Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors.docVIP

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Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors.doc

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Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

BarrierDisruptingEffectsofAlternariaAlternataExtract onBronchialEpitheliumfromAsthmaticDonors MarinaS.Leino1.,MatthewLoxham1*.,CorneliaBlume1,EmilyJ.Swindle1,NivenkaP.Jayasekera1, PatrickW.Dennison1,BettyW.H.Shamji2,MatthewJ.Edwards2,StephenT.Holgate1, PeterH.Howarth1,DonnaE.Davies1 1AcademicUnitofClinicalandExperimentalSciencesandtheSouthamptonNIHRRespiratoryBiomedicalResearchUnit,UniversityofSouthamptonFacultyofMedicine, SirHenryWellcomeLaboratories,SouthBlock,UniversityHospitalSouthampton,Southampton,UnitedKingdom,2NovartisInstitutesforBiomedicalResearch,Novartis HorshamResearchCentre,Horsham,UnitedKingdom Abstract SensitizationandexposuretotheallergenicfungusAlternariaalternatahasbeenassociatedwithincreasedriskofasthma andasthmaexacerbations.Thefirstcellstoencounterinhaledallergensareepithelialcellsattheairwaymucosalsurface. Epithelialbarrierfunctionhaspreviouslybeenreportedtobedefectiveinasthma.Thisstudyinvestigatedthecontribution ofproteasesfromAlternariaalternataonepithelialbarrierfunctionandinflammatoryresponsesandcomparedresponsesof in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non- asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severeasthmaticdonorswerechallengedapicallywithextractsofAlternariaandchangesininflammatorycytokinerelease andtransepithelialelectricalresistance(TER)weremeasured.ProteaseactivityinAlternariaextractswascharacterisedand the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment.In16HBEcells,AlternariaextractsstimulatedreleaseofIL-8andTNFa,withconcomitantreductioninTER; theseeffectswerepreventedbyheat-treatmentoftheextracts.Examinationoftheeffectsofproteaseinhibitorssuggested thatserineproteaseswerethepredominantclassofproteasesmediatingtheseeffects.ALIculturesfromasthmaticdonors exhibited a reduced IL-8 respo