Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

Introduction: Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Methods: Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.

Results: Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.

Conclusions: We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms. 

Carcinoma de células escamosas em epitélio nasal de um hamster chinês: relato de caso

The squamous cell carcinoma (SCC) is a neoplasm that affect pets, production animals and exotic animals, and it’s very common in tropical countries like Brazil, once it develops a sparsely pigmented, stratified squamous epithelium and on surfaces mucosa exposed to ultraviolet action. The SCC is quite infiltrative but rarely causes metastases. Its occurrence in the nasal epithelium is widely reported in cats. This case is a chinese hamster (Cricetulus griséus), female, young, who developed a nasal nodule, and their only clinical sign was intense itching. The animal underwent surgery to perform an incisional biopsy, which was referred to the Veterinary Pathology Service of UNESP, campus of Araçatuba (SP) for histopathological evaluation. The material showed features consistent with squamous cell carcinoma well differentiated.

Influence of treatment with intranasal corticosteroids on the nasal mucosa, weight, and corticosteroid concentration in rats

Background: The effect of intranasal corticosteroids on the nasal epithelium mucosa is an important parameter of treatment safety. This study was designed to examine whether treatment with topical corticosteroids in patients with allergic rhinitis causes atrophic nasal mucosal changes, when compared with systemic corticosteroids, in rats. Methods: Male Wistar rats were treated daily during 7 weeks with topical administration with 10 microliters of normal saline (control group), 10 microliters of mometasone furoate group, 10 microliters of triamcinolone acetonide (T group), and 8 mg/kg of daily subcutaneous injections of methylprednisolone sodium succinate (MP group). Body weight was evaluated weekly. At the end of the treatment, rats were killed by decapitation to collect blood for determination of corticosterone levels and nasal cavities were prepared for histological descriptive analyses. Results: Treatment with T and MP decreased body weight. Plasma corticosterone concentration was significantly reduced by MP treatment and presented a clear tendency to decrease after T treatment. Histological changes observed in group T included ripples, cell vacuolization, increase in the number of nuclei, and decrease in the number of cilia in the epithelial cells. Conclusion: Growth and corticosterone concentration were impaired by T and MP at the same proportion, suggesting a role of this hormone in body gain. With the exception of T, intranasal or systemic treatment with the corticosteroids evaluated in this study did not affect nasal mucosa. (Am J Rhinol Allergy 26, e46-e49, 2012; doi: 10.2500/ajra.2012.26.3702)

Olfactory glia enhance neonatal axon regeneration

Olfactory ensheathing cells (OECs) migrate with olfactory axons that extend from the nasal epithelium into the olfactory bulb. Unlike other glia, OECs are thought to migrate ahead of growing axons instead of following defined axonal paths. However it remains unknown how the presence of axons and OECs influences the growth and migration of each other during regeneration. We have developed a regeneration model in neonatal mice to examine whether (i) the presence of OECs ahead of olfactory axons affects axonal growth and (ii) the presence of olfactory axons alters the distribution of OECs. We performed unilateral bulbectomy to ablate olfactory axons followed by methimazole administration to further delay neuronal growth. In this model OECs filled the cavity left by the bulbectomy before new axons extended into the cavity. We found that delaying axon growth increased the rate at which OECs filled the cavity. The axons subsequently grew over a significantly larger region and formed more distinct fascicles and glomeruli in comparison with growth in animals that had undergone only bulbectomy. In vitro, we confirmed (i) that olfactory axon growth was more rapid when OECs were more widely distributed than the axons and (ii) that OECs migrated faster in the absence of axons. These results demonstrate that the distribution of OECs can be increased by repressing by growth of olfactory axons and that olfactory axon growth is significantly enhanced if a permissive OEC environment is present prior to axon growth.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice.

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

Short terms effects of air pollution from biomass burning in mucociliary clearance of Brazilian sugarcane cutters

Nasal mucociliary system is the first line of defense of the upper airways and may be affected acutely by exposure to particulate matter (PM) from biomass burning. Several epidemiologic studies have demonstrated a consistent association between levels of air pollution from biomass burning with increases in hospitalization for respiratory diseases and mortality. To determine the acute effects of exposure to particulate matter from biomass burning in nasal mucociliary transport by saccharin transit time (STT) test, we studied thirty-three non-smokers and twelve light smokers sugarcane cutters in two periods: pre-harvest season and 4 h after harvest at the first day after biomass burning. Lung function, exhaled carbon monoxide (CO), nasal symptoms questionnaire and mucociliary clearance (MC) were assessed. Exhaled CO was increased in smokers compared to non-smokers but did not change significantly after harvest. In contrast, SIT was similar between smokers and non-smokers and decreased significantly after harvest in both groups (p < 0.001). Exposure to PM from biomass burning did not influence nasal symptoms. Our results suggest that acute exposure to particulate matter from sugarcane burned affects mucociliary clearance in smokers and non-smokers workers in the absence of symptoms. (C) 2011 Elsevier Ltd. All rights reserved.

