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The use of non-bronchoscopic brushings to study the paediatric airway Lane, Catherine; Burgess, Scott; Kicic, Anthony; Knight, Darryl; Stick, Stephen Jun 8, 2005

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ralssBioMed CentRespiratory ResearchOpen AcceResearchThe use of non-bronchoscopic brushings to study the paediatric airwayCatherine Lane1, Scott Burgess1,2, Anthony Kicic1, Darryl Knight3 and Stephen Stick*1,2Address: 1School of Paediatrics and Child Health, University of Western Australia, Nedlands, 6009, Western Australia, Australia, 2Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, 6001, Western Australia, Australia and 3Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, V6Z 1Y6, British Columbia, CanadaEmail: Catherine Lane - catherinel@ichr.uwa.edu.au; Scott Burgess - sburgess@mater.org.au; Anthony Kicic - anthonyk@ichr.uwa.edu.au; Darryl Knight - dknight@mrl.ubc.ca; Stephen Stick* - Stephen.Stick@health.wa.gov.au* Corresponding author    AbstractBackground: The use of cytology brushes for the purpose of obtaining respiratory cells fromadults for clinical and research purposes is well established. However, the safety and utility of non-bronchoscopic brushings to study the paediatric airway has not been assessed. The purpose of thisstudy was to assess the practicality of using non-bronchoscopic brushing to sample epithelial cellsfrom children for investigation of epithelial function in health and disease using a wide range ofmolecular and cellular techniques.Methods: Non-bronchoscopic brushing was investigated in a non-selected cohort of healthy, andmildly asthmatic children presenting for surgery unrelated to respiratory conditions, at the majorchildren's hospital in Perth. Safety and side-effects of the procedure were assessed. Cell number,phenotype and viability were measured for all samples. The potential of these cells for use in long-term cell culture, immunohistochemistry, western blotting, quantitative PCR and gene arraying wasexamined.Results: Non-bronchoscopic brushing was well tolerated in all children. The only significant sideeffect following the procedure was cough: nursing staff reported cough in 20% of patients; parentsreported cough in 40% of patients. Cells sampled were of sufficient quantity and quality to allowcell culture in 93% of samples. Similarly, protein and RNA extracted from the cells was suitable forinvestigation of both gene and protein expression using micro-array and real-time PCR.Conclusion: Non-bronchoscopic brushing in children is safe and easy to perform, and is notassociated with any complications. Using this technique, adequate numbers of epithelial cells can beretrieved to allow cell culture, western blotting, real time PCR, and microarray analysis. Thepurpose of this study is to demonstrate the utility of non-bronchoscopic airway brushing to obtainand study epithelial cells and to encourage others so that we can accelerate our knowledgeregarding the role of the epithelium in childhood respiratory disease.Published: 08 June 2005Respiratory Research 2005, 6:53 doi:10.1186/1465-9921-6-53Received: 02 February 2005Accepted: 08 June 2005This article is available from: http://respiratory-research.com/content/6/1/53© 2005 Lane et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)Respiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53BackgroundThe use of cytology brushes for the purpose of obtainingrespiratory cells from adults for clinical and research pur-poses is well established. This technique is generallyreported to be safe, both in adults with pulmonary diseaseand in healthy volunteers [1]. Samples are usuallyobtained under direct vision using a bronchoscope. How-ever, we and others have recently used non-broncho-scopic brushing to sample airway epithelial cells fromchildren [2,3]. This method has several advantages overbronchoscopic brushing as it is simple and quick to per-form, and there is no need for a bronchoscope. Althoughwe have now successfully used this method in combina-tion with a variety of cellular and molecular techniques toinvestigate the role of the epithelium in childhoodasthma, initially, very little had been published on thetopic. In addition, the difficulty in obtaining target organtissue from children has meant that most informationregarding common childhood diseases such as asthmahas been derived from studies performed