The effects of physical exercise and smoking
habits on the expression of SPLUNC1 in nasal
lavage fluids from allergic rhinitis subjects
Kristina Irander, M.P. Borres and Bijar Ghafouri
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Kristina Irander, M.P. Borres and Bijar Ghafouri, The effects of physical exercise and
smoking habits on the expression of SPLUNC1 in nasal lavage fluids from allergic rhinitis
subjects, 2014, International Journal of Pediatric Otorhinolaryngology, (78), 4, 618-622.
http://dx.doi.org/10.1016/j.ijporl.2014.01.014
Copyright: Elsevier, Under a Creative Commons
license
http://www.elsevier.com/
Postprint available at: Linköping University Electronic Press
The
effects
of
physical
exercise
and
smoking
habits
on
the
expression
of
SPLUNC1
in
nasal
lavage
fluids
from
allergic
rhinitis
subjects
§
K.
Irander
a,
M.P.
Borres
b,c,
B.
Ghafouri
d,e,*
a
AllergyCenter,ENTSection,UniversityHospital,Linko¨ping,Sweden bDepartmentofWomen’sandChildren’sHealth,UppsalaUniversity,Sweden c
ThermoFisherScientific,Uppsala,Sweden d
DepartmentofMedicalandHealthSciences,DivisionofCommunityMedicineRehabilitationMedicine,FacultyofHealthSciences,Linko¨pingUniversity,and PainandRehabilitationCentre,CountyCouncilofO¨stergo¨tland,Linko¨ping,Sweden
e
OccupationalandEnvironmentalMedicine,DepartmentofClinicalandExperimentalMedicine,FacultyofHealthSciences,Linko¨pingUniversity,andCentre ofOccupationalandEnvironmentalMedicine,CountyCouncilofO¨stergo¨tland,Linko¨ping,Sweden
1. Introduction
Increasingattentionisbeingpaidtoagroupofproteinscalled palatelungandnasalepithelialclone(PLUNC),afamilyofrelated geneproductsincludingthreeshortproteins(SPLUNC1–3)andfive
long proteins (LPLUNC1–4, 6) [1]. The SPLUNC1 protein, also
knownasBPIfold-containingfamilyAmember1(BPIFA1)[2],isa
highlyabundantsecretedproteininupperairwaysandhasbeen
themoststudiedoneamongthefamilymembers[3].Accordingto
ARTICLE INFO Articlehistory:
Received15November2013
Receivedinrevisedform11January2014 Accepted14January2014
Availableonline23January2014 Keywords:
SPLUNC1protein Nasallavagefluid Allergicrhinitis Smokinghabits Physicalexercise Acousticrhinometry
ABSTRACT
Objective:Palate lungnasalepithelialclone(PLUNC) isafamilyofproteins,whichareproposedto participateintheinnateimmunedefenseagainstinfectionsintheupperaero-digestivetract.Theaimof thisstudywastoinvestigatetheexpressionofSPLUNC1inallergicrhinitissubjectswithconsiderations takentothemucosalfunctionandsmokinghabits.
Methods:Theparticipants,recruitedfromacohortfollowedfrominfancy,werere-examinedattheage of 18 years regarding allergy development. Based on medical histories and skin prick tests the participantswereclassifiedintogroupswithpersistentallergicrhinitis(n=18),intermittentallergic rhinitis(n=8)andhealthycontrols(n=13).Sevensubjects(3,2and2ineachgroup,respectively) reportedsmokinghabits.TheSPLUNC1levelsinnasallavagefluidswereanalyzedbyWesternblot. Changesinthevolumeofthepropernasalcavitybeforeandafterphysicalexercise(Vol2increase)were
analyzedbyacousticrhinometry.
Results:ComparedtothecontrolgrouptheSPLUNC1levelwassignificantlylowerinthepersistent allergygroup(3.83.4ODvs.1.31.5OD;p=0.02),butnotintheintermittentallergygroupwithout currentexposuretoallergens(3.64.7OD).NodifferenceswerefoundinVol2increasebetweenanyofthe
allergygroupsandcontrols.InsmokersVol2increasewassignificantlyreduced(p
<0.01)andtheSPLUNC1 levelswerelowercomparedtonon-smokers.AsignificantcorrelationwasfoundbetweenSPLUNC1and Vol2increase(p
<0.01;r=0.53)innon-smokers.
