rhinitis (AR) on the expression of matrix metalloproteinase (MMP)-9, MMP-2, and tissue inhibitor-1 of metalloproteinase (TIMP-1) in nasal polyposis., Study Design: A case-control study., Setting: A tertiary referral center., Subjects and Methods: The expression of MMP-9, MMP-2, and TIMP-1 was investigated in the nasal polyp tissue (NP) and maxillary sinus mucosa (MM) samples from 20 AR patients and 20 nonallergic patients undergoing endoscopic sinus surgery. Trichrome, periodic acid-Schiff, and hematoxylin-eosin staining were also performed and those expression levels were compared., Results: Infiltration of eosinophils was shown more intensely in NP rather than in MM, especially in the presence of AR. In the NP of AR patients, increased expression of MMP-9, MMP-2, and TIMP-1 was observed more prominently than in that of the control group. In case of MM, however, there was no significant difference between AR patients and the control group., Conclusion: The presence of AR may enhance the expression of MMP-9, MMP-2, and TIMP-1 associated with airway remodeling in nasal polyposis.

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

Endoscopic coagulation of the sphenopalatine artery (SPA) is indicated in patients with recurrent or severe posterior epistaxis. The present work aimed at studying the effects of the sphenopalatine artery coagulation on the nasal seromucinous glands. The study was carried out on 30 patients who were scheduled for endoscopic SPA coagulation. All patients signed informed consents that were approved by the Committee of Medical Ethics. Minute punch biopsies were taken from the inferior turbinates, under local anesthesia, just before the procedure as well as 12 weeks and 1 year postoperatively. The biopsies were stained with hematoxylin and eosin and PAS Alcian blue. Histochemical examination was done for the activity of the succinic dehydrogenase, alkaline phosphatase and cholinesterase enzymes. Morphometric analysis as well as analysis of the histological and histochemical results showed that SPA coagulation was followed by an early decrease in the size, number and secretory activity of the nasal seromucinous glands, which was associated with decreased activity of the alkaline phosphatase enzyme in the endothelial cells of the nasal blood vessels and decreased activity of the cholinesterase enzyme in the cholinergic nerve fibers. These changes, however, were temporary and reversible since examination of the late postoperative biopsies, at 1 year, showed normalization of the number, appearance, and histochemical activity of the glandular acini as well as the surrounding blood vessels and cholinergic nerve fibers.