in adults.We currently understand little about the molecular mech-anisms involved in the pathogenesis of asthma. The air-way epithelium is an especially attractive target in whichto identify new molecular mechanisms and therapeutictargets because it is critically involved in the developmentof asthma as the first cell of contact with the environment.This is particularly pertinent since it is likely that dysregu-lated epithelial repair in childhood asthma is a criticaldeterminant of disease progression in adults. To this end,the available evidence suggests that epithelial fragility anddysfunction as well as accumulation of sub-epithelialfibroblasts and remodelling of the airway wall in asthmacan occur early in childhood [4].The purpose of this report is to summarise our experienceswith non-bronchoscopic epithelial brushing techniquesand subsequent sample processing. We have detailed theuse of cells for gene and protein expression, and for cellculture. The use of primary culture systems has importantadvantages over the use of immortalized cell lines, andallows functional experiments to be conducted. We aim toencourage the use of these techniques to study normaldevelopmental processes in the lung and the early patho-physiology of respiratory diseases.MethodsChildren admitted to Princess Margaret Hospital for thepurposes of elective surgery for non-respiratory com-plaints were recruited for this study. Children were usuallyhaving minor gastrointestinal or ear, nose and throat sur-gery. The study was approved by the Princess MargaretHospital for Children Ethics Committee, and writtenmation sheet). The asthmatic and atopic backgrounds ofthe children were determined by allergen-specific IgE test-ing and a validated asthma and allergy questionnaire wasadministered to the parent or legal guardian [5]. Prior tosurgery, each child was anaesthetised and intubated. Anylon cytology brush (BC 25105, Olympus, Australia)was used to sample cells from the airway. The plasticsheath protecting the brush was removed and discarded,as retracting the brush into the tightly fitting sheath woulddislodge cells. The unprotected brush was inserted directlythrough the endotracheal tube, advanced until resistancewas felt, and rubbed against the epithelial surface to sam-ple cells. The brush was then withdrawn and the tip cut offinto 5 ml of culture media (RPMI-1640 containing 10%(v/v) heat inactivated foetal calf serum). This process wasrepeated at least once.A sub-group of twenty five of these children was studiedto assess how well the technique was tolerated. Respira-tory variables were monitored before, during, and afterthe brushing procedure. Symptoms following the brush-ing were recorded by contacting the parents within oneweek of the procedure. Children who underwent non-bronchoscopic brushing (12 male, mean age 10.2, SE0.71) were compared to 24 control children who wereanaesthetised and intubated only for similar procedures(15 male, mean age 10.9, SE 0.76).Sample ProcessingEpithelial cells were collected into RPMI-1640 containing10% (v/v) heat inactivated foetal calf serum. Sampleswere processed immediately. An aliquot of the cell sus-pension was diluted 1:2 with 0.4% trypan blue, andapplied to a haemocytometer. The total number of cells,and the percentage of viable cells, was determined undera light microscope, within 15 minutes of collection. Eachsample of cells obtained by non-bronchoscopic brushingwas processed to allow for use in multiple investigativetechniques; 1 × 106 cells were used for cell culture or pro-tein extraction, 0.5 × 106 cells to produce cytospin slidesfor immunocytochemical studies, and RNA was extractedfrom the remaining 1 × 106 cells.For culture, cells were washed once in RPMI-1640 mediaand the cell pellet resuspended in bronchial epithelialbasal media (BEBM, Clonetics, CA) supplemented withbovine pituitary extract (50 mM), insulin (5 mM), hydro-cortisone (0.5 mM), gentamicin (0.001%, v/v), ampho-tericin B (0.0005%, v/v), retinoic acid (0.1 µM),transferrin (10 mM), triiodothyronine (6.5 µM), epine-phrine (6.5 µM) and human recombinant epidermalgrowth factor (EGF: 0.5 µM). The cells were then seededinto a culture vessel (25 cm2 growth surface area) pre-Page 2 of 10(page number not for citation purposes)informed consent was obtained from the parents of thechildren prior to sampling (see appendix for parent infor-coated with a mixture of fibronectin, collagen and bovineserum