Conclusions: CurrentallergenexposurehasanimpactonSPLUNC1expressioninnasallavagefluid,why allergyoughttobeconsideredinstudypopulationswhereanalysesofSPLUNC1levelsareincludedinthe reports.Thenormalnasaldecongestionafterexercisewasnotaffectedbyallergyincontrasttosmoking habits. The correlation between SPLUNC1 levels and Vol2increase in non-smokers may indicate
involvementofSPLUNC1intheregulationofthenormalfunctionofthenasalmucosa.Complementary studiesareneededtoconfirmthesmoke-relatedreductionofSPLUNC1expressionandtoanalyzethe possibleparticipationofSPLUNC1inthenasalmucosaregulation.
ß2014TheAuthors.PublishedbyElsevierIrelandLtd.Allrightsreserved.
§
This isanopen-access articledistributedunderthetermsoftheCreative CommonsAttribution-NonCommercial-ShareAlike License,which permits non-commercialuse,distribution,and reproductioninany medium,providedthe originalauthorandsourcearecredited.
* Correspondingauthorat:RehabilitationMedicine,DepartmentofMedicineand HealthSciences(IMH),FacultyofHealthSciences,UniversityofLinko¨ping,SE-581 85Linko¨ping,Sweden.Tel.:+46101034657.
E-mailaddress:bijar.ghafouri@liu.se(B.Ghafouri).
ContentslistsavailableatScienceDirect
International
Journal
of
Pediatric
Otorhinolaryngology
j ou rna l h ome pa ge : w ww . e l se v i e r. co m/ l oc a te / i j porl
0165-5876/$–seefrontmatterß2014TheAuthors.PublishedbyElsevierIrelandLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.ijporl.2014.01.014
the immunohistochemical analyses SPLUNC1 is predominantly localizedinmucouscellsandductsofsubmucosalglands,butis alsofoundinsomeepithelialcells,andcoatsthesurfaceepithelial celllining[4].
ThefunctionsofthePLUNCproteinsarepartlydefined,butnew
information is continuously obtained. Great interest has been
focusedontheirfunctionasapartoftheinnateimmunedefense,
whichispresumedtobeduetothestructuralhomologybetween
thePLUNC proteins and mediators withknown effects against
Gram-negativebacteria,i.e.lipopolysaccharide-bindingand bac-tericidal/permeability-increasingproteins [5,6].The marked
hy-drophobicity and surfactant properties of the PLUNC proteins
interferewithbiofilmformationsbyairwaypathogens,andthese
propertiesaresuggestedtocontributetohostdefense[7].Therole ofantibacterialdefenseissupportedbyinvitrostudiesandanimal studies[8–13],aswellasinhumaninvivostudies[14–17].The functionandexpressionofPLUNCproteinshavealsobeenstudied inotherupperairwaydisorders.Incysticfibrosis[18,19]thelevels areincreased.Theirpotentialtoserveascancerbiomarkershas
been evaluated [4,20–24]. Furthermore, SPLUNC1 expressions
havebeenanalyzedinrelationtoexposuretoairborneindustrial pollutantswithknownirritatingeffectsintheairways,e.g.epoxy
chemicals[25]andcarbonnanotubes.[26].Reducedlevelshave
beenreportedintobaccosmokers[25,27].
UptonowknowledgeofnasalSPLUNC1expressioninallergic
rhinitissubjectsislimited.Inapilotstudy,includingsubjectswith intermittentallergicrhinitisduetopollenallergy,wepreviously
foundreducedSPLUNC1levelsin NLFduringthepollenseason
comparedtotheirlevelsoutofseasonandtonormalcontrols[28].
Theaim of this report, based on resultsfrom participantsin a
cohort study, was to gain further knowledge of SPLUNC1
expressionsin NLFfromallergicrhinitissubjects.In a previous
reportbasedonthiscohort,wefoundsmokinghabitstohavean
impactonthenasalmucosalfunction,asthenormaldecongestion afterphysicalexercisewasreducedinsmokers[29].Forthisreason
we foundan interest toinclude analysis of SPLUNC1levels in
relationtophysicalexerciseinnon-smokersandsmokersinthis report.
