2005The future of animal models of invasive aspergillosis

MedicalMycologySupplement12005,43,S115ÁS119

ThefutureofanimalmodelsofinvasiveaspergillosisT.F.PATTERSON

DepartmentofMedicine,UniversityofTexasHealthScienceCenteratSanAntonio,SanAntonio,Texas,USAThediagnosisofinvasiveaspergillosisremainsverydifficult,coupledwithlimited

treatmentoptions.Animalmodelshavebeenutilizedtoevaluateboththediagnosis

andtreatmentofinfectionandtoassessthepathogenicityandvirulenceofthe

organism.However,animalmodelshavenotbeenstandardizedandhavebeenused

inonlyalimitedfashionforgenomicevaluationinthisdisease.Extensiveefforts

areunderway

toexpandsignificantlytheAspergillusgenomicinformation.Thus,

thestandardizationofanimalmodelsofinvasiveaspergillosisiscriticaltocreatea

unifiedplatformtoenhanceevaluationofnewergenomicinformationandallow

assessmentofpathogenicityandvirulencefactors.Proposedmodels,supportedby

arecentlyawardedNationalInstitutesofHealth/NationalInstituteofAllergyand

InfectiousDiseasescontract,willbedevelopedincloseinteractionwiththe

extendedAspergilluscommunity(includingacademiaandindustry)toanswerkey

questionsinthisdisease.Thegoalofthisworkistoprovidetheframeworkto

evaluategenomictargetsinanimalmodelsinordertoimprovethediagnosisand

treatmentofinvasiveaspergillosisthatwillultimatelyresultinimprovedoutcomes

ofpatientswiththisfrequentlyfatalinfection.

KeywordsAspergillus,invasiveaspergillosis,animalmodels

Introduction

Animalmodelshavebeenutilizedtoevaluateboththe

diagnosisandtreatmentofinvasiveaspergillosis,and

havealsobeenusedtoassesspathogenicandvirulence

featuresoftheorganism[1Á3].However,thelackof

standardizationofanimalmodelsystemsinthisdisease

haslimitedtheirvalueinassessingbothvirulenceand

pathogenicityandhamperedeffortstoidentifynew

diagnosticandtherapeutictargets.Extensiveeffortsare

underwaytosignificantlyexpandtheAspergillusgeno-

micinformation;asaresult,thereisgreatpotentialfor

identifyingnewdiagnosticandtherapeuticstrategies

criticallyneededforthisoftenlethalinfection.One

approachtoimprovingAspergillusanimalmodelshas

beentheawardofacontractbytheNationalInstitutes

ofHealth/NationalInstituteofAllergyandInfectious

Disease(NIH/NIAID)toestablishandstandardizeanimalmodelsofinvasiveaspergillosis,includingmurineandlargeranimalspecieswithbothpulmonaryanddisseminatedinfection.Thisbriefreviewwilldiscussthefutureofanimalmodelsofinvasiveaspergillosis,thelimitationsofcurrentanimalmodels,andthechallengesfacedindevelopingstandardizedmodels.Theaimsandcriticalneedsforfuturemodels,particularlyinthedevelopmentofnewdiagnostictools,willalsobeoutlined.Thegoalofthisworkistoprovidetheframeworkforevaluatinggenomictargetsinordertoimprovethediagnosisandtreatmentofinvasiveaspergillosis,whichwillultimatelyresultinimprovedoutcomesforpatients.ObjectivesoffutureanimalmodelsThefuturedevelopmentofanimalmodelsofinvasive

aspergillosishasseveralkeyobjectives,asshownin

Table1.Theseincludethedevelopmentofstandardized

modelsthatallowreproducibilitybetweenlaboratories,

andbetweenserialexperimentsconductedlongitudin-

allyinthesamelaboratorywiththesameprotocols.A

numberofvariables,suchasdifferentroutesof

challenge(rangingfromintravenoustointranasalto

DOI:10.1080/13693780400029429Correspondence:T.F.Patterson,(ProfessorofMedicine),UniversityofTexasHealthScienceCenteratSanAntonio,MailCode7881,7703FloydCurlDrive,SanAntonio,TX78229-3900,USA.Tel:'12105676680;Fax:'12105673303;E-mail:patterson@uthscsa.edu.–2005ISHAM