albumin, and maintained at 37°C in a humidifiedRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53incubator. Twenty-four hours post-isolation, unattachedcells were collected. These cells were reseeded into thesame culture vessel, with fresh media containing UltroserG (2% v/v; BioSepra, CA), a serum substitute. The collec-tion and reseeding of viable unattached cells was repeatedat both 48 and 72 hours post isolation. Subsequent cul-tures were fed every second day and were usually passagedevery 13–16 days.Before the remaining cell suspension was used for proteinand RNA extraction, and to produce cytospin slides, themacrophages were removed by positive selection: the cellsuspension was added to a culture dish that had been pre-viously coated with CD-68 antibody (Dako, Australia).The plate was incubated for 20 minutes (37°C, 5% CO2)to allow the macrophages to adhere. The suspended epi-thelial cells were aspirated from the plate, and the macro-phages removed using trypsin (0.25%) for subsequentanalysis. The macrophage depleted cell suspension wasused to produce cytospins, extract protein, and extractRNA.Cytospin slides were prepared by centrifuging epithelialcells onto a glass slide in a cytocentrifuge (Hettich). Slideswere air dried, fixed in 4% paraformaldehyde for 10 min-utes, and then stored at -20°C until required. Immunocy-tochemical staining of the cytospins was used to confirmthe purity of the epithelial sample. Antibodies againstcytokeratin (a marker for tissue of epithelial origin), α -smooth muscle actin (a marker of myofibroblasts, myoep-ithelial cells and smooth muscle cells), smooth musclemyosin (a marker of smooth muscle cells), and vimentin(a marker of mesenchymal cells) were used to confirmepithelial cell phenotype.Immunocytochemical techniques provide only semi-quantitative data about protein expression, and are notsensitive enough to determine levels of protein expressionaccurately; therefore protein was extracted for analysiswith Western blotting. Protein was extracted from the pel-leted epithelial cells by lysing the cells in 200 µl of an SDSextraction buffer (20 mM Tris, 1 mM SDS, 1 mM DTT) inthe presence of protease inhibitors (Sigma). A commercialassay (Micro BCA Protein Assay, Pierce Biotechnology)was used according to the manufacturers instructions todetermine the concentration of total protein in the celllysate. For each sample, 50 µg of protein was subjected to12% SDS-PAGE, and immunoblotted with anti-β-actinantibody. Antibody binding was detected with ECL PlusWestern Blotting Detection Reagents (AmershamBiosciences).Total RNA was extracted from epithelial cells using theAustralia). RNA was prepared from 18 subjects accordingto a modified version of the protocol of Baugh et al [6]and hybridised to microarrays. For our first study, geneexpression profiles from 9 mild, asymptomatic asthmaticswere compared to 9 healthy children for a total of 18arrays. Expression of genes in cells from asthmatic andhealthy children was compared using Affymetrix HumanGenome U133 Arrays (HG-U95Av2), which examine theexpression of approximately 23,000 genes. Real-time PCRwas used to validate the array data for specific genes. Real-time PCR was conducted as previously described [3].Data was reported as mean (SE) and analysed by inde-pendent samples t-test. Significance was taken as p < 0.05.The proportion of basal cells was analysed using analysisof variance (ANOVA) to compare the difference betweenthe three phenotype groups.ResultsThe procedure is well toleratedThe only significant adverse event reported after the pro-cedure was cough (Table 1). The nursing staff recordedcough in 20% of patients undergoing brushing. Parentallyreported cough was higher, with 40% of parents reportingcough in those children undergoing the procedure. Coughpersisting longer than 4 hours was recorded in 32% ofpatients, and 28% had cough persisting to the followingday. Despite this, 100% of parents said that if asked by afriend "Should my child participate in this study?" theywould answer "yes". Children who underwent non-bron-choscopic brushing were, on average, ventilated for alonger period than the control group (27 vs. 22.3 min),but this was not statistically significant (p = 0.066, table1). There was no difference in any other clinical variablesrecorded, including the lowest level of oxygen saturation,and the time spent in recovery.Symptoms and respiratory variables were