The Continuing Challenges of Leprosy

7/14/2016 TheContinuingChallengesofLeprosy
ClinMicrobiolRev.2006Apr19(2):338381. PMCID:PMC1471987
doi:10.1128/CMR.19.2.338381.2006
TheContinuingChallengesofLeprosy
D.M.Scollard, *L.B.Adams,T.P.Gillis,J.L.Krahenbuhl,R.W.Truman,andD.L.Williams
LaboratoryResearchBranch,NationalHansen'sDiseasePrograms,LouisianaStateUniversitySVM,BatonRouge,Louisiana70803
*
Correspondingauthor.Mailingaddress:LaboratoryResearchBranch,NationalHansen'sDiseasePrograms,LSUSVM,SkipBertmanDr.,Baton
Rouge,LA70803.Phone:(225)5789841.Fax:(225)5789856.Email:dscoll1@lsu.edu.
Copyrightnotice
ThisarticlehasbeencitedbyotherarticlesinPMC.
ABSTRACT Goto:
Leprosyisbestunderstoodastwoconjoineddiseases.Thefirstisachronicmycobacterialinfectionthatelicitsan
extraordinaryrangeofcellularimmuneresponsesinhumans.Thesecondisaperipheralneuropathythatisinitiated
bytheinfectionandtheaccompanyingimmunologicalevents.Theinfectioniscurablebutnotpreventable,and
leprosyremainsamajorglobalhealthproblem,especiallyinthedevelopingworld,publicitytothecontrary
notwithstanding.Mycobacteriumlepraeremainsnoncultivable,andforoveracenturyleprosyhaspresentedmajor
challengesinthefieldsofmicrobiology,pathology,immunology,andgeneticsitcontinuestodosotoday.This
reviewfocusesonrecentadvancesinourunderstandingofM.lepraeandthehostresponsetoit,especially
concerningmolecularidentificationofM.leprae,knowledgeofitsgenome,transcriptome,andproteome,its
mechanismsofmicrobialresistance,andrecognitionofstrainsbyvariablenumbertandemrepeatanalysis.
Advancesinexperimentalmodelsincludestudiesingeneknockoutmiceandthedevelopmentofmolecular
techniquestoexplorethearmadillomodel.Inclinicalstudies,notableprogresshasbeenmadeconcerningthe
immunologyandimmunopathologyofleprosy,thegeneticsofhumanresistance,mechanismsofnerveinjury,and
chemotherapy.Innearlyalloftheseareas,however,leprosyremainspoorlyunderstoodcomparedtoothermajor
bacterialdiseases.
INTRODUCTION Goto:
Leprosyisbestunderstoodastwoconjoineddiseases.Thefirstisachronicmycobacterialinfectionthatelicitsan
extraordinaryrangeofcellularimmuneresponsesinhumans.Thesecondisaperipheralneuropathythatisinitiated
bytheinfectionanditsaccompanyingimmunologicevents,butwhosecourseandsequelaeoftenextendmany
yearsbeyondthecureoftheinfectionandmayhaveseverelydebilitatingphysical,social,andpsychological
consequences.Bothaspectsmustbeconsideredbyclinicians,researchers,andpolicymakerswhodealwithpersons
affectedbythisdisease.
Leprosyisnotgoingtodisappearanytimesoon.Effectivemultidrugregimensarenowusedworldwide,andthe
infectioninindividualsiscurable.However,althoughthereportednumberofregisteredcasesworldwidehas
declinedinthelasttwodecades,thereportednumberofnewcasesregisteredeachyearhasremainedthesame(at
500,000to700,000)overthesameinterval(42,237).Insomecountrieswhereleprosyisendemicthenumberof
newcasesactuallyappearstobeincreasing,whileinothersdecreasingtrendsarereported.Greatcautionmustbe
usedinreachingconclusionsfromtheseobservations,however,becausetheyarebasedentirelyonoperationaldata
whichreflecttheintensityofongoingworkmorethantheextentofanygivenproblem(112).Mathematical
modelingofthepotentialdeclineinleprosyincidenceandprevalence,usingvariouspremisesregardingefficacyof
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1471987/ 1/68
treatmentandprevention,suggeststhatthediseasewillremainamajorpublichealthproblemforatleastseveral
decades(259).
TheprecisemechanismoftransmissionofMycobacteriumlepraeisunknown.Nohighlyeffectivevaccinehasyet
beendeveloped,andextensivelaboratoryeffortshavenotyetproducedanypracticaltoolsforearlydiagnosisof
clinicallyunapparentdisease.
ThefullgenomeofM.lepraewasamongthefirsttobesequenced,andthisnewknowledgeisbeginningtobear
fruit.Molecularmicrobiologyhasbeguntoexplain,forexample,M.leprae'sfastidiousnatureandpredilectionfor
anintracellularlifestyle.Similarly,recenthumangeneticstudieshavebeenhighlyinformative,indicatingthat
immunitytoM.lepraeiscontrolledattwofundamentallevels:first,geneticdeterminantsofoverallsusceptibility
andresistancetothisorganismhavenowbeendescribed,andsecond,arangeofHLADrelatedimmuneresponses
havebeendemonstratedamongindividualswhoareinfected.
OnlyrecentlyhastheprobablemechanismofintracellularkillingofM.lepraebeenidentified.Theregulationof
cellmediatedimmunitytoM.lepraebycellularandcytokineinteractionscontinuestobeunraveled.Themajor
animalmodelsavailablearetheninebandedarmadilloandfootpadinfectionofnormalorimmunologicallycrippled
(nu/)mice.Thesemodels,however,areseriouslyflawedintheirabilitytorecapitulatemanyaspectsofthe
humandiseaseandareexceptionallyslow,difficult,andexpensivetoemploy.Leprosythereforeremainsamedical
andscientificchallengeofthefirstorder,eventhoughsupportforresearchonthisdiseasehasdeclinedsubstantially
asotherconditionshaveassumedgreaterglobalpriority.
Agreatdealofimportantnewinformationhasbeengeneratedbyrecentresearch.Brief,authoritativeoverviewson
progressinleprosyhavebeenpublishedinrecentyears,notablythoseofJacobsonandKrahenbuhl(167)and
BrittonandLockwood(42).Specializedreviewsofnarrowerscopearecitedintheappropriatesectionsbelow.
Here,wehaveattemptedtoprovideacriticalsummaryofcurrentknowledgefrombasicandclinicalresearch,
focusingparticularlyondevelopmentsfrom1990tothepresent.
BasicClinicalandImmunopathologicalFeaturesofLeprosy
Leprosypresentsawiderangeofclinicalandhistopathologicalmanifestations.Thisgreatdiversitypuzzledand
frustratedcliniciansandinvestigatorsuntilitwasappreciatedthatthisdiversitywasbasedontheabilityofthehost
todevelopacellularimmuneresponsetoM.leprae.Thefirstfullformulationofthisconceptwasdescribedby
Skinsnesasanimmunopathologicalspectrumin1964(384).Soonthereafter,apracticalclassificationscheme
basedonthesameprincipleswasproposedbyRidleyandJopling(319),enablingadegreeofglobaluniformityin
clinicalpracticethatgaverenewedimpetustoresearchonthisdisease.Inthesamedecade,thediscoveryby
immunologistsoffunctionallyandphenotypicallydistinctTandBlymphocytesubsetsandtheirrespectiverolesin
cellmediatedandantibodymediatedimmuneresponsesrevolutionizedimmunology.Scientistsrapidlydeveloped
anentirelynewsetoftoolsandsimultaneouslydiscoveredleprosyasachallenginghumandiseasethatappearedto
beanidealmodelwithwhichtoexaminetheoriesandmethodsrelatedtocellularimmunityinhumans.The
convergenceoftheseandotherfactorspromptedanextraordinaryburstofresearchonleprosyduringthelastthree
decadesofthe20thcentury(355).
ThefivepartRidleyJoplingclassificationidentifies,atoneextreme,patientswithahighdegreeofcellmediated
immunityanddelayedhypersensitivity,presentingwithasingle,welldemarcatedlesionwithcentral
hypopigmentationandhypoesthesia.Biopsiesoftheserevealwelldevelopedgranulomatousinflammationandrare
acidfastbacillidemonstrableinthetissuesthisistermedthepolartuberculoid(TT)(Fig.1).Attheotherextreme,
patientshavenoapparentresistancetoM.leprae.Thesepatientspresentwithnumerous,poorlydemarcated,raised
ornodularlesionsonallpartsofthebody,biopsiesofwhichrevealsheetsoffoamymacrophagesinthedermis
containingverylargenumbersofbacilliandmicrocoloniescalledglobi.Thisnonresistant,highlyinfectedformof
thediseaseistermedpolarlepromatous(LL).Themajorityofpatients,however,fallintoabroadborderline
categorybetweenthesetwopolarformsthisissubdividedintoborderlinelepromatous(BL),midborderline(BB),
andborderlinetuberculoid(BT).
FIG.1.
Immunopathologicspectrumofleprosy.Representativefieldsfromeachof
thehistopathologicaltypesofleprosyintheRidleyJoplingclassificationare
presentedintheupperpanel,inhematoxylinandeosinstainedsections
(magnification,63)....
Veryearlylesionsmaypresentasrelativelynonspecificperineuralinfiltratesinwhichrareacidfastbacillicanbe
demonstrated,butwithoutsufficientinfiltratestoclassifythemthesearecalledindeterminate.Thisclassification
shouldbeusedonlywhenthebiopsysampleshowsdefinitediagnosticevidenceofleprosy(nerveinvolvementand
acidfastbacilli),sinceadiagnosisofleprosymayoftenhavesignificantimpactonapatient'sfamily,employment,
andpsychologicalandsocialstatus.
Inspiteofnearlythreedecadesofintensiveresearchintotheimmunologyofleprosy,themechanismbywhichM.
lepraeisabletoelicittheentirerangeofhumancellularimmuneresponseshasstillnotbeenexplained.Most
clinicalimmunologicalinquirieshavefocusedontheimmunologicdefectoflepromatouspatients,i.e.,their
apparentlyspecificanergytoM.leprae.Thebroadresearcheffortsofrecentyearshave,however,providedan
increasinglydetaileddescriptionoftheimmunologicalcomponentsinskinlesionsacrosstheleprosyspectrum,
detailedbelowunderDevelopmentoftheImmuneResponse.
LeprominTest
Thelepromintestisoftenthecauseofconfusionandmisplaceddiagnosticexpectations.Theleprominskintestis
notdiagnosticofleprosyorexposuretoM.leprae.Thetestresponseismeasuredasinduration(inmm)4weeks
afterinjectionandisideallyalsoevaluatedbybiopsyandhistopathologicalexaminationofthetestsite.Thistest
providesameasureoftheindividual'sabilitytomountagranulomatousresponseagainstthemixtureofantigens
present.ResponsestoleprominarenotleprosyspecificmanyindividualswhohaveneverbeenexposedtoM.
lepraewilldevelopapositiveleprominreaction.
Leprosybacilli,derivedfromdifferentsourcesandsubjectedtodifferentpurificationprocedures,arethebasisfor
differenttypesofpreparationsusedforintradermalskintesting(227).Themostfrequentlyusedpreparation,andthe
oneforwhichtheresponseisbestcharacterized,isMitsudalepromin.Thisisasuspensionofwhole,autoclaved
leprosybacilli(357)thatisinjectedintradermally.Earlystudiesusedbacilliisolateddirectlyfromhuman
lepromatouslesions,butarmadilloderivedorganismshavebeenusedexclusivelysincethe1970s.Inrecentyears,
MitsudaleprominhasbeendistributedforresearchapplicationsbytheWorldHealthOrganization.Thisskintest
materialisnotapprovedbytheFoodandDrugAdministrationandisnotrecommendedorprovidedfordiagnostic
useintheUnitedStatesbytheNationalHansen'sDiseasePrograms.Studiesareunderwaytotrytoidentify
definedproteinantigensthatmightbeusefulasdiagnosticreagents(37,94),butnoneofthesehasyetbeen
determinedtobesatisfactorilysensitiveorspecificforthispurpose.
AlthoughtheresponsetoMitsudaleprominisnotleprosyspecific,anegativeresponseisassociatedwith
lepromatoustypesofleprosy,i.e.,withaninabilitytorespondtoM.lepraeandtoeliminatethebacilli.Apositive
lepromintest(at4weeks)isassociatedwiththeabilitytodevelopagranulomatousresponse,involvingantigen
presentingcellsandCD4+lymphocyteparticipationand,inleprosypatients,successfuleliminationofbacilli(121,
299).
Leprominisprobablytheonlywidelystudiedskintestantigenthatreflectstheabilityofanindividualtogeneratea
granulomatousresponsetomycobacterialantigens(asopposedtothe48to72hdelayedhypersensitivityresponse
totuberculinandotherskintests).Forthisreason,thepossibilityofgeneticinfluencesonleprominresponsiveness
hasbeenofinteresttogeneticistsconcernedwiththeinheritanceofimmunologicaspectsofthegranulomatous
response(8,30,107).
LeprosyinImmunocompromisedIndividuals
Unliketuberculosis,leprosyhasnotbeenobservedtobemorefrequentinpatientsinfectedwithhuman
immunodeficiencyvirus(HIV)inregionswherebothdiseasesareendemic(162,238,303).Ithasbeensuggested
thatthismaybeduetotherelativelylowvirulenceofM.lepraeorthatHIVinfectedindividualsmaydiebefore
leprosy(withitslongincubationtime)becomesclinicallyapparent(238).Nelson(286)hasrecentlyurgedthat
investigatorsexplorealternativeexplanations,however,sincetheapparentdissociationbetweenthetwodiseases
hascontinuedevenastheprevalenceofAIDShasincreased.
IncontrasttoallotherexperiencewithmycobacterialinfectionsinHIVpositiveindividuals,coinfectionwithM.
lepraeandHIVappearstohaveminimaleffectuponthecourseofeitherleprosyorHIV/AIDS.Thisisbest
illustratedbystudiesfollowingcohortsofinfectedpatients(162reviewedinreference221).
TheoccurrenceofleprosyreactionsinHIVpositivepatientswithleprosyhasbeenthefocusofseveralreports(18,
31,34,50,230,298,300),butsinceleprosyreactionsaffectahighpercentageofallleprosypatients(seeLeprosy
Reactions,below),itisnotclearthattheyareactuallymorefrequentormoresevereinHIVpositiveindividuals.
StudiesofcellphenotypesandcytokinesinleprosylesionsinpatientswithandwithoutHIVinfectionhavefound
nosignificantdifferencesbetweenthetwogroupswithrespecttotheseimmunologicparameters(136,298,329).It
ispossiblethattheveryslowgrowthofM.lepraeallowsthehostimmuneresponsetokeeppacewiththisinfection
toamuchgreaterdegreethanisthecasewithM.tuberculosis,M.avium,andtheothermycobacterialpathogensin
AIDS.Notably,however,infectionwithM.lepraemayelicitantibodiescrossreactivewithHIVscreeningassays
(13,15,205,372).
TreatmentofHIVinfectionwithhighlyactiveantiretroviraltherapyhasresultedintheemergenceofpreviously
unsuspectedleprosyinasmallnumberofreportedcases(77,230)andnotably,manyoftheseindividualshavealso
developedtype1reactions.ThissuggeststhatinfectionwithM.lepraemaybemorecommonthanhasbeen
documentedamongHIVpositiveindividualsinleprosyendemicregionsoftheworld,butthatearlyleprosylesions
areoverlookedwhenthesepatientsareconfrontedbytheothermajor,lifethreateningcomplicationsofAIDS.
Interestingly,immunosuppressionwithhumanizedmonoclonalantibodiestotumornecrosisfactoralpha(TNF)
hasresultedintherapiddevelopmentoflepromatousleprosyinatleasttwoindividualswhowerebeingtreatedfor
severearthritis(355a).Thesepatientsarepresumedtohavehadminor,undetectedleprosylesionspriortothe
administrationofimmunosuppressivetreatment.TheyrespondedpromptlytoantimicrobialtreatmentforM.leprae
withamultidrugregimen(seeChemotherapy,below),butdiddeveloptype1reactionsduringtheirrecovery,as
notedabovewithAIDSpatientsreceivinghighlyactiveantiretroviraltherapy.Similarly,asmallnumberofpatients
whohavebeenimmunosuppressedforrenalorhearttransplantationhavedevelopedleprosy(266Scollard,
unpublishedobservations),andthesehavealsorespondedwelltoantileprosytreatment.
Together,theevidenceindicatesthatbroadtherapeuticimmunosuppressiondoesrenderindividualshighly
susceptibletoinfectionwithM.leprae,butthatinHIVpositiveindividualsadegreeofhostresponsetoM.leprae
ismaintained,comparabletothatofnonHIVinfectedindividuals,evenasHIVinfectionprogressesandcirculating
CD4+cellnumbersdecline.Theexperiencewithhighlyactiveantiretroviraltherapysuggeststhatinleprosy
endemicareas,subclinicalandearlyclinicalinfectionwithM.lepraemaybemoreprevalentamongHIVpositive
individualsthanhasbeengenerallyrecognized.Inaddition,itappearsthattherestorationofimmunefunctionafter
highlyactiveantiretroviraltherapymaybeassociatedwiththedevelopmentoftype1reactions.
LaboratoryTestsfortheDiagnosisofLeprosy
Thegoldstandardforthediagnosisofleprosyisafullthicknessskinbiopsysampleobtainedfromtheadvancing
marginofanactivelesion,fixedinneutralbufferedformalin,embeddedinparaffin,andexaminedbyan
experiencedpathologist.Theprimarycharacteristicstoberecognizedarehistologicalpatternsofthehostresponse
inhematoxylinandeosinstainedsections(describedabove),theinvolvementofcutaneousnerves,andthe
identificationofacidfastbacilliwithinnervesusingtheFiteFaracomodificationofthecarbolfuchsinstain(70).In
tuberculoidlesions,wherebacillimayberareanddifficulttofind,thedifferentialdiagnosisofthegranulomatous
responsecommonlyincludescutaneoustuberculosis,sarcoidosis,andgranulomaannulare.Attheotherextreme,
bacilliareeasilydemonstratedintheinfiltratesofpolarlepromatousleprosy,butcaremustbeexercisedtoidentify
bacilliwithinnervesbecause,inimmunosuppressedindividuals,cutaneousinfectionswithothermycobacteriacan
mimicthefloridinfectionoflepromatousleprosy.
Anancillaryprocedure,theslitskinsmear,canbeusedforthesemiquantitativeenumerationofacidfastorganisms
ininfectedskinandisusefulinfollowupofpatientsduringandaftertreatment.Thistechniqueisreliableonly
whenperformedandinterpretedbyexperiencedtechnicians.
NoserologictestsareavailablefortheroutinelaboratorydiagnosisofHansen'sdisease,andnolaboratoriesinthe
UnitedStatesperformsuchassaysroutinely.Enzymelinkedimmunosorbentassaysandrelatedimmunoassayshave
beendevelopedtodetectantibodiestophenolicglycolipid1(PGL1)ofM.leprae(46,97),andthesehavebeen
usedinepidemiologicalstudies.Althoughtheyhavesomevalueinpopulationfollowupstudies,noneofthese
assayshasasatisfactorydegreeofsensitivityandspecificityfordiagnosticapplication.Thegreatestdrawbackto
theserologicdiagnosisofleprosyarisesfromthefactthatpatientsinwhomthediagnosisismostdifficult,TTand
BTpatientswithmoderatetohighgradecellularimmunitytoM.lepraeandonlysmallnumbersoforganisms,do
notreproduciblyproducedetectable,specificcirculatingantibodies.
Similarly,noskintestthatenablesthediagnosisofHansen'sdiseasehasbeendeveloped.Intradermalinjectionof
heatkilledM.leprae(thelepromintest,discussedabove)reflectstheabilityofanindividualtodevelopa
granulomatousresponsetothisorganism,butitdoesnotreflectinfectionbyorevenexposuretoM.leprae.Ithas
beenusedinepidemiologicalstudies,butithasnodiagnosticutilityinindividualcases,anditisnotavailableinthe
UnitedStates.
M.lepraeisnotcultivableinvitro(seeBasicCharacteristics,below),andlackofgrowthonstandardmycobacterial
isolationmediacanberegardedasonelaboratorycriteriondifferentiatingthisorganismfromothermycobacterial
pathogens.ThemajoradvanceinthelaboratorydiagnosisofHansen'sdiseaseinthelast15years,however,has
beenthedevelopmentofmethodsfortheextraction,amplification,andidentificationofM.lepraeDNAinclinical
specimensusingPCRandothermoleculartechniques.Thisisaninvaluableadditiontolaboratorydiagnosisandto
studiesofthebasicmicrobiologyofthisuncultivableorganism,althoughitiscostlyandhasnotyetbeenapproved
orbecomeavailableasaroutineclinicaltest.AdetaileddiscussionofPCRevaluationofspecimensforM.leprae
DNAispresentedbelow(seeMolecularIdentificationbyPCR,below).
MYCOBACTERIUMLEPRAE,THEETIOLOGICAGENTOFLEPROSY Goto:
BasicCharacteristics
Cellularmorphology. M.lepraeisanonmotile,nonsporeforming,microaerophilic,acidfaststainingbacterium
thatusuallyformsslightlycurvedorstraightrods(Fig.2).Agreatdealhasbeenlearnedaboutthenatureofthe
mycobacterialcellwallthroughbiochemicalandgeneticmanipulationofcultivablestrainssuchasM.tuberculosis,
M.avium,M.smegmatis,andM.bovisBCG.SimilarapproacheswithM.lepraehavebeenmeagerbycomparison,
butbasicchemicalstudieshaveconcludedthatthecellwallisacovalentlylinkedpeptidoglycanarabinogalactan
mycolicacidcomplexsimilarincompositiontoallmycobacterialcellwalls(79,98,425)(Fig.3).
FIG.2.
MorphologyofM.leprae.A.M.lepraeisweaklyacidfastbut,whenstainedwiththeFite
Faracomethod,itappearsasred,rodshapedorganismsshorterbeadedorgranularshapesare
observedwhenthebacilliaredeadordying.Theorganismsareseen...
FIG.3.
SchematicmodelofthecellenvelopeofM.leprae.Theplasmamembraneis
coveredbyacellwallcoremadeofpeptidoglycancovalentlylinkedtothe
galactanbyalinkerunitofarabinogalactan.Threebranchedchainsof
arabinanareinturnlinkedto...
Thecellwallcorecontainspeptidoglycan,composedofchainsofalternatingNacetylglucosamineandN
glycolylmuramatelinkedbypeptidecrossbridges,whichislinkedtothegalactanlayerbyarabinogalactan.Three
branchedchainsofarabinanareinturnlinkedtothegalactan,forming,alongwiththepeptidoglycanlayer,an
electrondensezonearoundM.leprae.Mycolicacidsarelinkedtotheterminiofarabinanchainstoformtheinner
leafletofapseudolipidbilayer.Theouterleafletiscomposedofaricharrayofintercalatingmycolicacidsof
trehalosemonomycolatesandmycoserosoicacidsofphthioceroldimycocerosatesaswellasphenolicglycolipids
(PGLs),formingtheelectrontransparentzone.Ithasbeenpostulatedthatmanyofthesesamemoleculestogether
withphosphatidylinositolmannosidesandphospholipidsarereleasedfromthecellwallaftersynthesis,forminga
capsulelikeregion.ThedominantlipidinthecellwallwhichgivesM.lepraeimmunologicalspecificityisPGL1.