2. Materialsandmethods
2.1. Subjectsandallergydiagnoses
Theparticipantswererecruitedfroma cohortfollowedfrom
infancytotheageof18yearsregardingallergydevelopment[30].
Diagnosesofallergyatthe18-yearfollow-upwerebasedonthe
historiesofallergicsymptomsandcarefulclinicalexaminations,all ofwhichwereperformedduringwintertimeoutofpollenseason. Theparticipantshadtobefreefromairwayinfectionsforatleast
10 days prior to theexamination. Asthis reportis focused on
SPLUNC1 expression in NLF in relation to nasal allergy, only
subjectssufferingfrom allergicrhinitiswereincluded,whereas
atopic subjectswith dermatitis but no airway symptoms were
excluded. Thus, allergic rhinitis with or without concurrent
bronchialorskinsymptomswasdiagnosedintwenty-sixsubjects. 2.2. Skinpricktestandallergysub-groups
Thediagnosisofallergicrhinitiswasverifiedbyaskinpricktest
withALK extracts (ALK,Sweden AB) includingpollen allergens
(birch,timothy,mugwort)andperennialallergens(horse,cat,dog,
Dpteronyssinus, Dfarinae,Alternaria,Cladosporium). Based on
theseresultsthesubjectswereseparatedintoapersistentallergic
rhinitis sub-group sensitized to perennial allergens with or
without sensitization to pollens (PAR group; n=18) and an
intermittentallergicrhinitissub-groupsensitizedtopollensonly
(IARgroup;n=8).Healthyandpricktestnegativesubjectsserved ascontrols(n=13).
2.3. Smokinghabits
The subjectswere askedto reportactive smoking habits as
occasional(1–2cigarettesperweek),low(1–9cigarettesperday), moderate(10–20cigarettesperday)andheavy(>20cigarettesper
day). A number of subjectswith smoking habits areshown in
Table1.
2.4. Symptomscores
Nasalsymptomsexperiencedonthedayofexaminationwere
registeredbytheparticipantsonvisualanalogscales,scoringfrom
0(nosymptoms)to10(disablingsymptoms).Thecombinedscores
of four rhinitis symptoms (itching, sneezing, secretion and
obstruction)werecalculated.
2.5. Acousticrhinometryandthephysicalexercisetest
Exerciseisknowntoresultinanincreasedvolumeoftheproper nasalcavityduetodecongestionofthenasalmucosa.Thisfunction
was evaluated by acousticrhinometry before and immediately
afteraphysicalexercisetest(refusedbytwosubjectsinthePAR
group). The individualshad to runon a treadmillfor 6minto
achieveapulserateof160beatsperminute.Acousticrhinometry
was performed using Rhin 2000 (S.R. Electronics A.S., Lynge,
Denmark).Themeanvalues fromthreerecordings andthesum
frombothnasalcavitieswerecalculatedusingthecomputerized
program with pre-determined calculations of volumes and
minimal nasal cross-sectional areas [31]. The value of Vol2
corresponds to the volume in the anterior part of the proper
nasal cavity (the distance between 2.20 and 5.40cm from the
nostrils,as calculated inRhin 2000),and theincrease afterthe exercise (Vol2increase) was calculated and chosen for statistical
analysis.
2.6. Nasallavagesampling
Nasallavagewasperformedusingsalinepre-warmedto378C. Thesubjectheldtheheadbentforwardwiththefacehorizontally, while theleftnasalcavity wasfilled withsaline,using a10ml syringeconnectedtothenostrilviaashorttubeandanasalolive. After5minapproximately5mlofthesalinecouldberecoveredby aspiration.Thesampleswerecentrifugedtoremovecellulardebris
and aliquots of the supernatants were stored at 208C in
Eppendorftubesuntilanalysis. 2.7. Totalproteinconcentrations
TotalproteinconcentrationsinNLFweredeterminedwith Bio-RadproteinassaysaccordingtoBradford[32].
Table1
SPLUNC1levelsinsmokersandnon-smokersintheallergyandcontrolgroups.The numberofsubjectswithsmokinghabitsisshown(n).Occasional(occ);moderate (mod);opticaldensity(OD).