comparedbetween asthmatic and non-asthmatic children undergo-ing non-bronchoscopic brushing (Table 2). The nursingstaff did not record cough in any of the asthmatic patients.Parentally reported cough was higher in the asthmaticsubjects (43% vs. 39%), but this was not statistically sig-nificant (p = 0.863). Parents of asthmatic children alsoreported a higher level of cough persisting to the follow-ing day (43% vs. 22%), however this was not statisticallysignificant (p = 0.322).Epithelial cell retrieval and processingThe mean cell retrieval from 151 brushings was 2.67 × 106cells (SE = 0.16 × 106, Figure 1A) and on average, 17.3%of cells were viable (SE = 2.15%, Figure 1B). The numberor viability of cells retrieved was not related to asthmaticPage 3 of 10(page number not for citation purposes)QIAGEN RNeasy kit (Vic, Australia). RNA quality andquantity was assessed using the Agilent Bioanalyser (Vic,or atopic status. The epithelial phenotype of the cells wasconfirmed by immunostaining (Figure 2). To confirm thatRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53the population of epithelial cells sampled did not varybetween phenotypes, the number of cells expressing thebasal cell marker isolectin B4 was determined. The pro-portion of basal cells was independent of phenotype (Fig-ure 3).In our hands, the cell culture success rate is such that 93%of samples exhibit some growth, 80% grow to confluence,and 80% survive a second passage. Initial cultures con-sisted of a heterogeneous cell population composed ofboth terminally differentiated epithelial cells as indicatedby the presence of cilia, but also non-ciliated epithelialcells. Subsequently passaged cultures were found to solelyexhibit the non-ciliated epithelial morphology. No differ-types. Established cultures have been successfully grownup to passage 7. When the cells reach 80% confluence intheir second passage (Figure 4), they are used for func-tional studies.Protein was extracted from the epithelial cells; the averageprotein yield was 74 µg (SE = 8.79 µg, N = 18) per 1 × 106cells. This was sufficient for detection of β-actin usingWestern blotting, demonstrating the feasibility of deter-mining protein expression from cells obtained by thenon-bronchoscopic brushing technique.RNA extraction yielded on average 2.9 µg of RNA (SE =0.21, N = 58, Figure 5A), as assayed by Agilent BioanalyserTable 1: Symptoms following non-bronchoscopic brushing. Symptoms following non-bronchoscopic brushing, and observations of respiratory parameters before, during, and after brushing. Cough was the only reported symptom, and all parents said they would recommend the study to a friend.Controls mean (SE) Sampled mean (SE) p-valueNumber of participants 24 25 -Cough recorded 0% 20% (8%) 0.022Parental reported cough - 40% (10%) -Cough > 4 h - 32% (9%) -Cough following day - 28% (46%) -Recommend to friend? - 100% (0%) -Length of Ventilation (mins) 22.3 (1.85) 27 (1.68) 0.066Lowest oxygen saturation 98.6 (0.12) 96.8 (1.02) 0.083Supplemental oxygen 79% (8%) 80% (8%) 0.944Highest respiratory rate 22.7 (0.77) 22.2 (0.58) 0.607Time in recovery (mins) 21.5 (2.22) 18.4 (1.40) 0.246Table 2: Symptoms following non-bronchoscopic brushing. Comparison of symptoms following non-bronchoscopic brushing, and observations of respiratory parameters before, during, and after brushing in asthmatic and non-asthmatic patients.Non-asthma mean (SE) Asthma mean (SE) p-valueNumber of participants 18 7 -Cough recorded 28% (11%) 0% (0%) 0.129Parental reported cough 39% (12%) 43% (20%) 0.863Cough > 4 h 28% (11%) 43% (20%) 0.489Cough following day 22% (10%) 43% (20%) 0.322Recommend to friend? 100% (0%) 100% (0%) -Length of Ventilation (mins) 28.9 (1.92) 22.1 (2.86) 0.071Lowest oxygen saturation 96.9 (1.33) 96.4 (1.46) 0.845Supplemental oxygen 83% (9%) 71% (18%) 0.524Highest respiratory rate 22.6 (0.86) 21.0 (0.72) 0.282Time in recovery (mins) 18.8 (1.73) 17.3 (2.50) 0.644Page 4 of 10(page number not for citation purposes)ences in cellular morphology have been observed betweencultures established from healthy and asthmatic pheno-(Vic, Australia). The ribosomal RNA ratio was used as ameasure of RNA quality; the average ratio was 1.52 (SE =Respiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/530.12, N = 58, Figure 5B). This was sufficient RNA to useboth for microarray analysis (1 µg) and real-time PCRanalysis (0.5 µg).Preliminary data from the microarrays showed that