Short terms effects of air pollution from biomass burning in mucociliary clearance of Brazilian sugarcane cutters

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Manifestações otorrinolaringológicas em crianças com AIDS

Introduction: The study of otolaryngologic manifestations in children HIV + can lead to early diagnosis of AIDS, allowing specific treatment, responsible by reduced morbidity and mortality. Objectives: Detect the otolaryngologic manifestations in children with AIDS and alert to the importance of the early diagnosis. Study design: Clinical prospective. Material and method: We evaluated 22 children with AIDS assisted at Faculdade de Medicina de Botucatu (São Paulo, Brasil). The medical records were reviewed and the children were submitted to otolaryngologic and hearing acuity exams. Results: We evaluated 12 boys and 10 girls, whose ages ranged from 8 months to 12 years. In B and C clinical classification were included 18 children who were using anti-retroviral medicaments. Physical examination mainly indicated cervical lymphadenopathy (18 cases), paleness of the nasal mucous membrane with abundant mucous secretion over the nasal epithelium (15 cases) and retraction of tympanic membranes (seven cases). The main otolaryngologic diagnoses were: rhinosinusitis (16 cases), oral candidiasis (13 cases), inadequate eustachian tube function (seven cases) and recurrent tonsillar infections (six cases). Conductive hearing loss were detected in 4 children. No child presented sensorineural hearing loss. Conclusions: The main otolaryngologic manifestations presented by the children with HIV virus were rhinosinusitis, oral candidiasis, inadequate eustachian tube function and recurrent tonsillar infections. The allergic aspect of the nasal mucous membrane and the cervical lymphadenopathy were frequent signs and could alert the otolaryngologyst to AIDS during the exam.

The percutaneous absorption of ionisable compounds

The effects of ionisation on transdermal drug delivery using excised human epidermis (HS) and silastic rubber (SR) as model permeation barriers were investigated in vitro using Franz-type absorption cells. Suspensions and solutions of salicylic acid (SA), the model ionogenic permeant, were used as donors and the variables studied were vehicle pH and trans-membrane pH-gradients. For solutions, the pH effect was related to the level of ionisation of the drug and the degree of saturation of the solution. With suspensions, the observed permeation rate was unaffected by pH. The penetration profiles through HS and SR were similar, although the overall flux through HS was about 70% of that observed through SR. Pretreatment of the membranes with various enhancer regimens, including oleic acid, Azone and N, N-dimethylamides in propylene glycol (PG) and isopropyl myristate (IPM) promoted the penetration of SA. SR was not a suitable model for enhancer pretreatment using IPM as a vehicle as the membrane was significantly disrupted by this vehicle. The results from comparable experiments with and without a trans-membrane pH-gradient did not have a significant effect upon flux or flux enhancement after pretreatment with the above enhancers. A theoretical model for the extraction coefficients of weak acids was derived using the partition coefficients of the ionised and unionised species, pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. The distribution of this solute between aqueous and oily phases, with and without added enhancer, was measured as a function of pH. The extraction coefficients determined were consistent with the model and showed that the behaviour of the system can be explained without referral to ion-pair mechanisms. Phosphonoacetate is an effective antiviral agent. However, as it is charged at physiological pH, its permeation across cell membranes is limited. To assess the improvement of the transport properties of this molecule, mono-, di- and tri-ester prodrugs were examined. These were assessed for stability and subsequent breakdown with respect to pH by HPLC. In vitro percutaneous absorption was observed using the triester, but not the ionic mono- or di-esters. The triester absorption could be potentiated using a range of enhancers with oleic acid being the most effective. Cyclodextrins (CD) have a role as absorption enhancers for peptide compounds across nasal epithelium. One potential mode of action is that CDs include these compounds, protect them from enzymic attack and thereby increase their residence time in the nasal epithelium. This study investigated the potential of CDs to protect ester prodrugs from enzymatic breakdown and prevent production of poorly transportable ionic species. Using a range of CD to ester molar ratios (10:1 to 2500:1) a small, but measurable, protection for the model esters (parabens) against esterase attack was observed. Possible mechanisms for this phenomenon are that CDs include the ester, making it unavailable for hydrolysis, the CDs may also affect the esterase in some way preventing access for the ester into the active site.

Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Novos sistemas terapêuticos para administração nasal de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Expression of the NF-κB inhibitor A20 is altered in the Cystic Fibrosis epithelium

Research Question: A20 is an LPS-inducible, cytoplasmic zinc finger protein, that inhibits TLR-activated NF-?B signalling by deubiquitinating TRAF6. A20 action is facilitated by complex formation with RNF11, Itch and TAX1BP1. This study investigates if the expression of A20 is altered in the chronically inflamed Cystic Fibrosis (CF) airway epithelium.

Methods: Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-?B subunits was analysed. Formation of the A20 ubiquitin editing complex was also investigated.

Results: In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-.

Conclusion: he expression of A20 is significantly altered in CF and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-?B-driven inflammation.

Reversibility of impaired nasal mucociliary clearance in smokers following a smoking cessation programme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)