RecentstudiessuggestthatPGL1isinvolvedintheinteractionofM.lepraewiththelamininofSchwanncells,
suggestingaroleforPGL1inperipheralnervebacillusinteractions(288).
AnnotationofM.leprae'sgenomeandcomparativegenomicstudieswithotherbacterialgenomeshaveproduced
insightintotheputativegenesneededtodirectthesynthesisofthiscomplexcellwallbiopolymer(38).Mostofthe
genesnecessarytobuildthepeptidoglycanarabinogalactanmycolatepolymerappeartobepresentintheM.leprae
genomeandfitareasonablestrategyforitsconstruction.Afewexceptionsaretwogenesinvolvedinpolyprenyl
phosphatesynthesis(dxsIIandidi),agene(fabH)involvedinmeromycolatesynthesis,andaglycosyltransferase
gene(pimB)involvedinthebiosynthesisofphosphatidylinositol,phosphatidylinositolmannosides,lipomannan,
andlipoarabinomannan.Itshouldbenotedthatmuchofthiscomparativework,whilespeculative,providesan
importantframeworkfromwhichtoinvestigatetheauthenticityoftheseputativepathways.
Growth. M.lepraehasneverbeengrownonartificialmediabutcanbemaintainedinaxenicculturesinwhat
appearstobeastablemetabolicstateforafewweeks(414).Asaresult,propagationofM.lepraehasbeen
restrictedtoanimalmodels,includingthearmadillo(415)andnormal,athymic,andgeneknockoutmice(222).
Thesesystemshaveprovidedthebasicresourcesforgenetic,metabolic,andantigenicstudiesofthebacillus.
GrowthofM.lepraeinmousefootpadsalsoprovidesatoolforassessingtheviabilityofapreparationofbacteria
andtestingthedrugsusceptibilityofclinicalisolates(364,414).TheviabilityofM.lepraeharvestedfromseveral
differentsourcesisnowknowntovarygreatly,andmanystandardlaboratorypractices,suchasincubationat37C,
rapidlyreducetheviabilityofthisorganism(414).However,M.lepraestoredat33Cin7H12mediumhasbeen
showntoremainviableforweeks.
Metabolism. TheprimaryreasonsforinvestigatingthemetabolicaspectsofM.lepraehavebeentodetermine
whetherspecialmediacouldbeformulatedtosupportinvitrogrowthofthebacilliandtolearnmoreabout
metabolicpathwaysthatcouldpotentiallybeexploitedfordevelopingnewantileprosydrugs.Earlyworkprovided
apictureofabacteriumwithsomebasicanabolicandcatabolicpathwaysneededforsurvivalinthehost,buta
thoroughassessmentofM.leprae'smetabolicpotentialwasstilllacking.Withthecompletedsequencingand
annotationofM.leprae'sgenome,animprovedunderstandingofM.leprae'smetaboliccapabilitiesnowexists(43,
105,432).
AnnotationofthegenomeidentifiedgenesshowingthatM.lepraehasthecapacitytogenerateenergybyoxidizing
glucosetopyruvatethroughtheEmbdenMeyerhofParnaspathway,supportingearlierbiochemicalobservations.
AcetylcoenzymeAfromglycolysisenterstheKrebscycle,producingenergyintheformofATP.Inadditionto
glycolysisforenergyproduction,genomeanalysisaswellasbiochemicalstudiesinM.lepraeandM.tuberculosis
suggestthattheseorganismsrelyheavilyuponlipiddegradationandtheglyoxylateshuntforenergyproduction.In
thisregard,M.lepraecontainsafullcomplementofgenesforBoxidationbut,comparedtoM.tuberculosis,very
fewgenescapableoflipolysis.AcetatehasbeenlosttoM.lepraeasacarbonsourcesinceonlypseudogenesare
presentforacetatekinase,phosphateacetyltransferase,andacetylcoenzymeAsynthase.
Overall,M.lepraehasmanyfewerenzymesinvolvedindegradativepathwaysforcarbonandnitrogenous
compoundsthanM.tuberculosis.Thisisreflectedinthepaucityofoxidoreductases,oxygenases,andshortchain
alcoholdehydrogenasesandtheirprobableregulatorygenes.Inaddition,othermajorproblemsassociatedwith
metabolismforM.lepraearethatthebacillihavelostanaerobicandmicroaerophilicelectrontransfersystemsand
thattheaerobicrespiratorychainisseverelycurtailed,makingitimpossibleforM.lepraetogenerateATPfromthe
oxidationofNADH.Incontrasttothereductionincatabolicpathways,theanaboliccapabilitiesofM.leprae
appearrelativelyunharmed.Forexample,completepathwaysarepredictedforsynthesisofpurines,pyrimidines,
mostaminoacids,nucleosides,nucleotides,andmostvitaminsandcofactors.Themaintenanceoftheseanabolic
systemssuggeststhattheintracellularnichethatM.lepraehasfoundforitselfmaynotcontainthesecompoundsor
transportsystems.
Genome,Transcriptome,andProteome
Genome. M.lepraethatwasoriginallypurifiedfromtheskinlesionsofamultibacillaryleprosypatientfromTamil
Nadu,India(TNstrain),andsubsequentlyexpandedinandpurifiedfromtheliverofaninebandedarmadillo
providedthesourceofDNAforsequencingoftheM.lepraegenome(74).Thegenomesequencewasgenerated
bysequencingacombinationofLorist6cosmidlibraryinserts(103)andsixfoldwholegenomeshotgunsequencing
ofM.lepraeinsertDNAinpUC18(74).AnnotationofM.leprae'sgenomehasrevivedinterestinbasic
investigationsofitsmetabolic,biochemical,andpathogenicpotential.
ComparisonofM.leprae'sgenomewiththatofitscloserelativeM.tuberculosis(Table1)suggeststhatM.leprae
hasundergoneanextremecaseofreductiveevolution(reviewedinreferences74and104).Thisisreflectedbyits
smallergenome(3.3MbforM.lepraeversus4.4MbforM.tuberculosis)andamajorreductioninG+Ccontent
(58%forM.lepraeversus66%forM.tuberculosis).M.leprae'sannotatedgenomecontainsonly1,614open
readingframespotentiallyencodingfunctionalproteins,comparedto3,993openreadingframespredictedinM.
tuberculosis(Table1).
TABLE1.
ComparativegenomicsofM.lepraeandM.tuberculosis
OneofthemoststrikingfeaturesofM.leprae'sgenomeisthatitpossesses1,133inactivatedgenes(geneslost
throughmutation,orpseudogenes),comparedtosixpseudogenesinM.tuberculosis(72).Inaddition,alarge
numberofgenesapparentlyhavebeenentirelydeletedfromthegenome.Theresultofthismassivegenelossleaves
M.lepraewithlessthan50%ofitsgenomeencodingfunctionalgenes,comparedtoM.tuberculosis,inwhich90%
ofthegenomeencodesfunctionalgenes,and34%ofM.leprae'sproteinsidentifiedinsilicoappeartobethe
productsofgeneduplicationeventsorsharecommondomains(105).
Downsizingofthegenomehasresultedintheeliminationofseveralmetabolicpathways,leavingapathogenwith
veryspecificgrowthrequirements,asdiscussedabove(Metabolism).ThelargestfunctionalgroupsofgenesinM.
lepraearethoseinvolvedingeneregulation,metabolismandmodificationoffattyacidsandpolyketides,cell
envelopesynthesis,andtransportofmetabolites(74,104,105).Defenseagainsttoxicradicalsisseverely
degenerative,asneitherkatGnorthenarGHJIclusterisfunctional.Inaddition,severalothergenesinvolvedwith
detoxificationarepseudogenesoraremissingfromthegenome.RedundancyasseenintheM.tuberculosisgenome
isoftenlostinM.leprae,asmostparaloguesseeninM.tuberculosisarepseudogenesinM.leprae.Noneofthe
additional142genesfoundonlyinM.lepraeappeartobeassociatedwithmetabolicpathways.
M.lepraeappearstohaveamajordeficiencyinitsabilitytoacquireironfromitsenvironment.Theentirembt
operonisdeleted,renderingitunabletomakeeitherthemembraneassociatedorexcretedformofmycobactinT.
Whilegenesknowntobeinvolvedinironacquisitionarenotobviousinitsgenome,thereislittledoubtthatM.
lepraeutilizesiron.Genesarepresentforcytochromec(ccsAB),aferredoxin(fdxCD),biosynthesisoftheheme
group(hemgenes),ahemogloblinlikeoxygencarrier(glbO),theironstoragebacterioferritinbfrA,andideR,the
ironregulationproteindependentonintracellulariron(432).TherearenointactpolymorphicG+Crichsequence
ormajorpolymorphictandemrepeatrelatedrepetitivesequencesinthegenomeofM.lepraeandonlyalimited
numberofprolineprolineglutamicacidandprolineglutamicacidproteins(72).AnnotationofM.leprae'sgenome
hasthusprovidedinsightintowhytheleprosybacillusisanobligateintracellularparasiteandhasprovidedthe
basisforfutureexperimentationtobetterunderstanditspathogenicity.
Transcriptome. Whilecomparativegenomeanalysisprovidesusefulcluestoidentifydeficitsingeneralcellular
metabolicpotentialandcellularcomposition,itoffersonlyastartingpointfromwhichfunctionalstudiescan
proceed.BecauseofourinabilitytocultivateM.lepraeaxenically,extremelylimitedquantitiesofbacterialproteins
canbepurifiedforanalysis.TranscriptionalanalysisofM.lepraegenesprovidesaperspectivethatis
complementarytoproteinanalysisbyidentifyingactivelytranscribedgenes,therebyexpandingourknowledgeof
genesexpressedduringinfectionthatmightotherwisebemissedwhenexaminingM.leprae'sproteome.
Withlessthan50%codingcapacityand1,133pseudogenes,thosegenesthatareexpressedhelpdefinetheminimal
genesetnecessaryforinvivosurvivalofthismycobacterialpathogenaswellasgenespotentiallyrequiredfor
infectionandpathogenesisasseeninleprosy.Toidentifygenestranscribedduringinfection,genetranscriptsfrom
M.lepraegrowinginathymicnudemicehavebeensurveyedusingreversetranscriptionPCRandcrossspecies
DNAmicroarraytechnologieswithanM.tuberculosismicroarray(440).Transcriptsweredetectedfor221open
readingframes,whichincludegenesinvolvedinDNAreplication,celldivision,SecAdependentproteinsecretion,
energyproduction,intermediarymetabolism,andirontransportandstorageandgenesassociatedwithvirulence
(seesupplementalTableS1athttp://www.leprosyila.org/Mycobacterium.html).
TheseresultssupporttheviewthatM.lepraeactivelycatabolizesfattyacidsforenergy,producesawidearrayof
secretoryproteins,utilizesthelimitedarrayofsigmafactorsavailable,producesseveralproteinsinvolvediniron
transport,storage,andregulation(intheabsenceofrecognizablegenesencodingironscavengers),andtranscribes
severalgenesassociatedwithvirulenceinM.tuberculosis.Transcriptlevelsofnineofthesepotentialvirulence
genes(aceA,esat6,fbpA,relA,sigA,sigE,soda,aphC,andmce1A)werecomparedinM.lepraederivedfromthe
lesionsofmultibacillaryleprosypatientsandfrominfectednudemousefootpadtissueusingquantitativerealtime
reversetranscriptionPCR.Genetranscriptlevelswerecomparableinthetwoisolatesforallbutoneofthegenes
studied(esat6),suggestingthatprofilingofM.lepraegenesfromanimalmodelssuchasthenudemouseshouldbe
continued.Identifyinggenesassociatedwithgrowthandsurvivalduringinfectionshouldleadtoamore
comprehensiveunderstandingofM.leprae'sabilitytocausedisease.
Proteome. Thefunctionalcomplementofanygenomeistheproteome,whichconsistsoftheproteinsexpressedby
agivenorganismunderdefinedconditions.UnderstandingthebasisofM.leprae'sgrowth,virulence,and
immunogenicityhasalwaysbeenthefocusofproteomicdiscovery,withthegoalofdevelopingstrategiesfor
improvedtreatmentandcontrolofleprosy.Earlyworkwasdrivenbyeffortstoproducevaccinesanddiagnostic
reagents.PriortothepublicationofthefullDNAsequenceandannotationoftheM.lepraegenomein2001(74)
proteinanalysisofM.lepraereliedprimarilyontraditionalsubcellularfractionationofpurifiedbacilliandgenomic
libraryscreeningusingimmunologicreagents.Ofthetwostrategies,immunologicscreeningofgenomiclibraries
provedmorefruitfulandexpandedthenumberofpurifiedandcharacterizedproteinstoover40(41,131,338,
407).ImmunologicscreeningtookadvantageofthefactthatserumandTcellsfromleprosypatientsorhealthy
individualspreviouslysensitizedtoM.lepraewereabundant,providingpowerfulprobesforantigenicproteinsof
M.lepraeexpressedinEscherichiacoli.
Amajordrawbacktoimmunologicscreeningwasthatnonimmunogenicproteinsweremissed.ClarkCurtissused
analternativegenomicscreeningapproachinwhichM.lepraegeneswereexaminedforexpressioninE.coli.This
ledtothediscoveryofa46kDaproteinfromM.lepraecapableofcomplementingacitratesynthasemutantofE.
coli(165)andthedemonstrationthatM.lepraepromoteractivitywaspossibleinE.coli(356).Unfortunately,
whilegeneticcomplementationinE.colihadbeenverysuccessfulforidentifyinggenesinotherentericbacteria,it
appearedthatproblemsassociatedwitheitherM.lepraegeneexpressioninE.coliortherelativelylarge
evolutionarydistancebetweenthetwobacterialimitedfurtherapplicationoftheapproach.
BrennanandcoworkershavecombinedsubcellularfractionationofM.lepraewithimmunologicscreeningand
chemicalanalysisofpurifiedproteinsusingmicrosequencingandmassspectrometrytoexpandourunderstanding
ofthecellularlocationandfunctionofmanyM.lepraeproteins.Proteinshavebeenidentifiedfrommajorcellular
compartments,includingcytosol,membrane,andcellwall,andacomprehensivelistoftheseproteinsandtheir
characteristicswaspublishedearlier(248).
MuchofthisworkhasnowbeenassimilatedintoalargerframeworkestablishedfromthecompletionoftheM.
lepraegenomesequence.AnnotationoftheM.lepraegenomeindicatesthatapproximately1,600openreading
framesarescatteredamongadecayinggenome,includingapproximately1,100pseudogenes,withgeneremnants
makinguptheremaining23.5%ofthegenome.BycomparativegenomicanalysiswithM.tuberculosisandother
knowngenesequences,itappearsthatapproximately50%ofM.leprae'sopenreadingframescanbeassigned
putativefunctions.TheotherhalfofM.leprae'sgenesareconsideredtoencodehypotheticalproteins,withasmall
percentagedesignatedunknowngenes.
ThepowerofthisnewportraitofM.lepraehelpsintegratewhatthebacteriumcanandcannotdo.Forexample,
bioinformaticanalysesofmetabolicpathwaysindicatedthatM.lepraehaslostmanymetabolicpathways,together
withtheirregulatorycircuits,particularlythoseinvolvedwithcatabolicpotential(104).Aidedbythismetabolic
picture,researcherscannowinvestigategeneexpressionasitrelatestovariousmetabolicconditionseitherin
limitedculturewithM.lepraeorbystudyinggenefunctionincultivablemycobacteriaintowhichspecific
mutationshavebeenintroduced.Inthiswayitmaybepossibletodeterminetheconditionsthatenhanceorsuppress
M.lepraeviabilitywhenitisheldinaxenicculture.
AnotherimportantelementofestablishingtheproteomeofM.lepraeimpactsearlierworkinvolvingantigenic
analysisofM.lepraewiththepurposeofidentifyingproteinsusefulfordiagnosticsandvaccines.Forexample,a
groupofM.lepraeproteinsthathavegoneunderstudieduntilrecentlyissecretedproteins.Purifiedbacillifrom
armadillo,mouse,andhumantissuesbydefinitionlack(oraresignificantlyreducedintheirconcentrationof)
secretedproteins.BioinformaticapproacheshaveidentifiedthebasicgenesnecessaryforafunctionalSecA
dependentsecretorysysteminM.leprae(74).Inaddition,Williamsandcoworkers(440)haveshownthatallgenes
intheSecAdependentpathwayaretranscribedduringgrowthofM.lepraeinnudemousefootpadsandthatsome
25proteinswithpotentialforsecretionaretranscribed(seeTranscriptome,above).Therefore,M.lepraehasthe
potentialtoproducesecretedproteins,mostofwhichhavenotbeenstudiedforimmunogenicpotential.Afull
assessmentoftheseproteinsmaygiverisetoimportantantigensusefulfordevelopingmuchneededearly
diagnostictestsaswellastherapeuticandprophylacticvaccines.
Finally,byvirtueofitsrelativelysmallgenesetandpossiblysmallerproteome,M.lepraehasbecomeanimportant
modelforconceptualizingtheminimalgenesetneededforobligateintracellularparasitism.Itisunlikelythatallof
M.leprae'sopenreadingframesareexpressedduringallphasesofgrowthandparasitism.Teasingapartthese
intricaterelationshipsshouldprovideimportantinsightsintomechanismsofvirulence,suchasnerveinfection,as
wellasidentifyingproteinsexpressedduringearlyinfection,whichcouldgiverisetobetterdiagnosticreagentsand
potentiallyavaccinetoimproveourchancesofmanagingleprosy.
MolecularIdentificationbyPCR
DefinitiveidentificationofM.lepraeissometimesproblematic,sincetheorganismisnotcultivable.Thisproblemis
confoundedtodaybytheincreasedprevalenceofothermycobacterialinfectionsoftheskin.Rapidmoleculartype
assayshavebeendevelopedfordetectionofM.lepraedirectlyfrompatientspecimensusingavailablegeneticdata
(reviewedinreferences132,208,and348).
TheseassayshavebeenbasedprimarilyontheamplificationofM.lepraespecificsequencesusingPCRand
identificationoftheM.lepraeDNAfragment.Thistechniquehasbeenappliednotonlytoskinbiopsysamplesbut
alsotoseveraldifferenttypesofspecimens,asindicatedinTable2.However,oneshouldnotinferfromthisthat
anytissueorspecimenissuitableforPCRbaseddetectionofM.leprae(seeTable3).
TABLE2.
ApplicationofPCRforthedetectionofM.lepraeinhumanspecimens
TABLE3.
M.lepraegenesusedinthedevelopmentofPCRassays
ManydifferentM.lepraegeneshavebeenutilizedinthedevelopmentofPCRassaysfordetectionofM.lepraein
clinicalspecimens,assummarizedinTable4.RNAanalysisusing16SrRNAandreversetranscriptionPCRhas
theaddedbenefitofmeasuringviabilityposttreatment(146,228).PCRhasthusgeneratednewapproachestothe
detectionandidentificationofM.lepraeand,coupledwithmutationdetectionanalyses,hastheabilitytoprovide
rapiddrugsusceptibilityresultsfromspecimenstakendirectlyfromthepatient.
TABLE4.
IndicationsandsuitablespecimensforM.lepraePCR
Onthebasisofextensiveassessmentofthesetestsinfieldstudies,PCRbasedandreversetranscriptionPCRbased
techniqueshaveshownaspecificityof100%andasensitivityrangingfrom34to80%inpatientswith
paucibacillaryformsofthediseasetogreaterthan90%inpatientswithmultibacillaryformsofthedisease.
AutomationofPCRbasedassayshasallowedtheirimplementationinmanyreferencelaboratories,chieflyin
countrieswithendemicleprosy.Therefore,PCRcanprovideanexcellentadjuncttoclinicalandhistopathological
diagnosisofleprosy.
EpidemiologyandStrainIdentification
Understandingtheepidemiologyofleprosyisaprerequisiteforeffectivecontrolofthedisease.SinceM.leprae
cannotbeculturedinvitro,ithasbeenvirtuallyimpossibletoassessexposure,onsetofinfection,andvarious
aspectsofdiseaseprogression.Asaconsequence,thesequenceofeventsthatmustoccurforsuccessful
transmissionofleprosyispoorlyunderstood.Geneticmarkersmayholdthekeytoestablishingspeciesandstrain
specificmarkersforassessingexposuretoM.lepraeandtracingtransmissionpatterns.Thesetoolsshouldbe
helpfulforimprovingourunderstandingoftheepidemiologyofleprosy.
Overthelasttwodecades,awiderangeofmoleculartestshavebeenappliedtorevealgenotypicvariationinM.
leprae.TheresultsofinitialstudiessuggestedthatthegenomeofM.lepraewashighlyconserved.Restriction
fragmentlengthpolymorphismanalysisofM.lepraeisolatesusingacombinationofrestrictionenzymesandprobes
andsequencingoftheinternaltranscribedspacerregionofthe16S23SrRNAoperonyieldednopolymorphic
DNAsequences(69,92,436).ApolymorphicstructureinthepolAgene(119)andvariationinaGACATCrepeat
intherpoTgene(252)havebeendescribed,butthevalueoftheseelementsfordifferentiatingpossibleM.leprae
strainsappearstobelimited.
IncompletingthesequenceoftheM.lepraegenome,Coleetal.(74)identifiedseveraltandemrepeatsthatcould
proveusefulfordiscriminatingM.lepraestrains.Recently,Shinetal.reportedevidencefordiversityamongM.
lepraeisolatesobtainedfromseveralpatientbiopsysamplesinthePhilippines,basedonthefrequencyofTTC
repeatslocateddownstreamofaputativesugartransporterpseudogene(371).InadditiontotheTTClocus,insilico
analysisofthegenomesequenceindicatesthatM.lepraehasseveralothertandemrepeatlociwhichmayprovide
thegeneticdiversitynecessaryforcreatingatypingschemecapableofansweringimportantquestionsrelatedtothe
epidemiologyofleprosy.Recentstudiesemployingfourdifferentvariablenumbertandemrepeatmarkershave
successfullydifferentiatedM.lepraestrainsusedinthelaboratoryandsuccessfullygroupedidenticalsamplesand
passagesamplestestedinblindpanels(412).
Farlessdiversityisseenwithregardtosinglenucleotidepolymorphismswithinthegenome.TheM.lepraesingle
nucleotidepolymorphismfrequency(1per28kb)isamongthelowestseenforahumanpathogen,andonlythree
informativesinglenucleotidepolymorphismshavebeenidentified.Amongthe64possiblepermutationsofdifferent
basesateachofthesepolymorphisms,only4areobserved.Thevariationinsinglenucleotidepolymorphism
genotypeishighlycorrelatedwiththegeographicoriginofthestrain,andanalysisofstrainsfromdifferent
continentshasbeenusefulinpredictingtheevolutionandglobalspreadofleprosy.Thediseaseappearstohave
originatedineasternAfricaortheNearEastandspreadwithsuccessivehumanimmigrations(272).Europeansand
NorthAfricansappeartohaveintroducedleprosyintoWestAfricaandtheAmericaswithinthelast500years.