Table1ObjectivesofAspergillusanimalmodels

.Standardizedmodel(s)

.Recapitulateshumandisease

.Reasonablecosts

.Reproducibility

.Easeofuse

.Amenabletostudiesincluding

ÁEvaluationofnoveldiagnostics

ÁEvaluationofhostresponse

ÁEvaluationoforganismvirulencefactorsthroughmolecularmanipulations

ÁInvivoexpressionanalysis

ÁEvaluationoftherapeuticcompounds

aerosolchallenge),markedlyaffecttheoutcomeofstudies[1].Inaddition,thelackofstandardizationofvariousfactors,suchasinfectinginoculum,underlyingimmunosuppression,andvirulenceofstrainstudied,havemadecomparisonbetweenlaboratoriesdifficult

[4].Thus,amajorobjectiveforfuturemodelsistohavea‘referencestandard’modelÁor,morelikely,modelsÁthathavebeenvalidatedbetweenlaboratoriesandhavebeenshowntoproduceknowncoursesofinfectionordiseaseaswellasextentofinfectionwhichcanthenbeusedforcomparativepurposes.

Anothermajorgoalforfuturemodelsisforthecourseofdiseaseinthemodeltoaccuratelyreflectclinicalinfection.Itisparticularlyimportantfordiagnosticstudiesthatthemodelrecapitulateclinicalinfection,whichappearsmostlikelytooccurafterchallengethroughanaerosolroute.However,inmostanimalmodels,widelydisseminatedinfectionoccursmoreconsistentlywithanintravenouschallenge,andinsomesettings,suchasanimmunosuppressedrabbitmodel,pulmonaryinfectionalsoresultsfollowingintravenouschallengewhichproducesacourseofinfectionthatmimicswidelydisseminatedinfection

[5,6].Insomestudies,thesemodelsusingintravenouschallengeanddisseminatedinfectioninlargerspecies(rabbitsandguineapigs)havebeenshowntobeparticularlyusefulinevaluatingthekineticsofdiag-nosticmarkersaswellastherapeuticstudies[7Á11].

Otherissuesarepracticalandpragmatic.TheseincludecostÁsmallerspecies,suchasmice,allow

screeningstudiesbuttheserialassessmentofasmallernumberoflargerspeciesextensivelystudiedwithmultiplesurrogateendpointsmayreducetheutilizationofanimalresourcesandalsooverallcosts[12,13].Otherissuestoconsiderinfuturemodelsincludeeaseofuse,reproducibilityand,importantly,theamenabilityoftheanimalmodelstotheevaluationofnoveldiagnostics(largerspeciesofferserialsamplesbutsmalleranimalsallowmoreextensivenumberstobestudied),theevaluationofhostresponsesandassessmentoftherelationshipbetweenvirulenceoftheorganism,theeffectoftherapeuticcompoundsonresponseandcourseofinfection,andinvivogeneexpression.Becauseofthecriticalneedidentifiedforthedevelopmentofnewdiagnosticmarkersininvasiveaspergillosis,aswellastherefinementandbetterunderstandingofcurrentlyavailablemethods,theNIH/NIAIDrecentlyawardedacontract(NIH-NIAID-N01-AI-30041).Theobjectivesofthiscontractaretoestablishandstandardizeanimalmodelsofinvasiveaspergillosisthatwillallowthereliablequan-tificationofinfectiousburdenandtodefinesurrogatemarkersofinfectionanddiseaseprogression.ThesestudiesareaimedatimprovingthediagnosisandtherapyofinvasiveaspergillosisbyprovidingtheextendedAspergillusresearchcommunitywiththeresourcestoaddresssomeofthekeyquestionsinthefield,includinggeneticapproachestodiagnosis,animalmodelexperimentsandtrainingtoestablishtheseapproaches.KeyissuesforconsiderationinfuturemodelsAnumberofvariableshavebeenidentifiedascriticalforprovidingreferencestandardsforthisdisease(Table2).ThesefactorsincludestandardizationoftheinoculumÁrealizingthatanaerosolwillmostsimilarlymimicclinicalinfectionasopposedtointranasaldropletinstillation,intratrachealdelivery,orintrave-Table2Keyissuesforstandardization.Aspergillusstrainselection.InoculumstandardizationÁRouteofchallenge.ModelcharacterizationÁColonization/infectionÁLocalinfection/dissemination.HostselectionÁOutbred/inbredspeciesÁAnimalspecies(mice,guineapig,rabbit).ImmunosuppressionÁSteroidsÁNeutropeniaÁOthermethodsÁNon-immunosuppressed.Diseaseprogression.TissueburdenÁColonyformingunits(CFU)ÁSurrogatemarkerskAntigen(s):Galactomannan,otherskDNAkChitinkb-D-glucanÁOthermarkers.GeneexpressionÁEvaluationofnewgenomictargets

