The Functional Diversity of Nitric Oxide Synthase Isoforms in Human Nose and Paranasal Sinuses: Contrasting Pathophysiological Aspects in Nasal Allergy and Chronic Rhinosinusitis.

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.

Differential expression of angiotensin-converting enzyme-2 in human paranasal sinus mucosa in patients with chronic rhinosinusitis.

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-1ß (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.

Increased nasal matrix metalloproteinase-1 and -9 expression in smokers with chronic rhinosinusitis and asthma.

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.

Nasal odorant metabolism: enzymes, activity and function in olfaction.

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.

Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death.

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.

Genetic polymorphism of antioxidant enzymes in eosinophilic and non-eosinophilic nasal polyposis.

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.

Drug-metabolizing Enzymes and Efflux Transporters in Nasal Epithelium: Influence on the Bioavailability of Intranasally Administered Drugs.

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.

Upregulation of KLHDC4 Predicts a Poor Prognosis in Human Nasopharyngeal Carcinoma.

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.

Human neutrophil elastase induces MUC5AC overexpression in chronic rhinosinusitis through tumour necrosis factor-α converting enzyme.

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).

Macrolides increase the expression of 11ß-hydroxysteroid dehydrogenase 1 in human sinonasal epithelium, contributing to glucocorticoid activation in sinonasal mucosa.

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 [11ß-hydroxysteroid dehydrogenase (11ß-HSD1 and 11ß-HSD2)], steroid-synthesizing enzymes (3ß-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 3ß-HSD, CYP21, 11ß-HSD1 and CYP11B1 increased in human epithelial cells treated with clarithromycin and azithromycin, whereas the expression levels of 11ß-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 3ß-HSD, CYP11A1, CYP21, CYP11B1 and 11ß-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.

Proinflammatory mediators alter expression of nuclear factor kappa B-regulating deubiquitinases in sinonasal epithelial cells.

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.

Local and systemic proteolytic responses in chronic rhinosinusitis with nasal polyposis and asthma.

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.

Corticosteroid treatment regulates mucosal remodeling in chronic rhinosinusitis with nasal polyps.

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

Exhaled nitric oxide and nasal tryptase are associated with wheeze, rhinitis and nasal allergy in primary school children.

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-1ß) were not associated with symptoms or with nasal allergy. In conclusion, FeNO and nasal tryptase reflect allergic inflammation in the respiratory system.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

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 ß-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.

Essential role of the cytochrome P450 enzyme CYP2A5 in olfactory mucosal toxicity of naphthalene.

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.

Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation.

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.

Inducible nitric oxide synthase (iNOS) in sinonasal polyp pathogenesis.

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_glandular and epithelial_apical iNOS values decreased. In the ethmoid and maxillary sinuses, iNOS_D_. 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.

Respiratory epithelial adenomatoid hamartoma: a poorly recognized entity with mast cell recruitment and frequently associated with nasal polyposis.

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.