EXPERIMENTALMODELSOFLEPROSY Goto:
OvercomingObstaclestoLeprosyResearch
Rees(315a)dividedexperimentalleprosyresearchinanimalmodelsintotwoeras:1874to1960,thedarkages,
andpost1960,i.e.,aftertheShepardmousefootpadmodel.Anexhaustiveyetincompletelistofanimalspecies
testedasmodelsforleprosybeginswithrabbitsinfectedbyHansenandincludesdogs,cats,pigeons,chickens,
paddybirds,canaries,parrots,lovebirds,eels,tadpoles,frogs,toads,pigs,turtles,snakes(includingrattlesnakes),
goldfish,rainbowperch,varioussaltwaterfish,rats,blackmice,whitemice,dancingmice,chipmunks,golden
hamsters,albinohamsters,gerbils,avarietyofnonhumanprimates,andguineapigs(190).Experimentsemployeda
confusingmyriadofprotocolswithwidelyvariantreportsofsuccess,butthesewereusuallyabortiveoratbest
inconclusiveandunconfirmedfindings.Noserialpassagewasreportedand,inhindsight,becauseoftherelative
indestructibilityofevendeadM.leprae,theminimalsuccessesreportedcouldhavebeenduetolocalleprominlike
responses.
OneobstacletothedevelopmentofananimalmodelforleprosyhasbeenthepoorqualityoftheM.leprae
inoculum,whichusuallyconsistedoffreshorfrozenhomogenatesofnodulesandlesionsfromuntreatedhuman
lepromas.AnimportantresearchemphasisintheNationalHansen'sDiseaseProgramlaboratoriesoverthepastfew
yearshasbeentheproduction,characterization,andprovisionofviableMycobacteriumlepraeforourown
researchersandqualifiedinvestigatorsaroundtheworld.Alarge(>200mice)colonyofM.lepraeinfectedathymic
nu/numiceismaintainedforthispurpose.AprotocolofrigorouslyprogrammedpassageofM.leprae,with
radiorespirometry(theoxidationof14Clabeledpalmiticacid)asameasureofviability,hasenabledtheharvest,on
aroutine(weekly)schedule,of4to6billionbacillithatare80to90%viable,aresourceunprecedentedinalmost
130yearsofleprosyresearch.WiththeseorganismswehaveconfirmedthepreferenceofM.lepraeforcooler
temperatures(4Cforstorage,26Cto33Cformetabolicactivity)andtherapidlydeleteriouseffectsofincubation
at37Corasinglefreezingthawingcycle(414).
WhereasradiorespirometrymeasuresthemetabolicactivityofasuspensionofM.leprae,andthishasbeenshown
tobecorrelatedwithgrowthinthemousefootpad,anovel,twocolorfluorescenceviabilitystainingassay
(MolecularProbesBacLightbacterialviabilitykit)hasrecentlybeenadaptedtoprovidearapid(1h),reliable,
quantitative,directcountviabilityassaythatmeasuresthecellwallintegrityofindividualbacilli(229).This
confirmspreviousfindingsregardingbiophysicaloptimaformaintainingM.leprae.
Thesecondimpedimentintheearlyattemptstodevelopaleprosymodelinanimalswasfailuretorecognizethe
prolongedgrowthcycleofM.lepraeandnotacknowledgingitspreferenceforcoolerbodysites.Anewerawas
enteredwithdescriptionofthemousefootpadmodelbyShepardin1960(363).PassageofM.lepraeinfectionwas
achieved,drugevaluationandrudimentaryimmunologystudiesbecamefeasible,andthebasisforsubsequent
explorationofM.lepraeinfectioninvariousimmunocompromised,transgenic,andknockoutmurinemodelswas
established.
MouseFootpadsandNudeMice
Shepard'sdemonstrationofthemultiplicationofM.lepraeinthefootpadsofCarworthFarmswhitemice(363)
openednewopportunitiesforinvestigationintobasicimmunologicalmechanismsofhostresistanceaswellas
screeningofantileprosydrugsanddrugcombinationsanddetectionofdrugresistantstrainsofM.leprae.
TheimportanceoftheTlymphocyteinhostresistancewasrevealedinexperimentalM.lepraeinfectionof
neonatallythymectomizedorcongenitallyathymicmiceandrats(75,208,315).Inimmunocompetentmice,an
inoculumofafewthousandbacilligrowslocallyandplateausatapproximately1millionorganismsperfootpad.
Thereisvirtuallynodiseaseinthesefootpads,andthehistopathologicalchangesareminor,consistingofsmall
granulomascontainingafewlymphocytesandveryfewbacilli.Furthermore,thereisessentiallynodissemination
ofinfection.Inathymicnu/numice,however,localfootpadmultiplicationofM.lepraeappearstobeunimpeded,
reaching1010ormorebacilliperfootpad.
Histopathologically,theinfectedfootpadtissuebecomesanenormousforeignbodytypemacrophage(M)
granuloma,orleproma,andthecellsareengorgedwithbacilli(61)(Fig.4).Unlikethecourseinan
immunologicallyintactmouse,somedisseminationdoesoccur,andifobservedforalongenoughinterval,evidence
ofgrowthintheoppositehindfootpadorforefeetisseen.Thus,developmentofthismousemodelwasasecond
majormilestoneinleprosyresearchfor,inadditiontoitsimmunologicalsignificance,theathymicnu/numouse
footpadallowedtheroutinecultureoflargenumbersofhighlyviableM.lepraeforexperimentaluse(208,229,
414)(seeBasicCharacteristics,above).Morerecently,otherimmunosuppressedstrainsofmiceandratshavebeen
reportedtoallowenhancedgrowthofM.leprae,includingseverecombinedimmunodeficiencymice,whichlack
bothTandBcells(22).
FIG.4.
CultivationofM.lepraeinmousefootpads.A.Enlargednudemouse
footpad6monthsafterinfectionwith5107liveM.leprae.B.Heavily
infectedmacrophagesharvestedfrommousefootpad(magnification,
1,000).
GeneKnockoutMice
TheimportanceoftheproductionofTh1cytokineresponsesinhostresistancetointracellularinfections,including
M.tuberculosisandopportunisticmycobacteria,wasconfirmedwiththerecentlyidentifiedhumangenetic
deficienciesincytokinereceptors(420).Clinicalevidenceforanalteredcourseofleprosyinsuchindividualshas
notbeendemonstrated,althoughthisismostprobablyduetotheprotractedcourseofthediseaseandtherelative
avirulenceofM.leprae.However,experimentalM.lepraeinfectionsincytokineknockout(KO)micehave
substantiatedtheimmunologicalimportanceofthesecytokinesacrosstheleprosyspectrumandrevealed
compensatorymechanismsofhostresistancetoM.leprae.
Aninvaluablemeansforstudyingimmunologicalparametersofhostdefenseisviagenetransfertechnologyinthe
murinesystem.Throughavarietyofmechanismsaspecificgenecanberenderedeithertotallyorconditionally
inactive(244).Theresultingknockoutmodelsallowinvestigationatthelevelofthedirecteffectsduetothelossof
thefunctionalgeneaswellasthecompensatorymechanismsoperationalinitsabsence.Numeroustargetedgene
KOstrainsarenowcommerciallyavailable,includingthoseunabletoproducespecificcytokinesandchemokines,
theirreceptors,immunemodulators,orcellsurfacemarkers.Inaddition,newgenerationsofmice,includingtissue
specificKO,conditionalKO,multipleKO,andknockinmutants,arecontinuallybeingdeveloped.
WehaveexaminedseveralstrainsofKOmiceinourstudiesonM.lepraegrowthandgranulomadevelopmentin
experimentalleprosy.Perhapsthebestcharacterizedistheinduciblenitricoxidesynthase(iNOS)KO(NOS2/)
strain.MisolatedfromNOS2/miceareincapableofproducingreactivenitrogenproductsandtheycannot
inhibitM.lepraemetabolicactivityinvitro,althoughtheyarefullycompetentinproducingreactiveoxygen
products(3).WheninoculatedintoNOS2/mousefootpads,M.lepraeinitiallygrewtoslightlyhigherlevelsthan
seeninwildtypecontrols,butthereafterthecourseofinfectionwassimilar.Thegranulomasformedinwildtype
miceinresponsetoM.lepraeinfectionconsistedofonlysmall,focalcollectionsofmononuclearcellsincontrast,
thegranulomasformedinNOS2/micecontainedlarge,dense,organizedcollectionsofepithelioidcellsand
lymphocyteswhichinfiltratedtheperineuriumanddestroyedmusclebundles(6).Inaddition,Th1typecytokine
expressionwassignificantlyaugmentedinNOS2/mice,andgranulomasinlivertissuedemonstratedapattern
similartothatseeninhumanleprosylesions(268)withCD4+Tcellsdistributedthroughoutthelesion,surrounded
byCD8+Tcells(D.A.Haggeetal.,submittedforpublication).Overall,theM.lepraeinfectedNOS2/mouse
modelexhibitsfindingssimilartothoseofBTleprosyinhumans.Interestingly,incontrasttoNOS2/mice,
macrophagesfrommicethataresuperoxidedeficientduetoadefectivegp91subunitofthephagocyteoxidase
(phox91/)(93)efficientlykillM.lepraeinvitro,andinfectedphox91/micedevelopafootpadinduration
similartothatofwildtypemice(Adams,Scollard,andKrahenbuhl,unpublished).
Gammainterferon(IFN)KO(IFN/)micealsoexhibitedenlargedfootpadsandenhancedcellularinfiltration
uponinfectionwithM.leprae(7).Thefootpad,however,containedepithelioidMandscatteredlymphocytesthat
werenotassembledintoorganizedgranulomas.GrowthofM.lepraewasenhancedapproximately1logoverthat
inwildtypemiceandaTh2typecytokineprofilewasgenerated.Overall,thegeneralfeaturesexhibitedbythe
IFN/modelweresimilartothoseofBBBLleprosy.
StudiesinotherKOmousemodelsarecurrentlyunderway.GrowthofM.lepraeinmicedeficientininterleukin
10(IL10)wassimilartothatseeninimmunocompetentmice,whereasbacillarygrowthwasaugmentedinmice
deficientinIL12,TNF,tumornecrosisfactorreceptor,lymphotoxin,CD4,andCD8(Adams,unpublished
data).Itisimportanttonote,however,thatM.lepraegrowthintheseKOfootpadmodels,includingtheIFN/
model,didnotreachtheenormouslevelsseenintheTcelldeficientmousemodels.Thus,eventhoughthese
cytokinesandcelltypesareimportantfortheexpressionofcellmediatedimmunity,compensatorymechanismsare
abletolimitbacillarygrowth.Immunoregulationinthesemiceisbeingstudiedfurtherbyadditionalconditional
approaches,suchastreatmentwithcompetitiveinhibitorstocreateasecondKOorrestorationoftheKOin
infectedmice.Theseimmunoregulatorymechanismsmaybecrucialinthemanifestationoftheunstableborderline
areasoftheleprosyspectrum.
NineBandedArmadillo
Armadilloswereoriginallyadaptedtocaptivityinordertostudytheirunusualreproductivetraits,whichinclude
bothdiapausicdevelopmentandpolyembrony(394).In1968,KirchheimerandStorrsbeganexperimentingwith
armadillostopotentiallyexploittheircoolbodytemperature(30to35C)andshowedthatarmadillosareuniquely
susceptibletoM.leprae(216,217).
Theninebandedarmadillo(Dasypusnovemcintus)istheonlyimmunologicallyintactanimalthatregularly
developsfullydisseminatedM.lepraeinfections.Intravenousinoculationwith108to109bacilliregularlyresultsin
10,000foldincreasesinthenumberofM.lepraeoveraspanofabout18months,andarmadilloshavebeenthe
hostsofchoiceforinvivopropagationofM.lepraeformorethan30years.Withhighburdensofbacilliintheir
reticuloendothelialtissues,armadilloscanyieldgramquantitiesofM.leprae(185,413).
Approximately65%ofallarmadillosexperimentallyinoculatedwithM.lepraewilldevelopafullydisseminated
infection.Infectedarmadillosexhibitfewdiscernibleclinicalsigns.Histopathologicalexaminationofinfected
animalstypicallyrevealsheavyinfiltrationofM.lepraeladenmacrophagesthroughouttheliver,spleen,andlymph
nodes,aswellasnotableinvolvementofthelips,tongue,nose,nasalmucosa,skin,bonemarrow,eyes,lungs,
peripheralnervoussystem,gonads,andothertissues.Likehumans,armadilloscanupgradeanddowngradetheir
responsetoM.lepraeoverthecourseofinfection,but90%oftheanimalsthatexhibitsignsofsystemic
disseminationwilleventuallysuccumbtotheirleprosy(181).Intravenousinoculationpromotesthemostrapidand
severeinfections.However,respiratoryinstillationandpercutaneousandintraperitonealinoculationarealsoknown
tobeeffective(413).
Freerangingarmadillosareexposedtoanumberofatypicalmycobacterialspeciesintheenvironmentandmay
developnonspecificantibodyresponsescrossreactivewithantigenssharedbyM.leprae(413).Armadillo
immunoglobulinM(IgM)ishighlycrossreactivewithhumanIgM,andarmadilloIgGreactswellwithproteinA
orG.
ThegranulomatousresponseofarmadillostoM.lepraeishistopathologicallyidenticaltothatseeninhumans.
Armadilloderivedlepromin(leprominA)canbeusedeffectivelytoindexthecellmediatedresponseofindividual
animalsandtoclassifytheirtypeofleprosyaccordingtotheRidleyJoplingscale.Thereactionsofindividual
animalscanrangefrompolarlepromatoustopolartuberculoid.Lepromatousarmadillosdeveloptheverylarge
burdensofbacillineededtoobtainM.lepraefromtheirtissueswithhighpurityandareselectedmostcommonly
forpropagationpurposes(182186),However,bothleprominpositiveandleprominnegativearmadilloscanresist
challengewithM.leprae,andarmadillosareusefulmodelsforstudyingbothsusceptibilityandthevariable
resistancetoleprosythatmayresultfromtreatmentorvaccination(185,187189).
In1973Kirchheimershowedthat80%ofthearmadillossensitizedwithheatkilledM.lepraecouldresistinfectious
challenge(213).SubsequentvaccinationstudieswithBCGdemonstratedeffectiveprotectionofarmadillosagainst
M.leprae.ThearmadilloistheonlyanimalmodelthatcandemonstrateeffectiveprotectionagainstM.lepraewith
BCG,equivalenttoratesobservedforBCGinseveralhumanvaccinationtrials.Studieswiththeseanimalscould
potentiallybenefitoureffortstogeneratemoreefficaciousantituberculosisandantileprosyvaccines(29,188,214,
215).Unfortunately,thenumberofarmadillosanddurationoftimerequired(upto1,140days)foreffective
challengestudieswitharmadilloscurrentlylimittheutilityofarmadillosasvaccinemodels.
Inadditiontothearmadillo'svalueasasourceoforganismsandanimmunologicalmodel,theinfectionof
peripheralnervesinthearmadilloconstitutesauniquemodeloflepromatousnerveinvolvementinhumans(350,
351)(reviewedinMechanismsofNerveInjury,below).
Recently,theHumanGenomeConsortiumcompletedthesequencingofthearmadillogenomeaspartofa
comparativegenomicsinitiative.ArmadillosarethemostcommonmodernrepresentativesofXenathra,afamilyof
mammalsthatdivergedfromtherodentprimatetreeintheCretaceousperiod.Theavailabilityofextensive
sequenceinformationonthearmadillo(http://www.ncbi.nlm.nih.gov/BLAST)islikelytorapidlyexpandthe
availabilityofnewimmunologicalprobesandreagentsforusewitharmadillosandwilladvancetheiruseasmodels
forresistance,vaccination,andnerveinjury.
WildninebandedarmadillosinthesouthcentralUnitedStatesarehighlysusceptiblenaturalhostsof
Mycobacteriumleprae.Surveysconductedoverthelast30yearsonmorethan5,000animalsconfirmthatthe
infectionispresentamongarmadillosinArkansas,Louisiana,Mississippi,andTexas.LittleevidenceforM.leprae
infectionisfoundamongarmadilloselsewhereintheU.S.range,andonlyafewreportsrelatefindingtheinfection
amonganimalsinCentralorSouthAmerica.However,theissuehasreceivedonlyscantattentioninother
countries.ArmadillosonlyrecentlyexpandedtheirrangeintotheUnitedStates,andleprosywaspresentinTexas
andLouisianapriortothearrivalofarmadillos.Theecologicalrelationshipbetweenhumansandarmadilloswith
M.lepraeinthisregionremainsunclear.However,infectedarmadillosconstitutealargereservoirofM.leprae,and
theymaybeasourceofinfectionforsomehumansinthiscountry,andperhapsinotherlocationsacrossthe
animal'srange(415).
Theimpactofarmadilloleprosyonhumansisdifficulttomeasure.Anumberofanecdotalreportshaveassociated
handlingarmadilloswithindividuals'developingleprosy,andcasecontrolstudieshaveyieldedconflictingresults
(45,239).AmongLouisianaresidentsdevelopingleprosy,noassociationwitharmadillocontactwasfound(110),
butamongMexicanbornpatientswhopresentedinLosAngelesandwhohadlivedinareaswheretheycould
havebeenexposedtoarmadillos,anincreasedlikelihoodforlepromatoustypeleprosywasreported(408).Leprosy
remainsrareintheUnitedStates,andthedegreeofriskattributabletoarmadillosisquitelow.Nonetheless,
armadillosarealargenaturalreservoirandcanbeaneffectivevehicleforexposuretolargenumbersofM.leprae
(112).However,understandingtheactualimpactofarmadillosonhumaninfectionwilllikelyrequiretheevolution
ofbettermoleculartechniquesthatcantracktransmission.
HOSTRESPONSETOM.LEPRAE Goto:
GeneticInfluencesonLeprosyinHumans
BeforeHansen'sdiscoveryoftheleprosybacillusin1874,leprosywaswidelyregardedasaninheriteddisease.
Evidencefromstudiesoftwinswithleprosyandoffamilyclusteringofcasescontinuedtosuggestthatsome
inheritedinfluencewasafactorinsusceptibilitytothisdisease.Anappreciationoftheroleofimmunityinthe
differentclinicalandpathologicalmanifestationsofleprosyaswellassubsequentadvancesinthefieldof
immunologyprovidedafoundationforfocusedinquiriesconcerningthegenesthatmightinfluencesusceptibilityto
M.leprae.
Theideathatatleasttwodifferentgenesmightcontrolthehumanimmuneresponsetoleprosywasproposedinthe
1970s(89)andsupportedbysubsequentinvestigations(1,427).Aconvincingbodyofevidencenowexiststo
indicatethatdifferentgenesdoinfluencethehumanimmuneresponsetoM.leprae,operatingattwolevels(Fig.5).
Thefirstlevel,overallsusceptibility/resistancetotheinfection,isamanifestationofinnateresistancemediatedby
cellsofthemonocytelineage.Ifinnateresistanceisinsufficientandinfectionbecomesestablished,genetic
influenceisexpressedatthesecondlevel,i.e.,influencingthedegreeofspecificcellularimmunityanddelayed
hypersensitivitygeneratedbytheinfectedindividual.Suchacquiredimmunityismediatedprimarilythroughthe
functionofTlymphocytes,incooperationwithantigenpresentingcells(seeAdaptiveImmunity,below).The
associationofgenesinvolvedinbothinnateandacquiredimmunitytoleprosyhasrecentlybeenreviewedby
MarquetandSchurr(249),andgeneticdefectsindifferentcomponentsofthetype1cytokinepathwaythataffect
humanresistancetomycobacteriahavebeenreviewedbyvandeVosseandcolleagues(420).
FIG.5.
TwostagemodelofgeneticinfluenceonhumanimmunitytoM.leprae.
InfectionbyM.lepraeprobablyoccursthroughaskinornasalroute,by
mechanismsthatarenotyetdefined.Thevariousgenesandlocilistedare
discussedinthetextunderGenetic...
GeneticinfluencesoninnateresistancetoM.leprae.(i)PARK2/PACRG. Oneofthemostextraordinaryadvancesin
theunderstandingofleprosyhasbeentheidentificationbyMiraandcolleagues(262)ofalocuswithinthegene
PARK2/PACRGthatisassociatedwithoverallsusceptibilityofhumanpopulationstoM.leprae.Thisisthefirst
exampleoftheuseofpositionalcloningtoidentifyahumangeneassociatedwithsusceptibilitytoaninfectious
disease(48).IntheirinitialassociationscanofalinkagepeakidentifiedthroughagenomescanofaVietnamese
patientpopulation,theinvestigatorsidentifiedalocuswithinthisgenethatwashighlyassociatedwithleprosy
(leprosyperse),regardlessofthesubtypeofthisdisease.Theseresultswereconfirmedbyasecondanalysisof
Brazilianfamilieswithoneormorepersonsaffectedbyleprosy.ThespecificlocusisapromoterregionofPARK2
andacoregulatedgene,PACRG,locatedonchromosome6q25q27.PARKINwasnamedforitsassociationwith
anearlyonsetformofParkinson'sdisease,andthefindingthatalocuswithinthisgeneisalsoassociatedwith
susceptibilitytoleprosyisveryunexpected.
Functionally,thespecificlocusidentifiedcodesforthesynthesisofaligaseintheubiquitinproteasomepathwayof
intracellularproteindegradation(454).Recentworkhasrevealedsomemechanismsbywhichthispathway
regulatestheprocessingofproteinantigenswithinmacrophages,therebyaffectingantigenpresentationto
lymphocytesandtheresultingimmuneresponse(278,429).Theexactmechanismbywhichthisgeneinfluences
overallsusceptibilitytoleprosy,however,remainstobedetermined.
(ii)NRAMP1. Thefirstevidenceofageneticdeterminantofoverallsusceptibilityorresistancethatmightrelateto
leprosywasthedemonstrationbySkameneandcolleaguesofagenecontrollingsusceptibilityandresistanceof
micetointracellularpathogens(383).Locatedatasinglelocusonmousechromosome1,thisgenewasinitially
designatedBCG.Basedonitsfunctioninmiceitwastermednaturalresistanceassociatedmacrophageprotein1
(NRAMP1)andisnowdesignatedSLC11A1.Functionally,murineNRAMPproteinsinfluencepathogenviability
and/orreplicationwithinmacrophagesbytransportingironandotherdivalentcationsacrossthephagosomal
membrane(139).AlthoughtheprecisefunctionofhumanNRAMP1hasnotbeendefinitelyestablished,thehuman
gene,locatedonchromosome2q35,ishighlyhomologouswiththemousegene.
