Myasthenia gravis: past, present, and future
Bianca M. Conti-Fine, ⌠, Monica Milani, Henry J. Kaminski
J Clin Invest. 2006;116(11):2843-2854. https://doi.org/10.1172/JCI29894.
Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure ofneuromuscular transmission, which results from the binding of autoantibodies to proteinsinvolved in signaling at the neuromuscular junction (NMJ). These proteins include thenicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved inAChR clustering. Much is known about the mechanisms that maintain self tolerance andmodulate anti-AChR Ab synthesis, AChR clustering, and AChR function as well as thosethat cause neuromuscular transmission failure upon Ab binding. This insight has led to thedevelopment of improved diagnostic methods and to the design of specificimmunosuppressive or immunomodulatory treatments.
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Acquiredâmyastheniaâgravisâ (MG)â isâanâuncommonâdisorderâ(200â400âcasesâperâmillion;âref.â1).âItsâsymptomsâareâcausedâbyâaâcharacteristicâmuscleâweaknessâthatâworsensâafterâuseâofâaffectedâmuscles.âInâaboutâtwo-thirdsâofâpatients,âtheâextrinsicâocularâmusclesâ(EOMs)âpresentâtheâinitialâsymptoms.âTheâsymptomsâusuallyâprogressâ toâtheâotherâbulbarâmusclesâandâ limbâmus-cles,âresultingâinâgeneralizedâMGâ(gMG).âInâaboutâ10%âofâMGâpatients,âsymptomsâremainâlimitedâtoâtheâEOM,âandâthisâcondi-tionâisâtermedâocularâMGâ(oMG).
MGâfulfillsâtheâstrictâcriteriaâofâanâAb-mediatedâautoimmuneâdisorder:â(a)âAbsâareâpresentâatâtheâsiteâofâpathology,âtheâneuro-muscularâjunctionâ(NMJ);â(b)âIgâfromâMGâpatientsâorâanti-AChRâAbsâfromâexperimentalâanimalsâcauseâMGâsymptomsâwhenâinject-edâintoârodents;â(c)âimmunizationâofâanimalsâwithâAChRârepro-ducesâtheâdisease;âandâ(d)âtherapiesâthatâremoveâAbsâdecreaseâtheâseverityâofâMGâsymptoms.
Historical perspectiveTheâfirstâdescribedâcaseâofâMGâisâlikelyâthatâofâtheâNativeâAmericanâChiefâOpechancanough,âwhoâdiedâinâ1664,âasâreportedâbyâVirgin-ianâchroniclers:ââTheâexcessiveâfatigueâheâencounteredâwreckedâhisâconstitution;âhisâfleshâbecameâmacerated;âhisâsinewsâlostâtheirâtoneâandâelasticity;âandâhisâeyelidsâwereâsoâheavyâthatâheâcouldânotâseeâunlessâtheyâwereâliftedâupâbyâhisâattendantsâ.â.â.âheâwasâunableâtoâwalk;âbutâhisâspiritârisingâaboveâtheâruinsâofâhisâbodyâdirectedâfromâtheâlitterâonâwhichâheâwasâcarriedâbyâhisâIndiansââ(2).âInâ1672,âtheâEnglishâphysicianâThomasâWillisâdescribedâaâpatientâwithâtheââfatiguableâweaknessââofâlimbsâandâbulbarâmusclesâcharacteris-ticâofâMGâ(3).âInâtheâlateâ1800s,âtheâfirstâmodernâdescriptionsâofâpatientsâwithâmyasthenicâsymptomsâwereâpublishedâ(3),âandâtheânameâmyasthenia gravisâwasâcoinedâbyâfusingâtheâGreekâtermsâforâmuscleâandâweaknessâtoâyieldâtheânounâmyastheniaâandâaddingâtheâLatinâadjectiveâgravis,âwhichâmeansâsevereâ(4).
AttemptsâatârationalâtreatmentsâofâMGâbeganâinâtheâ1930s.âAâmajorâstepâforwardâoccurredâinâ1934âwhenâMaryâWalkerârealizedâthatâMGâsymptomsâwereâsimilarâtoâthoseâofâcurareâpoisoning,â
whichâwasâtreatedâwithâphysostigmine,âaâcholinesteraseâinhibi-tor.âSheâshowedâthatâphysostigmineâpromptlyâimprovedâmyas-thenicâsymptomsâ(5),âmakingâanticholinesteraseâdrugsâaâstapleâinâMGâmanagement.âBecauseâthymusâpathologyâisâcommonâinâMGâpatients,âasâfirstânotedâinâtheâlateâ1800sâ(3),âinâ1937,âBlalockâremovedâaâmediastinalâmassâfromâaâyoungâwomanâwhoâhadâMGâ(3);âtheâpatientâimprovedâpostoperatively.âLater,âBlalockâreportedâotherâmyasthenicâpatientsâwhoâimprovedâafterâthymusâremovalâ(3),âestablishingâthymectomyâasâaâtreatmentâforâMG.
Inâ1959â1960,âSimpsonâandâNastuckâproposedâindependentlyâthatâMGâhasâanâautoimmuneâetiologyâ(6,â7)âbasedâonâseveralâobser-vations:â(a)âMGâpatientsââseraâcompromiseâcontractionâinânerve-muscleâpreparations;â(b)âtheâlevelâofâserumâcomplementâcorrelatesâinverselyâwithâtheâseverityâofâMGâsymptoms;â(c)âinfantsâofâmyas-thenicâmothersâmayâpresentâtransientâmyasthenicâsymptomsâ(neo-natalâMG);â(d)âinflammatoryâinfiltratesâmayâoccurâinâmusclesâofâMGâpatients,âandâpathologicâchangesâareâcommonâinâtheirâthymi;âandâ(e)âMGâmayâbeâassociatedâwithâotherâputativeâautoimmuneâdisorders.
Inâ 1973,â Patrickâ andâ Lindstromâ demonstratedâ thatâ rabbitsâimmunizedâwithâpurifiedâmuscle-likeâAChRâdevelopedâMG-likeâsymptomsâ(experimentalâautoimmuneâMGâ[EAMG])â(8).âAfterâthatâseminalâdiscovery,âmanyâstudiesâdemonstratedâanâautoimmuneâresponseâagainstâmuscleâAChRâinâMGâandâtheâroleâofâanti-AChRâAbsâinâcausingâtheâstructuralâandâfunctionalâdamageâofâtheâNMJ.âTheseâfindingsâpromotedâtheâuseâofâimmunosuppressantsâinâMG.âInâtheâ1970s,âprednisoneâandâazathioprineâbecameâestablishedâtreatmentsâforâMGâ(9),âandâplasmaâexchangeâwasâintroducedâasâanâeffectiveâacuteâtreatmentâforâsevereâMGâ(10),âfurtherâprovingâthatâcirculatingâfactorsâcausedâMGâsymptoms.
Pathogenesis of MG: current state of the artStructure and function of the NMJ.âTheâ terminalâarborizationâofââÎąâmotorâneuronâaxonsâfromâtheâventralâhornsâofâtheâspinalâcordâandâbrainstemâprovidesâtheânerveâterminalsâthatâformâtheâNMJâ(Figureâ1).âTheseâmyelinatedâaxonsâreachâtheâmusclesâthroughâperipheralânerves;âthenâeachâaxonâdividesâintoâbranchesâthatâinner-vateâmanyâindividualâmuscleâfibers.âAsâitâapproachesâitsâtargetâfiber,âeachâbranchâlosesâtheâmyelinâsheathâandâfurtherâsubdividesâintoâmanyâpresynapticâboutons,âwhichâcontainâACh-loadedâsynapticâvesiclesâandâfaceâtheâsurfaceâofâtheâmuscleâfiberâatâtheâNMJâ(Figureâ1).âTheâsynapticâboutonâandâtheâmuscleâsurfaceâareâseparatedâbyâtheâsynapticâcleft,âaâ20ânmâthickâspaceâthatâcontainsâacetylcholin-esteraseâ(AChE)âandâotherâproteinsâandâproteoglycansâinvolvedâinâ
Myasthenia gravis: past, present, and futureBianca M. Conti-Fine,1 Monica Milani,1 and Henry J. Kaminski2
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA. 2Departments of Neurology and Neurosciences, Case Western Reserve University, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA.
Myastheniagravis(MG)isanautoimmunesyndromecausedbythefailureofneuromusculartransmission,whichresultsfromthebindingofautoantibodiestoproteinsinvolvedinsignalingattheneuromuscularjunction(NMJ).TheseproteinsincludethenicotinicAChRor,lessfrequently,amuscle-specifictyrosinekinase(MuSK)involvedinAChRclustering.Muchisknownaboutthemechanismsthatmaintainselftoleranceandmodulateanti-AChRAbsynthesis,AChRclustering,andAChR functionaswellasthosethatcauseneuromusculartransmissionfailureuponAbbind-ing.Thisinsighthasledtothedevelopmentofimproveddiagnosticmethodsandtothedesignof
specificimmunosuppressiveorimmunomodulatorytreatments.
Nonstandardabbreviationsused:âAChE,âacetylcholinesterase;âAPL,âalteredâpeptideâligand;âEAMG,âexperimentalâautoimmuneâMG;âEOM,âextrinsicâocularâmuscle;âEPP,âendplateâpotential;âgMG,âgeneralizedâMG;âIVIg,âintravenousâIg;âMG,âmyastheniaââgravis;âMuSK,âmuscle-specificâtyrosineâkinase;âNMJ,âneuromuscularâjunction;ââoMG,âocularâMG.