geneexpression in epithelial cells from asthmatic subjects wassignificantly different to that in healthy subjects (Figure6). Expression of genes in the asthmatic cohort rangedfrom 6-fold up-regulated to 5-fold down-regulated com-pared to expression in the healthy controls.DiscussionWe have demonstrated that non-bronchoscopic brushingis safe and well tolerated in children. The main symptomreported following brushing was cough, which wasreported more frequently by parents than the nursingstaff. The procedure was acceptable to participants, asnone of the patients experienced symptoms that wouldcause the parents not to recommend the study to a friend.Respiratory variables and symptoms in children undergo-ing brushing were no different in asthmatics compared tonon-asthmatics (table 2). The level of cough reported byparents was increased in asthmatic children, however thisdifference was not significant. The difference was mostlikely due to increased observation of their child's symp-toms, as there was no difference in the level of coughThe non-bronchoscopic brushing technique was able toharvest useful quantities of epithelial cells, with a meanviability of 17.3%. Studies in adult asthmatics, harvestingcells under direct vision, report average viability rangingfrom 25–30%, with a significantly lower viability in theasthmatic epithelial cells [1,7,8]. In this study, the viabil-ity of the epithelial cells was not significantly different inthe asthmatics. This may reflect the mild nature of asthmaexperienced by these children, as previous studies havereported the level of viability is correlated with the severityof the disease [8].This report outlines a broad range of techniques that canbe used to study a single sample of airway epithelium,thus maximising the information obtained from onebrushing of the airway. Previous studies conducted inchildren have detailed only limited use of the sample [2].We report that it is possible to obtain cells of sufficientquality and quantity to allow investigation using cell cul-ture, immunohistochemistry, western blotting, real-timePCR, and microarray. We have used immunocytochemi-cal staining to examine expression of protein, and suffi-cient protein can be extracted to perform Western blotanalysis. We have cultured the epithelial cells, and canmaintain and propagate the cultures successfully overseven passages. Whilst non-bronchoscopic brushings ofA: Histogram of number of cells sampled in 151 non-bronchoscopic brushings of the paediatric airwayFigure 1A: Histogram of number of cells sampled in 151 non-bronchoscopic brushings of the paediatric airway. The number of cells retrieved ranged between 0.1 million and 11.8 million (mean 2.7 million). B: Histogram of the percentage of viable cells in 43 non-bronchoscopic brushings of the airway. The mean viability was 17.3%.Page 5 of 10(page number not for citation purposes)reported by nursing staff in asthmatic and non-asthmaticchildren.the paediatric airway are easily and safely obtained, it isimportant to maximise the amount of informationRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53obtained from each brushing. To this end, we have out-lined a program of non-bronchoscopic brushing thatutilises a wide range of techniques, allowing a broadapproach to the study of the airway epithelium inchildren.ConclusionThe purpose of this study was to demonstrate how a singlesample of airway epithelial cells obtained by non-bron-choscopic brushing can be used to study gene and proteinexpression and provides sufficiently viable cells to allowcell cultures to be established for functional analysis. Thetechnique is particularly useful for studying the paediatricairway because it is simple and minimally invasive, andcan be used to overcome a major obstacle to our under-standing of paediatric respiratory disease namely a paucityof target organ tissue. We have presented data that can beused by other groups to establish similar programs,Authors' contributionsCL carried out the protein and RNA analysis, was involvedin the cell culture work, and drafted the manuscript. SBdesigned and carried out the paediatric complicationsstudy, and participated in sample processing. AK refinedthe method for culture of airway epithelial cells. SS con-ceived of the non-bronchoscopic brushing program. DKestablished the cell culture program and with SS, partici-pated in the design and coordination of this study, andcontributed