SPLUNC1(OD) SPLUNC1(OD)
Smokers Smokinghabits
occ/low/mod(n) Non-smokers (n) PARgroup 0.00.0 2/1/0 1.61.5 15 IARgroup 3.04.3 0/1/1 3.85.1 6 Controlgroup 1.91.2 2/0/0 4.13.6 11 Overallgroup 1.42.3 4/2/1 2.93.3 32
2.8. WesternblotanalysisofSPLUNC1levels
Iodoacetamide, dithiothreitol (DTT), sodium dodecyl sulfate
(SDS)andCHAPSwereacquiredfromSigma(Steinheim,Germany).
TEMED, Tween-20, 40% acrylamide solution,2% bis acrylamide
solutionandammoniumpersulfatewerepurchasedfromBio-Rad
(Hercules,CA,USA).Urea(proanalysis)wasfromFluka(Buchs,
Switzerland),andacetonitrileandaceticacidfromRiedel-deHae¨n (Seelze,Germany).Allotherchemicalswereofanalyticalgrade.
ProteinsfromnasallavagefluidwereseparatedusingSDS-PAGE, withagradientgelrangeT:5–20%andC:1.5%andastackinggelwith T:5%andC:5%onMini-ProteanIIelectrophoresiscellfromBio-Rad
Laboratories. Samples, 0.3
m
g of protein, were mixed 1:1 withcocktail(10%(w/v)SDS,150mMDTT,1%(w/v)bromophenolblue,
0.5mMTris–HClpH6.8,glycerol).Aspositivecontrolnasallavage
fluid,knowntocontainPLUNC,wasused.Thesampleswereboiled
3minbefore loaded in the wells on the SDS-PAGE and run in
electrodebuffer(0.16%(w/v)Tris,0.72%(w/v)glycine,0.05%(w/v)
SDS).TheSDS-PAGEwasrunforapproximately30minin100V,
60mAandthenelevatedto200Vuntilfinished.
SDS-PAGE gelswereblottedonImmun-BlotPVDFMembrane
using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad
Laboratories). Membranes were blocked in Tris-buffered saline
(40mMTris–HCl,500mMNaCl,pH7.5)with5%non-fatdriedmilk
overnight.MembraneswerewashedwithTween-20Tris-buffered
saline(TTBS:40mMTris–HCl,500mMNaCl,0.05%Tween-20)and
incubatedwithprimaryantibodyagainstSPLUNC1(goatpolyclonal, R&DSystems,MN,USA)inTTBSwith2%non-fatdriedmilkover
night.Themembraneswere washedwith TTBSand followedby
incubationwith HRP-conjugated secondaryantibody (anti-goat/
sheepIgG,SIGMA,MI,USA)for1h.Thelatterwashprocedurewas repeatedoncepursuedbydetectionofantigen/antibodyconjugate
withECL(GEHealthcare)anddevelopedonX-rayfilm.TheX-ray
filmswerevisualizedusingacooledCCD(charged-coupleddevice) cameradigitizingat13401040pixelsresolution(Fluor-S Multi-Imager,Bio-RadLaboratories,CA,USA)incombinationwithanalysis
softwareQuantityOneVersion 4.3.1 (Bio-RadLaboratories).The
amountofproteininabandwasassessedasopticaldensity(OD). 2.9. Statisticalanalyses
StatisticswereanalyzedbyusingtheGraphPadPrismsoftware
program.Thenon-parametric methodofMann–WhitneyU test
was used in calculations of differences between two groups.
Results are presented as mean values1 standard deviation.
Spearmanrankcorrelationtestwasusedintheanalysesofcorrelation betweentwoparameters.Atwo-tailedp-valueof0.05wasregarded assignificant.
2.10. Ethicalconsiderations
The study was approved by the Ethical committee at the
University Hospital in Linko¨ping, Sweden (03694). A written
informedconsentwasobtainedfromeachoftheparticipants.The studywasperformedaccordingtotheprinciplesintheDeclaration ofHelsinki.