Conflictofinterest:âTheâauthorsâhaveâdeclaredâthatânoâconflictâofâinterestâexists.
Citationforthisarticle:âJ. Clin. Invest.â116:2843â2854â(2006).âdoi:10.1172/JCI29894.â
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Figure 1Structure of the NMJ. As it enters the muscle and approaches its target fibers, each Îąâmotor neuron axon divides into branches that innervate many individual muscle fibers. Each branch loses its myelin sheath and further subdivides into many presynaptic boutons, which contain ACh-loaded synaptic vesicles and face the surface of the muscle fiber at the NMJ. The synaptic bouton and the muscle surface are separated by the synaptic cleft, which contains AChE and proteins and proteoglycans involved in stabilizing the NMJ structure. The NMJ postsynaptic membrane has characteristic deep folds, and the AChR is densely packed at the fold top. When the nerve action potential reaches the synaptic bouton, ACh is released into the synaptic cleft, where it diffuses to reach and bind the AChR. ACh binding triggers the AChR ion channel opening, permitting influx of Na+ into the muscle fiber. The resulting EPP activates voltage-gated Na+ channels at the bottom of the folds, leading to further Na+ influx and spreading of the action potential along the muscle fiber. Other proteins, including Rapsyn, MuSK, and agrin, which are involved in AChR clustering, are also present on the muscle membrane in close proximity to the AChR. MASC, myotube-associated specificity component; RATL, rapsyn-associated transmembrane linker. Figure modified with permission from Lippincott Williams and Wilkins (126).
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stabilizingâtheâNMJâstructure.âTheâNMJâpostsynapticâmembraneâhasâcharacteristicâdeepâfolds:âtheâAChRâisâdenselyâpackedâ(aboutâ12,000âmoleculesâperâÎźm2)âinâhighlyâorderedâhexagonalâlatticesâofâmoleculesâatâtheâtopsâofâtheâfolds.
MuscleâAChRâmoleculesâareâtransmembraneâproteinsâformedâbyâ5âsubunits:â2âidenticalâÎąâsubunits,âwhichâcontributeâimportantâstructuralâelementsâtoâtheâACh-bindingâsites,âandâ3âdifferentâbutâhomologousâsubunits,âtermedâβ,âÎłâ(orâÎľ;âseeâbelow),âandâδ.âMuscleâexpressesâ2âdevelopmentallyâregulatedâAChRâisoforms.âEmbryonicâmuscleâexpressesâAChRsâformedâbyâÎą,âβ,âÎł,âandâδâsubunits.âAfterâinnervation,âexpressionâofâtheâÎłâsubunitâgeneâisâsubstitutedâbyâthatâofâtheâhomologousâÎľâsubunitâgeneâtoâyieldâtheâadultâAChRâiso-form,âaâcomplexâofâÎą,âβ,âδ,âandâÎľâsubunits.âSomeâadultâmuscles,ânotablyâtheâEOM,âstillâexpressâembryonicâAChRâ(11).
Whenâtheânerveâactionâpotentialâreachesâtheâsynapticâbouton,âtheâdepolarizationâopensâvoltage-gatedâCa2+âchannelsâonâtheâpresynap-ticâmembrane.âThisâCa2+âinfluxâtriggersâfusionâofâsynapticâvesiclesâwithâtheâpresynapticâmembraneâandâAChârelease.âQuantal contentâofâaânerveâimpulseârefersâtoâtheânumberâofâAChâvesiclesâ(quanta)âreleasedâbyâthatâimpulse.âTheâAChâdiffusesâintoâtheâsynapticâcleftâ(whereâitâcanâbeâhydrolyzedâbyâAChE)âandâreachesâandâbindsâtoâAChR,âtherebyâtriggeringâtheâopeningâofâitsâcationâchannelsâandâinfluxâofâNa+âintoâtheâmuscleâfiber.âTheâresultingâendplateâpotentialâ(EPP)âactivatesâvoltage-gatedâNa+âchannels,âleadingâtoâfurtherâinfluxâofâNa+âandâspreadingâofâtheâactionâpotentialâalongâtheâmuscleâfiber.
Theâpostsynapticâtransmembraneâprotein,âmuscle-specificâtyro-sineâkinaseâ(MuSK)â(Figureâ1),âisâtheâmainâautoantigenâinâsomeâMGâpatientsâ(12).âMuSKâexpressionâinâbothâdevelopingâandâmatureâmuscleâisâsimilarâtoâthatâofâAChR.âInâmatureâmuscle,âMuSKâisâpres-entâprominentlyâonlyâatâtheâNMJ,âwhereâitâisâpartâofâtheâreceptorâforâagrin.âAgrinâisâaâproteinâsynthesizedâbyâmotorâneuronsâandâsecretedâintoâtheâsynapticâbasalâlamina.âTheâsignalingâmediatedâbyâagrin/MuSKâinteractionâtriggersâandâmaintainsârapsyn-dependentâclusteringâofâAChRâandâotherâpostsynapticâproteinsâ(13).âRapsyn,âaâperipheralâmembraneâproteinâexposedâonâtheâcytoplasmicâsur-faceâofâtheâpostsynapticâmembrane,âisânecessaryâforâclusteringâofâAChR,âwithâwhichâitâcoclusters.âRapsynâandâAChRâareâpresentâinâequimolarâconcentrationsâatâtheâNMJ,âandâtheyâmayâbeâphysicallyâassociated.âRapsynâcausesâclusteringâofâNMJâproteinsâotherâthanâtheâAChR,âincludingâMuSK.âMiceâlackingâagrinâorâMuSKâfailâtoâformâNMJsâandâdieâatâbirthâofâprofoundâmuscleâweakness,âandâtheirâAChRâandâotherâsynapticâproteinsâareâuniformlyâexpressedâalongâtheâmuscleâfibersâ(14).
NMJ properties that influence susceptibility to muscle weakness in MG.âTheâEPPâgeneratedâinânormalâNMJsâisâlargerâthanâtheâthresholdâneededâtoâgenerateâanâactionâpotential.âThisâdifferenceâmayâvaryâinâdifferentâmuscles,âasâdiscussedâbelow.âNeuromuscularâtransmissionâsafety fac-torâisâdefinedâasâtheâratioâbetweenâtheâactualâEPPâandâtheâthresholdâpotentialârequiredâtoâgenerateâtheâmuscleâactionâpotential.âItsâreduc-tionâisâtheâelectrophysiologicalâdefectâthatâcausesâMGâsymptoms.
Theâquantalâcontentâofâanâimpulse,âtheâconductionâpropertiesâandâdensityâofâpostsynapticâAChR,âandâtheâactivityâofâAChEâinâtheâsynapticâcleftâallâcontributeâtoâtheâEPPâ(15).âAlso,âtheâpostsynapticâfoldsâ(Figureâ1)âformâaâhigh-resistanceâpathwayâthatâfocusesâend-plateâcurrentâflowâonâvoltage-gatedâNa+âchannelsâinâtheâdepthsâofâtheâfolds,âtherebyâenhancingâtheâsafetyâfactor.âAâreductionâinâtheânumberâorâactivityâofâtheâAChRâmoleculesâatâtheâNMJâdecreasesâtheâEPP,âwhichâmayâstillâbeâadequateâatârest;âhowever,âwhenâtheâquantalâreleaseâofâAChâisâreducedâafterârepetitiveâactivity,âtheâEPPâmayâfallâbelowâtheâthresholdâneededâtoâtriggerâtheâactionâpotential.
NMJâpropertiesâvaryâamongâmusclesâandâmayâinfluenceâmuscleâsusceptibilityâtoâMGâ(15).âThisâisâwellâillustratedâbyâtheâNMJâofâtheâEOMs,âwhichâareâespeciallyâsusceptibleâtoâdevelopingâmyasthenicâweakness.âTheâNMJsâofâEOMâdifferâfromâthoseâofâskeletalâmuscleâinâseveralâways.âTheyâhaveâlessâprominentâsynapticâfolds,âandâthere-foreâfewerâpostsynapticâAChRsâandâNa+âchannels,âandâaâreducedâsafetyâfactorâ(16).âTheyâareâsubjectâtoâveryâhighâneuronalâfiringâfrequency,âmakingâthemâproneâtoâfatigue.âAlso,âtheyâexpressâlessâintrinsicâcomplementâregulators,âmakingâthemâmoreâsusceptibleâtoâcomplement-mediatedâinjuryâ(17).âInâskeletalâmuscles,âfast-twitchâfibersâhaveâNMJsâwithâgreaterâquantalâcontents,âaâgreaterâdegreeâofâpostsynapticâfoldingâ(18),âandâhigherâpostsynapticâsensi-tivityâtoâAChâthanâslow-twitchâNMJsâ(19),âandâtheyâhaveâincreasedâNa+âcurrentâinâtheâNMJâregionâ(20).âTheseâpropertiesâmayâmakeâfast-twitchâskeletalâmuscleâfibersâlessâsusceptibleâtoâmyasthenicâfailureâthanâslow-twitchâfibers.
Effector mechanisms of anti-AChR Abs.âAnti-AChRâAbsâaffectâneuro-muscularâtransmissionâbyâatâleastâ3âmechanisms:â(a)âbindingâandâactivationâofâcomplementâatâtheâNMJ;â(b)âacceleratedâdegradationâofâAChRâmoleculesâcrosslinkedâbyâAbâ(aâprocessâknownâasâanti-genicâmodulation);âandâ(c)âfunctionalâAChRâblockâ(Figureâ2).