Anassociationofthisgenewithoverallsusceptibilitytoleprosywasfirstreportedinastudyoffamilieswith
multiplecasesofleprosy(2).SubsequentstudieshavealsosuggestedthatNRAMP1maybeassociatedwith
differentleprosytypesinsomepopulations,possiblythroughitsinfluenceontheexpressionofmajor
histocompatibilitycomplex(MHC)classIImolecules,regulationofexpressionofTNFA,andinductionofnitric
oxidesynthase(33).Theability(orinability)ofanindividualtodevelopagranulomatousresponsetoan
intradermalinjectionofkilledM.leprae(theMitsudaskintest)hasbeenlinkedtoNRAMP1insomestudies(8,
108)butnotinothers(152),possiblyduetothefrequencyofdifferentpolymorphismsindifferentpopulationsorto
methodologicaldifferencesinthestudies.
Geneticinfluencesonacquiredimmuneresponsesinleprosy.(i)HLA. Earlystudiesusingserotypingtechniques
attemptedtofindassociationsbetweenleprosyandhumanleukocyteantigens(HLAs)inmajorhistocompatibility
complexclassII.ThesehavebeenreviewedbySergeantson(358)andOttenhoffanddeVries(296).Overall,
severaloftheseserotypingstudiessuggestedanassociationofHLADR2andDR3withtuberculoid
(paucibacillary)leprosy.AlthoughsomestudiesindicatedanassociationofHLADR2withbothtuberculoidand
lepromatousleprosy,noevidenceconvincinglydemonstratedanassociationofthelepromatousresponsewithany
otherHLADloci.Subsequentmoleculargeneticstudieshaveborneoutmanyoftheearlysuggestionsthatthe
HLAregiondoesplayadeterminingroleintheresponsetoM.leprae.Advancesinthetechnologyofmolecular
geneticsandinmathematicalmethodsofinterpretationofthedatahaveextendedtheseinvestigationsfarbeyond
thecapabilitiesoftheearliertechniques.
(ii)Chromosome10p13. Agenomewidelinkagescanof244familiesinsouthernIndiarevealedsignificantlinkage
ofaseriesofmicrosatellitemarkersonchromosome10p13withsusceptibilitytoleprosy(377).Mostofthepatients
inthesefamilieshadtuberculoid(paucibacillary)leprosy,anditisnotclearwhetherthelocithatwereidentifiedare
associatedwithoverallsusceptibilitytoleprosyoronlywiththetuberculoidtypeofleprosy.
Thetransporterassociatedwithantigenprocessing(TAP)isaproteincomposedoftwopolypeptides,
(iii)TAP.
TAP1andTAP2.Theirrespectivegenes,locatedonchromosome6p21,liewithintheMHCclassIIregion
betweenHLADPandDQ(388).Functionally,TAPproteinstransportpeptidestotheendoplasmicreticulumin
antigenpresentingcells,wheretheyarejoinedtoMHCclassImoleculesforantigenpresentation.TheTAP2gene
hasbeenassociatedwithtuberculoidleprosy(306),butbecausethisgeneislocatedsoclosetootherHLAgenes,
interpretationoftheresultshasbeendifficultandthesignificanceofthisfindingisuncertainandawaitsmore
detailedstudies.
(iv)TNFA.Tumornecrosisfactoralpha,producedprimarilybymacrophagesandcausingactivationofmacrophages
andTcells,playsamajorroleinnonspecificinflammationandinnateresistanceandisalsooneofthemost
powerfulstimulantsofcellmediatedimmunity.Inleprosy,TNFisgenerallyassociatedwithresistancetoM.
leprae.Forexample,serumlevelsofTNFareelevatedinpatientswithresistant(tuberculoid)diseaseandwith
type1reactions,andexpressionofthiscytokineisalsoincreasedlocallyinskinlesionsinthesemanifestationsof
leprosy(seeLeprosyReactions,below).
TheTNFAgeneislocatedintheMHCclassIIIregiononchromosome6p21.Severalpolymorphismsofthisgene
havebeenidentified,especiallyinthepromoterregion.BecauseofthewiderangeofinfluenceofTNFon
cellularimmunity,thesepromoterpolymorphismsareofgreatinterestaspossiblemodulatorsofthedegreeofhost
responseandthereforeofclinicaltypesofleprosy.Thus,severalgeneticstudieshavereportedassociationsofTNFA
alleleswithdifferenttypesofleprosy.InanIndianpopulation,anassociationofoneallelewithlepromatousleprosy
wasobserved(326)inaBrazilianpopulation,anotherallelewasassociatedwithtuberculoiddisease(335).The
latterstudyalsofoundthisalleletobeprotectiveagainstleprosyperse,i.e.,againsttheoveralllikelihoodof
acquiringleprosyofanytype.AssociationsofsomeTNFAalleleswiththestrengthofskintestresponsestoM.
leprae(theMitsudatest)havealsobeenreported(108,273).Together,theclinical,experimental,andgenetic
evidencesuggeststhattheTNFAgeneisinvolvedinacomplexmannerintheregulationofhumanimmune
resistancetoM.leprae.
(v)TLRs. ColorfullynamedaftertheircounterpartinDrosophilamelanogaster,humanTolllikereceptors(TLRs)
arecellsurfacemoleculesthatplayanimportantroleintherecognitionofpathogens.BecauseactivationofTLRs
resultsinthereleaseofseveralchemicalmediatorsofimmunity,TLRgenesalsoexertanimportantinfluenceonthe
earlyeventsinspecificimmuneresponses(154).EvidencefromstudiesofleprosypatientsindicatesthatTLR2
controlstheproductionofcytokines,cellsignaling,andotheraspectsofresistancetoM.leprae(35,195,196,223).
(vi)VDR.AfterearlierstudiessuggestedthatpolymorphismsofthehumanvitaminDreceptorgene(VDR)were
associatedwithsusceptibilitytotuberculosis(323),astudyofleprosypatientsindicatedthatdifferentallelesofthis
genewereassociatedwithtuberculoidandlepromatousleprosy(325).TheVDRgene,locatedonchromosome
12q12,encodesanintracellularreceptorproteinwhichbindstheactivemetaboliteofvitaminD,1,25(OH)2D3.
BindingtothisreceptorleadstotheactivationofmonocytesandinfluencesthefunctionofbothCD4+andCD8+T
lymphocytes(153).
DevelopmentoftheImmuneResponse
Innateimmunity. Thehostdefenseeventsthatoperateearlyininfectionduringtheindeterminatephaseareperhaps
theleastunderstoodaspectsoftheimmunologyofleprosy.Aneffectiveinnateimmuneresponseincombination
withthelowvirulenceoftheleprosybacillusmayunderlieresistancetothedevelopmentofclinicaldisease.
Dendriticcells(DC)likelyplayakeyroleinmodulatingtheearly
(i)Antigenpresentingcellsanddendriticcells.
innateimmuneresponsetoM.leprae(87).AtthesiteofM.lepraeinvasionofthehost,e.g.,thenasalmucosaor
skinabrasion,andintheabsenceofanadaptiveimmuneresponse,theDCmaybethefirstcelltoencounterthe
bacilli.UptakeofthebacillibyDCandsubsequentlocalproductionofcytokinesandchemokinescouldregulate
inflammationandmanipulatetheensuingcourseoftheadaptivecellmediatedimmunityintoaTh1orTh2
responsetoM.leprae.DChavebeenfoundtobeveryeffectivepresentersofM.lepraeantigen(242,265,336).
MHCclassIandIIexpressionwasdownregulatedinmonocytederivedDCinfectedwithM.lepraebacilli(151),
butDCstimulatedwithM.lepraemembraneantigensupregulatedMHCclassIIandCD40ligandassociatedIL12
production(242).ThissuggeststhatwholebacillimaysuppresstheinteractionofDCandTcells.
DCinfectedwithM.lepraeexpressedPGL1onthecellsurface.PGL1hasexhibitedimmunosuppressive
properties,andmaskingofDCexpressedPGL1withspecificantibodyupregulatedboththeproliferativeresponse
andIFNproductionbyTcellsstimulatedwithM.lepraeinfectedDC(151).
BothIL12andIL10areproducedbyDC,andIL10andantiIL12havebeenreportedtoinhibitthe
lymphoproliferativeresponsefollowingpresentationofM.lepraebyDC(336).MacrophagederivedDChave
beenshowntobeevenmoreefficientantigenpresentingcellsfurthermore,theywerehighlysusceptibletokilling
+
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1471987/
+ 17/68
byM.lepraemembranespecificCD8+cytotoxicTcells(212).HigherlevelsofCD1+DCarefoundinTTlesions
thaninLLlesions(379).
LangerhanscellsareasubsetofDCthatinitiateimmuneresponsesintheskin.LLpatientshavesignificantlyfewer
Langerhanscellsintheskin,regardlessofwhetherthebiopsysamplewastakenfromhealthyskinoralesion,
comparedtouninfectedcontrolsorTTpatients(133).Incontrast,patientswithTTlesionshaveincreasednumbers
ofLangerhanscellsinthelesions,suggestinganactiveinfiltrationofthesecellstothesesites.Langerhanscells
foundintheepidermisofleprosylesionscoexpresshighlevelsofCD1aandlangerin(161),andM.lepraereactive,
CD1arestrictedTcellclonesderivedfromleprosypatientsrespondedtoantigenpresentedbyLangerhanscelllike
DC.Theantigenpresentedwaslikelyarabinomycolate,aglycolipidcomponentofthemycobacterialcellwall.
Administrationofrecombinantcytokinessuchasgranulocytemacrophagecolonystimulatingfactor(201)andIL2
(199)intoLLlesionshasbeenshowntoinduceaninfiltrationofLangerhanscellsintothesites.
Examinationofleprosybiopsysampleshasrevealedthatmonocytesanddendriticcellsintuberculoidlesions
expressedTolllikereceptorsTLR1andTLR2muchmorestronglythanthoseinlepromatouslesions(223).In
addition,invitrostudiesshowedthattheM.leprae19kDaand33kDalipoproteinscouldactivatemonocytesand
monocytederiveddendriticcellsthroughTLR2.Thecytokineprofilepresentinthelesionalsoappearedtobe
correlatedwithTLRfunction:Th1typecytokinesweregenerallyassociatedwithTLR1andTLR2activation,and
Th2typecytokineswereassociatedwithinhibitionofactivation.Interestingly,specificcytokinescouldregulate
theTLRthroughtwoindependentmechanismsinvitro,viamodulationofTLRexpressionorbyaffectingTLR
activation.
(ii)Patternrecognitionreceptors.Duringtheinnateimmuneresponse,pathogenassociatedmolecularpatterns
displayedonmanymicroorganismsarerecognizedbypatternrecognitionreceptorsexpressedonimmunecellsat
thesitesofinitialexposure.OneclassofpatternrecognitionreceptorcontainsthecalciumdependentorCtype
lectins,whichbindspecificcarbohydratemoietiesonpathogensandfacilitateinternalizationforantigenprocessing
andpresentation.AsecondcategoryofpatternrecognitionreceptoriscomprisedofTolllikereceptors.Engagement
ofthesereceptorscantriggerreleaseofantimicrobialproductswhichcanexertapreliminaryassaultonthe
pathogenaswellassignalexpressionofcostimulatorymoleculesandproductionofcytokineswhichinducethe
adaptiveimmunesystem.Athirdclassofreceptorimportantformycobacterialuptakecontainsthecomplement
receptors.
Themannosereceptor(alsocalledCD206),areceptorbelongingtotheCtypelectin
(iii)Ctypelectinreceptors.
superfamily,bindscarbohydratemoietiesonavarietyofpathogens(9).Itisexpressedprimarilyoncellsofthe
myeloidlineage,especiallymatureM,althoughnotonmonocytes,andonsomesubsetsofdendriticcells.The
Mhasbeenshowntoplayaroleinuptakeofvirulentmycobacteria(342),andamajormycobacterialligandis
likelylipoarabinomannan(304)which,onvirulentstrainsofM.tuberculosisaswellasM.leprae,containsterminal
mannosecapsonthearabinosesidechainsofthemolecule(38).Mannosecappedlipoarabinomannancan
modulateseveraleffectorfunctionsofmononuclearphagocytes,includingTNF,prostaglandinE2,andnitrite
production(5,14,24,59),aswellasMactivationformicrobicidalcapacity(5).Ithasalsobeenreportedthat
uptakeofmycobacteriaviathemannosereceptordoesnotelicitarespiratoryburst(20).
AnotherCtypelectinisthedendriticcellspecificintercellularadhesionmoleculegrabbingnonintegrin(DCSIGN
alsocalledCD209).DCSIGNisexpressedondendriticcellsandalsorecognizespathogensviathebindingof
mannosecontainingstructures(422).InstudiesusingM.tuberculosis,DCSIGNwasshowntobethemajor
receptoronDCforthebacilli,withcomplementreceptorsandthemannosereceptorplayingaminorrole(400).
Again,theprimarymycobacterialligandforDCSIGNwasmannosecappedlipoarabinomannan(220,242,400,
422).SomeinvestigatorshaveproposedthatvirulentmycobacteriamaysubvertDCfunctionviaDCSIGNby
suppressingDCmaturation,possiblythroughtheinhibitionofIL12production(290)andtheinductionofIL10
(125).EngagementofDCSIGNmayalsoinhibitTLRsignaling(422).
Langerin(CD207)isaCtypepatternrecognitionreceptorexpressedbyLangerhanscells.Langerinoligomerizes
astrimersatthecellsurfaceandpossessesasinglecalciumdependentcarbohydraterecognitiondomainwith
specificityformannose,Nacetylglucosamine,andfucose(219).
LangerinisnecessaryfortheformationofBirbeckgranules,thepentalaminarendosomalstructuresfound
exclusivelyinLangerhanscells.Exogenouscarbohydrateligandsareendocytosedvialangerinandtransportedto
theBirbeckgranulesforprocessing.Langerinmayplayaroleintheuptakeofnonpeptidemycobacterialantigens
(161).
MammalianTolllikereceptorsarecrucialfortherecognitionofmicrobialpathogensbyM
(iv)Tolllikereceptors.
anddendriticcellsduringinnateimmunity.TLRsarephylogeneticallyconservedtransmembraneproteinsthat
containrepeatedleucinerichmotifsintheirextracellulardomains.Thecytoplasmicsignalingdomainislinkedto
theIL1receptorassociatedkinase,whichactivatestranscriptionfactorssuchasNFKBtoinducecytokine
production.TenTLRshavebeenidentified,ofwhichTLR2TLR1heterodimers,TLR2homodimers,andTLR4
appeartobesignificantintherecognitionofmycobacteria.TLRshavebeenfoundtobenecessaryfortheoptimal
productionofIL12(39),aproinflammatorycytokineresponsiblefortheinductionofTh1typeimmunity,aswell
asTNF(417),acytokineimportantincellularactivationandgranulomaformationbutalsoimplicatedinthe
tissuedestructionassociatedwithleprosyreactions.
RecentworkhassubstantiatedanimportantroleforTLRsintherecognitionandsubsequentimmuneresponseto
M.leprae,particularlythroughdendriticcells(discussedabove).KangandChae(193)firstnotedthecorrelationof
aCtoTsubstitutionatnucleotide2029ofTLR2,whichresultedinthechangeofArgtoTrpataminoacidresidue
667,withthelepromatousformofleprosy.Subsequently,uponstimulationofcellsexpressingthismutationwithM.
lepraeorM.lepraeantigens,thismutationwasshowntobeassociatedwithadefectiveactivationofNFB(35)
anddecreasedproductionofIL12,IL2,IFN,andTNFbutincreasedgenerationofIL10(195,196)
comparedtowildtypecells.
SchlesingerandHorwitz(343)establishedthatcomplementreceptors1and3onthesurfaceof
(v)Creceptors.
monocytesandCR1,CR3,andCR4onMarekeymediatorsofphagocytosisofM.leprae.Inaddition,uptakeof
PGL1,amajorsurfaceglycolipidofM.leprae,wasfacilitatedbycomplementcomponentC3(343).Uptakevia
complementreceptorsdoesnotelicitarespiratoryburst(seebelow)thus,thisisonemechanismwhereby
pathogenicmycobacteriacaneludethetoxicoxygenradicalswhichcanbegeneratedduringphagocytosis.
Adaptiveimmunity:developmentofcellmediatedimmunity. CellsoftheTcelllineageplayanessentialrolein
resistancetoM.leprae,asevidencedbytheprolificlocalfootpadmultiplicationofthebacilliinneonatally
thymectomized(315)andcongenitallyathymic(75)mice.However,LLpatientsarenotimmunocompromised
hostsandarenotpronetocancerortheopportunisticinfectionsthatafflictpersonswithimmunodeficiency
diseases.TheimmunologicalanergyassociatedwithLLisspecificfortheantigensofM.leprae.
(i)Protectiveanddestructiveeffectsofcellmediatedimmunity. Ithasbeenestimatedthat>95%ofpersonsare
resistanttoleprosy.Uponexposure,protectionprobablyoccursearly,withnoovertsignsofdisease.Thereisno
testcurrentlyavailabletoreliablydetectexposuretoM.lepraeortodiagnosepreclinicalinfection.Individualswith
clinicalleprosy,eventhoseclassifiedwithpaucibacillarydiseaseandhavinghighlevelsofcellmediatedimmunity,
possesslivingorganismsintheirtissues.Theprotectiveaspectsofcellmediatedimmunityinpaucibacillaryleprosy
arelargelydefinedascontrollingthemultiplicationoforganisms.Thecollateraldamagetotissuescausedbythe
granulomatousinflammationaccompanyingcellmediatedimmunitymayhaveserious,longtermconsequences,
suchasinjurytoperipheralnerves.
(ii)Tlymphocytepopulations. (a)MHCrestrictedCD4+andCD8+cells.Byimmunohistologicalstaining,TT
lesionsexhibitedmostlyCD4+helpercellswithaCD4+/CD8+ratioof1.9:1(269).AlthoughtheCD4+/CD8+
ratioinnormalperipheralbloodisalso2:1,thereappearedtobeapreferentialmigrationinto,proliferationin,or
retentionofselectedcellsinthevarioustypesofleprosylesionsinthatcellsoftheThelper/memoryphenotype
outnumberedthenaivephenotype14foldinTTlesions.TcytotoxiccellswerenumerousinTTlesions.These
cellsmayplayaroleinmediatingtheMlocalization,activation,andmaturationthatleadtorestrictionor
eliminationofthepathogen.Interestingly,CD4+cellsweredistributedthroughoutthelesion,whereasCD8+cells
werestationedattheperipheryintheTTlesion(268).
LLlesions,incontrast,displayedaCD4+/CD8+ratioof0.6:1,andunlikeTTlesions,theCD8+Tcellswere
distributedthroughoutthelesionratherthanattheperiphery.Usingmonoclonalantibodieswhichcoulddistinguish
Tcellsubsets,theauthorsfoundthattheCD4+cellspresentwereprimarilyofanavephenotypeandtheCD8+
cellswerepredominantlyofasuppressorsubsetthus,theyproposedthattheseCD8+suppressorcellsmayserveto
downregulateMactivationandsuppresscellmediatedimmunity.However,arolefortherecentlydescribed
Foxp3expressingCD4+CD25+regulatoryTcells(159)inthevariousformsofleprosyhasyettobedetermined
butmayprovecriticalinthedevelopmentofLL.
(b)CD1restrictedTcells.CD1moleculesbindligandsviahydrophobicinteractionsinastructurallyunique,deep
antigenbindingpocketthatisdesignedtoaccommodatethehydrocarbonchainsoflipids.Therearetwodistinct
groupsofCD1molecules.GroupI,comprisedofCD1sa,b,c,ande,isfoundinhumansystemsbutnotin
rodents.GroupIIcontainsCD1dofhumansandCD1ofrodents.Allhaveevolvedtopresentlipidandglycolipid
antigensratherthanpeptides,andhumanCD1moleculespresentnonpeptidecomponentsofmycobacteriato
specificCD1restrictedTcells.
InvitroandinvivostudieshaveindicatedanimportantrolefortheCD1systemofmycobacteriallipidantigen
presentationinimmunitytoM.leprae.MycobacteriumreactivedoublenegativeTcelllinesderivedfromaskin
lesionofaleprosypatientrespondedtosubcellularfractionsofmycobacteriainthepresenceofCD1expressing
antigenpresentingcells(378).LipoarabinomannandepletedsolublecellwallfractiondidnotinducedetectableT
cellproliferation.RecognitionofpurifiedlipoarabinomannanfromM.lepraewasrestrictedbyCD1b,andTcells
lysedlipoarabinomannanpulsedmonocytesinaCD1brestrictedmanner.LipoarabinomannanalsoinducedtheseT
cellstosecretelargeamountsofIFN.Uponexaminationofleprosypatients,fewCD1+cellswerefoundinLL
leprosylesions.Incontrast,therewasastrongupregulationofCD1+cellsinthegranulomatouslesionsofpatients
withTTleprosyorreversalreaction(324).ThesecellswerealsoCD83+,amarkerfordendriticcells,indicatinga
strongcorrelationbetweenCD1expressionandcellmediatedimmunityinleprosy.Interestingly,administrationof
granulocytemacrophagecolonystimulatingfactor,acytokinewhichcanpromotedendriticcellactivation,toLL
leprosypatientsinducedinfiltrationofCD1+cellsintothelesions(332).
(iii)Cytotoxiccells.(a)Tcells.CD8+andCD4+TcellscanfunctionasclassIandclassIIrestrictedcytotoxicT
cells,respectively,andbotharecapableoflysingM.lepraeinfectedM(65,149,192).Lysisoftargetcellsby
cytotoxicTlymphocytesismediatedbyperforinandcytotoxicgranulessuchasgranzymeB,aserineprotease
locatedincytotoxicTcellsandNKcells(369).Uponcontactwiththetargetcell,perforinisreleasedbycytotoxic
Tcellsandformsporesinthetargetcellmembrane,allowinggranzymeBtoenterthecell,whereitactivates
caspasesandleadstotargetcelldeath.GranulysinisanotherdefensiveantimicrobialproteinusedbyTlymphocytes
andisexpressedintuberculosis(393)andleprosy(293).Inleprosylesionsthepresenceofgranulysincorrelated
withthepolarformsofthediseaseandwasobservedmorefrequentlyinTTskinlesionsthaninLLskinlesions.
Perforinwasequallydistributedincellsacrossthespectrum.NeitherNKcells,M,nordendriticcellsexpressed
granulysin.
LysisofM.lepraeinfectedMtargetcellsmaycontributetoprotectioninleprosyasanadjuncttotheongoing
attemptsatintracellularkillingorinhibitionbyIFNactivatedM.Exvivoandinvitrodatafrommicehave
demonstratedthatthelongtermintracellularpresenceofliveM.lepraeimpairedtheafferentandefferentfunctions
oftheinfectedM,especiallytheirabilitytobecomeactivateduponstimulationwithIFN(373,375,376).M.
lepraereleasedfromtheseheavilyinfectedMafterlysisbyTcellswouldbephagocytizedbynewlyarrived
activatedM,whicharemoreabletocopewiththebacillithantheprevioushostcells,andsubjectedtoanother
roundofattackbypowerfulantimicrobialmechanisms.