3. Results
3.1. Proteinconcentrationsinnasallavages
ThetotalproteinconcentrationsintheNLFwere250190
m
g/ ml(PARgroup),330260m
g/ml(IARgroup)and180100m
g/ml (control group). No significant statistical differences were found betweengroups.3.2. Symptomscores
Thecombinedrhinitissymptomscoreswerelow,andthescore
value of 2.84.1 in the PAR group was not significantly high
comparedtothevaluesof1.21.7intheIARgroupand1.62.8in thecontrolgroup.
3.3. SPLUNC1levelsinrelationtoallergicrhinitis
TheSPLUNC1proteincouldbedetected bytheWesternblot
analysisasadistinctbandat25kDa (Fig.1). Themeanlevel of
SPLUNC1,analyzedinthesameamountoftotalprotein(0.3
m
g)fromeachoftheNLFsamples,wassignificantlylowerinthePAR
groupcomparedtothecontrolgroup(1.31.5ODvs.3.83.4OD;
p=0.02). The mean level in IAR group (3.64.7 OD) was not
statisticallydifferentfromthelevelinthecontrolgroup(Fig.2). 3.4. SPLUNC1levelsinrelationtosmokinghabits
The smokers were equally distributed between the three
groups.ThelevelsofSPLUNC1wereingeneralnumericallylower
Fig.2.SPLUNC1levelsintheallergygroupsandthecontrolgroup.Opticaldensity (OD);notsignificantdifference(n.s.).
Fig.1.ArepresentativewesternblotimageforSPLUNC1innasallavagefluidfrom healthy controls, subjects with persistentallergic rhinitis and subjects with intermittent allergicrhinitis. The quantification data from the allergy group (persistent;n=18andintermittent;n=8)andhealthycontrols(n=13)areshown inFig.2.
insmokerscomparedtonon-smokersinallgroups(Table1).The numberofsmokersintheseparateallergygroupswastoolowfor statisticalcalculations.
3.5. Vol2increaseinrelationtoallergy,smokinghabitsandSPLUNC1
levels
IntheoverallgrouptheVol2increasewas2.11.4cm3.Allergy
wasnotfound tohaveanyimpact onthisincreaseincontrastto smokinghabits.ThelevelofVol2increasewassignificantlylowerinthe
smokinggroupcomparedtothenon-smokinggroup(0.51.1cm3;
n=7 smokers vs. 2.41.3cm3; n=30 non-smokers; p<0.01). A
significant correlation was found between Vol2increase and the
SPLUNC1levelsinnon-smokers(p<0.01;r=0.53;n=30)(Fig.3). 4. Discussion
This study showed that allergy has an impact on SPLUNC1
expression.ThelevelsofSPLUNC1weresignificantlylowerinthe
PAR group, being currently exposed to airway allergens, as
comparedtothelevelinthehealthycontrolgroup.TheSPLUNC1
levelintheIARgroup,beingoutoftheirpollenseasonwithno currentallergenexposure,wasnotdifferentfromthecontrols.Itis ofinteresttonotice,thatthisinfluenceonSPLUNC1levelsisfound
despitequitemodestsymptomsofnasalallergywithscoresonly
slightlyandnotsignificantlyhigherinthePARgroupcomparedto
theIARgroupand thecontrols. Theresult ofreduced levelsof
SPLUNC1in currently allergen exposed allergic subjects are in
accordance withtheresultsin our previous pilotstudy,where
SPLUNC1levelsinNLF wereanalyzedby proteomictechniques
[28], showing significantly reduced SPLUNC1 levels in pollen
allergicsubjectsduringpollenseason,butnormalizedvaluesoutof
seasonascomparedtocontrols.Thus, twodifferentmethodsof
SPLUNC1 analysis have verified significant reductions of nasal
SPLUNC1 levels in allergic rhinitis subjects during periods of
current allergen exposure. A relation between the severity of
rhinitissymptomsandthelevelofSPLUNC1wouldincreasethe
acceptanceofSPLUNC1involvementinallergicrhinitis.Arelation wassupportedaccordingtotheresultsinourpreviousstudy[28], wherethesignificantlyhighersymptomscorelevelduringallergy
season in rhinitis subjects was associated with a significant
reductionoftheSPLUNC1levelinthesesubjectsascomparedto
healthy controls. This relation was not found in this study,
probablyduetothemodestlevelsofsymptomsinthePARsubjects.