TheâNMJsâofâMGâpatientsâandâEAMGâanimalsâcontainâactiva-tionâfragmentsâofâcomplementâcomponentâ3â(C3),âtheâterminalâandâ lyticâcomplementâcomponentâ9â (C9),âandâtheâmembraneâattackâcomplexâ(MAC)â(21).âDifferentâlinesâofâindirectâevidenceâsuggestâthatâcomplementâactivationâatâtheâNMJâmightâbeâtheâpri-maryâcauseâofâAChRâlossâandâfailureâofâneuromuscularâtransmis-sionâ(Figureâ2A):â(a)âcomplementâdepletionâprotectsâanimalsâfromâEAMGâ(22);â(b)âadministrationâofâAbsâthatâblockâcomplementâcomponentâ6â(anti-C6)â(23)âorâaâcomplementâinhibitorâ(solubleâCR1)â(24)âprotectsârodentsâfromâEAMG;â(c)âmiceâwithâaâreducedâcomplementâfunctionâbecauseâofâaâgeneticâdeficitâofâcomplementâcomponentsâareâresistantâorâlessâsusceptibleâtoâEAMGâinductionâthanâmiceâwithânormalâcomplementâ(25);â(d)âIL-12âdeficientâmice,âwhichâsynthesizeâTh1-driven,âcomplement-fixingâAbsâpoorlyâ(26),âdevelopâminimalâEAMGâsymptomsâafterâAChRâimmunizationâinâspiteâofârobustâanti-AChRâAbâsynthesis;âmoreover,âtheirâNMJsâcontainâAbsâbutânotâcomplement,âsuggestingâthatâanti-AChRâAbsâthatâdoânotâactivateâcomplementâdoânotâeffectivelyâcompromiseâneuromuscularâtransmission.
Cellsâareâprotectedâfromâactivationâofâautologousâcomplementâonâtheirâsurfacesâbyâtheâso-calledâintrinsicâcomplementâregulators.âTheseâincludeâtheâdecay-acceleratingâfactorâ(DAFâorâCD55),âtheâmembraneâcofactorâproteinâ(MCPâorâCD46),âandâtheâmembraneâinhibitorâofâreactiveâlysisâ(MIRLâorâCD59)â(27â29).âConsistentâwithâanâimportantâroleâofâcomplementâinâEAMG,âpassiveâtransferâofâEAMGâwithâanti-AChRâAbsâcausesâmoreâsevereâmuscleâweaknessâinâDAF-deficientâmiceâthanâinâwild-typeâmiceâ(30).
AntigenicâmodulationâisâtheâabilityâofâanâAbâtoâcross-linkâ2âanti-genâmolecules,âtherebyâtriggeringâaâcellularâsignalâthatâcausesâaccel-eratedâendocytosisâandâdegradationâofâtheâcross-linkedâmoleculesâ(Figureâ2B).âIgGâfromâMGâpatientsâcausesâantigenicâmodulationâofâmuscleâAChRâinâvivoâandâinâvitroâ(31).âIfâacceleratedâdegradationâisânotâcompensatedâbyâincreasedâAChRâsynthesisâ(32),âitâwillâleadâtoâaâreductionâofâtheâavailableâAChRâmoleculesâatâtheâNMJâandâmyasthenicâsymptoms.âThisâpropertyâcanâbeâusedâasâaâdiagnosticâtestâforâMGâ(32).âHowever,ânotâallâanti-AChRâAbsâcauseâantigenicâmodulationâbecause,âevenâthoughâallâIgGâAbsâhaveâ2âantigen-bind-ingâsites,âtheâepitopeâlocationâonâtheâAChRâsurfaceâmayârestrictâtheâabilityâofâAbsâtoâcross-linkâaâsecondâAChRâmoleculeâ(33).
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FunctionalâAChRâblockâdueâtoâAbâbindingâtoâtheâACh-bindingâsiteâ(Figureâ2C)âisâanâuncommonâpathogenicâmechanismâinâMG,âyetâitâmayâbeâclinicallyâimportant.âThisâisâbecauseâtheâpresenceâofâAbâtoâtheâACh-bindingâsiteâofâtheâAChRâcausesâacute,âsevereâmuscleâweaknessâinârodentsâwithoutâeitherâinflammationâorânecrosisâofâtheâNMJâ(34).âManyâMGâpatientsâhaveâlowâlevelsâofâanti-AChRâAbsâthatârecognizeâtheâACh-bindingâsiteâ(35);âtheseâmightâblockâtheâAChRâinâspiteâofâtheirâlowâconcentrationâandâcontributeâtoâacuteâmyasthenicâcrises.
Role of CD4+ T cells in MG.âPathogenicâanti-AChRâAbsâareâhigh-affinityâIgGs,âwhoseâsynthesisârequiresâthatâactivatedâCD4+âTâcellsâ
interactâwithâBâcells,âresultingâinâlow-affinityâanti-AChRâAbs.âThisâtriggersâsomaticâmutationsâofâtheâIgâgenes,âleadingâtoâsynthesisâofâhigh-affinityâAbs.âBâcellsâsecretingâlow-affinityâanti-AChRâAbsâareâcommon;âforâexample,âaboutâ10%âofâmonoclonalâIgGsâinâmultipleâmyelomaâpatientsâbindâmuscleâAChRsâ(36).âMyelomasâareârarelyâassociatedâwithâMG,âperhapsâbecauseâofâtheâlowâaffinityâofâtheirâanti-AChRâAbsâ(36).
MGâpatientsâhaveâAChR-specificâCD4+âTâcellsâwithâTâhelperâfunctionâinâtheâbloodâandâthymusâ(37),âandâtheirâsymptomsâimproveâafterâthymectomyâ(38)âorâtreatmentâwithâanti-CD4âAbsâ
Figure 2Effector mechanisms of anti-AChR Abs. (A) Ab binding to the AChR activates the complement cascade, resulting in the formation of membrane attack complex (MAC) and localized destruction of the postsynaptic NMJ membrane. This ultimately leads to a simplified, altered morphology of the postsynaptic membrane of the NMJ of MG patients, which lacks the normal deep folds and has a relatively flat surface. (B) Abs cross-link AChR mol-ecules on the NMJ postsynaptic membrane, causing endocytosis of the cross-linked AChR molecules and their degradation (antigenic modulation). This ultimately leads to a reduced number of AChR molecules on the postsynaptic membrane. (C) Ab binding the ACh-binding sites of the AChR causes functional block of the AChR by interfering with binding of ACh released at the NMJ. This results in failure of neuromuscular transmission.
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(39).âMoreover,âinâMGâpatientsâwithâAIDS,âaâreductionâinâCD4+âTâcellsâcorrelatesâwithâmyasthenicâsymptomâimprovementâ(40).âStudiesâ inâexperimentalâ systemsâdirectlyâdemonstratedâthatâCD4+âTâcellsâareânecessaryâforâtheâdevelopmentâofâMGâsymp-toms.âSCIDâmiceâengraftedâwithâbloodâlymphocytesâfromâMGâpatientsâproduceâantiâhumanâAChRâAbsâandâdevelopâMGâsymp-tomsâonlyâifâtheâgraftedâcellsâincludeâCD4+âTâcellsâ(41).âAlso,âmiceâgeneticallyâdeficientâ inâfunctionalâCD4+âTâcellsâdoânotâdevelopâEAMGâ(42).âHealthyâsubjectsâmayâhaveâAChR-specificâCD4+âTâcells,âwhichâdoânotâcauseâaâclinicallyâsignificantâautoim-muneâresponse,âprobablyâbecauseâofâmechanismsâofâimmuno-logicalâtolerance,âwhichâfailâinâautoimmunity.
BloodâCD4+âTâcellsâfromâbothâgMGâandâoMGâpatientsârespondâtoâtheâAChRâinâvitro.âThoseâfromâgMGâpatientsârespondâtoâallâtheâAChRâsubunitsâ(43),âandâtheirâepitopeârepertoireâexpandsâasâtheâdiseaseâprogressesâ(44).âAâfewâAChRâsequencesâwereârecognizedâbyâmostâgMGâpatientsâ(e.g.,âseeâref.â45).âCD4+âTâcellâlinesâspecif-icâforâtheseââuniversalââAChRâepitopes,âwhenâgraftedâintoâSCIDâmice,âsupportâanti-AChRâAbâproductionâbyâBâcells,âwhichâresultâinâMGâsymptomsâ(41).âTheâresponsesâtoâAChRâandâAChRâepitopesâofâCD4+âTâcellsâfromâoMGâpatientsâwereâweakerâandâlessâstableâoverâtimeâthanâinâthoseâofâgMGâpatientsâ(44).âAlso,âCD4+âTâcellsâfromâindividualâoMGâpatientsârarelyârecognizeâallâAChRâsubunits,âevenâwhenâtheâdiseaseâhasâlastedâforâmanyâyearsâ(44).âItâisânotâclearâwhetherâCD4+âTâcellsâfromâoMGâpatientsârecognizeâtheâembryonicâÎłââorâtheâadultâÎľâsubunitâorâbothâ(44).