(b)Naturalkillercells.NKcellsexertspontaneousnonMHCrestrictedcytotoxicityagainstavarietyofneoplastic
andpathogenbearingtargetcells,andalthoughCD3,theysharemanycharacteristicswithcytotoxicCD3+T
cells.WhileNKcellnumbersinthebloodweresimilaracrosstheleprosyspectrum,amarkeddecreasein
circulatingNKhasbeenreportedwhenpatientswereundergoingerythemanodosumleprosum(ENL)reactions
(seebelow)(160).ThissituationreversedwhentheENLreactionsubsided.NKcellsappeartoberecruitedtoLL
lesionsinjectedwithIL2,wheretheymayberesponsibleforthesubsequentlocalclearanceofthebacilli(199).
ThecytotoxicityofNKcellsandtheirmoreactiveIL2stimulatedlymphokineactivatedkiller(LAK)celllacks
antigenspecificitybutisdirectedagainstM.lepraeinfectedmacrophages(66)andSchwanncells(392).
(iv)Macrophages. TheMistheprimaryhostcellforM.leprae.Intheabsenceofaneffectiveadaptiveimmune
response,theserelativelynontoxicbacillicanmultiplyinMtoover100organismspercell(145).Invitro,M.
lepraecanbemaintainedinametabolicallyactivestateforweeksinMsupplementedwithIL10andculturedat
33C(122).TheMalsoplaysanimportantroleinthehost'sdefenseagainstM.leprae,beingakeyplayerinboth
theafferentandefferentlimbsoftheimmuneresponse.Antigenprocessingandpresentationandmonokine
secretionarethreemajorfunctionsoftheMintheafferentstage.TheprimaryefferentfunctionofMiskilling
thisintracellularpathogen.
TLRshavealsorecentlybeenshowntobeimportantinthedifferentiationofmonocytesintomacrophagescapable
ofantimicrobialfunctionsordendriticcellshavingprimarilyantigenpresentingcapabilities(224).Activationof
TLR2,usingmycobacterialantigen,onmonocytesisolatedfromTTpatientsinduceddifferentiationintoboth(DC
SIGN+)MandCD1b+dendriticcells.Incontrast,whenperipheralbloodmonocytesfromLLpatientswere
stimulatedinsuchamanner,thecellsdifferentiatedintoDCSIGN+MbutnotCD1b+dendriticcells.Thispattern
ofexpressionwaslikewiseobservedinleprosylesions.TheimplicationofthesefindingsisthatuponinitialM.
lepraestimulationofmonocytesviaTLRs,bothtuberculoidandlepromatouspatientsmaygeneratesimilarinnate
responsestoM.leprae,butlepromatouspatientsareunabletoproceedtotheadaptiveresponseseenintuberculoid
patients.Ifconfirmedandexpanded,thismayofferimportantinsightintothemechanismsthatunderliethebroad
spectrumofhumanimmuneresponseinthisdisease.
(a)MechanismsofmacrophagekillingofM.leprae.AlthoughtheviabilityofM.lepraeissupportedinnormal
mouseM,IFNactivatedMcandrasticallyinhibitorkillM.lepraeinvitro(307).Thesefindingsconfirmedthe
demonstrationthatinnormalM,phagosomelysosomefusionwasblockedbylive,butnotdead,M.leprae(373)
and,moreimportantly,thatinactivatedM,phagosomesharboringM.lepraefusedwithsecondarylysosomes.
TwoimportantantimicrobialpathwaysbywhichMcaninhibitorkillinvadingpathogensarethegenerationof
reactiveoxygenintermediatesandofreactivenitrogenintermediates.
UponphagocytosisofmicroorganismsbyM,arespiratoryburstensuesinwhichthereisagreatincreaseinthe
consumptionofoxygencatalyzedbyNADPHoxidaseandtheproductionofsuperoxide.Otherreactiveoxygen
intermediates,includinghydrogenperoxide,hydroxylradical,andsingletoxygen,aresubsequentlygenerated.
Thesetoxicoxygenproductsareanimportantantimicrobialdefensemechanismofphagocytecells,especially
againstextracellularpathogens.M.leprae,however,isonlyaweakstimulusoftheMoxidativeburst(155),
possiblyduetoadownregulationofsuperoxidegenerationbyPGL1(58).M.lepraealsopossessesasuperoxide
dismutase(406)andexpressesbothSodCandSodAbyreversetranscriptionPCR(440).Thus,leprosybacilli
appeartobewellequippedtohandleantimicrobialreactiveoxygenintermediatesgeneratedbythehostM.
Reactivenitrogenintermediates,primarilynitricoxide,arederivedfromtheterminalguanidinonitrogenofL
argininebyahighoutput,inducibleformofnitricoxidesynthase(iNOS)producedbyactivatedM.Theabilityof
activatedmurineMtoinhibitM.lepraemetabolicactivityisdependentonthegenerationofsuchreactivenitrogen
intermediates,asactivatedMculturedinthepresenceofcompetitiveinhibitorsoftheenzyme,suchasL
monomethylarginineoraminoguanidine,hadnodetrimentaleffectonbacterialmetabolism(4).Furthermore,
activatedMfromNOS2knockoutmicecouldnotkillM.leprae(6).
TheroleofreactivenitrogenintermediatesasaMeffectormechanisminhumansissomewhatcontroversialasit
hasbeendifficulttogetinvitroculturedcellstogeneratehighlevelsofnitriteinasconsistentamannerasinmurine
cells(430).However,severalstudieshaveshownthatiNOSisexpressed,asdetectedbyimmunohistochemistry,at
thesiteofdiseaseinpatientsinfectedwithintracellularpathogens,includingM.leprae.KhanolkarYoungetal.
(210),usingantibodiesagainstiNOS,foundthatiNOSwashighlyexpressedinthelesionsoftuberculoidleprosy
patientsandwasincreasedtoevenhigherlevelsduringthereversalreaction(seebelow).Moreover,thelevelsof
iNOSsubsidedoverthecourseofprednisolonetreatment(233).
BecausedetectionofiNOSbyimmunohistochemistrydoesnotnecessarilyindicateactualproductionofreactive
nitrogenintermediates,lesionshavealsobeenstainedfornitrotyrosine,thestableendproductofthenitrosylationof
tyrosineresiduesinproteinsbyperoxynitrite(345).ItwasfoundthatiNOSandnitrotyrosinewereexpressedin
borderlineleprosypatientsbothwithandwithoutthereversalreaction.Inaddition,elevatedlevelsofnitrateswere
measuredintheurineofleprosypatientsundergoingreversalreactions,andtheselevelsdecreaseduponhighdose
prednisolonetreatment(344,345).
MethodshaverecentlybeendevelopedtoisolategranulomaMfromthefootpadsofM.lepraeinfectedmice(145,
373).Thismodel,whichenablesthestudyofMfromtheactualsiteofinfectioninexperimentalleprosy,has
alloweddeterminationofculturecharacteristics,cytokineproduction,andcellsurfacephenotypicmarkersbyflow
cytometryofthesegranulomaderivedcells.InitialstudiesoffootpadgranulomaMfromM.lepraeinfected
athymicnu/numiceindicatedthattheMwerephenotypicallyindistinguishablefromnormalperitonealMexcept
thattheycontainedenormousnumbersofM.leprae(374,375).TheseM.lepraeinfectedfootpadderived
granulomaMwerealsofunctionallysimilartoperitonealMexceptforonenotabledefect:theywererefractory
toactivationbyIFNforbothmicrobicidalandtumoricidalactivity.Inaddition,therewasnoIFNinduced
augmentationofclassIIMHCexpressionorphorbolmyristateacetateinducedsuperoxideproductionintheM.
lepraeengorgedgranulomaM.TheseresultsprovidefurthersupportthatM.lepraeisapotentmodulatorofM
effectorfunctionsandthatitsinfluenceislargelyrestrictedtothemicroenvironmentofthegranuloma.Thisworkis
nowbeingextendedtoevaluationofM.lepraeinfectedgeneknockoutmice.
Recentevidencefromourlaboratory,usinganinvitrosysteminwhichM.lepraeinfectednu/numousefootpad
granulomatargetMarecoculturedwithfreshuninfectedeffectorM,suggeststhatMmayplayaroleincell
toxicityinleprosylesions(145).WhennormaleffectorMwerecoculturedwithtargetM,theeffectorM
acquiredthebacillifromtheinfectedtargetM.Moreover,iftheeffectorMwereactivatedwithIFN,theycould
killthetargetcellderivedM.leprae.Killingofthebacilliinthissystemwasnotarapidprocessbutrequired3to5
daysofcocultureforoptimaleffect.Furthermore,itwasdependentoncellcellcontactandproductionofreactive
nitrogenintermediates,butdidnotrequireconcomitantIFNorTNFproduction.Theexactmechanismby
whichtheeffectorMacquiredM.lepraefromthetargetMisnotyetknown.However,innumeroussystems,
especiallythoseinvolvingtumormodels,Mhavedemonstratedcytotoxiccapabilitiesusingavarietyoffunctions,
includingdirectlysis,engulfment,andinductionofapoptosisornecrosis.Currentstudiesareaimedatdetermining
whichmechanism(s)anewMmayemploytoeffectcellularturnoverinaleprosylesion.
(v)Cytokinesinleprosy. TheTh1/Th2paradigm,basedonfunctionaldiscriminationofThelpercellsaccordingto
theirpatternofcytokineproduction,assertsthatTh1andTh2cellspromoteacellularandhumoralimmune
response,respectively(277).Thisfunctionaldifferentiationhasofferedanattractivehypothesistoexplainthe
differencesbetweentuberculoidandlepromatousresponsestoM.leprae.
Severalmajorstudiesoflocalimmuneresponsesinleprosyskinlesionshavebeenpublished(Table5).Theseare
difficulttocomparebecausetheyhaveuseddifferentdesigns,differentmethodsofquantification,anddifferent
conventionstoexpresstheirresults.Overall,however,thesestudieshavegenerallyrevealedapredominanceofIL
2,TNF,andIFNtranscriptsintuberculoidlesionsandIL4andIFNinlepromatousones,geneexpression
profilesconsistentwithTh1andTh2patterns,respectively(16,113,281,380,448).CD4+clonesisolatedfrom
TTlesionssecretedprimarilyIFN,whereasaCD4+clonefromanLLlesionproducedpredominantlyIL4(381),
andCD8+clonesisolatedfromLLpatientslikewisegeneratedlargeamountsofIL4(328).
TABLE5.
Cytokinegeneexpressioninnonreactionalleprosya
FurtherstudieshavealsoindicatedthatIL12andIL18promoteresistancetoM.lepraeandarehighlyexpressed
intuberculoidlesions(123,380).Moststudieshavegroupedpatientsonlyintotuberculoidorlepromatous
groupings,however,anditisnotclearwhethervariationsincytokineproductioncorrespondwellwiththevarious
degreesofcellularimmunityrepresentedintheborderlineportionoftheleprosyspectrum.
Thestudiesnotedabovehaveexaminedbiopsiesfromwellestablishedlesions,usuallypresentforatleast2years.
Theimmunologicactivitywithinearlierlesions,inpatientswithonlyasinglelesion,hasrecentlybeenevaluatedby
Stefanietal.(391).Theseearlylesions,histologicallyconsistentwithTTorBTdisease,alsodisplayedaTh1like
patternofcytokinegeneexpression(Table5).
CirculatingleukocytesandTcelllinesfromtuberculoidpatientsstimulatedbyM.lepraeinvitrohavealso
generallybeenfoundtoproduceaTh1cytokinepattern(Table5),whileleukocytesandTcelllinesfrom
lepromatouspatientsgenerallyproduceaTh2cytokinepattern(263,285).However,leukocytesfrom
approximately40%ofallpatientsinonesuchstudyproducedamixedTh0cytokineprofile,i.e.,IFN,IL2,and
IL4(263).ItispossiblethatsomeofthepatientswhosecellsproducedtheTh0patternwereintheborderline
portionoftheleprosyspectrum(BLorBT)alternatively,thehumanimmuneresponsetoM.lepraemaynot
correspondentirelywiththeTh1/Th2model.FractionatedM.lepraeantigenshavealsobeenfoundtostimulate
IFNinvitrowithleukocytesfromtuberculoidpatients(95).
Experimentalimmunotherapywithintradermalinoculationofcytokineshasprovidedadditionalinformationabout
theirrolesinimmunologicaleventswithinleprosyskinlesions(197).Bothshortandlongtermintradermal
administrationofIFNresultedinaninfluxofmononuclearcellsandanincreaseintheCD4/CD8ratiointhe
lesions,butdidnotreversethespecificnonresponsivenessofcirculatingleukocytestoM.leprae(200).M.leprae
exposureinvitrodidnotelicitIFNincirculatingmononuclearcellsoflepromatouspatientstheadditionofIL2
reversedthisinmost(butnotall)ofthesepatients'peripheralbloodmononuclearcells(291).Inlepromatous
patients,intradermalinjectionsofIL2generatedapparentincreasesincellmediatedimmunitywithintheskin
lesions(199)andresultedinincreasedlevelsofantibodiestoM.lepraeantigens(198),butdidnotenhance
systemiccellmediatedimmunitytoM.leprae.
Insummary,studiesofcytokinegeneexpressioninleprosylesionsthusfarhavegivenusamoredetailed
descriptionoftheimmunologicalparametersofthepolartypesofleprosy,confirmingandsupportingtheoriginal
conceptthattuberculoidlesionsaremanifestationsofdelayedhypersensitivityandcellularimmunityandthat
lepromatousonesresultwhenimmunerecognitionoccurs(asindicatedbyantibodyproduction)butthehostis
unabletodevelopcellularimmunitytoM.leprae.However,thesestudieshavenotyetrevealedthemechanismsby
whichthecellularimmuneresponseissoextraordinarilytitratedtoproducetheentireleprosyspectrum.Researchin
thisareacontinuesonthepremisethattheanswerwillbefoundinanunderstandingofthecomplex
immunoregulatorymechanismsofcytokineproductionandinhibition.Someoftheanswerstothesequestionsmay
alsobefoundinstudiesofthegeneticallyinheritedabilitytorespondtoM.lepraeandotherpathogens(seeGenetic
InfluencesonLeprosyinHumans,above).
LEPROSYREACTIONS Goto:
Reactionsareacuteinflammatorycomplicationsoftenpresentingasmedicalemergenciesduringthecourseof
treatedoruntreatedHansen'sdisease.Twomajorclinicaltypesofleprosyreactionsoccurtogethertheymayaffect
30to50%ofallleprosypatients(28,226,352).BecauseM.lepraeinfectsperipheralnerves,theinflammation
associatedwithreactionsisamedicalemergency,asseverenerveinjurymaydeveloprapidly,withsubsequentloss
ofsensation,paralysis,anddeformity.
Thecause(s),mechanisms,andtreatmentofthesereactionsremainhighlyproblematic,forbothcliniciansandbasic
scientists.Thedifferenttypesofreactionsappeartohavedifferentunderlyingimmunologicmechanisms,butthese
arepoorlyunderstoodinspiteofasubstantialbodyofdetaileddescriptiveinformation,andthefactorsthatinitiate
themareunknown.
ClinicalPresentation,Diagnosis,andHistopathology
Type1reactionsoccurinpatientsintheborderlineportionofthespectrum(i.e.,BL,BB,andBT).Theyarealso
knownasreversalreactions,becauseearlyobservationssuggestedthatafterthereactionhadsubsided,clinicaland
histopathologicalevidenceindicatedthattheimmunityinthelesionshadincreased(upgrading)ordecreased
(downgrading)(320).Downgradingisrarelyseeninthecurrenteraofantimicrobialtreatment,andtheevidence
regardingtype1reactionsreviewedhereisbasedonstudiesofupgradingreactions.
Thesereactionspresentasindurationanderythemaofexistinglesions,frequentlywithprominentacraledemaand
oftenwithprogressiveneuritis,causingsensoryandmotorneuropathy(Table6).Inseverereactionsthelesionsmay
ulcerate.Type1reactionsusuallydevelopgradually,andtheirnaturalcoursemaylastformanyweeks.
TABLE6.
Comparisonofclinicalfeaturesoftype1andtype2leprosyreactions
Type2reactions,alsoknownaserythemanodosumleprosum,occurinmultibacillarypatients(LLandBL).These
patientsexperienceanabruptonsetofcropsofverytender,erythematousnodulesthatmaydevelopontheface,
extremities,ortrunk,withoutpredilectionforexistinglesions(Table6).Systemically,thesepatientsoftenalso
experiencefever,malaise,andsomedegreeofneuritiswithsensoryandmotorneuropathy.Iridocyclitisand
episcleritis,orchitis,arthritis,andmyositismayalsoaccompanythisreaction.Inseveretype2reactions,someofthe
cutaneouslesionsmayulcerate.Thenaturalcourseoftype2reactionsis1to2weeks,butmanypatients
experiencemultiplerecurrencesoverseveralmonths.
TheLuciophenomenonisanacute,severe,necrotizingvasculitisoccurringprimarilyinpatientsofMexican
ancestry(96).Fortunately,thiscomplicationisrare,becauseitisassociatedwithhighmorbidityandmortality.
Thesereactionshavebeenassociatedwiththepresenceofhighlevelsofcryoglobulins,andM.lepraeantigens
havebeenassociatedwithsomeofthese(100,305),buttheroletheymightplayinthesereactionsisunclear.
Characteristically,endothelialcellsareunusuallyheavilyinfectedandmaybeseeninvariousstagesof
degeneration.Lucioreactionsmaybeaccompaniedbyaprofoundanemia,andseverecasesmayrequireintensive
woundcareanddebridementcomparabletothatusedinthemanagementofsevereburns.Thesereactionsrequire
intensiveinpatientmanagement,andintheUnitedStates,consultationwiththeNationalHansen'sDiseaseProgram
isrecommended.
ImmunologicalFeaturesofReactions
Evidenceregardingthemechanismsofleprosyreactions. Alltypesofleprosyreactionsarebelievedtobe
immunologicallymediated,butthemechanismsresponsibleforeachtypeofreactionremainpoorlyunderstood.
Althoughtype1and2reactionstogetheraffect40to50%ofallpatientsatleastonceinthecourseoftheirdisease,
noclinicalorlaboratorytestscanaccuratelypredictwhoismostlikelytodevelopareactionorwhenitmightoccur.
Thefactorsprecipitatingreactionsandtheearliesteventsinthedevelopmentofreactionsarethereforeunknown.
Substantialevidencenowindicatesthattype1reactionsaretheresultofspontaneous
(i)Type1(reversal)reactions.
enhancementofcellularimmunityanddelayedhypersensitivitytoM.lepraeantigens,butthecausesand
mechanismsofthisenhancementremainpoorlyunderstood.Earlyfunctionalstudiesoflymphocytesdemonstrated
increasedlymphocyteproliferationinresponsetoM.lepraeantigensinvitroduringtype1reactions(26,135).
SubsequentimmunophenotypingstudiesrevealedthatthenumberandpercentageofCD4+Tcellsareincreasedin
reactingskinlesions(267,269,353reviewedinreference270).MeasurementofsolubleIL2receptorlevelsin
patientserafoundhighlevelsintype1reactionswhenthepatientsfirstpresentedfortreatmentandfoundthatthese
levelsdeclinedsteadilyduringtreatment(416).
Duringtype1reactions,increasesinexpressionofthegenesforseveralproinflammatorycytokines,includingIL1,
IL2,IL12,IFN,andTNF,havenowbeendocumentedinseveralstudies(Table7).Thisactivationispresent
bothlocally,inreactingskinlesions,andsystemically,inserumandincirculatingleukocytes.Thepatternof
cytokineexpressionhassuggestedtomanyinvestigatorsthattype1leprosyreactionsrepresentaspontaneously
enhancedTh1response.However,thesestudieshavenotbeenabletoclearlydifferentiatewhichchanges
observedareconsequencesofreactionandwhich(ifany)reflectinitiatingevents,andseveralofthereportshave
notclearlydistinguishedimmunologicalfrominflammatoryphenomena.
TABLE7.
Cytokinegeneexpressionchangesduringtype1andtype2reactionsa
Clinicalstudieshavedeterminedthattheserumlevelsofsomeofthesecytokinesdeclineduringthecourseof
successfulprednisolonetreatmentoftype1reactionsbutshowlittleornoreductionduringthetreatmentoftype2
reactions.Suchadeclinehasbeendocumented,forexample,forindicatorsofinflammationsuchasneopterinand
iNOS(114,233),aswellasforcytokinesandreceptorsmoreindicativeofimmunologicfunctionsuchassoluble
IL2receptor,IFN,IL6,IL10,IL12,andIL13(21,233,416).Theseobservationssuggestthatinadditionto
thenonspecificantiinflammatoryeffectsofprednisolone,itmayhavesomeinhibitoryeffectontheunderlying
immunologicmechanismsofthesereactions,althoughitisnotcertainthatthisisthecase.
Theeventsorconditionsthattriggertype1leprosyreactionsareunknownnotably,onlyabout15to30%ofthe
patientsatrisk(i.e.,BL,BB,andBT)areaffected.Type1reactionshavebeenobservedtofollowimmunization
withothermycobacteriainsomecircumstances(433,452),andvariousenvironmentalfactorshavebeen
consideredbutnotconfirmedtobeassociatedwiththeonsetofthesereactions.Thepossibilitythatgeneticfactors
mightpredisposesomepatientstodeveloptype1reactionshasnotyetbeenexamined(seeGeneticInfluences,
above).Notably,experimentalintradermalinoculationofIL2orofIFNdidnotprecipitatetype1reactions(198,
332).Inaddition,althoughthalidomidewasreportedtoinhibitTNFinseveralexperimentalconditions(410),it
hasnobenefitinthetreatmentoftype1reactions.
(ii)Type2leprosyreaction(erythemanodosumleprosum). Type2reactionsoccurinpatientswithpoorcellular
immunitytoM.leprae,abundantbacilli(i.e.,antigen)incutaneousandperipheralnervelesions,andastrong
polyclonalantibodyresponsewithhighlevelsofcirculatingimmunoglobulins.Theacutelesionsarecharacterized
byaneutrophilicinfiltratesuperimposeduponachroniclepromatouspattern,observationsthathavelong
dominatedthinkingaboutthisreaction.Basedprimarilyonhistologicalevidence,Wemambuandcolleagues
proposedthatENLrepresentsanArthuslikephenomenonmediatedbyimmunecomplexes(431).Immunoglobulin
andcomplementdepositionhavebeendemonstratedintheskinlesions,andserumcomplementisdecreasedin
thesepatients,consistentwiththishypothesis,andsomemycobacterialconstituentshavebeenidentifiedinsomeof
thesecomplexes(322).However,neithercirculatingnorfixedimmunecomplexeshavebeenreproducibly
demonstratedinENLlesions.Thedemonstrationofimmunecomplexeswithinclinicallesionsinotherdiseaseshas
alsobeendifficultandproblematic,however,andtheimmunecomplextheoryofENListhusneitherprovednor
disproved.
Otherstudieshaveidentifiedpossibleevidenceofcellularimmuneactivationintype2reactions,including
increasesincirculatingIFN,TNF,andIL12(Table7)(449).IncreasesinmRNAlevelsforthesecytokines
havealsobeenobservedinbiopsiesofskinlesions,indicatingthatcellularimmuneactivationisoccurringlocally.