Adverseeffectson thenasalmucosadue tosmokeexposure
werefound, eventhoughthesmokinghabitsweremodest.The
normalexerciserelatedincreaseofthenasalcavityvolumewas
significantlylowerinsmokerscomparedtonon-smokers;thisis
previously reported [29]. Analysis of SPLUNC1 expression in
relation tosmoking habits showednumericallylower levels of
SPLUNC1insmokerscomparedtonon-smokersinlinewithother
studies[25,27].Thedifferencedidnotreachstatisticalsignificance,
probably due to the low number of participants, which were
recruitedfromacohortdesignedforlongitudinalfollow-upsand
not permitting substitution of subjects excluded for various
reasons. The impact of smoking on the nasal mucosa was
expressedinamoreobviouswayintheanalysisofSPLUNC1in
relation to Vol2increase. These two parameters were found to
correlatesignificantly,butonlyinnon-smokingindividuals.This
relation was not detected in smokers, apparently due to the
reductionofSPLUNC1levelsaswellasoftheVol2increasevalues.
TheassociationbetweenSPLUNC1andthenormaldecongestionof
the nasal mucosa has to our knowledge not been described
previouslyandfurtherstudiesareneededinordertoexplainthe
mechanisms behind theresults. We can only speculate on the
implication of this correlation, whether it is an indicationof a
participation of SPLUNC1 in the normal function of the nasal
mucosa,orwhethertheelasticityofthemucousmembraneaswell
asthelevelofSPLUNC1isaffectedinparallelbysomecommon
factor. Such a factor might be neutrophil elastase, which is
describedtoreduceSPLUNC1[33].
Inconclusion,ouranalysesofSPLUNC1expressionsinNLFhave
shownnewandvaluableinformation.Currentallergenexposure,
even at low levels causing modest clinical symptoms, has a
significantimpactonSPLUNC1levelsinallergicrhinitissubjects. Thus,itcouldbeofimportance,eveninnon-allergicupperairway
disorders, to consider current respiratory allergy in study
populations, where nasal SPLUNC1 levels are compared
inter-individually. However, SPLUNC1 cannot be regarded as an
adequate biomarker of allergy in single subjects, due to
over-lappingvaluesbetweenallergicandhealthyindividuals.
Smoking habits were found to have adverse effects on the
SPLUNC1levelsandmucosalfunction.Thepossibleinvolvementof SPLUNC1inthenormalfunctionofthenasalmucosa,indicatedby
the significant correlation in non-smokers between SPLUNC1
levelsandtheincreaseinnasalvolumes,needsfurtheranalyses
includingthewayitisimpairedbytobaccosmoke.
Competinginterests
Nocompetingfinancialinterestsexist.
Conflictsofintereststatement
Theauthorshavenoconflictsofinteresttoreport. Acknowledgments
SpecialthanksalsotoLenaLindell,NinaTimelinandLisbeth Hja¨lle,Linko¨pingUniversity,forexcellentassistance.
Financial support was provided by Stiftelsen Astma- och
Allergifo¨rbundets Forskningsfond and Swedish Association of
Otorhinolaryngology,HeadandNeckSurgery.
References
[1]C.D.Bingle,E.E.LeClaire,S.Havard,L.Bingle,P.Gillingham,C.J.Craven, Phyloge-neticandevolutionaryanalysisofthePLUNCgenefamily,ProteinSci.13(2004) 422–430.
[2]C.D.Bingle,R.L.Seal,C.J.Craven,SystematicnomenclatureforthePLUNC/PSP/ BSP30/SMGBproteins asasubfamilyoftheBPIfold-containingsuperfamily, Biochem.Soc.Trans.39(2011)977–983.
[3]L.Bingle,C.D.Bingle,DistributionofhumanPLUNC/BPIfold-containing(BPIF) proteins,Biochem.Soc.Trans.39(2011)1023–1027.
Fig.3.Thecorrelationbetweentheincreaseofthenasalvolumeafterexercise (Vol2increase
)andtheSPLUNC1levelsinnon-smokersandsmokers.Opticaldensity (OD).