Theâpathogenicâroleâofâanti-AChRâCD4+âTâcellsâinâMGâandâEAMGâexplainsâtheâimportantâroleâofâMHCâclassâIIâmolecules,âwhichâpres-entâtheâantigenâepitopesâtoâtheâspecificâCD4+âTâcells.âInâmice,âsus-ceptibilityâtoâEAMGâcorrelatesâwithâtheâclassâIIâmoleculeâallelesâthatâtheyâexpressâ(46).âMoreover,âaâmutationâofâtheâgeneâencodingâtheâβâsubunitâofâtheâI-AbâmoleculeâconvertsâtheâhighlyâsusceptibleâC57BL/6âstrainâofâmiceâintoâtheâEAMG-resistantâBM12âstrainâofâmiceâ(47).âMGâpatients,âlikeâpatientsâwithâotherâautoimmuneâdis-eases,âexpressâsomeâMHCâ(HLA)âallelesâwithâhigherâfrequencyâthanâexpectedâinâtheâgeneralâpopulation.âHLAâgeneâproductsâfoundâfre-quentlyâinâMGâpatientsâincludeâtheâB8âandâA1âclassâIâmolecule,âtheâDR3/DW3âclassâIIâmolecule,âandâcertainâDQâalleleâproducts.âSomeâstudiesâhaveâusedâmiceâthatâexpressâindividualâDRâorâDQâallelesâtransgenically,âtoâdetermineâwhetherâsomeâDRâorâDQâmoleculesâinfluenceâtheâdevelopmentâofâEAMG.âThoseâstudiesâconfirmedâthatâexpressionâofâtheâDQ8âandâDR3âmoleculesâcorrelatedâwithâEAMGâsusceptibilityâandâexpressionâofâtheâDQ6âmoleculeâcorre-latedâwithâresistanceâ(48,â49).
Role of CD4+ T cell subtypes and cytokines in MG and EAMG.âDifferenti-atedâCD4+âTâcellsâareâclassifiedâintoâsubtypesâbasedâonâtheâcytokinesâtheyâsecrete.âAmongâthem,âTh1âandâTh2âcellsâhaveâdifferentâandâatâtimesâopposingâfunctionsâ(50)â(Figureâ3).âTh1âcellsâsecreteâproin-flammatoryâcytokines,âsuchâasâIL-2,âIFN-Îł,âandâTNF-Îą,âwhichâareâimportantâinâcell-mediatedâimmuneâresponses.âTh2âcellsâsecreteâantiinflammatoryâcytokines,âsuchâasâIL-4,âIL-6,âandâIL-10,âwhichâareâalsoâimportantâinducersâofâhumoralâimmuneâresponses.âMoreover,âIL-4âstimulatesâdifferentiationâofâTh3âcells,âwhichâsecreteâTGF-βâandâareâinvolvedâinâimmunosuppressiveâmechanismsâ(51).âBothâTh1âandâTh2âcytokinesâmayâinduceâAbâsynthesis.âHowever,âtheyâsupportâtheâsynthesisâofâdifferentâIgâtypes.âInâmiceâ(andâlikelyâinâhumans)âTh1âcellsâinduceâIgGâsubclassesâthatâbindâandâactivateâcomplementâefficientlyâwhereasâTh2âcellsâinduceâIgâisotypesâandâIgGâsubclassesâthatâfixâcomplementâpoorlyâorânotâatâall.âInârats,âbothâTh1âandâTh2âcellsâinduceâcomplement-fixingâIgGâsubclassesâ(52).
MGâpatientsâhaveâabundantâanti-AChRâTh1âcellsâinâtheâbloodâthatârecognizeâmanyâAChRâepitopesâ(e.g.,âseeâref.â53)âandâinduceâsynthesisâofâpathogenicâanti-AChRâAbsâwhenâgraftedâtogetherâwithâBâcellsâandâmacrophagesâfromâtheâsameâpatientâintoâSCIDâmiceâ(41).âMGâpatientsâalsoâhaveâanti-AChRâTh2âandâTh3âcellsâinâtheâbloodâ(54).âMiceâcongenitallyâlackingâorâoverexpressingâaâcytokineâhaveâbeenâusedâtoâstudyâtheâroleâofâcytokinesâinâEAMG.âThoseâstudiesâsuggestedâthatâTh1âcellsâandâtheirâcytokinesâareâneededâforâEAMGâdevelopment,âprobablyâbecauseâtheyâinduceâexpressionâofâcomple-ment-bindingâpathogenicâanti-AChRâAbsâ(e.g.,âseeârefs.â26,â55).TheâresistanceâtoâEAMGâinductionâconferredâbyâgeneticâdeficiencyâinâTNF-ÎąâorâTNFâreceptorâproteinsâ(56)âisâovercomeâbyâtreatmentâwithâIL-12,âconfirmingâthatâsensitizationâandâdifferentiationâofâTh1âcellsâisâimportantâforâEAMGâdevelopmentâ(57).âOtherâstudiesâdemonstratedâtheâimportantâroleâofâTh1âcellsâinâEAMGâbyâchang-ingâtheâconcentrationâofâTh1âcytokinesâinârodentsâwithânormalâgenesâforâcytokinesâandâtheirâreceptors.âForâexample,âtreatmentâofâratsâwithâantiâTNF-ÎąâAbsâsuppressesâEAMGâdevelopmentâ(58),âandâtreatmentâofâmiceâwithâaâsolubleârecombinantâformâofâtheâhumanâTNFâreceptor,âableâtoâoutcompeteâmouseâTNF-Îąâforâbindingâtoâtheâmouseâreceptor,âsignificantlyâimprovesâsymptomsâofâestablishedâEAMGâ(59).âMoreover,âestrogenâenhancesâEAMGâdevelopmentâinâmiceâbyâpromotingâaugmentedâIL-12âproductionâbyâAChR-specificâTh1âcells,âsuggestingâthatâestrogensâmediateâsexâdifferencesâinâauto-immunityâbecauseâofâaâTh1-mediatedâmechanismâ(60)â(Figureâ3).âProinflammatoryâTh1âcytokinesâinduceâexpressionâofâMHCâclassâIIââmoleculesâinâmuscle,âtherebyâfacilitatingâpresentationâofâmuscleâAChRâepitopesâandâfurtherâexpansionâofâactivatedâanti-AChRâCD4+âTâcellsâ(61).âIncreasedâIFN-ÎłâproductionâmayâexplainâtheâincreasedâexpressionâofâIFN-Îłâinducedâchemokinesâandâmonokinesâandâtheirâreceptorsâinâmuscle,âthymus,âandâlymphânodesâinâMGâpatientsâandâratsâwithâEAMG.âAâdecreaseâinâchemokineâexpressionâcorrelatesâwithâdecreasedâseverityâofâsymptomsâ(62).
Anti-AChRâTh2âcellsâhaveâcomplexâandâcontrastingârolesâ inâEAMG.âTheyâcanâbeâprotectiveâ(e.g.,ârefs.â63,â64),âbutâtheâTh2âcytokinesâIL-5,âIL-6,âandâIL-10âalsoâfosterâEAMGâdevelopmentâ(e.g.,ârefs.â65â67).âTheâresistanceâtoâEAMGâofâmiceâgeneticallyâdeficientâinâIL-6âisâassociatedâwithâaâreducedâformationâofâgerminalâcentersâinâtheâspleenâandâaâreducedâsynthesisâofâanti-AChRâIgGâAbsâwhileâtheâanti-AChRâIgMâresponseâisânormal,âsuggestingâaâdefectâinâTâcellâhelpâandâinâtheâswitchingâfromâIgMâtoâIgGâisotypesâ(67).
OtherâCD4+âTâcellâsubtypesâmayâhaveâaâroleâinâMG.âCD4+âTâcellsâthatâexpressâtheâCD25âmarkerâandâtheâtranscriptionâfactorâFoxp3âareâknownâasâTregsâandâareimportantâinâmaintainingâselfâtoler-anceâ(Figureâ3).âTregsâinâMGâpatientsâmayâbeâfunctionallyâimpairedâ(68).âInâaddition,âtheânumberâofâcirculatingâTregsâhasâbeenâshownâtoâincreaseâafterâthymectomy,âandâtheâincreaseâcorrelatedâwithâsymptomâimprovementâ(69).
Role of NK and NKT cells in MG and EAMG.âCD1-dârestrictedâNKTâcellsâmayâbeâinvolvedâinâmaintainingâselfâtolerance.âInâEAMGâandâMG,âNKTâcellsâandâTregsâmayâcooperateâinâregulatingâtheâanti-AChRâresponse.âInâAChR-immunizedâmice,âactivationâofâNKTâcellsâbyâaâsyntheticâglycolipidâagonistâinhibitsâEAMGâdevelopment;âtheseâtherapeuticâeffectsâareâlikelyâmediatedâbyâtheâincreaseâinânumberâandâmodulatoryâfunctionâofâTregsâinducedâbyâtheâglycolipidâ(70).
NKâcellsâcanâalsoâinfluenceâtheâdevelopmentâofâEAMGâandâpos-siblyâMG.âInâmice,âNKâcellsâareânecessaryâforâEAMGâdevelopmentâ(71).âTheââpermissiveââroleâofâNKâcellsâinâEAMGâisâdueâtoâtheirâsecretingâIFN-Îł,âtherebyâpermittingâandâenhancingâtheâsensitiza-tionâofâTh1âcells.âIL-18âisâanâimportantâgrowthâandâdifferentiationâ
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factorâforâbothâNKâcellsâandâTh1âcells,âespeciallyâinâcooperationâwithâIL-12â(55).âThus,âIL-18âmayâbeâespeciallyâimportantâinâMGâandâEAMGâpathogenesis.âThisâisâsupportedâbyâtheâfindingâthatââIL-18âdeficientâmiceâareâresistantâtoâEAMGâandâpharmacologicâblockâofâIL-18âsuppressesâEAMGâ(55,â71).âTheâfindingâthatâMGâpatientsâhaveâincreasedâserumâlevelsâofâIL-18,âwhichâareâhigherâinâgMGâthanâinâoMGâpatientsâandâtendâtoâdecreaseâwithâclinicalâimprovement,âsupportsâaâroleâforâIL-18âinâhumanâMGâ(72).