Incontrast,increasesintheexpressionofIL6,IL8,andIL10mRNAsandsustainedexpressionofIL4andIL5
mRNAs,allcytokinesassociatedwithneutrophilchemotaxis,antibodyproduction,andreducedcellmediated
immunity,wereobservedinENLlesions.
Thefactorsthattriggertype2reactionsareevenmorepoorlyunderstoodthantheimmunologicmechanismsofthe
reactionitself.Otherinfectionsorviralillness,fever,immunization,andpsychologicalstresshaveallbeeninvoked,
butnoconvincingevidencehassupportedanyofthese.Pregnancyappearstohaveaninhibitoryeffectontype2
reactions,whereasthereactionmayrecurseverelyinthepostpartumperiodinwomenwithlepromatousleprosy
(101,236).Anecdotalreportssuggestthatinsomewomen,theseverityofatype2reactionfluctuatesduringthe
menstrualcycle,andstillotheranecdotalevidencehassuggestedthatthetype2reactionismorefrequentand
severeamongchildrenwhodevelopleprosyattheonsetofpuberty(81).
Theonlystimulusknowntoinitiateatype2reactionistheintralesionalinjectionofIFN(332).Inexperimental
immunotherapeutictrials,multipleintradermalinjectionsofIFNover6to12monthselicitedthisreactionin6of
10lepromatouspatientsstudiedall6ofthesehadpolarLLdisease.Thefourpatientswhodidnotdevelopthe
reactionhadBLorsubpolarLLtypesofleprosy,inwhichtheabilitytodevelopcellmediatedimmunitytoM.
lepraeisgreatlyreducedbutisnotaltogetherabsent.Theobservationthattype2reactionsdevelopedinpatients
whohadthemostprofoundinabilitytodevelopcellmediatedimmunitytoM.lepraemaysuggestthat,insuch
patients,theeffectofIFNistoactivateimmunologicmechanismsthat,becausetheycannotgeneratecellmediated
immunity,areinsteadchanneledintopathwaysthatleadtoenhancedhumoralimmuneactivity,i.e.,Th2cytokine
responses.Notably,intradermalinjectionsofIL2inlepromatouspatientsdidnotelicitatype2reaction(198).
Evidencefromtherapeutictrialsregardingmechanismsofreactions. Reactionsarepoorlyunderstood,andtheir
managementisoftendifficultandperplexing.Corticosteroidsarethemainstayofthetreatmentoftype1reactions
highdosesareoftenrequired,sometimesforprolongedperiodsoftime(40),withtheattendantriskofseriousside
effects(397).Fortype2reactions,thalidomideisthetreatmentofchoice(405).(Fordecades,thetype2reaction
wastheonlyclearindicationfortheuseofthalidomide,andthedrugmightwellhavebeenforgottenentirelyhadit
notbeensovaluableforthetreatmentofthesereactions.)Theantiinflammatorypropertiesofclofazimineare
usefulintreatingtype2reactionsalso.Corticosteroidsarewidelyusedforthetreatmentofthesereactions(235,
349),sometimesbecausetheydonotrespondwelltothalidomide,andmoreoftenbecausethedrugishighly
restrictedorunavailable.Antimicrobialtherapyshouldbemaintainedthroughouttreatmentforbothtypesof
reaction.
Thegreatdifficultiesencounteredintreatingreactionsprovideanilluminatingexampleoftheimportanceof
understandingthemechanismsofdisease:i.e.,sincethecause(s)andmechanismsofreactionsarepoorly
understood,treatmentislargelyempiricalandisoftensuboptimal.Anumberofotherimmunosuppressiveagents
havethereforebeentestedfortheireffectonreactionsinthesearchforadditionalregimensthatmightbeeffective
individuallyoratleastofferasteroidsparingeffect.Sincetheseagentsactatdifferentpointsinthedevelopmentof
animmunologicalresponseandsomeinformationisavailableconcerningtheirmechanismsofaction,areviewof
thesetrialsisofinterestintryingtounderstandtheimmunologicalmechanismscausingreactions(Table8).
TABLE8.
Effectsofimmunosuppressiveagentsonleprosyreactionsc
Corticosteroids,duetotheirgeneralantiinflammatoryeffects,arehighlyeffectiveclinicallyforbothtype1and
type2reactions.Itisnotclear,however,thatthistreatmenthasasignificanteffectupontheunderlyingmechanisms
ofeithertypeofreaction(282,283).Reducedlevelsofproinflammatorycytokineshavebeenobservedin
peripheralbloodmonocytesfrompatientstreatedwithcorticosteroidsfortype1reactions(245),butotherstudies
havesuggestedthatalterationsincytokinelevelsarenotalwaysobservedinpatientswhodoreceivegoodclinical
benefitfromantiinflammatorytreatment(12,275,404).
Thalidomidewasdeterminedtobeextraordinarilyeffectiveinthetreatmentoftype2reactionsbeforeitsteratogenic
propertieswererecognized(367).Themechanismbywhichthalidomideexertsthisstrongantiinflammatoryeffect
intype2reactionsisnotclearlyunderstoodeventoday,however,inspiteoftheflurryofinterestintheapparent
inhibitoryactionofthalidomideonTNF(410).Theeffectsofthalidomidearestrikinglydifferentoverawide
rangeofconcentrations,andattheconcentrationsusedclinicallyithasalsobeenobservedtoenhancethe
productionofIL2(360).EarlystudiesnotedthatthalidomideinhibitedtheIgMresponse(361),anditisalso
knowntopromoteapoptosisinneutrophils(19),bothofwhicharepotentiallysignificanteffectsinthecontextof
type2reactions.
Cyclosporinehasbeenusedtotreattype2reactions,withmixedresults(260,421,418).Cyclosporineisapotent
suppressorofcellularimmunity,blockingthetranscriptionofIL2andseveralothercytokinesbyinterferingwith
thecalcineurindependenttranslocationofthenuclearfactorofTcellactivation(NFAT)tothenucleus(27,211,
250).Theevidencethatcyclosporineisbeneficialinsomeseverecasesoftype2reactionmayindicatethatthe
mechanismsunderlyingthesereactionsarenothomogeneous.Tcellrelatedmechanismsmaybeinvolvedinthe
pathogenesisofsevereorrecurrentENL,butthebroaderexperience,inwhichcyclosporinewasoflittlebenefit,
suggeststhatsuchTcellfunctionsmaynotbeamajorfeatureofmosttype2reactions.
Azathioprineisapurineantagonistandiswelldocumentedtoinhibitlymphocyteproliferation,althoughitsprecise
mechanismofimmunosuppressionisunknown(27).Whenfollowedbyprednisolone,ithasbeenfoundtoprovide
resultscomparabletothosewithprednisolonealoneinthetreatmentoftype1reactions(246),thuspossibly
providingasteroidsparingregimeninthetreatmentofthisreaction.Azathioprinealonedidnotprovidesuperior
resultsinthisstudy,however,suggestingthatthebroadimmunosuppressionassociatedwiththisagentdidnot
interferewithsomeofthebasicmechanismsunderlyingthereaction.Azathioprinealonehasnotbeenassessedin
thetreatmentoftype2reactionsbuthasalsobeenreportedtobeusefulincombinationwithprednisoloneinthe
managementofintractabletype2reactionsthatdonotrespondwelltoprednisolonealone(243).Thishasnotbeen
evaluatedinacontrolledstudy,however.
Methotrexatehasrecentlybeenreportedtobeeffectivewhenusedincombinationwithcorticosteroidsinpatients
whosetype2reactioncouldnotbecontrolledwithcorticosteroidsalone(202).Thisremainstobeconfirmedin
controlledstudies.
Pentoxifylline,likethalidomide,hasbeenobservedtoproduceareductionincirculatinglevelsofTNFinpatients
withtype2reactionsaswellasinhibitTNFmRNAinskinlesions(275,331,304).However,pentoxifyllinehas
notprovidedaclinicalbenefitcomparabletothatofthalidomide(82,276),andsomeevidencehassuggestedthat
theinhibitionofTNFmaynotbethemajorbeneficialeffectofthalidomideinENL(276).
Mycophenolatemofetil,aninhibitorofBandTcellproliferationthatblockstheproductionoftheguanosine
nucleotidesrequiredforDNAsynthesis(27),hasshownnobenefitintype2reactionsinonesmallstudy(47),
althoughtheeffectofhigherdoseshasnotyetbeenstudied.Thisagenthasnotbeentestedintype1reactions.
Insummary,inhibitionoflymphocyteproliferationbyseveralpotentantimetaboliteshashadlittleornoconsistent
effectinthetreatmentofeithertypeofleprosyreaction.Similarly,clinicalinhibitorsofTNF,IL2,andother
cytokineshavehadminimaleffectsonreactionsinmostcases.Themechanismoftheremarkablybeneficialeffect
ofthalidomideontype2reactionsremainsunexplained.Thereisnoconvincingevidencetodatethattheanti
inflammatorytreatmentsusedactuallyinterrupttheunderlyingimmunologicalprocesses.Theoverallbeneficial
effectsofcorticosteroidsonbothtypesofreactionareprobablyduelargelytotheirantiinflammatorymechanisms.
Althoughcorticosteroidandthalidomidetreatmentsgreatlyalleviatesufferingandmitigatenerveinjury,itisquite
possiblethattheimmunologicaleventsthatfuelreactionssimplyruntheircourseandabateindependentlyofthe
antiinflammatorytreatmentitself.
MECHANISMSOFNERVEINJURY Goto:
Amongbacterialpathogens,infectionofperipheralnervesisauniquepropertyofM.leprae.Infectionofperipheral
nervesisthesinequanonofleprosy,butmanyclinicaldetailsregardingthefrequencyandextentofnerveinjury
haveonlyrecentlybeendescribed,andthemechanism(s)underlyingnerveinjuryinleprosyisverypoorly
understood(354).
NeuropathyinleprosyarisesnotonlyfromtheinfectionofperipheralnervesbyM.lepraebutalsofromthe
inflammatoryandimmunologicresponsestothispathogen.Thisneuropathyisoftendevastatingtothepatient's
healthandwellbeing,throughthedevelopmentofanesthesia,paralysis,andpotentialcripplingdeformitiesof
fingersandtoesduetoulnar,medianradial,peroneal,orposteriortibialneuritisoroculardamageinthecaseof
facialnerveinvolvement.Studiesofthepathogenesisofneuropathyinleprosyhavebeenseverelyhamperedbythe
lackofgoodexperimentalmodelsandbecausebiopsyofthemostactivelyinflamedsitesofaffectedperipheral
nervetrunksisnotpossible.
Althoughlocalizedanesthesiaisaseriousandwellknownconsequenceofleprosy,recentevidencealsoindicates
thatalargepercentageofpatientsexperienceneuropathicpain(142,396),sometimeslongaftertheyappeartobe
otherwisecuredofinfection.Littlestudyhasbeendoneconcerningthemechanismsofneuropathicpaininleprosy,
andtheremainderofthisdiscussionofnerveinjurywillconcentrateonmechanismsrelatedprimarilytoinjury
resultinginanesthesiaand,ultimately,paralysis.
Recentadvancesinthestudyofnerveinjuryinleprosyhavebeenmostprominentinfiveareas:improvementsin
clinicalsensorytestingwithmonofilaments,recognitionthatthemechanismsoflocalizationofM.lepraetonerves
mayinvolvethevascularendothelium,directexaminationofimmunologicalparametersinbiopsysamplesof
affectednervesinleprosypatients,identificationofthemolecularmechanismsofM.lepraebindingtoSchwann
cells,anddevelopmentofgreatlyimprovedSchwanncellculturemodelsforinvitrostudiesoftheconsequencesof
M.lepraeinfection.
Accurate,reproduciblemeasurementofsensoryfunctionusingcalibratedSemmesWeinsteinmonofilamentshas
beenamajoradvanceinthestudyofnerveinjuryinleprosy(231).Suchtestingcanclearlyidentifylossof
protectivesensationbeforeitresultsinulcerationandotherinjuryandcanidentifyearlyneuropathy,withsubtle
functionalimpairment,thatisotherwiseoftenoverlooked.Thishascontributedtoadvancesinthepreventionof
disabilityinleprosy(419).Usingthismethod,a5yearfollowupstudyofnervefunctionimpairmentinover200
patientshasdemonstratedthatnerveinjurycontinuestobeaproblemevenaftertheinfectionistreatedandcured
(316).
Althoughneuritisandneuropathyhaveoftenbeenstudiedanddiscussedinthecontextofleprosyreactions,which
mayexacerbateneuritis,themoresensitiveassessmentsofsensorylosshavealsodemonstratedthatnervefunction
impairmentoccursindependentlyofreactions(419).Withthesesensitivemethods,itisnowevidentthatearlynerve
functionimpairmentoccursearlierinlepromatousthanintuberculoidpatients(258,316,339).Inaddition,astudy
ofprophylactictreatmentwithprednisolonehasshownthatitreducesnervefunctionimpairmentthatismeasurable
at4months,butthisimprovementwasnotevidentafter1year(385).Thus,theabilitytoclinicallyevaluate
differentdegreesofneuropathyandcorrelatethiswithresponsestointerventionhasveryimportantimplicationsfor
bothclinicalandbasicresearchonthemechanismsofnerveinjuryinleprosy.
Overtsensoryandmotorneuropathiesthatpromptpatientstoseekmedicalattentionoftenoccurearlierandmore
intenselyinthosepatientswhoselesionscontainfewbacilli(BTandTTtypes),mostprobablybecausethe
granulomatousinflammatoryresponsetoM.lepraeinthesepatientsleadstoinjurytoadjacentnerves.Incontrast,
lepromatouspatientsoftendevelopovertneuropathymoreslowly,eventhoughtheSchwanncellsand
macrophagesoftheirperipheralnervesaremoreheavilyinfectedwithM.leprae.Afterprolonged,untreated
infection,however,thenervesofalltypesofpatientsareatriskofchronicinflammationandfibrosisthatbecomes
thefinalcommonpathwayofinjury,potentiallyresultinginanesthesiawithparalysisofintrinsicandextensor
musclesofthehandsandfeet.Theaffectedlimbsarethenathighriskofinjuryormutilation,processesthat
acceleratethephysiologicresorptionofboneandresultinlossofdigits.Involvementofthefacialnerveleaves
patientsatriskofcornealanesthesia,abrasion,andblindness.
Fourrelatedaspectsofnerveinjuryinleprosymustbeconsideredinunderstandingthepathogenesisofneuritisin
leprosy:localizationofM.lepraetoperipheralnerves,infectionofSchwanncells,immunologicresponses,and
inflammation.Ofthese,theinfectionofSchwanncellsisthemostobviouslyremarkableandhasreceivedbyfarthe
greatestexperimentalattention,andtheotheraspectshavesufferedespeciallyfromtheinherentdifficultiesin
obtainingmaterialforinvestigation,sincebiopsyofaffectednervesisseldomclinicallyindicatedandisotherwise
unethical.Thearmadilloappearstoprovideamodelforsomeaspectsofleprosyneuropathyinhumans,asnoted
below,butalsohasposedmajorlimitationsasanexperimentalanimal.
LocalizationofM.lepraetoPeripheralNerves
ThefirstessentialstepinleprosyneuritisisthelocalizationofM.lepraetoperipheralnerves.Eversinceautopsy
dissectionsinthe1890s(85,130),whichfollowedaffectedperipheralnervesfromtheskinlesiontothespinalcord,
theinfectionofperipheralnerveshasbeenunderstoodtobeanascendingneuropathyoriginatinginsensory
cutaneousnervesandtravelingproximallytoinvolvelargernervetrunkscarryingmixedsensoryandmotorfibers
(327).ThishasbeenextrapolatedtoimplythatM.lepraeinitiallybindstoexposedSchwanncellsinthedermisand
thenmovesproximallywithinthenerve,swimminglikefishupastream(209).
However,recentstudiesofperipheralnervesinexperimentallyinfectedarmadilloshavesuggestedthatM.leprae
infectsnervesfromtheoutsidein,firstaggregatinginepineuriallymphaticsandbloodvesselsandthenenteringthe
endoneurialcompartmentthroughitsbloodsupply(347,351).
AmodelillustratingthishypothesisoflocalizationofM.lepraetoperipheralnervesispresentedinFig.6
(347).Thisviewofthepathogenesisofinfectionofperipheralnervesraisessignificantimplicationswithrespectto
bothunderstandingtheprocessandpossiblepointsofpreventiveortherapeuticintervention.Ifseveralstepsare
requiredfortheultimateentryofM.lepraeintoSchwanncells,thenthereareseveralpotentialsitesofintervention,
e.g.,bindingtoendothelialcells,entryintotheendothelium,exitfromendothelialcellsintotheendoneurium,and
bindingtoSchwanncells.If,ontheotherhand,M.lepraeentersnervesexclusivelyviathesinglestepofdirect
bindingtoexposedSchwanncellsinthedermis,thenthisistheonlyopportunityforpreventiveortherapeutic
intervention,andthelikelihoodofdevelopingsuchinterventionsiscorrespondinglydecreased.
FIG.6.
ProposedmodelofinfectionofperipheralnervebyM.lepraeviablood
vessels.Acutaneousnervewiththreefasciclesisrepresentedhereto
illustratetheproposedstepsinthepathogenesisofinfectionofperipheral
nervesbyM.leprae.(A)Initially,...
TheSchwanncell,theprincipalsupportcellintheperipheralnervoussystem,appearstobethemajortargetofM.
lepraeinperipheralnerves.Inpatientswithadvancedleprosy,bothmyelinatedandnonmyelinatedSchwanncells
areinfectedbyM.leprae(163,179,180),althoughsomereportshavesuggestedsomepreferencefor
nonmyelinatingSchwanncells(311).Invitro,wehaveobservedasimilarlybriskandheavyinfectionofbothcell
types(144).Someinvestigators,however,havereportedexclusiveinfectionofnonmyelinatingcellsinvitro(309).
M.lepraeInteractionswithSchwannCells
AdhesiontoSchwanncells. SeveralpotentialmechanismsofbindingofM.lepraetotheSchwanncell(SC)have
beenelucidated(310,308,398).AntibodiesdirectedagainstthepolysaccharideandlipidcomponentsofM.leprae
inhibitedadhesiontoSCs,whilethosedirectedagainstbothsurfaceandcytoplasmicproteinepitopesdidnotshow
anysucheffect(67),indicatingthattheassociationofM.lepraewithSCsmaybemediatedbymorethanoneofits
cellsurfacemolecules.RecentstudieshavedemonstratedthatM.lepraespecificallybindstodystroglycaninthe
presenceoftheGdomainofthe2chainoflaminin2(310).Using2lamininsasaprobe,amajorproteininthe
M.lepraecellwallfraction(MLLBP21)thatbinds2lamininsonthesurfaceofSCshasbeenidentified(370).
Phenolicglycolipid1ofM.lepraehasalsobeendemonstratedtobindspecificallytolaminin2inthebasallamina
ofSCaxonunits(288).PGL1,therefore,appearstobeinvolvedintheinvasionofSCsbyM.lepraeinalaminin
2dependentpathway.Importantly,however,evidenceclearlyindicatesthatthismechanismofbindingtotheSC
surfacevia2lamininsisnotuniquetoM.leprae.Othermycobacterialspecies,includingM.tuberculosis,M.
chelonae,andM.smegmatis,havebeenshowntoexpressan2lamininbindingcapacity(247)andthesespecies
readilyinteractwiththeST8814Schwannomacellline.Thissuggeststhattheabilitytobind2lamininsis
conservedwithinthegenusMycobacterium.OtherstudieshavealsodemonstratedtheabilityofmyelinP0tobind
M.leprae(398).
IngestionbySCs. AfterM.lepraeadherestotheSCsurface,itisslowlyingested,asdescribedinrecentstudies
usingprimarydenervatedratSCculturesandSCneuroncocultures(144)(Fig.7).AfteringestiontheSCappeared
tobeincapableofdestroyingthisintracellularparasitewhenculturesweremaintainedat33C(optimalconditions
forM.lepraeviabilityandtemperatureofperipheralnerves)(414).InvitrostudiesofingestionofM.lepraebya
humanSchwannomacellline(ST8814)foundthatseveralproteinkinaseswereessentialforingestionbutthat
cyclicAMPdependentkinaseswerenot(11).Inthesestudies,acidificationofvesiclescontainingirradiatedM.
lepraeproceedednormallybutwasminimalwhenliveM.lepraewasused,suggestingthatviableM.leprae
interfereswithnormalendocyticmaturation.
FIG.7.
TransmissionelectronmicrographsofMycobacteriumlepraeinfectedrat
Schwanncell(SC)neuroncocultures.Infectedcultureswereobtainedby
exposureofprimarySchwanncellstoM.lepraefor48h.Aftercultivation
for12daysat33C,they...
EffectsofSCsonM.leprae. SCsapparentlyprovideanenvironmentsuitableforthepreservationandproliferation
ofM.leprae.StudiesusinghighlyviablesuspensionsofnudemousederivedM.lepraehavedemonstratedthatthe
viabilityofthebacilliinratSCsiscomparabletothatpreviouslydescribedforbacilliwithinmacrophagesinvitro
andthatsurvivalofthisorganismwithinSCsisgreaterat33Cthanat37C(144).ThissurvivalwithinSCsin
vitroisconsistentwiththelongstandinghistopathologicalobservationsthatM.lepraeappearstopersistandgrow
withinSCsinhumannerves.
EffectsofM.lepraeonSCs. TheeffectofM.lepraeontheSChasbeenthesubjectofmanystudiesinvitro.
Notably,however,optimalconditions(highlyviablebacilliandcoolercultivationtemperatures)havenotbeenused
inmoststudiesofthisinteraction,possiblycontributingtothevarietyofconflictingreportsintheliterature(264,
279,382,392).InfectionofSCswithwhole,viableM.lepraehasnotbeenobservedtocauseSCloss(144),and
evenappearedtofavorSCsurvivalratherthanapoptosis(311).However,humanSCsexpressTolllikereceptor2
bothinvitroandinvivo,andbindingofanM.lepraederivedlipoproteintoTLR2onSCshasbeenreportedto
resultinapoptosis(294).TheseinvestigatorsalsoidentifiedSCsthathadundergoneapoptosisinbiopsiesofhuman
lesions.Thesignificanceoftheseobservationswithrespecttoclinicalnerveinjuryremainsuncertain.
M.lepraealsoappearstohavenoeffectonintact,matureSCaxonunits,butdidalterSCexpressionofasmall
numberofgenesexamined(thoseforglialfibrillaryacidicprotein,transforminggrowthfactor1,NCAM,ICAM,
Ncadherin,andL1)(144).However,transcriptlevelsforallbutoneofthesegenes,thatencodingNcadherin,
variedlessthantwofold.Therefore,thefunctionalsignificanceofthesealterationsremainstobedetermined.