[4]L.Bingle,S.S.Cross,A.S.High,W.A.Wallace,D.A.Devine,S.Havard,etal.,SPLUNC1 (PLUNC)isexpressedinglandulartissuesoftherespiratorytractandinlung tumourswithaglandularphenotype,J.Pathol.205(2005)491–497. [5]C.D.Bingle,S.-U.Gorr,Hostdefenseinoralandairwayepithelia:chromosome20
contributesanewproteinfamily,Int.J.Biochem.CellBiol.36(2004)2144–2152. [6]Y.P.Di,FunctionalrolesofSPLUNC1intheinnateimmuneresponseagainst
Gram-negativebacteria,Biochem.Soc.Trans.39(2011)1051–1055.
[7]L.Gakhar,J.A.Bartlett,J.Penterman,D.Mizrachi,P.K.Singh,R.K.Mallampalli, etal.,PLUNCisanovelairwaysurfactantproteinwithanti-biofilmactivity,PLoS ONE5(2010)e9098.
[8]H.W.Chu,J.Thaikoottathil,J.G.Rino,G.Zhang,Q.Wu,T.Moss,etal.,Functionand regulationofSPLUNC1proteininmycoplasmainfectionandallergic inflamma-tion,J.Immunol.179(2007)3995–4002.
[9]H.-D.Zhou,X.-L.Li,G.-Y.Li,M.Zhou,H.-Y.Liu,Y.-X.Yang,etal.,EffectsofSPLUNC1 proteinonthePseudomonasaeruginosaandEpstein–Barrvirus,Mol.Cell. Bio-chem.309(2008)191–197.
[10]L.Lukinskiene,Y.Liu,S.D.Reynolds,C.Steele,B.R.Stripp,G.D.Leikauf,etal., AntimicrobialactivityofPLUNCprotectsagainstPseudomonasaeruginosa infec-tion,J.Immunol.187(2011)382–390.
[11]D.Jiang,S.E.Wenzel,Q.Wu,R.P.Bowler,C.Schnell,H.W.Chu,Humanneutrophil elastasedegradesSPLUNC1andimpairsairwayepithelialdefenseagainst bacte-ria,PLoSONE8(2013)e64689.
[12]Y.Liu,J.A.Bartlett,M.E.Di,J.M.Bomberger,Y.R.Chan,L.Gakhar,etal.,SPLUNC1/ BPIFA1contributestopulmonaryhostdefenseagainstKlebsiellapneumoniae respiratoryinfection,Am.J.Pathol.182(2013)1519–1531.
[13]S.Sayeed,L.Nistico,C.StCroix,Y.P.Di,MultifuntionalroleofhumanSPLUNC1in Pseudomonasaeruginosainfection,Infect.Immun.81(2013)285–291. [14]L.Fornander,B.Ghafouri,E.Kihlstro¨m,B.A˚kerlind,T.Scho¨n,C.Tagesson,etal.,
InnateimmunityproteinsandanewtruncatedformofSPLUNC1in nasopharyn-gealaspiratesfrominfantswithrespiratorysyncytialvirusinfection,Proteomics Clin.Appl.5(2011)513–522.
[15]L.M. Teran,S.Ru¨ggeberg,J.Santiago,F.Fuentes-Arenas, J.L.Herna´ndez,A.R. Montes-Vizuet,etal.,ImmuneresponsetoseasonalinfluenzaAvirusinfection: aproteomicapproach,Arch.Med.Res.43(2012)464–469.
[16]S.Seshadri,D.C.Lin,M.Rosati,R.G.Carter,J.E.Norton,L.Suh,etal.,Reduced expressionofantimicrobialPLUNCproteinsinnasalpolyptissuesofpatientswith chronicrhinosinusitis,Allergy67(2012)920–928.
[17]Y.A.Tsou,M.T.Peng,Y.F.Wu,C.H.Lai,C.D.Lin,C.J.Tai,etal.,DecreasedPLUNC expression innasalpolypsis associatedwithmultibacterial colonizationin chronicrhinosinusitispatients,Eur.Arch.Otorhinolaryngol.(2013)(Epubahead ofprint).
[18]L.Bingle,F.A.Barnes,S.S.Cross,D.Rassl,W.A.Wallace,M.A.Campos,etal., Differential epithelialexpression of the putative innate immune molecule SPLUNC1incysticfibrosis,Respir.Res.8(2007)79.