Other autoantigens in MG.âUpâtoâ20%âofâMGâpatientsâdoânotâhaveâanti-AChRâ Absâ andâ areâ consequentlyâ knownâ asâ seronegativeâpatientsâ(73).âManyâseronegativeâpatientsâ(31%â41%âinâmostâstud-ies)â(74)âdevelopâAbsâagainstâMuSK.âLowerâfrequenciesâofâpatientsâwithâanti-MuSKâAbsâamongâseronegativeâMGâpatientsâmayâoccurâinâparticularâethnicâgroupsâorâgeographicâlocationsâ(e.g.,âChinese,âNorwegians)â(74,â75);âthisâmightâreflectâenvironmentalâorâgeneticâsusceptibilityâfactors.âMGâpatientsâwithâanti-MuSKâAbsâneverâhaveâanti-AChRâAbs,âwithâtheânotableâexceptionâofâaâgroupâofâJapaneseâpatientsâinvestigatedâinâoneâstudyâ(74,â76).
Theâagrin/MuSKâsignalingâpathwayâlikelyâmaintainsâtheâstruc-turalâandâfunctionalâintegrityâofâtheâpostsynapticâNMJâapparatusâalsoâinâadultâmuscle.âAnti-MuSKâAbsâandâIgGâfromâMGâpatientsâwhoâhaveâanti-MuSKâAbsâdisruptâAChRâaggregationâinâmyotubesâfromâMuSK-immunizedâanimalsâandâblockâagrin-inducedâAChRâclusteringâ(12,â77).âAlso,âimmunizationâofâanimalsâwithâMuSKâfragmentsâinducesâmyasthenicâsymptomsâ(77).âThisâsuggestsâthatâanti-MuSKâAbsâaffectâtheâagrin-dependentâmaintenanceâofâAChRâclustersâatâtheâNMJ,âultimatelyâleadingâtoâreducedâAChRânumbers.âComplement-mediatedâdamageâmightâalsoâbeâresponsibleâforâlossâofâAChRsâandâNMJâdamage.âHowever,âsomeâMGâpatientsâwithâanti-MuSKâAbsâdoânotâexperienceâAChRâlossâatâtheâNMJâ(78),âper-hapsâbecauseâanti-MuSKâAbsâareâmainlyâIgG4,âwhichâdoânotâbindâcomplementâefficientlyâ(79).âMoreover,âsomeâstudiesâhaveâfoundâthatâanti-MuSKâAbsâdoânotâcauseâsubstantialâAChRâloss,âcomple-mentâdeposition,âorâmorphologicâdamageâatâtheâNMJâ(78).âAnti-MuSKâAbsâmayâhaveâotherâpathogenicâmechanisms.âInâthisâregard,âaârecentâstudyâthatâinvestigatedâtheâeffectsâofâanti-MuSKâAbâcon-
Figure 3Cytokine network and cells involved in the pathogenesis and immunoregulation of MG. Th1 cytokines stimulate production of IgG subclasses that bind and activate complement effectively, whereas Th2 cytokines stimulate the production of Ig classes and IgG subclasses that do not. The Th2 cytokine IL-4 is also a differentiation factor for Th3 cells, immunosuppressive cells that secrete TGF-β. The Th1 cytokine IFN-Îł stimulates expression of MHC class II molecules on the muscle cell membrane, thus facilitating presentation of muscle AChR. The IL-18 secreted by APCs favors the differentiation of Th1 cells both directly and indirectly through the action of NK cells. CD1-dârestricted NKT cells can activate Tregs, thereby inhibiting autoimmune processes. See text for further details.
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tainingâseraâfromâMGâpatientsâonâmuscleâcellâculturesâfoundâthatâsomeâseraâinhibitâcellâproliferationâbyâcausingâcellâcycleâarrestâandâcauseâdownregulationâofâtheâexpressionâofâAChRâsubunits,ârapsyn,âandâotherâmuscleâproteinsâ(80).
Someâseronegativeâpatientsâwhoâdoânotâhaveâeitherâanti-AChRâorâanti-MuSKâAbsâmightâhaveâaâplasmaâfactorâthatâactivatesâaâsecondâmessengerâpathwayâinâtheâmuscle,âresultingâinâphosphorylationâandâinactivationâofâtheâAChRâ(81).MGâpatientsâmayâalsoâsynthe-sizeâAbsâagainstânonâmuscle-specificâproteins,âsuchâasâmyofibril-larâproteinsâ(82).âSomeâofâthoseâAbs,âespeciallyâanti-myosinâAbsâandâantiâfastâtroponinâAbs,âmayâcross-reactâwithâtheâAChRâ(83).âMGâpatientsâwithâthymomaâhaveâAbsâagainstâtitinâ(84)âandâtheâryanodineâreceptorâ(85).
Diagnosis of MGTextbookâdescriptionsâsuggestâthatâclinicalâdiagnosisâofâMGâshouldâbeâstraightforward.âHowever,âthisâisânotâalwaysâtheâcase.âDelayedâorâmissedâdiagnosesâoccurâfrequently.âThisâisâbecauseâMGâisârela-tivelyârareâandâthereforeâunfamiliarâtoâpractitioners,âandâitsâfluc-tuatingâmuscularâweaknessâcanâbeâpuzzling.âOnceâsuspected,âtheâdiagnosisâofâMGâreliesâonâserologicalâtestsâthatâdetectâanti-AChRâorâanti-MuSKâAbsâandâsometimesâAbsâagainstâotherâmuscleâproteinsâ(actin,âstriationalâprotein)âandâelectrodiagnosticâtestsâthatâdetectâcharacteristicâdefectsâinâneuromuscularâtransmissionâ(Tableâ1).
Theâdiscoveryâofâanti-AChRâAbsâyieldedâusefulâdiagnosticâtests.âTheâdemonstrationâofâserumâanti-AChRâAbsâprovesâtheâdiagno-sisâofâMG.âHowever,âtheirâabsenceâdoesânotâexcludeâitâbecauseâanti-AChRâAbsâareâdetectableâonlyâinâ80%â90%âofâgMGâpatientsâandâ30%â50%âofâoMGâpatientsâ(86).âTheâmostâcommonlyâusedâanti-AChRâAbâassayâmeasuresâtheâserumâlevelsâofâanâautoanti-bodyâthatâprecipitatesâmuscleâAChRâextractedâfromâappropriateâ
humanâcellâlinesâorâamputatedâtissue.âTheâAChRâisâdetectedâbyâtheâbindingâofâaâradiolabeled,âirreversibleâcholinergicâantagonist,âÎą-bungarotoxinâ(86).âThus,âthisâassayâcannotârevealâanti-AChRâAbsâagainstâtheâcholinergicâsite,âwhichâcanâbeâassayedâbyâtestingâtheâabilityâofâtheâpatientsââIgGâtoâcompetitivelyâinhibitâtheâbind-ingâofâcholinergicâligandsâ(86).âYetâanotherâserologicâtestâmea-suresâtheâabilityâofâtheâpatientsââseraâorâIgGâtoâinduceâantigenicâmodulationâofâtheâAChRâinâcellâculturesâ(86).âInâpatientsâwithâMGâsymptomsâwhoâdoânotâhaveâdetectableâanti-AChRâAbsâ(73),âserologicâdiagnosisâofâMGâcanâbeâattemptedâbyâdeterminingâtheâpresenceâofâanti-MuSKâAbsâ(12).âAboutâ5%âofâMGâpatientsâhaveâneitherâanti-AChRânorâanti-MuSKâAbs.
Standardâelectrodiagnosticâtestsâthatâutilizeârepetitiveâstimula-tionâofâperipheralânervesâareâcommonlyâusedâtoâdetectâneuromus-cularâtransmissionâdefectâinâMG.âTheyâareârelativelyâsensitiveâandâreliableâ(Tableâ1).âTheâmostâsensitiveâelectrodiagnosticâtestâforâMGâisâsingle-fiberâelectromyography,âwhichârevealsâdeficitsâofâneuro-muscularâtransmissionâinâ95%â99%âofâMGâpatientsâandâexcludesâtheâdiagnosisâofâMGâwhenâitâyieldsânormalâresultsâ(87).âItâselectivelyârecordsâactionâpotentialsâfromâaâsmallânumberâ(usuallyâ2âorâ3)âofâmuscleâfibersâinnervatedâbyâaâsingleâmotorâunit.âTheâamountâofâAChâreleasedâatâtheâNMJâatâdifferentâtimesâhasâaâsmallâvariability,âresultingâinâcomparableâvariationsâinâtheâriseâofâEPPâandâtheâmuscleâfiberâpairâinterpotentialâintervals.âThisâvariabilityâisâhighlyâsensitiveâtoâneuromuscularâtransmissionâabnormalitiesâandâisâincreasedâinâMGâpatients.âNeuromuscularâblockingisâtheâfailureâofâtransmis-sionâofâoneâofâtheâpotentials,âwhenâoneâofâtheâmuscleâfibersâfailsâtoâtransmitâanâactionâpotentialâbecauseâtheâEPPâdoesânotâreachâtheânecessaryâthreshold.âTheâAmericanâAssociationâofâNeuromuscularâ&âElectrodiagnosticâMedicineâhasâdevelopedâguidelinesâforâelectro-diagnosticâtestingâforâevaluationâofâMGâ(88).