Incontrasttotheseobservations,Rambukkanaandcolleagues,alsousingaratSCaxoncoculturesystem,have
describedrapiddemyelinationfollowingadherenceofM.lepraetoSCsintheabsenceofimmunecells,interpreted
tobeacontactdependentmechanismdependentonPGL1,acomponentoftheM.lepraecellwall(311).Similar
findingsinTandBcelldeficient(Rag1/)miceledtheseauthorstoconcludethatattachmentofM.lepraetothe
myelinatedSCsurfaceissufficienttoinducerapiddemyelinationofthesecells,thussuggestingamechanismfor
demyelinationofnervesinleprosy(forareview,seereference309).Theseconclusions,however,areat
considerableoddswithwelldocumentedclinicalandhistopathologicalobservations.First,patientswithuntreated
lepromatousleprosymayhavebillionsofbacilliintheirbodiesbutshowlittleornodemyelination.Secondly,rapid
demyelination(ifitoccursinclinicalleprosy)isalatemanifestationofthediseaseratherthananearlyone(295).
ImmuneresponsesandSCs. Finally,theimmuneresponsemayalsobedirectedatM.lepraeinfectedSCs.Isolated
humanSCculturesappeartobeabletoprocessandpresentM.lepraeantigenstoCD4+Tcells(387).Theinfected
SCswerehighlysusceptibletokillingbyCD4+cytotoxicTcellclonesderivedfromleprosypatients.Longterm
culturesofhumanSCsalsoexpressMHCclassIandII,ICAM1,andCD80surfacemoleculesinvolvedinantigen
presentation.ThesecellsprocessandpresentM.lepraeandsomeofitsproteinandpeptideantigenstoMHCclass
IIrestrictedCD4+TcellsandareefficientlykilledbytheseactivatedTcells.
Asalongtermconsequenceoftheseandother,unknownmechanisms,SCsareultimatelyfunctionallyimpairedor
destroyedininfectednerves.Theendresultisademyelinationneuropathy(164,399).Segmentaldemyelinationis
seenadjacenttoareasofinfection,asdiscussedabove.Axonalatrophyandaccompanyingdemyelinationhavealso
beendescribedinleprousnerves,andthishasbeenassociatedwithabnormalphosphorylationofhighand
mediumweightneurofilaments(340).
Insummary,M.lepraehasauniqueabilitytoinfectperipheralnerves,probablyenteringviatheirvascular
endotheliumbymechanismsnotyetdetermined.Oncetheyhavegainedaccesstotheendoneurialcompartment,M.
lepraeorganismsbindtoSCsviaseveralbindingmoleculesonthesurfacemembrane.Thebacilliareingestedby
SCs,andviableorganismsappeartobeabletointerferewithnormalendocyticmaturationandthus,probably,with
potentialkillingmechanisms.WithinSCsinvitro,M.lepraeisabletosurviveforalimitedtimeat33C.M.leprae
doesnotappeartoinduceSCdeath,byapoptosisorotherwise,andinfectedSCsareabletoassociatenormally
withaxonsinvitro.Infectiondoes,however,producemeasurablechangesintranscriptionofsomeofthegenesthat
havebeenexaminedthusfar.
InfectedSCsarealsoabletoprocessandpresentantigentoTcells,andthusmaybecometargetsofimmune
responses.Immunologicallydriveninflammationisprobablyresponsibleformuchoftheclinicallyapparentnerve
injury,becausenervefunctionimpairmentoccursmorerapidlyandmoreseverelyinpatientswithastrongcellular
immuneresponse(i.e.,tuberculoiddisease).Thelimitedevidenceavailableindicatesthattheimmunological
mechanismsoperatingwithinnervesaresimilartothosewhichhavebeendescribedinmuchmoredetailinthe
skin.Inlepromatouspatients,withminimalimmunologicalresponsetoM.leprae,nervesmaybeheavilyinfected
withonlymildtomoderateimpairmentoffunction.Ultimately,however,selectedperipheralnervesinallformsof
leprosyundergodemyelination.Withouteffectivetreatment,manysuchnerveswillbecomecompletely
nonfunctional,leavingthepatientwithaninsensate,paralyzedhandorfoot.Understandingthemanyundeciphered
mechanismsunderlyingnerveinjuryandapplyingavailableclinicalandrehabilitationtoolstopreventandminimize
nerveinjuryareamongthegreatestchallengesandhighestprioritiesinleprosyresearchtoday.
CHEMOTHERAPY Goto:
CurrentTherapiesandDrugResistance
Inthe1950s,dapsone(diaminodimethylsulfone)wasintroducedasstandardchemotherapyforleprosyandwas
usedworldwidefortreatmentofbothmultibacillaryandpaucibacillaryformsofthedisease.Longterm
monotherapywithdapsoneresultedinpoorcomplianceinmanyareas,ultimatelyleadingtotheemergenceof
dapsoneresistantleprosy,resultingintreatmentfailuresandresistancelevelsreportedtobeashighas40%insome
areasoftheworld(446,365).
Fortunately,additionalantimicrobialagentssuchasrifampinandclofazimineweredevelopedandintroducedfor
thetreatmentofleprosy(44,232).Althoughrifampinprovedtobeapowerfulantileprosydrug,useofrifampin
aloneorincombinationwithdapsoneforthetreatmentofdapsoneresistantleprosyledtotherapiddevelopmentof
rifampinresistantorganisms(137,177).Otherdrugswithantileprosyactivitywerealsoevaluated.Clofazimine
provedtobeonlyweaklybactericidalagainstM.lepraeandthereforewasnotsuitableasmonotherapyforleprosy
(169,177).
ToovercometheproblemofdrugresistantM.lepraeandtoimprovetreatmentefficacy,theWorldHealth
Organizationrecommendedmultidrugtherapyforleprosyin1981.Theinitialrecommendationforpatientswith
multibacillaryleprosywastogivedailydapsoneandclofaziminewithmonthlyrifampinandclofaziminefor2years
oruntiltheskinsmearwasnegative.Theserecommendations,aswellasdiagnosticcriteria,havebeenmodified
severaltimessince1981.CurrentlytheWorldHealthOrganizationrecommendscountinglesionstodistinguish
paucibacillaryfrommultibacillarydisease,lessthanfivelesionsbeingclassifiedaspaucibacillaryandfiveormore
lesionsasmultibacillary.Since1998theyhavealsorecommendedtreatingmultibacillarypatientsforonly12
monthsandpaucibacillarypatientsforonly6months(reviewedinreference349)(Table9).Inaddition,aWorld
HealthOrganizationcommitteerecommendedthatpatientswithasinglelesionbetreatedwithasinglecombination
doseofrifampin(600mg),ofloxacin(400mg),andminocycline(100mg)(312www.who.int/lep/romfaq3.htm),
butthisregimenremainsverycontroversial.Theserecommendationsarisefromeffortstoreducetheresources
allocatedtoleprosyindevelopingcountriesandarethesubjectofconsiderabledebate.Optimaldiagnostic
evaluationemployingskinsmearsorbiopsies,classifyingthelesionsontheRidleyJoplingscale,andconservative,
longerdurationoftreatmentwithmultipleantimicrobialsarerecommendedintheUnitedStatesandmostdeveloped
countries(349).
TABLE9.
Antimicrobialagentsforleprosya
Multidrugtherapyhasbeenverypracticalandsuccessfulfortreatmentofbothmultibacillaryandpaucibacillary
leprosy(171,444,447),andtheoverallnumberofregisteredcasesworldwidehasfallendramatically(171,445).
However,evenwiththesepowerfuldrugcombinations,thenumberofnewlyregisteredcaseshasnotfallen
consistently,anddrugresistancestilloccurs.
Arecentreportdemonstratedthat19%of265M.lepraeisolatesfrombiopsiedsamplesofleprosypatientswere
resistanttovariousconcentrationsofdapsone,rifampin,orclofazimineand6.23%wereresistanttomorethanone
druginthemousefootpadsusceptibilityassay(102).Inaddition,severalinvestigatorshaveidentifiedmultidrug
resistantstrainsofM.leprae(reviewedinreference434).Ofloxacinandminocyclinehavebeenaddedtothedrug
arsenalforthetreatmentofleprosy(126,172,175,176seealsohttp://www.who.int/lep/romfaq3.htm).Current
treatmentrecommendationsintheUnitedStateshavebeensummarizedelsewhere(276a,349),andLockwoodhas
providedarigorousevidencebaseddiscussionofthetreatmentofleprosy(234).
Withorwithoutthedevelopmentofdrugresistance,relapseoccursinsomecasesevenaftermultidrugtherapy.The
reportedextentofrelapsevariesgreatly,dependingonseveraloperationalfactorsandonthedurationoffollowup.
Becauseoftheveryslowgrowthofthisorganism,followupofatleast10yearsisnecessarytoobtainareasonable
assessmentofrelapse,andduringthelast20yearsrecommendationsconcerningtreatmentdurationanddrug
combinationshavechangedseveraltimes,furthercomplicatingthisassessment.Thelargeststudy,althoughshorter
than10yearsinduration,isa6yearfollowupof47,276patientsintheChinesenationalprogram(63),which
revealedanoverallrelapserateof0.73/1,000personyears,significantlygreaterforpaucibacillarypatients
(1.04/1,000personyears)thanformultibacillarypatients(0.61/1,000personyears).
InsouthernIndia,arelapserateequivalentto20/1,000personyearswasobservedamongmultibacillarypatients
givenfixedduration(2year)multidrugtherapy,reducedto10/1,000personyearsinpatientstreateduntilsmear
negative(134).A10yearprospectivestudyinthePhilippines(55)observedanoverallrelapserateequivalentto
2.8/1,000personyears.Significantdifferenceswerenotedintheratesofrelapseinmultibacillarypatientsfollowed
atareferralcenter(9%)versusfieldclinics(3%).Importantly,inboththesouthernIndiaandPhilippinestudies,
higherratesofrelapsehavebeenobservedinpatientswithahighbacterialindex(BI)(4)atthetimeofdiagnosis,
underscoringtheadvicethatsuchpatientsrequirelongertreatment(128).Inthelongeststudytodate,a16year
followupofpatientsinKarigiri,India(292),arelapserateequivalentto0.7/1,000personyearswasobserved
amongmultibacillarypatientswhohadreceivedmultidrugtherapy.Relapsesoccurred14to15yearsafterrelease
fromtreatmentandagainweremorefrequentinpersonswithahighinitialBI.
MolecularMechanismsofDrugResistance
Dapsone(4,4diaminodiphenylsulfone)isasyntheticsulfone,isstructurallyandfunctionallyrelatedtothe
sulfonamidedrugs,andtargetsdihydropteroatesynthase,akeyenzymeinthefolatebiosynthesispathwayin
bacteria,byactingasacompetitiveinhibitorofpaminobenzoicacid(317,359).Dapsonehasalsobeenshownto
targetthefolatebiosyntheticpathwayofM.leprae(225).
SpecificmutationswithinthehighlyconservedpaminobenzoicacidbindingsiteofE.colidihydropteroate
synthase,encodedbyfolP,resultinthedevelopmentofdapsoneresistance(80).NewevidencefromtheM.leprae
genomesequencingprojectindicatedthatM.lepraepossessestwofolPhomologues(folP1andfolP2)(74).
ThroughsurrogategeneticstudieswithM.smegmatis,therelationshipbetweendapsoneresistanceandthe
dihydropteroatesynthaseofM.lepraehasbeenestablished(439).Missensemutationswithincodons53and55of
thesulfoneresistancedeterminingregionoffolP1resultinthedevelopmentofhighleveldapsoneresistanceinM.
leprae(Table10).
TABLE10.
Mutationswithindrugtargetgenesassociatedwithdrugresistancein
Mycobacteriumleprae
Rifampin(3{[(4methyl1piperazinyl)imino]methyl}rifamycin)isthekeybactericidalcomponentofall
recommendedantileprosychemotherapeuticregimens.Asingledoseof1,200mgcanreducethenumberofviable
bacilliinapatient'sskintoundetectablelevelswithinafewdays(232).Thetargetforrifampininmycobacteriaand
E.coliisthesubunitoftheRNApolymeraseencodedbyrpoB(156,178,403,441).Comparisonofthededuced
primarystructuresofsubunitproteinsfromseveralbacteriatothatofM.lepraedemonstratedthatM.leprae
sharessixhighlyconservedfunctionalregionscommontothisenzymeinbacteria.
MycobacterialresistancetorifampincorrelateswithchangesinthestructureofthesubunitoftheDNA
dependentRNApolymerase,primarilyduetomissensemutationswithincodonsofahighlyconservedregionof
therpoBgenereferredtoastherifampinresistancedeterminingregion(280,403,441).RifampinresistanceinM.
lepraealsocorrelateswithmissensemutationswithinthisregionofrpoB(156).SubstitutionswithincodonSer425
havebeenshowntobethemostfrequentmutationsassociatedwiththedevelopmentoftherifampinresistant
phenotypeinM.leprae(Table10).
Clofazimine[3(pchloroanilino)10(pchlorophenyl)2,10dihydro2(isopropylimino)phenazine]isasubstituted
iminophenazinewithantimycobacterialactivityforwhichthemechanismhasnotbeenfullyelucidated(175).
Clofazimineattainshighintracellularlevelsinmononuclearphagocyticcells,itsmetaboliceliminationisslow,ithas
anantiinflammatoryeffect,andtheincidenceofresistancetoitinM.lepraeislow.Itishighlylipophilicand
appearstobindpreferentiallytomycobacterialDNA(171).BindingofthedrugtoDNAappearstooccur
principallyatbasesequencescontainingguanine,explainingclofazimine'spreferencefortheG+Crichgenomesof
mycobacteriaoverhumanDNA.
Lysophospholipidsappeartomediatetheactivityofclofazimineinsomegrampositivebacteria(83).However,itis
unclearwhetherthismechanismofactionisoperationalinM.leprae.Sinceclofaziminemayactthroughseveral
differentmechanisms,itisnotdifficulttounderstandwhyonlyafewcasesofclofazimineresistantleprosyhave
beenreportedovertheyears(84,256,368).
Clarithromycinisasemisyntheticmacrolidethatdiffersfromerythromycininitsmethylsubstitutionatthenumber6
positionofthemacrolidering(reviewedinreference424).ItdisplayssignificantbactericidalactivityagainstM.
lepraeinhumans(127,173).Inpatientswithlepromatousleprosy,dailyadministrationof500mgofclarithromycin
kills99%ofviableM.lepraewithin28daysand>99.9%by56days.Althoughthemechanismofactionofthis
antibioticagainstM.lepraeisunknown,itisthoughttobesimilartothatoferythromycin,whichactsbyinhibiting
proteinsynthesisbybindingtotheribosome.
Clarithromycinresistanceinbacteriaandmycobacteriaappearstobeduetoadecreaseinbindingofthedrugto
ribosomesandisassociatedwithmissensemutationswithinthe23SrRNAgene(257,424,428).Thishasnotyet
beenestablishedtobethecasewithM.leprae,however,andinarecentstudynomutationswereobservedwithin
the23SrRNAgeneinclarithromycinresistantM.lepraestrains(450).
Minocycline(7dimethylamino6demethyl6deoxytetracycline)istheonlymemberofthetetracyclinegroupof
antibioticstodemonstratesignificantactivityagainstM.leprae,presumablyduetoitslipophilicproperty,which
mayenhancecellwallpenetration(126,176).MinocyclineisbactericidalforM.leprae,anditsactivityisadditive
whenitiscombinedwithdapsoneandrifampin.ThemechanismofactionofminocyclineagainstM.lepraeis
unknownbutisthoughttobesimilartothatofalltetracyclines,whichactbyinhibitingproteinsynthesis.
Tetracyclinesbindreversiblytothe30Sribosomalsubunit,blockingthebindingofaminoacyltRNAtothemRNA
ribosomecomplex(402).
Resistancetotetracyclinemaybemediatedbythreedifferentmechanisms:anenergydependenteffluxof
tetracyclinebroughtaboutbyanintegralmembraneproteinribosomalprotectionbyasolubleproteinorenzymatic
inactivationoftetracycline.ThemolecularmechanismofminocyclineresistancehasnotbeenstudiedinM.leprae
primarilybecausethisdrughasonlyrecentlybeenusedwidely(inthetreatmentofsinglelesion,paucibacillary
leprosy),andresistantstrainshavenotbeenidentified.
Ofloxacin(4fluoroquinolone)isafluorinatedcarboxyquinolonethathasmoderateantiM.lepraeactivity(175,
176).ThemechanismofactionofofloxacinonM.lepraeisunknown,butinotherbacteriaitappearstoinhibit
DNAreplicationbyinhibitingtheDNAgyrase,atetramercontainingtwoAsubunits(GyrA)andtwoBsubunits
(GyrB)(99).
MutationswithinahighlyconservedregionofgyrA,thequinoloneresistancedeterminingregion,areassociated
withthedevelopmentofofloxacinresistanceinmostresistantstrainsofmycobacteria(53,401).Thequinolone
resistancedeterminingregionofM.lepraegyrAishighlyhomologoustothatofM.tuberculosis,andmissense
mutationswithinthisregionhavebeenfoundinofloxacinresistantstrainsofM.leprae(Table10).
DevelopmentofDrugResistanceinM.leprae
LackingdirectevidenceforthemechanismsofM.leprae'sresistancetomostoftheantileprosydrugs,ourcurrent
understandingisbasedonstudiescarriedoutinM.tuberculosis(280)andotherbacteriaandlimitedstudieswithM.
lepraegenesinsurrogatehosts.FromthesestudiesonecanpredictthatdrugresistanceinM.lepraeisattributable
tochromosomalmutationsingenesencodingdrugtargets,thesemutationsoccurspontaneouslyasaresultoferrors
inDNAreplication,andthesemutantsarefurtherenrichedinapopulationbyinappropriateorinadequatedrug
therapy.
BecauseM.lepraecannotbecultivatedinvitro,thefrequencyofdrugresistantmutantsinapopulationisprimarily
inferredfromstudieswithM.tuberculosis.Forexample,thefrequencyofdapsoneresistantmutantsinapopulation
ofM.lepraeisestimatedtobe106andthefrequencyforrifampinorofloxacinresistanceisestimatedat107to
108(158,280).RatesofclofazimineresistanceinM.lepraeareunknownbutappeartoberelativelylow.Since
untreatedmultibacillarypatientscanharborlargebacterialloads(>1011M.leprae),itisfeasiblethatapatientcould
containupto105dapsoneresistantorganismsandthousandsofrifampinorofloxacinresistantorganisms.
Inappropriatetherapy(noncomplianceorinadequatetherapy)forthesepatientshasthepotentialtoenrichthe
subpopulationsofdrugresistantM.leprae,leadingtothespreadofoneormoreresistantphenotypes.Indeed,drug
resistantisolatesofM.lepraehavebeenfoundinmanypartsoftheworld(49,51,52,84,86,102).
DetectionofDrugResistantLeprosy
LeprosypresentsaveryspecialproblemforthedetectionofresistancebecauseoftheinabilitytocultureM.leprae
axenically.ConventionaldrugsusceptibilitytestingofM.lepraefromclinicalspecimensreliesontheabilityto
cultivateM.lepraeinthehindfootpadsofmicebythemethoddescribedbyShepardandChang(364).This
methodrequirestherecoveryofasufficientnumberofviableorganismsfromapatienttoinoculatethefootpadsof
20to40mice(dependingonthenumberofdrugstobetested),witheachfootpadreceiving5103organisms.
Resultsareavailableafter6monthsto1year.Whilethisassaygivesdefinitiveinformationpertainingtothe
susceptibilityofanM.lepraeisolatetostandardantileprosydrugs,itiscumbersome,veryexpensive,andvery
slow.
ThefirstrapiddrugscreeningassaysforM.lepraeweredevelopedbasedonradiorespirometrytechniques
(BACTECandBuddemeyer)andhavebeenusedsuccessfullytoidentifynewantileprosydrugs(117).However,
theuseofthesetechniquesfordrugsusceptibilitytestinginleprosyislimitedbyastringentrequirementforvery
largenumbers(107)ofviableorganismsfromeachpatient.
Molecularassaysforresistancewouldsimplifysusceptibilitytestingandprovideameansformonitoringresistance
globally.Toreducethenumberoforganismsneededandtominimizethetimerequiredfordrugsusceptibility
testingofM.leprae,severalprotocolsbasedongenotypicidentificationofdrugresistantmutantshavebeen
developed.ThesetechniquesarebasedontheamplificationofspecificDNAfragmentsfromcrudebiological
specimens(e.g.,skinbiopsyspecimensfromleprosypatients)usingPCRamplificationanddetectionofmutations
associatedwithdrugresistancewithintheseDNAfragmentsbydirectDNAsequencing,singlestrand
conformationpolymorphismanalysis,heteroduplexanalysis,andsolidphasereversehybridizationanalysis,similar
tothelineprobeassay(Table11).
TABLE11.
TargetgenesforM.lepraedrugresistancePCRbasedassays
PCRdirectDNAsequencing. SequencingofthePCRampliconisthemostdefinitiveofallofthenucleicacidbased
mutationdetectionprotocolsbecauseitdetectstheactualnucleotidechangesinthetargetgeneinwhichmutations
associatedwithantibioticresistancearefound.Inaddition,theassaycanbedesignedtobespeciesspecific,
providingdirectevidenceforthepresenceofaparticularpathogeninthespecimenbeingtested.PCRdirectDNA
sequencinghasbeenusedtoidentifyrifampin,dapsone,andofloxacinresistantmutantsofM.leprae(Table11).
TheseassaysarebasedonPCRamplificationoftheappropriatetargetDNAdirectlyfromskinbiopsyspecimens
usingoligonucleotideprimersthatarespecificfortherifampin,sulfone,orquinoloneresistancedeterminingregion
ofM.leprae.TheDNAsequenceofthesePCRproductsisthendeterminedandexaminedforthepresenceof
mutationspreviouslyassociatedwithdrugresistance.TheSer425LeumutationintheBsubunitoftheRNA
polymeraseisthemostfrequentlydetectedmutationassociatedwithrifampinresistanceinM.leprae(Table10).
PCRdirectDNAsequencingcanbeperformedinawellequippeddiagnosticlaboratorywitheithermanualor
automatedDNAsequencingsystemsandrequiresapproximately1to2daystoobtaindrugsusceptibilityresults
directlyfromclinicalspecimens.
PCRSSCP. APCRsinglestrandconformationpolymorphism(SSCP)assayhasbeendevelopedtodetectrifampin
resistantM.lepraeinhumanspecimens(156,157).Toaccomplishthis,therifampinresistancedeterminingregion
targetwasamplifiedbyPCRandthedoublestrandedPCRproductswereheatedtodissociatethemintosingle
strandsandthenseparatedbydenaturinggelelectrophoresisunderstringentlycontrolledtemperatureconditions.
GelswerethenstainedtoobserveDNAfragmentmobilitypatterns,calledSSCPprofiles.DNAstrandsfrom
rifampinsusceptibleorganismsmigrateatarateproportionaltotheirmolecularweightandconformationandgivea
reproducibleSSCPprofile.TheDNAfragmentpatternsobservedwithSSCParehighlyreproducibleandyield
profilesuniquetospecificmutations.