[19]A.L.Garland,W.G.Walton,R.D.Coakley,C.D.Tan,R.C.Gilmore,C.A.Hobbs,etal., MolecularbasisforpH-dependentmucosaldehydrationincysticfibrosisairways, Proc.Natl.Acad.Sci.U.S.A.110(2013)15973–15978.
[20]S.Beniloch,J.M.Galbis-Caravajal,C.Alenda,F.M.Peiro´,M.Sanches-Ronco,J.M. Rodriguez-Paniagua,etal.,Expressionofmolecularmarkersinmediastinalnodes fromresectedstage1non-small-celllungcancer(NSCLC):prognosticimpactand potentialroleasmarkersofoccultmicrometastasis,Ann.Oncol.20(2009)91–97. [21]P.A.Vargas,P.M.Speight,C.D.Bingle,A.W.Barett,L.Bingle,ExpressionofPLUNC familymembersinbenignandmalignantsalivaryglandtumours,OralDis.14 (2008)613–619.
[22]A.Vidotto,T.Henrique,L.S.Raposo,J.V.Maniglia,E.H.Tajara,Salivaryandserum proteomicsinheadandneckcarcinomas:beforeandaftersurgeryand radio-therapy,Canc.Biomarkers8(2011)95–107.
[23]W.A.Gonza´lez-Arriagada,A.R.Santos-Silva,F.A.Ito,P.A.Vargas,P.M.Speight,L. Bingle,etal.,ExpressionpatternofPLUNCproteinsasanauxiliarytoolforthe diagnosisofhigh-grademucoepidermoidcarcinomaofthesalivarygland,J.Oral Pathol.Med.41(2012)589–597.
[24]P.Chen,X.Guo,H.Zhou,W.Zhang,Z.Zeng,Q.Liao,etal.,SPLUNC1regulatescell progressionand apoptosis through the miR-141-PTEN/p27 pathway,butis hinderedbyLMP1,PLoSONE8(2013)e56929.
[25]B.Ghafouri,E.Kihlstro¨m,B.Sta˚hlbom,C.Tagesson,M.Lindahl,PLUNC(palate, lungandnasalepithelialclone)proteinsinhumannasallavagefluid,Biochem. Soc.Trans.31(2003)810–814.
[26]Y.P.Di,A.V.Tkach,N.Yanamala,S.Stanley,S.Gao,M.R.Shurin,etal.,Dualacute pro-inflammatoryandanti-fibroticpulmonaryeffectsofSPLUNC1afterexposure tocarbonnanotubes,Am.J.Respir.CellMol.Biol.49(2013)759–767. [27]K.Steiling,A.Y.Kadar,A.Bergerat,J.Flanigon,S.Sridhar,V.Shah,etal.,
Compari-sonofproteomicandtranscriptomicprofilesinthebronchialairwayepithelium ofcurrentandneversmokers,PLoSONE4(2009)e5043.
[28]B.Ghafouri,K.Irander,J.Lindbom,C.Tagesson,M.Lindahl,Comparative proteo-micsofnasalfluidinseasonalallergicrhinitis,J.ProteomeRes.5(2006)330–338. [29]K.Irander,M.P.Borres,J.P.Palm,Acousticrhinometry,spirometryandnitricoxide inrelationtoairwayallergyandsmokinghabitsinanadolescentcohort,Int.J. Pediatr.Otorhinolaryngol.75(2011)177–181.
[30]K.Irander,M.P.Borres,An18-yearfollow-upofallergydevelopmentrelatedtonasal metachromaticcellfindingsduringinfancy,Allergol.Int.59(2010)193–200. [31]E.Millqvist,M.Bende,Referencevaluesfor acousticrhinometryinsubjects
withoutnasalsymptoms,Am.J.Rhinol.12(1998)341–343.
[32]M.M.Bradford,Arapidandsensitivemethodforthequantitationofmicrogram quantitiesofproteinutilizingtheprincipleofprotein-dyebinding,Anal.Biochem. 72(1976)248–254.
[33]D.Jiang,S.E.Wenzel,Q.Wu,R.P.Bowler,C.Schnell,etal.,Humanneutrophil elastasedegradesSPLUNC1andimpairsairwayepithelialdefenseagainst bacte-ria,PLoSONE8(2013)e64689.