to the manuscript.AppendixParent Information SheetDoes raised exhaled nitric oxide reflect unrecognised airway inflammation in healthy children?You are being invited to take part in a research study.Before you decide, it is important for you to understandwhy the research is being done and what it will involve.Immunocytochemical confirmation of epithelial cell phenotypeFigure 2Immunocytochemical confirmation of epithelial cell phenotype. A: Pan-cytokeratin, B: α-smooth muscle actin, C: Smooth mus-cle myosin, D: Vimentin.Page 6 of 10(page number not for citation purposes)bench-mark for quality assurance and respond to com-mon questions by human research ethics committees.Please take time to read the following informationcarefully. Ask us if there is anything that is not clear or ifRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53you would like more information. Take time to decidewhether or not you wish to take part.GENERAL INFORMATION:Nitric oxide (NO) is a molecule that is involved in manyraised in asthmatics. Many studies have demonstratedthat exhaled NO might be a useful marker of airwayinflammation in asthma thus aiding diagnosis and moni-toring of disease activity. However, children who respondto skin allergy tests (atopic) and who do not have any res-piratory symptoms also have raised eNO. We do not knowwhy eNO is raised in healthy atopic children but it mayalso be due to inflammation in the airways that is notpresently causing respiratory problems. If we can deter-mine what causes raised eNO in healthy atopic childrenwe will better understand how this test will help us mon-itor airway disease."What will happen if I agree for my child to take part?"The present study will use standard diagnostic techniquesto investigate whether inflammation that might nototherwise be recognised is the cause of raised NO levels inthe breath of children with atopy. In order to study thisissue we will recruit children, WITH and WITHOUTATOPY, who are at Princess Margaret Hospital for daysurgery.We will use the following strategy:• You will be asked to complete a questionnaire aboutyour child's symptoms of allergy and asthma and medica-tions that may effect eNO levels.Percentage of basal cells in brushings obtained non-bronchoscopically from the paediatric airwayFigure 3Percentage of basal cells in brushings obtained non-bronchoscopically from the paediatric airway. A: Histogram of the percent-age of basal cells in each brushing. On average, 10.04% of cells were basal cells. B: Percentage of basal cells retrieved in brush-ings from each phenotype. The percentage of basal cells retrieved was not different depending on phenotype.Phase-contrast micrograph of cultured epithelial cellsFigure 4Phase-contrast micrograph of cultured epithelial cellsPage 7 of 10(page number not for citation purposes)physiological processes in the body including inflamma-tion. NO is detectable in exhaled breath (eNO) and isRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53• If your child is old enough they will be asked to blowinto a machine to determine their eNO levels. This mayoccur either before the operation or at the time of theirnext appointment.• During surgery we will obtain a brushing of the surfaceof the windpipe.• A blood sample will be taken for allergy testing to com-mon allergens. It will also be used for gene testing for NOgenes and asthma genes.Measurements we will make:1. Prior to surgeryExhaled nitric oxide – To measure eNO we will ask yourchild to take a deep breath in and blow into a machinewhile trying to maintain a constant expiratory flow.2. During surgeryBrushings – To collect cells from the wall of the airwayswe will pass a fine brush through the larynx (voice box)into the windpipe and rub along the wall of the windpipea few centimetres below the vocal cords. If you wish, wewill demonstrate the techniques and equipment usedBlood sample – Blood will be collected once your child isasleep.Risks:The brushing is a simple test and takes less than 5 minutesto complete. In adults it is performed without an anaes-thetic. The test will not be carried out if your child's anaes-thetist or surgeon believes the test will interfere with yourchild's treatment. We have routinely performed hundredsof these tests without incident. A dry cough is the onlyadverse symptom reported, it seems to occur in approxi-mately half of the children involved in our study.Benefits:We will be able to provide