Table 1Diagnostic tests for MG
Test Sensitivity Comments
ClinicaltestsEdrophonium Detectable in 80%â90% of MG patients Edrophonium testing is safe. However, because of known adverse effects, alternative nonpharmacological tests have been developed.Sleep Criteria poorly definedIce-pack test Criteria poorly defined
AssaysofserumAbsAnti-AChR Detectable in approximately 80%â90% of May also be observed in patients with Lambert-Eaton syndrome and gMG patients and 30%â50% of oMG patients motor neuron disease, thymoma patients without MG, and relatives of MG patients.Anti-MuSK Detectable in approximately 30%â40% of anti-AChR Abânegative gMG patients and rarely in oMG patientsAnti-striational protein Detectable in 80% of thymomatous MG More common in older patients. First autoantibody detected in MG. patients and 30% of nonthymomatous However, poor specificity makes this test nondiagnostic. MG patients
ElectrodiagnostictestsRepetitive stimulation Positive in approximately 90% of gMG of peripheral nerves patients and 30%â60% of oMG patientsSingle-fiber Positive in 95%â99% of MG patients Despite its sensitivity, single-fiber electromyography is not the test of electromyography choice because it is dependent on operator skills and patient cooperation. Also, results are abnormal in neuropathies and motor neuron and muscle diseases.
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Therapeutic MG managementTheâcurrentâmanagementâofâMGâincludesâtheâuseâofâanticholines-teraseâdrugsâforâtemporaryâimprovementâofâneuromuscularâtrans-mission,âremovalâofâanti-AChRâAbsâbyâplasmaâexchangeâorâspecificâimmunoadsorptionâprocedures,âuseâofânonspecificâimmunosup-pressantsâorâimmunomodulatorsâtoâcurbâtheâanti-AChRâresponse,âandâthymectomyâ(Tableâ2).âNoâcurrentâtreatmentâtargetsâtheâautoim-muneâdefectâofâMGâselectively.âHowever,âtheâadvancesâinâunderstand-ingâtheâpathogenesisâofâautoimmunityâandâMGâsuggestâthatânewâapproachesâthatâwillâcurbâorâevenâeliminateâtheâanti-AChRâresponseâspecificallyâwillâbeâforthcomingâ(seeâTableâ2âandââTheâfutureâ).
Anticholinesteraseâdrugsâ improveâmyasthenicâ symptomsâ inânearlyâallâpatients,âbutâtheyâfullyârelieveâtheâsymptomsâinâonlyâaâfew.âThus,âmostâpatientsârequireâadditionalâimmunosuppressiveâtreatmentâ(seeâbelow).âAânovelâapproachâtoâlong-termâtherapeuticâinhibitionâofâAChEâactivityâinâMGâpatientsâisâbasedâonâtheâobser-vationâthatâinâtheâNMJâofâbothâMGâpatientsâandâEAMGâanimals,âthereâ isâenhancedâtranscriptionâandâalteredâsplicingâofâAChEâpre-mRNA,âwithâaccumulationâofâaânormallyârareâreadthroughâAChE-Râvariantâ(89).âTheâcommonlyâoccurringâsynapticâAChE-Sâvariantâformsâmembraneâmultimers.âInâcontrast,âAChE-Râexistsâasâ solubleâ monomersâ thatâ lackâ theâ carboxyterminalâ cysteineâneededâforâmembraneâattachment.âThus,âAChE-RâpermeatesâtheâsynapticâspaceâandâdegradesâAChâbeforeâitâreachesâtheâpostsyn-apticâmembrane,âtherebyâcompromisingâAChRâactivation.âTheseâobservationsâpromptedâtheâdesignâandâuseâofâEN101,âanâantisenseâoligonucleotideâthatâsuppressesâtheâexpressionâofâAChE-R.âEN101ânormalizesâneuromuscularâtransmissionâinâEAMGâbyâmodulat-
ingâtheâsynthesisâofâAChEâvariants,âtherebyâaffectingâtheârateâofâAChâhydrolysisâandâtheâefficacyâofâAChRâactivationâ(90).âEN101âisâundergoingâhumanâtrials.
Despiteâtheâlackâofâlargeâcontrolledâtrials,âcorticosteroidsâareâtheâimmunosuppressiveâagentâmostâfrequentlyâusedâforâtheâtreatmentâofâMGâandâtheâmostâconsistentlyâeffectiveâ(91).âTheyâareâadminis-teredâatâhighâdosesâforâseveralâmonthsâandâatâlowâdosesâforâyears.âAnti-AChRâAbâlevelsâdecreaseâinâtheâfirstâmonthsâofâtherapy.âMostâpatientsâobtainâaâclinicalâbenefit,âwhichâmayâbeârelatedâtoâreduc-tionâofâlymphocyteâdifferentiationâandâproliferation,âredistribu-tionâofâlymphocytesâintoâtissuesâthatâareânotâsitesâofâimmunore-activity,âchangesâinâcytokineâexpressionâ(primarilyâofâTNF,âIL-1,âandâIL-2),âinhibitionâofâmacrophageâfunctionâandâofâantigenâpro-cessingâandâpresentation,âorâaâpossibleâincreaseâinâmuscleâAChRâsynthesis.âTheâshortcomingâofâcorticosteroidâtreatmentâisâtheâfre-quentâsteroid-relatedâcomplications.âThisâhasâmotivatedâtheâuseâofâotherâimmunosuppressants,âeitherâasââsteroid-sparingââagentsâorâasâaâsubstituteâforâcorticosteroids;âaâproportionâofâMGâpatientsâcanâbeâtreatedâsuccessfullyâwithoutâcorticosteroids.
Azathioprine,âaâpurineâanalog,âreducesânucleicâacidâsynthesis,âtherebyâinterferingâwithâTâandâBâcellâproliferation.âItâhasâbeenâutilizedâasâaâsingleâimmunosuppressantâagentâinâMGâsinceâtheâ1970s,âandâlarge,âretrospectiveâreportsâsupportâitsâefficacyâ(92).âAârandomized,âdouble-blindâtrialâdemonstratedâitsâefficacyâalsoâasâaâsteroid-sparingâagent.âItsâmajorâdisadvantageâisâtheâdelayedâclini-calâresponse,âwhichâmayâtakeâupâtoâ15âmonthsâ(93).
Cyclophosphamideâadministeredâ intravenouslyâandâorallyâisâanâeffectiveâtreatmentâforâMGâ(94);âmoreâthanâhalfâofâtheâ
Table 2Current and potential therapies for MG management
Approach Therapeutics Mechanismofaction
CurrentModulation of neuromuscular Cholinesterase inhibitors Prolong ACh activity transmission EN101 Antisense nucleotide that suppresses the synthesis of AChE-R, the soluble and most effective form of synaptic AChEImmunomodulation Thymectomy Probably multiple effects Plasma exchange Removal of Ab IVIg Multiple effects Immunoadsorption Removal of anti-AChR AbGeneral immunosuppression Prednisone Multiple effects Azathioprine Purine analog. Inhibits T and B cell proliferation Cyclosporine, Tacrolimus Blocks T cell activation and growth Mycophenolate mofetil Inhibits guanosine nucleotide synthesis and selectively inhibits activated T cells Etanercept Recombinant soluble TNF receptor that competitively inhibits TNF-Îą binding
Potential Complement inhibition Anti-C6 Abs Abs that block complement component 6 and protect rodents from EAMG CR1 Soluble recombinant receptor that competitively inhibits complement Induction of tolerance to AChR Multiple approaches Administration of AChR or portion of sequence by tolerance-inducing routes (e.g., oral, nasal) T cell vaccination Use of modified APCs loaded with AChR epitopesDepletion of AChR-specific B Multiple approaches For example, AChR toxin to eliminated B cells, Fas ligand conjugates targeted or T cells to T cellsInterruption of MHC class II, Multiple approaches For example, APL (peptide epitope analogs that cannot activate CD4+ T cells) epitope peptide, T cell receptor, and CD4+ complex
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patientsâbecameâasymptomaticâafterâ1âyearâofâtreatment.â Itsâdelayedâeffectâandâundesirableâsideâeffectsâ(hairâloss,âandâlessâfrequently,ânausea,âvomiting,âanorexia,âandâskinâdiscoloration)âlimitsâitsâuseâtoâtheâmanagementâofâpatientsâwhoâdoânotârespondâtoâotherâimmunosuppressiveâtreatments.
Cyclosporineâisâaâcyclicâundecapeptideâthatâblocksâtheâsynthesisâofâcytokinesâ(andâespeciallyâIL-2),âIL-2âreceptors,âandâotherâpro-teinsâcriticalâinâtheâfunctionâofâCD4+âTâcells.âItsâefficacyâinâMGâwasâfirstâsuggestedâbyâaâsmall,ârandomized,âplacebo-controlledâstudy,âwhichâhasânotâbeenâfollowedâbyâsimilarâstudiesâonâlargerâgroupsâofâpatientsâ(95).âHowever,âlargerâretrospectiveâstudiesâhaveâsupportedâitsâuseâasâaâsteroid-sparingâagentâ(96).