–2005ISHAM,MedicalMycology,43,S115ÁS119

nouschallenge.Additionally,anappropriateaerosoldeliverycanyieldamorehomogeneouspulmonaryinfectioncomparedtointranasalinstillation,asdemon-stratedthroughquantitativePCRevaluation,allowingtheentiremurinelungtobeutilizedforvariousdiagnostictestswiththeknowledgethattheinfectiousburdenisconsistentlydistributed[14].However,alter-nativeroutesmaywellhaveimportantadvantages,suchasproductionofdisseminatedinfection(forintrave-nouschallenge)orreproducibleinoculationintotheairways(forintranasalorintratrachealchallenge)

[1,15,16].Otherfeaturesincludingvirulenceofthestrainand/orspecieswillalsoneedtobeaddressed.Forexample,thecourseofinfectionchosenforstudywillvary,includingcolonizationvs.localinvasiveinfectionvs.disseminateddisease.CurrentstudiesfrequentlyutilizetheAspergillusfumigatusstrainAF293whichisthestrainusedforsequencing,butotherspeciesofferdistinctvirulencepropertiesandperhapsvaryingdiagnosticchallenges[17,18].

Anothermajorissueforconsiderationishostselec-tion.Inadditiontospeciesselection,asdiscussedabove,animalscanbeselectedasinbredstrainsvs.outbredspecies.Inbredstrainspresumablyofferlesshostgeneticvariability,buttheyareobviouslylimitedintermsofavailabilityandinanimalspeciesforwhichtheyexist.Theydoallowdistinctassessmentofsusceptibilityasdemonstratedinexperimentsdescribedbelow.However,notallinbredstrainswillbeidenticalinvariousregionsoftheworld,theyarenotalwaysreadilyavailableandaredistinctlymorecostly.Furthermore,theargumentcanalsobemadethatthediseaseunderstudyoccursinanoutbredclinicallyheterogenouspatientpopulation.Nevertheless,advan-tagesanddisadvantagesofeachcanbedescribed.Insettingswherespecifichostfactorsseemlesscriticaltooutcome,screeningstudiesmaybesufficientwithoutbredmice,sothatvalidationofthemodelinoutbredaswellasinbredstrainsisanimportantgoaloffuturemodels.

Otherhostissuesforconsiderationincludetheroleofimmunosuppression.Increasingly,infectedpatientshavelesstraditionalriskfactorsforinvasiveaspergil-losis[19Á21].Thus,futuremodelswilllikelyneedto

includeevaluationofnotonlyneutropenia(whichhasbecomelesscommonasariskfactor)butalsootherimmunosuppressiveagentsincludingcorticosteroids,asthesevariousimmunosuppressiveregimensmayimpactdiagnostictestperformanceinclinicalinfectionaswellasanimalmodels[22].

Finally,oneofthemostproblematicissuesinassessingtissueburdenofAspergillushasbeenthequantificationofthetissueburden.Semi-quantitative–2005ISHAM,MedicalMycology,43,S115ÁS119