PCRsolidphasehybridization. APCRsolidphasehybridizationassayhasrecentlybeendevelopedforthe
detectionofrifampinresistantM.leprae(158).AninitialPCRstepwithabiotinylatedandanunlabeledprimer
producesan83bp,biotinylatedfragmentoftheM.lepraerifampinresistancedeterminingregion.Theamplified
PCRproductisthenhybridizedtoasetofDNAcaptureprobeswhichhavebeenimmobilizedatspecificsitesona
BiodyneCmembrane.TheimmobilizedcaptureprobesaresmallDNAfragmentsthatarehomologoustoshort
segmentsoftherifampinresistancedeterminingregionoftherpoBgenefromarifampinsusceptiblestrainofM.
lepraeorspecificmutantstrains.ThestringencyofthehybridizationreactionisdesignedsothatthePCRproduct
willonlybindtoprobeswith100%sequencehomology.Theresultanthybridsaredetectedbychemiluminescence
usingastreptavidinperoxidaseconjugate.Thegenotypeofthetestorganismisdeterminedbythecaptureprobes
whichhybridize.
PCRheteroduplexanalysis. APCRheteroduplexbasedassaywasinitiallydevelopedtodetectthepresenceof
drugresistantM.tuberculosisfromsputumspecimensusingauniversalheteroduplexgenerator(UHG)(438).A
similarapproachhasbeenusedtodevelopaPCRUHGassaytodetectthepresenceofdapsoneresistantM.leprae
inskinbiopsyhomogenatesoflepromatousleprosypatients(437).TheassayrequiresPCRamplificationofthe
sulfoneresistancedeterminingregionoffolP1andthemixingofthesePCRproductswithauniversalheteroduplex
generator(UHGDDS141),asynthetic141bpsulfoneresistancedeterminingregionDNAfragmentthatcontains
severalbasepairmismatchesflankingcodonsthatareassociatedwithdapsoneresistance.WhenUHGDDS141is
denaturedbyheatandslowlyannealedtodenaturedsulfoneresistancedeterminingregionPCRproductsfromM.
leprae,theresultantheteroduplexesformuniquestructureswhich,whenanalyzedbyelectrophoresis,provide
enhancedmutationdetectionoverstandardheteroduplexdetection.Enhancedmutationdetectionoccursbecause
largeareasofunmatchednucleotides(bubbles)inthenewlyformedduplexesgreatlyaffectthemobilityofthe
resultantDNAfragments.Whentheheteroduplexesareseparatedbyelectrophoresisonpolyacrylamideminigels,
uniqueheteroduplexprofilesareobservedforsusceptibleandresistantgenotypes.PCRUHGrequires
approximately6htocompleteanduses6%precastnondenaturingTrisborateEDTAminigelsanda
nonradioactivedetectionformat.
PREVENTION:THEQUESTFORALEPROSYVACCINE Goto:
Vaccinologyhasgrownfromanempiricalsciencewithlittleinthewayofbiologicalunderstandingofeventstoa
highlystructuredsciencedrawingondetailedimmunologicalstudiesatthecellularandmolecularlevelsofboththe
hostandtheinfectiousagent.Thecells,cytokines,andregulatorypathwaysactiveinthehost'simmuneresponseto
M.lepraeandothermycobacterialpathogenscontinuetobeelucidated,buildingafoundationforour
understandingofthecausesofimmunopathogenesisandprotectiveimmunity.AnnotationoftheM.lepraegenome
andbioinformaticprocessingofthedatahavechangedthewayweinvestigatepotentialantigensfornewvaccines.
Giventhepotentialfordevelopinganeffectivevaccineforleprosybasedonthesenewtools,thedebatecontinues
astowhetherthereisaneedforavaccineintheoverallstrategytocontroloreradicateleprosy.
Asdiscussedabove,thecurrentstrategyforcontrollingleprosyisbasedontheimplementationofeffectivedrug
regimenssetforthbytheWorldHealthOrganization.Unfortunately,recentepidemiologicaldatasuggestthatthis
strategyappearstohavehadlittleeffectonreducingtheannualincidenceofnewcasesofleprosy.Additionally,
recentreportshaveshownthatrelapseratesof16to39%amongmultiibacillarypatientswithhighBIsare
appearing4to10yearsaftercompletionof2yearmultidrugtherapy(128,134,168).Acknowledgingtheseclinical
realitiesrequiresanobjectiveassessmentofcurrentcontrolstrategiesandremindsusthatotherintervention
strategiesmaybenecessarytoeradicateleprosy.Improvedstrategiesmayrequirenewapplicationofoldtools,such
asspecialprogramsdesignedtoimprovedrugdistributionandtreatmentcompliance,butshouldalsoincludebasic
researchdesignedtodeveloptestsforearlydiagnosisaswellaspreandpostexposurevaccinesforleprosy.
Fromthestandpointofdiseasecontrol,vaccinesaresimilartodrugsinthattheymaybeappliedasprophylactic
(preexposure)ortherapeutic(postexposure)measures.Vaccines,however,haveapotentialaddedadvantageby
producingarelativelylonglivedimmunologicalmemorycomponent.Accordingly,aneffectiveprophylactic
vaccineforleprosycouldbreaktransmissionbyconferringuponrecipientsimmediateaswellasextended
protectionfrominfectionwithM.leprae.Aprophylacticvaccineshouldalsoprotectagainstbothdrugsusceptible
anddrugresistantstrains,helpingcurbtheemergenceofdrugresistance.Usedasatherapeuticmeasureforleprosy
control,apostexposurevaccinecouldimproveapatient'sresponsetomultidrugtherapybyhasteningacureand
potentiallyreducingtheincidenceofrelapsecases.Whilemostoftheseconceptsremainhypothetical,evidenceis
availablethatantileprosyvaccinescanprovidevariouslevelsofprotectionaswellaslimitedbeneficialtherapeutic
effects.
ThevaccinestudiedmostinleprosyisM.bovisBCG.ExperiencewithBCGvaccinationforleprosyremains
enigmaticinthatlevelsofprotectionvaryfrom20to80%(Table12).Theseresultsarenotunexpectedconsidering
BCG'svariableefficacyagainsttuberculosis(71).Fine(111)hasreviewedmanyoftheissuessurroundingthe
curiousvariabilityseenwithBCGvaccinationfortuberculosis.Hespeculatesthatfactorssuchasbiological
differencesinBCGstrains,exposuretoenvironmentalmycobacteria,andineffectiveboostingagainstreinfection
withorreactivationoftuberculosismaygiverisetotheobservedvariabilityinprotectionseenwithBCG
vaccination.ItislikelythatsomeorallofthesefactorsmayplayaroleinthevariabilityseeninBCGvaccine
efficacyagainstleprosy.
TABLE12.
SummaryofestimatesofefficacyofBCGandothervaccinesagainst
leprosya
Becauseleprosyisarelativelyuncommondiseaseentity,evenincountrieswiththehighestprevalence,casecontrol
studieshavebeenparticularlyusefulinobtaininginformationconcerningvaccineefficacy.Tworecentcasecontrol
studies,inIndia(455)andinBrazil(78),provideclearevidencethatBCGprotectsagainstleprosy.IntheBrazilian
study,theinvestigatorsarguethattheirresultssuggestthatneonatalBCGvaccinationmayhaveanimportantimpact
ontransmissionofleprosyandthatenvironmentalmycobacteriamaynotimpactvaccineefficacy,atleastinthe
AmazonregionofBrazil.Zodpeyandcoworkersshowedanoverallvaccineefficacyof54%,withthegreatest
protectiveeffectseenformultibacillaryleprosy(68%),suggestingthataneffectivevaccineforleprosy,likeBCG,
appearstohaveitsgreatesteffectonthediseaseform(lepromatous)mostlikelytotransmitM.lepraewithinthe
community.
ThreerelativelyrecentvaccinetrialshavestudiedthehypothesisthatcombiningBCGwithkilledM.leprae
improvesvaccineefficacy.TheprimaryreasonfortestingthishypothesisistodeterminewhetherM.lepraespecific
antigensareabletoimprovetheefficacyofBCG.Theearliestofthethreestudies(76)wasconductedinVenezuela
andconcludedthattheprotectiveefficacyofBCGwasproportionaltothenumberofdoses(i.e.,thenumberof
BCGscars)andthatprotectionagainstleprosywasnotimprovedwhenheatkilledM.lepraewascombinedwith
BCG.Similarly,inthedoubleblind,controlledtrialdoneinMalawi(204),noadvantagewasobservedby
includingheatkilledM.leprae(HKML)alongwithBCG.However,amongscarpositiveindividuals,asecond
BCGvaccinationgavefurtherprotectionagainstleprosy(about50%)overafirstBCGvaccination.
Incontrast,thevaccinetrialdoneinsouthernIndiashowednopositiveeffectofvaccinatingBCGscarpositive
individuals,butenhancedprotectionoverthatwithBCGalonewasseeninindividualsreceivingBCGplusHKML
(140).AdditionalarmsinthesouthernIndiavaccinetrialincludedtwoatypicalmycobacteria,Mycobacteriumw
(207)andICRCbacillus(313).Bothvaccinesarenonlivingpreparations,andbothweresuperiortoBCGalone
withrespecttopercentprotectionatthesecondandthirdresurveysofthetrial.Interestingly,ICRCaloneandBCG
plusHKMLgaveapproximatelythesamelevelofprotection(64and65%,respectively),suggestingthatakilled
mycobacterialvaccinecontainingM.lepraecrossreactiveantigensisaseffectiveasalive,attenuatedBCGvaccine
inthispopulationandsetting.So,whilethecontroversycontinuesoverwhatelementsofaleprosyvaccineare
superiorinaparticularsetting,thereislittleornocontroversyoverthepositiveeffectsofvaccinationtoreduce
leprosyincidence.
Lessisknownabouttheapplicationofleprosyvaccinesfortherapeuticpurposes.Anumberofreportsusing
Mycobacteriumwasimmunotherapysuggestthatwhengivenasadjuncttherapytomultidrugtherapy,significant
clinicalimprovementdoesresult(88,206,453).Forexample,DeSarkaretal.(88)observedsignificant
improvementinbothclinicalandhistopathologicalassessmentsoflesionsinpatientsreceivingMycobacteriumw
plusmultidrugtherapy.Inaddition,patientsvaccinatedwithMycobacteriumwdemonstratedreducedbacillary
indicesovertimecomparedtopatientsreceivingonlymultidrugtherapyfor12months.Animportantfindinginthis
study,corroboratingfindingsfromanearlierstudy(453),wasthatpatientsinthevaccinegroupexperienceda
higherpercentage(30%)oftype1reactionscomparedtothecontrolgroup(10%).Fortunately,neuritiswasnot
increasedwithvaccinationinpatientswithorwithoutreactions,afindingalsoreportedintheearlierstudy(453).In
contrasttotheincreasedincidenceoftype1reactionsfollowingvaccinationwithMycobacteriumw,theincidence
oftype2reactionswassimilarincontrolsandvaccinatedpatients.Thiscontrastswiththeexperience,notedabove,
thatcytokinetreatmentsusingIFNledtoupgradingofLLandBLlesionsandwereaccompaniedbyahigh
incidenceofENL(332).Thesefindingsunderscorethechallengeofproducinganeffectivepostexposureleprosy
vaccinewiththepurposeofincreasingcellmediatedimmunitywithoutinducingharmfulsequelae.
Anotherimportantpieceofthepuzzlethatispropellinginvestigatorstosearchfornewmoleculeswithvaccine
potentialisthecompletionofthegenomesequencesofM.leprae(74)andM.tuberculosis(73).Priortothe
completesequencingoftheM.lepraegenome,theonlyreliablesourceofM.lepraeantigenswashighlypurified
bacillioriginatingfrominfectedarmadillotissues.WhilemanymajorcompoundsofM.lepraehavebeen
discoveredandstudiedindetailusingthismaterial,purifiedproteinshavebeenavailableinverylimitedquantities
andofpoorqualitybycontemporarystandards,makingthemdifficulttouseforvaccinedevelopment.Inaddition,
certainsecretedproteinsarealmostsurelylostuponpurificationofthebacillifrominfectedarmadillotissues.
NewapproachestoidentifyinggenesfromcompletedM.lepraegenomesequencesarebeingappliedusing
standardizedbioinformaticstools(253).Thesetoolscanidentifyproteinswithspecialfeatures,suchasuniqueor
sharedaminoacidsequencehomologieswithproteinsofM.tuberculosisorothermycobacterialspecies,andthe
presenceofspecializedpeptidesignaturessuggestingtheircellularlocationandpossiblesecretionacrossthecell
membrane.ProteinsofinterestcanbeprioritizedbasedonpotentialBcellorTcellepitopes,althoughstrict
associationsbetweenbioinformaticstoolsandantigenicepitopesremainunderdeveloped.Finally,theproteins
selectedforfurtherstudycanbepurifiedasrecombinantproteinsforanendlesssupplyoftheproteinfor
immunologicandvaccinestudies(255,289,386,442).
Inadditiontonewlyavailablesearchalgorithmsforgenesofinterest,newvehiclesfordeliveryofproteinantigens
havebeenidentifiedfromresearchonrecombinantDNAoverthelast20years.Muchofthistechnologyisbeing
usedtocreatevaccinestobeadministeredinconcertwithBCG,eitherasrecombinantBCGoverexpressingoneor
moreantigenicproteinsorinaprimeboostscenariowhereantigenisgivenfirstinanadjuvant(priming)andthen
followedbyBCGvaccinationtoboosttheinitialresponse.SinceBCGisgivenatbirthinmanycountries,the
standardprimeboostscheduleforleprosyandtuberculosisvaccineswouldlikelybereversed.Supportingthis
reversalofprimingandboostingwithBCGarethreestudiesusingdifferentproteinvaccinesfortuberculosisthat
haveshownfavorableprotectiveresponsesinanimalmodels(36,254,271).Applicationofthisstrategyfor
designingleprosyvaccinesshouldnotpresentanyuniquehurdlesnowthatM.lepraeantigensofinterestcanbe
clonedandexpressedinlargequantities.
RecallingtheresultsofearliervaccinetrialswithBCGremindsusthatitisafairlypotentvaccineforleprosyin
manysettings.NewlydesignedvaccinesfortuberculosisthatmayutilizeBCGalteredthroughgeneticengineering
orthroughaprimebooststrategymayormaynotprovidebetterprotectionagainstleprosythanisaffordedby
currentBCGvaccines.Accordingly,newlyconfiguredvaccinesfortuberculosisshouldbetestedforefficacy
againstM.lepraechallengetoshowthatbenefitsforleprosycontrolthroughvaccinationfortuberculosisarenot
lostintheprocess.
Whiletheimmunogenicityofvariouscandidateproteinscanbetestedinvitrousinghumancells,animalmodelsin
whichtotesttheefficacyofanewvaccinearelimitedtothemouseandarmadillo.Shepard'smousefootpadmodel
isthegoldstandardforleprosyvaccinestudies(366).Theinfectionthatdevelopsinthemousefootpadmaybestbe
describedaslimitedmultiplicationatthesiteofinfectionandmaybesomewhatanalogoustoindeterminateor
tuberculoidleprosyinhumans.Sincemanyindeterminatecasesselfhealandtuberculoidleprosyinhumans
involvesnervedamage(notseeninmice),thismodelfordefiningvaccineefficacyhasimportantlimitations.The
modeldoes,however,enablescreeningoutofvaccineswithlittleornoprotectiveefficacyagainstachallengewith
liveM.leprae.Vaccinesthatareeffectiveinthemousemodelcouldthenbetestedinthearmadillo,ananimalthat
manifestsmostofthecharacteristicsofhumanleprosy.
Finally,vaccineefficacymustbetestedinhumansfollowingappropriatesafetyandpotencytrials.Thesetrialsmust
belargeduetotherelativelylowincidenceofleprosyinmostsettings,whichcontinuestobeanobstaclefor
assessingvaccinesasdiseasemanagementtoolsinleprosy.Therapeuticvaccinetrialscouldbemorefocusedand
evaluatedinsmaller,definedgroupsofpatients.Withtheadventofabetterunderstandingofthemolecularnature
ofM.lepraeandthehumanresponsetoinfection,newvaccineswillbecomeareality.Thechallengeforthe
researchandpublichealthsystemsistounitethepoliticalwillandfinancialresourcestotestoneormoreofthenew
vaccinesforleprosy.
SUMMARYANDCONCLUSIONS Goto:
Basedonoperationaldata,withitsinherentbiases,thenumberofnewcasesofleprosyidentifiedannually
worldwidehasprobablynotchangedoverthelasttwodecades,althoughthenumberofregisteredcaseshas
declinedasaresultoftreatmentandremovalfromregistries.Thisdiseaseposesmajorchallengestoour
understandinginmicrobiology,immunology,pathology,treatment,andprevention,requiringcontinuedemphasis
onbasicresearchandclinicalmanagementinthefield.Reviewedabovearemajoradvancesthathavebeenmadein
thebasicunderstandingofleprosysince1990,includingthefollowing.
ThegenomeofM.lepraehasbeensequenced,andthisorganismhasbeenshowntobeabletosynthesizefarfewer
proteinsthantheothermajorhumanpathogenicmycobacterium,M.tuberculosis.Thus,althoughM.lepraestill
cannotbecultivatedaxenically,thenewmolecularabilitytoassessitsabilitytotranscribeandsynthesizevarious
proteinsinresponsetodifferentenvironmentsandstresseswilllikelyprovidevaluableinformationaboutits
mechanismsofpathogenicityinthenearfuture.
PCRanalysisoftissuesforM.lepraeDNAnowprovidesavaluablemeansforidentifyingthisorganism.
MutationsintheM.lepraegenomethatareassociatedwithresistancetoseveralofthedrugsusedagainstthis
pathogenhavebeenidentified,andDNAanalysistodetectthesemutationsislikelytoreplacethemousefootpad
technique.
Majoradvanceshavebeenmadeinexperimentalmodelsofleprosy.Theavailabilityofknockoutmicedeficientin
selectedimmunologicabilitieswillenabledissectionoftherolesofdifferentcytokinesandTcellsubsetsinthe
responsetothisinfection.Thearmadillogenomehasbeenpartiallysequencedandisbeingannotatedthiswillsoon
enableinvestigatorstoidentifyandsynthesizeimmunologicallyrelevantcytokinesandprobesforuseinthisanimal
model.
Withinmacrophages,M.lepraeisnowknowntobekilledbyreactivenitrogencompounds.Avarietyof
mechanismsofinnateandadaptiveimmunityhavebeenidentifiedandpostulatedtoplayaroleinthedevelopment
ofcellularimmunityinleprosy.Noneofthesecanyetexplaintheremarkablespectrumofcellularimmune
responsestothisorganisminhumansubjects,however.
GeneticinfluencesonimmunitytoM.lepraeinhumansappeartooperateattwolevels:somemechanismsactat
thelevelofoverallsusceptibility,andothersfunctionatthelevelofacquiredimmunity.Oneleprosysusceptibility
genehasbeenidentified,andseveralgenespossiblyinfluencingadaptiveimmunityhavealsobeendescribed.
Reactionsinleprosyremainpoorlyunderstood.Immunopathologicalstudieshavegenerallyfoundthattype1
(reversal)reactionscorrespondtoanupregulationofTh1typeimmuneresponses,andthattype2reactions(ENL)
correspondtoanenhancementoftheTh2typeofresponse.Thesefindingsarenotyetverysatisfying,however,
sincethereareseveralunresolveddiscrepanciesintheseassociations,andnoinformationthusfarindicateswhat
triggersreactionsorwhytheyaffectsomepatientsbutnotothers.
Themechanismsofnerveinjuryinleprosyremainpoorlyunderstood,althoughrecentstudieshaveshedlighton
themechanismsoflocalizationofM.lepraetonervesandonthemolecularmechanismsofbindingandingestionof
M.lepraebySchwanncells.Amajorfrontierinclinicalleprosyisthepreventionofdisabilitybyactiveand
persistentattentiontonervefunctionimpairment,sincenerveinjurymayprogressevenaftercompletionof
chemotherapy.
Severaleffectiveantimicrobialagentsarenowavailabletotreatleprosy,andthisinfectioniscurable.Molecular
methodscannowidentifyseveralmutationsinM.lepraeassociatedwithantimicrobialresistance.Themedically
conservativeapproachtotreatmentrecommendsusingmultipleagents(multidrugtherapy)forseveralmonthsor
years,dependingontheclassificationofthedisease.TheWorldHealthOrganizationhasrecommendedmuch
shortertreatmentprotocols,andthesehavebeenthesubjectofmuchcontroversyamongphysicianstreatingpatients
withleprosy.
BCGprovidesalowbutmeasurabledegreeofprotectionagainstM.leprae,butnohighlyeffective,specific
vaccinehasyetbeendeveloped.Noinfectiousdiseasehasbeeneliminatedusingdrugtreatmentalone,withoutan
effectivevaccine,butthedifficultiesofimplementationofaleprosyvaccinealsoposeprofoundchallengestothe
useofsuchavaccineifoneisdeveloped.
FutureofLeprosyTreatmentandResearch
Globaleliminationofleprosyhasbeentheoverarchinggoaloflaboratoryresearchandhealthpolicyfornearly20
years.Thiswasnotaccomplishedby2000or2005anddoesnotappearlikely,probablyduetoacomplexmixture
ofsocial,economic,andbiologicalfactorsthatcannotberesolvedinthelaboratoryalone.Eliminationofan
infectiousdiseaserequiresahighlyeffectivevaccinedevelopingonewasthecentralfocusofleprosyresearch
duringthe1980sandearly1990s.Thishasnotbeensuccessfulthusfar,althoughvaccineeffortscontinue.
Witheliminationstillinmind,thecurrentprimarygoalisearlydiagnosis,inordertotrytointerrupttransmission
withtreatmentasearlyaspossible.Thisisthemajorfocusofthemostconcertedlaboratoryresearcheffortstoday,
employingadvancedmoleculartools.Evenifthisshouldbecometechnicallyfeasibleinthenearfuture,this
conceptfacesenormouschallengestoverificationbeforetreatmentofasymptomaticindividualscanbe
recommended.Implementingtreatmentofasymptomaticpersonswouldbeevenmoredifficultatthiswriting,in
manyareaswithendemicleprosy,evenpatientswithovertdiseasearefindingthatresourcesfordiagnosisand
treatmentarebeingsystematicallyreduced.
Analternativeparadigmtoeliminationofleprosyislivingwithleprosybutrenderingitharmless,anideaadvanced
byYoYuasaoftheSasakawaMemorialHealthFoundation(451).Recognizingthehighcostandapparentfutility
ofeliminationcampaignsinthemosthighlyleprosyendemicregionsoftheworld,thisapproachcallsforimproved
toolsformanagementoftheinfectionanditscomplicationsandbettermethodsforthepreventionandtreatmentof
nerveinjury.Bothoftheseparadigms,aswellasthetensionbetweenthem,reflectthecontinuingchallengesof
leprosy.
ACKNOWLEDGMENTS Goto:
ThephotographsinFig.2weretakenbyG.McCormick,NHDP.Figure6wascreatedbyT.Thomassie,NHDP.
ThisworkwassupportedinpartbyNIHgrantsAI050027andAI045725fromtheNationalInstituteofAllergy
andInfectiousDiseases,NIAIDcontractY1AI264601,andgrantsfromtheHeiserFoundationandfrom
AmericanLeprosyMissions.
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