Tacrolimus,âaâmacrolideâantibiotic,âisâsimilarâtoâcyclosporineâinâitsâbiologicalâactivity.âNoâlargeâcontrolledâstudiesâonâtheâefficacyâofâtacrolimusâinâMGâareâavailable.âHowever,âaâlargeâretrospec-tiveâstudyâofâtreatment-resistantâpatientsâsupportsâitsâuseâasâaâsteroid-sparingâagentâ(97).
InâtheâUS,âmycophenolateâmofetilâisâincreasinglyâusedâbothâasâaâsteroid-sparingâagentâandâasâaâstand-aloneâimmunosuppressant.âUnfortunately,âtheâinvestigationsâthatâsupportâitsâuseâstudiedâsmallâpatientâgroupsâorâwereâofâaâretrospectiveânature;âthereforeâtheâvalueâofâtheirâconclusionsâisâlimitedâ(98).âThoseâstudiesâsug-gestâthatâmycophenolateâhasâlimitedâtoxicity,âconsistentâwithâitsâselectiveâinhibitoryâactivityâofâguanosineânucleotideâsynthesisâinâactivatedâTâandâBâcells,âwhichâshouldâlimitâitsâeffectsâonâotherâcellâtypesâ(99).âRandomized,âcontrolledâtrialsâtoâevaluateâmycopheno-lateâmofetilâusefulnessâinâMGâtreatmentâareâunderâway.
ForâsomeâMGâpatients,âsymptomsâdoânotâimproveâwithâtheâuseâofâcorticosteroidsâorâoneâofâtheâimmunosuppressiveâagentsâdescribedâabove,âaloneâorâinâcombination,âandâtheyâmayâdevelopâintolerableâsideâeffectsâpromptingâconsiderationâofâotherâtherapeuticâoptions.âMGâpatientsâresistantâtoâtherapyâhaveâbeenâsuccessfullyâtreatedâwithâcyclophosphamideâinâcombinationâwithâboneâmarrowâtrans-plantâ(100)âorâwithârituximab,âaâmonoclonalâAbâagainstâtheâBâcellâsurfaceâmarkerâCD20â(101).âEtanercept,âaâsoluble,ârecombinantâTNFâreceptorâthatâcompetitivelyâblocksâtheâactionâofâTNF-Îą,âhasâbeenâshownâtoâhaveâaâsteroid-sparingâeffectsâinâstudiesâonâsmallâgroupsâofâpatientsâ(102).âPlasmaâexchangeâandâi.v.âIgâ(IVIg)â(dis-cussedâbelow)âcanâbeâusedâasâchronicâtherapyâalthoughâtheirâben-efitâhasânotâbeenâdocumentedâinârigorouslyâdesignedâstudies.
Plasmaâexchangeâ(plasmapheresis)âandâIVIgâareâusedâforâacuteâmanagementâofâsevereâmuscularâweakness.âPlasmapheresisâinvolvesâreplacingâ1â1.5âtimesâtheâplasmaâvolumeâwithâsaline,âalbumin,âorâplasmaâproteinâfraction,âleadingâtoâaâreductionâofâserumâAChRâAbâlevelsâ(103).âForâIVIgâtherapy,âIgâisolatedâfromâpooledâhumanâplasmaâbyâethanolâcryoprecipitationâisâadministeredâforâ5âdaysâ(0.4âg/kg/day).âFewerâinfusionsâatâhigherâdosesâareâalsoâused.âTheâmechanismâofâactionâofâIVIgâisâcomplexâandâlikelyâincludesâinhibi-tionâofâcytokines,âcompetitionâwithâautoantibodies,âinhibitionâofâcomplementâdeposition,âinterferenceâwithâbindingâofâFcâreceptorâonâmacrophagesâandâIgâreceptorâonâBâcells,âandâinterferenceâwithâantigenârecognitionâbyâsensitizedâTâcellsâ(104).âMoreâspecificâtech-niquesâtoâremoveâpathogenicâanti-AChRâAbsâutilizingâimmunoad-sorptionâhaveâbeenâdevelopedârecently,âwhichâofferâaâmoreâtargetedâapproachâtoâMGâtreatmentâ(105).
TheâobservationsâthatâleadâtoâtherapeuticâablationâofâtheâthymusâinâMGâhaveâbeenâmentionedâabove.âHowever,âtheâclinicalâefficacyâofâthymectomyâhasâbeenâquestionedâbecauseâtheâevidenceâsupport-ingâitsâuseâisânotâsolidâ(106).âRetrospectiveâstudiesâyieldedâwidelyâdifferentâconclusions,âbutâtheyâincludedâpatientsâwhoseâthymiâwereâ
removedâbyâdifferentâsurgicalâapproaches,âandâtheyâusedâdiffer-entâdefinitionsâofâremissionâandâlimitedâstatisticalâanalysisâ(107).âMoreover,âcorticosteroidsâandâotherâimmunosuppressiveâagentsâareâcommonlyâusedâbyâMGâpatientsâundergoingâthymectomy,âmakingâitâdifficultâtoâascertainâwhetherâthymectomyâprovidesâadditionalâbenefits.âUsually,âthymectomyâisâperformedâonâpatientsâearlyâinâtheâcourseâofâtheirâdiseaseâandârestrictedâtoâpatientsâyoungerâthanâ60.âAâreviewâbyâtheâAmericanâAcademyâofâNeurologyâconcludedâthat,âwhileâthymectomyâshouldâbeâconsideredâaâtreatmentâoption,âitsâbenefitsâinânonthymomatousâMGâhaveânotâbeenâfirmlyâestablishedâ(106)âandâthatâaâprospective,âcontrolled,ârandomizedâstudyâwithâstandardizedâmedicalâtherapyâforâallâpatientsâisâneeded.âAlso,âtheâNIHâisâsponsor-ingâaâclinicalâtrialâtoâdetermineâwhetherâtheâextendedâtranssternalâthymectomyâreducesâcorticosteroidârequirementsâforâpatientsâwithâAChRâAbâpositiveânonthymomatousâgMGâ(108).
Thymectomyâmayânotâbeâaâviableâtherapeuticâapproachâforâanti-MuSKâAbâpositiveâpatientsâbecauseâtheirâthymiâlackâtheâgerminalâcentersâandâtheâinfiltratesâofâlymphocytesâthatâcharacterizeâthymiâfromâpatientsâwhoâhaveâanti-AChRâAbs.âThisâsuggestsâthatâaâdiffer-entâpathologicâmechanismâoccursâinâanti-MuSKâAbâpositiveâandâanti-AChRâAbâpositiveâMG.
Lackâofâwell-designedâinvestigationsâwithâappropriateâstatisticalâpowerâunderminesâtheârationaleâforâuseâofâtheâcurrentâimmuno-therapiesâforâMG.âMoreover,âthereâareânoârigorousâtrialsâthatâcom-pareâtheâefficacyâofâtheâvariousâimmunosuppressiveâapproaches.âDifferentâfactorsâcontributeâtoâthisâsituation,âincludingâtheâvari-ableâclinicalâpresentationsâandâtimeâcourseâofâMGâandâitsâlowâinci-dence;âaâlackâofâagreed-uponâclinicalâscalesâtoâmeasureâtheâoutcomeâofâtheâtreatment;âandâaâsuboptimalâcooperationâamongâtheâmajorâcentersâthatâcareâforâMGâpatientsâduringâclinicalâtrialsâ(109).
The futureComplementâinhibitionâisâanâattractiveâfutureâtherapeuticâapproachâforâMGâbecauseâitâisâeffectiveâinârodentâEAMGâ(e.g.,âref.â24).âMore-over,âanti-C5âinhibitorsâshowâshort-termâsafetyâandâareâeffectiveâinâaâvarietyâofâhumanâdisorders,âincludingâmyocardialâinfarctionâ(110),âcoronaryâarteryâbypassâgraftâsurgeryâ(111),âandâlungâtransplanta-tionâ(112).âThus,âtherapeuticâapproachesâbasedâonâinhibitionâofâcomplementâactivationâwillâlikelyâbeâtriedâforâMGâinâtheâfuture.
However,âtheâultimateâgoalâforâMGâtreatmentâisâtoâeradicateâtheârogueâanti-AChRâautoimmuneâresponseâspecificallyâandâreestab-lishâtoleranceâtoâtheâAChRâwithoutâaffectingâtheâotherâfunctionsâofâtheâimmuneâsystemâorâcausingâotherâadverseâeffects.âSuchâtargetedâimmunosuppressiveâapproachesâ(Tableâ2)âareâstillâfarâfromâclinicalâuse.âHowever,âtheirâsuccessâinâEAMGâsuggestsâthatâapproachesâforâspecificâmodulationâofâtheâautoimmuneâanti-AChRâresponseâmayâbecomeâpartâofâMGâpatientâcareâinâtheânextâdecade.âWeâwillâsum-marizeâhereâtheâdifferentâapproachesâthatâhaveâprovenâsuccessfulâforâtheâpreventionâandâtreatmentâofâEAMGâinducedâbyâimmunizationâwithâAChR.âWeâwillâalsoâanalyzeâtheâpossibleâtechnicalâandâbiologicalâlimitationsâtoâtheirâapplicationâforâtheâtreatmentâofâhumanâMG.
ApproachesâthatâhaveâprovenâsuccessfulâinârodentâEAMGâincludeâtheâfollowing:â(a)âadministrationâofâAChRâorâpartsâofâitsâsequenceâinâaâmannerâknownâtoâinduceâtolerance;â(b)âdepletionâofâAChR-specificâBâcellsâorâTâcells;âandâ(c)âinterferenceâwithâformationâofâtheâcomplexâbetweenâMHCâclassâIIâmolecules,âepitopeâpeptide,âTâcellâreceptor,âandâCD4âmolecule.