colonycountshavebeenusedsuccessfullyinexperi-mentstoeffectivelyassesstissueburdenofAspergillus[5,23].However,theutilityoftheseculturesforhyphalorganismsislimitedastheyaredifficulttoreproducereliablybetweenlaboratories,asgrindingthehyphaemayproduceincreasedtissuecounts.Thesecountscanevenbefalselyelevatedwithantifungalagentssuchastheechinocandins,whichfragmenttheorganismtoincreasethecolonyformingunitsalthoughthetissueburdenisactuallyreduced[13,24,25].Thus,extensiveeffortsforassessingtissueburdeninfuturemodelsarefocusedontheuseofsurrogatemarkers.Theseeffortswillnotonlyprovideseveralendpointstoassesstissueburdenbutcouldalsoallowareductioninthenumbersofanimalsrequiredintheexperimentaldesignbyprovidingmultipleendpointsofassessment[12].Theseadditionalsurrogatescanincludegalactomannan,1-3,b-D-glucan,chitin,quantitativePCRtechniques,andothers[26,27].Inaddition,theincorporationofnovelmarkers,suchaslung-injuryscoresorhighresolutionchestcomputedtomography,canbeusefulintheassessmentoftissueburden[12,28].SequencingtheA.fumigatusgenomemayalsorevealnewpotentialsurrogatemarkertargets.Animportantaimforfuturemodelsisthestandardizationoftissueburdenusingthesesurrogatemarkers.FutureinhalationalmodelsTheutilityandreproducibilityofaerosolchallengewithA.fumigatusiscurrentlyundergoingevaluation[14,29].Forexample,inthemodeldevelopedbySheppardetal.[28]aneasilyreproduciblemethodofaerosolchallengeofimmunosuppressedmiceinanacrylicchamberproducedreproduciblecolonycountsandaconsistentcourseofinfection.Intra-laboratorystudiestovalidatethisapproachappearpromising,withasimilarcourseofinfectionandsimilarburdenofpulmonarydiseasebeingdemonstrated[unpublisheddata].Whilethereproducibilityofthiseasilytransportableaerosolsystemisencouraging,additionalstudiesutiliz-ingalarge-scaleinhalationalMadisonchamberarealsoinprogress.Thisinhalationalchamberprovidesaccuratedeliveryofaerosolinoculum(ofavarietyoforganisms,includingMycobacteriumtuberculosis,Ba-cillusspp.andYersenia,aswellasfungi)thatcanbedeliveredsimultaneouslytoanextensivenumberofmicebutalsomultiplelargeranimalssuchasrabbitsorguineapigs[30].Theadvantagesofsuchasystemincludeprecisedeliveryofadryaerosolinoculum,butalsosimultaneouschallengeofanimalswithanumberofvariables(suchastheroleofimmunosuppression,specificmousestrain,etc.)thatlimitsexperimental

variabilityandpotentiallyreducesthenumberofanimalsrequiredforstudy[31].

FuturegeneticstudiesusinganimalmodelsThepost-genomiceraofA.fumigatusisextremelyexcitingforthepotentialidentificationofnewdiag-nosticandtherapeutictargetsaswellasfornewinsightsintothepathogenesisandvirulenceoftheorganism.PreliminarystudiesbyZaasetal.[29,32]illustratethepotentialofthesestudies.Theseinvesti-gatorsusedanaerosolchallengeofavarietyofinbredmicetoassesstheroleofhostgeneticbackgroundonsusceptibilitytoAspergillus[32].Interestingly,ingcom-putationalgenetics,achromosomalregionwasthenidentifiedthatcorrelatedwithresistancephenotype.Candidategeneswereidentifiedfromthoseregionandstudiesarecurrentlyongoingtorevealthepotentialsignificanceofthosefindings.Theseresultsdemon-stratethepotentialofcarefullydesignedanimalmodelstoplayanimportantroleinthefutureevaluationintheexperimentalassessmentofinvasivepulmonaryasper-gillosis.

Conclusion

AnimalmodelshavebeenusefulforassessingthepathogenesisandvirulenceofAspergillusandhavesignificantlyaidedthedevelopmentofnewdiagnosticandtherapeuticmodalities.Futuremodelsofexperi-mentalaspergillosisareaimedatassistingintheidentificationanddevelopmentofnewdiagnosticandtherapeutictargetsthatmayresultfromsequencingoftheAspergillusgenome.Thesewillbeofmajorbenefittostudiesaimedultimatelyatimprovingthediagnosisandtreatmentofthisoftenlethalinfection.Acknowledgements

ThisworkhasbeensupportedwithFederalfundsfromtheNationalInstituteofAllergyandInfectiousDis-eases,NationalInstitutesofHealth,underContractNo.N01-AI-30041.

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