you with information regardingthe allergic status of your child.All information that is collected about your child duringthe course of the research will be kept strictly confidential.Any information about your child that leaves the hospitalwill have your/his/her name and address removed so thatyou/he/she cannot be recognised from it.It is up to you to decide whether or not to take part. If youdecide to take part you will be given this informationQuantity and quality of RNA extracted from brushings obtained non-bronchoscopically from the paediatric airwayFigure 5Quantity and quality of RNA extracted from brushings obtained non-bronchoscopically from the paediatric airway. A: Histo-gram of the amount of RNA extracted. On average, 2.9 µg of RNA was extracted from 2 ml of cell suspension. B: Histogram of the quality of RNA. The ribosomal RNA ratio was used as a measure of RNA quality; on average the RNA quality was 1.52.Page 8 of 10(page number not for citation purposes)before you decide to go ahead. sheet to keep and be asked to sign a consent form. If youdecide to take part you are still free to withdraw at anyRespiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53time and without giving a reason. This will not affect thestandard of care your child receives.If you have any complaints about any aspect of the studyyou can contact the Executive Director Medical Services ofPMH (Dr Geoff Masters) on 9340 1550Thank you for reading this information sheet.If you have any further questions with regard to thisstudy they can be discussed withDr Stephen Stick (Telephone 9340 8830)Dr Scott Burgess (Hospital switch board – Telephone9340 8222 page 2025 at any time)AcknowledgementsThis work was supported by a grant from the NH&MRC Australia (303145). We gratefully acknowledge the assistance of Professor Charles Mackay and Trina So (Garvan Institute of Medical Research, Sydney, Aus-tralia) in the hybridisation of the microarrays, and Professor David Erle (University of California, San Francisco) in the preliminary analysis of the microarray data.References1. Romagnoli M, Vachier I, Vignola AM, Godard P, Bousquet J, Chanez P:Safety and cellular assessment of bronchial brushing in air-way diseases.  Respir Med 1999, 93(7):461-466.Scatter plot of average gene expression (x axis) vs. log of fold change in gene expressionFigure 6Scatter plot of average gene expression (x axis) vs. log of fold change in gene expression. Those genes which are differentially regulated more than 1.5 fold are highlighted.Page 9 of 10(page number not for citation purposes)2. Doherty G, Christie S, Skibinski G, Puddicombe S, Warke T, DeCourcey F, Cross A, Lyons J, Ennis M, Shields M, et al.: Non-bron-Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Respiratory Research 2005, 6:53 http://respiratory-research.com/content/6/1/53choscopic sampling and culture of bronchial epithelial cells inchildren.  Clin Exp Allergy 2003, 33(9):1221-1225.3. Lane C, Knight D, Burgess S, Franklin P, Horak F, Legg J, Moeller A,Stick S: Epithelial inducible nitric oxide synthase activity is themajor determinant of nitric oxide concentration in exhaledbreath.  Thorax 2004, 59(9):757-760.4. Knight DA, Lane CL, Stick SM: Does aberrant activation of theepithelial-mesenchymal trophic unit play a key role inasthma or is it an unimportant sideshow?  Curr Opin Pharmacol2004, 4(3):251-256.5. Asher M, Keil U, Anderson H, Beasley R, Crane J, Martinez F, MitchellE, Pearce N, Sibbald B, Stewart A, et al.: International Study ofAsthma and Allergies in Childhood (ISAAC): rationale andmethods.  Eur Respir J 1995, 8(3):483-491.6. Baugh LR, Hill AA, Brown EL, Hunter CP: Quantitative analysis ofmRNA amplification by in vitro transcription.  Nucleic Acids Res2001, 29(5):E29.7. Campbell AM, Chanez P, Vignola AM, Bousquet J, Couret I, Michel FB,Godard P, Paul-Lacoste P, Vachier I, Roux S, et al.: Functional char-acteristics of bronchial epithelium obtained by brushingfrom asthmatic and normal subjects.  Am Rev Respir Dis 1993,147(3):529-534.8. Vignola AM, Campbell AM, Chanez P, Bousquet J, Paul-Lacoste P,Michel FB, Godard P, Vachier I, Roux S, Loubatiere J, et al.: HLA-DRand ICAM-1 expression on bronchial epithelial cells inasthma and chronic bronchitis.  Am Rev Respir Dis 1993,148(3):689-694.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 10 of 10(page number not for citation purposes)

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