Antigenâpresentationâunderâspecialâcircumstancesâmayâleadâtoâantigen-specificâtoleranceâinâadultâanimalsâratherâthanâactivatedâCD4+âTâcells.âEarlierâstudiesâshowedâthatâinârats,âpresentationâofâ
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AChRâepitopesâbyâunsuitableâAPCsâ(fixedâBâcellsâthatâhadâbeenâincubatedâwithâAChRâunderâconditionsâfavoringâAChRâuptakeâandâprocessing)âcausedâunresponsivenessâofâtheâAChR-specificâCD4+âTâcellsâtoâfurtherâstimulationâwithâAChRâ(113).âMoreârecently,âseveralâstudiesâhaveâdemonstratedâthatâDCs,âespeciallyâafterâtreat-mentâwithâTGF-β,âIFN-Îł,âorâIL-10,âwhenâinjectedâintoâratsâwithâdevelopingâorâongoingâEAMG,âsuppressedâorâamelioratedâtheâmyasthenicâsymptomsâ(114â116).âTheâeffectâwasâcorrelatedâwithâaâreducedâproductionâofâanti-AChRâAbsâwithoutâaâreducedâprolif-erativeâresponseâofâTâcellsâtoâtheâAChR.âApproachesâbasedâonâtheâuseâofâtolerance-inducingâAPCs,âwhichâshouldâpresentâallâAChRâepitopesâandâthereforeâinfluenceâallâAChR-specificâTâcells,âmightâbeâusefulâforâtheâtreatmentâofâMG.âShouldâpulsingâofâtheâAPCsâwithâhumanâAChRâbeâneeded,âbiosyntheticâhumanâAChRâsubunitsâcouldâbeâusedâasâantigens.
MucosalâorâsubcutaneousâadministrationâofâAChRâorâsyntheticâorâbiosyntheticâAChRâpeptidesâtoârodentsâââapproachesâknownâtoâinduceâantigen-specificâtoleranceâinâadultâanimalsâââpreventedâorâdelayedâEAMGâdevelopmentâ(e.g.,âseeârefs.â117â119).âDependingâonâtheâdoseâofâtheâantigenâadministered,âanergy/deletionâofâanti-gen-specificâTâcellsâ(atâhighâdoses)âand/orâexpansionâofâcellsâpro-ducingâimmunosuppressiveâcytokinesâ(TGF-β,âIL-4,âIL-10)â(atâlowâdoses)âareâmajorâmechanismsâinâmucosalâtoleranceâinduction.âTheâuseâofâmucosalâtolerizationâproceduresâinâhumanâMG,âhowever,âisâproblematicâbecauseâthoseâproceduresâcanâbeâaâdouble-edgedâswordâ(120);âtheyâreduceâAChR-specificâCD4+âTâcellâresponsesâbutâmayâalsoâstimulateâAChR-specificâBâcellsâtoâproduceâAbs,âtherebyâworseningâtheâdisease.âAlso,âaâlargeâamountâofâhumanâAChRsâwouldâbeârequired,âwhichâmayâbeâdifficultâtoâobtain.
ConjugatesâofâaâtoxinâwithâAChRâorâsyntheticâAChRâsequences,âwhenâadministeredâtoâanimalsâwithâEAMG,âeliminatedâBâcellsâproducingâanti-AChRâAbsâ(121).âThisâ isâprobablyâbecauseâtheâAChRâmoietyâofâtheâconjugateâdocksâontoâtheâmembrane-boundâAbsâofâAChR-specificâBâcells,âwhichâcanâthenâbeâkilledâbyâtheâtoxicâdomain.âThisâapproachâhasâ2âcaveats.âFirst,âtheâtoxinâmayâdamageâotherâcells.âSecond,âanti-AChRâCD4+âTâcellsâcanârecruitânewâBâcellsâtoâsynthesizeâmoreâanti-AChRâAbs.
AChR-specificâCD4+âTâcellsâcanâbeâspecificallyâeliminatedâinâvitroâbyâAPCsâgeneticallyâengineeredâtoâexpressârelevantâportionsâofâtheâAChR,âFasâligandâ(toâeliminateâtheâactivatedâAChR-specificâTâcellsâwithâwhichâtheyâinteract),âandâaâportionâofâFas-associatedâdeathâdomain,âwhichâpreventsâself-destructionâbyâtheâFasâligandâ(122).âItâisânotâknownâyetâwhetherâthisâstrategyâcanâbeâsafelyâusedâtoâmodulateâEAMGâinâvivo.
ActivationâofâCD4+âTâcellsârequiresâinteractionâandâstableâbind-ingâofâseveralâproteinsâonâtheâsurfacesâofâtheâCD4+âTâcellâandâofâtheâAPC.âInâexperimentalâsystems,âinterferingâwithâformationâofâthisâcomplexâusuallyâreducedâtheâactivityâofâautoimmuneâCD4+âTâcells.âThisâmayâbeâobtainedâbyâadministeringâorâinducingâAbsâthatârecognizeâtheâbindingâsiteâforâtheâantigenâofâtheâTâcellâreceptorâ
(knownâasâTâcellâvaccination)â(123).âTâcellâvaccinationâisâalreadyâusedâinâclinicalâtrialsâforâtheâtreatmentâofâmultipleâsclerosis,ârheu-matoidâarthritis,âandâpsoriasisâ(124).âItâisâeffectiveâinâEAMG,âandâitâisâaâpromisingâfutureâstrategyâforâtheâtreatmentâofâMGâ(124).âTheâmechanismsâofâactionâofâTâcellâvaccinationâareâcomplex,âandâtheyâlikelyâincludeâtheâinductionâofâmodulatoryâCD4+âandâCD8+âTâcellsâ(124).âAnotherâapproachâusedâsyntheticâpeptideâanalogsâofâanâepi-topeârecognizedâbyâautoimmuneâCD4+âTâcellsâthatâbindâtheâMHCâclassâIIâmoleculesâbutâcannotâstimulateâtheâspecificâCD4+âcells.âTheseâareâknownâasâalteredâpeptideâligandsâ(APLs).âAPLsâcom-peteâwithâpeptideâepitopesâderivedâfromâtheâautoantigen,âtherebyâturningâoffâtheâautoimmuneâresponse.âAPLsâmightâalsoâstimulateâmodulatoryâantiinflammatoryâCD4+âTâcellsâorâanergizeâtheâpatho-genicâCD4+âTâcellsâ(125).âTheârichâepitopeârepertoireâofâanti-AChRâCD4+âTâcellsâinâMGâpatientsâreducesâtheâtherapeuticâpotentialâofâapproachesâthatâinterfereâwithâactivationâofâspecificâCD4+âTâcells;âtargetingâonlyâaâfewâepitopesâmayânotâsignificantlyâreduceâtheâanti-AChRâresponse.âMoreover,âtheseâtreatmentsâareâlikelyâtoâproduceâonlyâtransientâimprovementâthatâceasesâwhenâadministrationâofâtheâantiâTâcellâAbâisâdiscontinued.â
MGâandâEAMGâhaveâofferedâuniqueâopportunitiesâtoâinvesti-gateâtheâmolecularâmechanismsâofâanâAb-mediatedâautoimmuneâdisease.âManyâfactorsâhaveâcontributedâtoâmakingâMGâtheâbestâunderstoodâhumanâautoimmuneâdisease.âTheseâincludeâtheâsim-plicityâofâtheâpathogenicâmechanismâinâMG,âwhereâNMJâfailureâexplainsâallâsymptoms;âtheâdeeperâunderstandingâofâtheâstructureâandâtheâfunctionâofâtheâNMJâandâitsâmolecularâcomponents,âmostânotably,âtheâAChR;âandâtheâincreasingâunderstandingâofâtheâmech-anismsâthatâmodulateâimmuneâresponsesâandâmaintainâtolerance.âHopefullyâincreasingâknowledgeâofâtheâimmunobiologyâofâMGâwillâformâaâfoundationâforâdesigningânewâandâspecificâtherapeuticâapproachesâaimedâatâcurbingâtheârogueâautoimmuneâresponseâandâreestablishingâimmunologicalâtoleranceâwithoutâinterferingâwithâtheâotherâimmuneâfunctions.
Ifâthisâexpectationâisâfulfilled,âMG,âwhichâhasâbeenâaâbenchmarkâtoâunderstandingâautoimmunityâinâhumans,âwillâbecomeâaârefer-enceâpointâforâtheâdesignâofâspecificâimmunosuppressiveâtreat-mentsâofâotherâautoimmuneâAb-mediatedâdiseases.
AcknowledgmentsTheâauthorsâareâsupportedâbyâgrantsâfromâtheâNIHâ(NS23919âtoâB.M.âConti-Fine;âEY013238âandâR24EY014837âtoâH.J.âKaminski)âandâfromâtheâMuscularâDystrophyâAssociationâofâAmericaâ(toâM.âMilaniâandâB.M.âConti-Fine).
Addressâcorrespondenceâto:âBiancaâM.âConti-Fine,âDepartmentâofâBiochemistry,âMolecularâBiology,âandâBiophysics,âUniversityâofâMinnesota,â6-155âJacksonâHall,â321âChurchâSt.âSE,âMinneapolis,âMinnesotaâ55455,âUSA.âPhone:â(612)â624-6796;âFax:â(612)â625-2163;âE-mail:â[email protected].
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