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Methionine and Threonine Requirements of Dutch Rabbits Fed
20 Mar 2019 Six male Dutch rabbits were housed individually in a dormito- ry-type cage and they were randomly fed graded levels of dietary methionine.
Spontaneous Aortic Lesions in Rabbits I. Morphologic Characteristics
Dutch adult rabbits older than six months were used. Approximately one-half of the adult NZW aortas were obtained from the Pel-Freez Corp- oration.
The Dutch pattern in rabbits is caused by a gene p which is rabbit in
on the fifth chromosome of the rabbit in that order. The Dutch pattern in rabbits is caused by a gene p which is almost completely recessive to self-colour
4-H Rabbit Manual
d) Participating in an open show circuit with purebred rabbits Dutch patterns can also appear in other breeds of rabbit. ENGLISH SPOT.
Autoantibody Production in Rabbits
Thesera of rabbitsinjected with rat liver kidney
Evaluating Pain and Analgesia Effectiveness Following Routine
28 Feb 2022 We aimed to determine if the rabbit grimace scale (RbtGS) could be used as a viable rapid assessment tool in two breeds of rabbit
Further notes on Dutch and English rabbits
Castle and myself as to the genetieal interpretation of the various grades of ;Dutch rabbits and of their relation to the self-eolmu'ed animah As our eontro)
The anatomy of the reproductive system in male Dutch rabbits (
Organs were obtained from 39 adult Dutch-belted rabbits that averaged 2023 g in bodyweight. Organ weights (means and stan- dard deviations) were as follows:
ANOTHER CASE OF A BLACK-BLUE MOSAIC IN THE DUTCH
A MOSAIC RABBIT. Figure 3. This is a female Black Dutch rabbit with a semicircular patch of blue hair (dilute black) adjacent to the white belt.
DEVELOPMENT OF GLUCURONYL TRANSFERASE AND OTHER
Dutch rabbits were found to be suitable animals for the study of the develop- ment of liver glucuronyl transferase activity during the neonatal period
842_4immunology00444_0030.pdf Immunology,1963,6,19.AutoantibodyProductioninRabbitsII.ORGAN-SPECIFICAUTOANTIBODYINRABBITSINJECTEDWITHRATTISSUESG.L.ASHERSON*ANDD.C.DUMONDEtRheumatismResearchUnit(M.R.C.),CanadianRedCrossMemorialHospital,Taplow,Maidenhead,Berkshire(Received7thJune1962)Summary.Theseraofrabbitsinjectedwithratliver,kidney,heart,muscle,spleenandbraininFreund'scompleteadjuvantfixedcomplementwithrabbittissue.Thiscomplement-fixingactivitywasattributedtoautoantibodieswhichwereabletofixcomplementinvitrowiththetissueoftherabbitinwhichtheyoccurred.Absorption,geldiffusionandantibodyandantigentitrationsindicatedthatsomeoftheanti-liver,anti-kidney,anti-heart,anti-muscleandanti-brainseracontainedorgan-specificautoantibody.Theseraalsocontainedautoantibodyreactingwithwidelydistributedantigen(s),whichwasrelativelylabileat65°.Theanti-kidneyandanti-brainserareactedwithdistinctantigenswhichwereextractedfromrabbitkidneyandbrainwithamixtureofchloroformandmethanol.ThenaturalautoantibodyofKiddandFriedewaldwasusuallylabileat65°andbehavedlikeamacroglobulinonsucrosegradientcentrifugation.Serataken1weekafterimmunizationwithrattissuecontainedheat-labilemacroglobulinantibody.However,serataken1monthafterimmunizationalsocontainedsmallmolecularweightantibodywhichwasstableat65°.INTRODUCTIONAntibodyagainstseveralrabbittissueshasbeendemonstratedaftertheinjectionofvarioustissuesfromotherspeciesintotherabbit.Theseincludelens,uveaandcornea,brain,heart,adrenal,thyroidandnucleoprotein(seeAshersonandDumonde,1962).Itwasthereforeofinteresttoinvestigatewhethertheinjectionofdifferentrattissuesintorabbitsledtotheformationofdifferentautoantibodies.Thispapershowsthattheinjectionofratliver,kidney,brain,heart,muscleandspleeninFreund'scompleteadjuvantcausesautoantibodyformationinrabbits,thatsomeoftheseantibodiesareorgan-specificandthatbothmacroglobulinandsmallmolecularweightautoantibodiesoccur.*BeitMemorialFellow.Presentaddress:NationalInstituteforMedicalResearch,London,N.W.7.tMemberoftheM.R.C.ScientificStaff.19
G.L.AshersonandD.C.DumondeMATERIALSANDMETHODSANIMALSDutchrabbitsandwhiteadultratswereused.IMMUNIZATIONOrgansfromfreshlykilledratswerehomogenizedin0-25MsucroseandthenemulsifiedwithanequalvolumeofFreund'scompleteadjuvant(Difco)togiveafinalconcentrationof1in4ofwettissuew/v.Eachrabbitreceived0.5ml.oftheemulsionintoeachofthefourfootpads.At10weekstherabbitsreceivedfurtherfootpadinjectionsandat14weeks05g.wetweightoftissuehomogenizedin0*25MsucroseintraperitoneallyfollowedbyIml.of1/10homogenateintravenously.Theanimalswerebledoutandkilledat15weeks.SERATheserawereinactivatedatadilutionof1/4inveronal-bufferedcalciummagnesiumsalinepH7.i2(OxoidbarbitoneCFTdiluenttablets)forhalfanhourat560unlessotherwisestated.Thephraseheat-stableantibodyreferstoantibodydetectedinserainactivatedat650.COMPLEMENTFIXATIONThemethodpreviouslydescribed(AshersonandDumonde,1962)wasusedbut45minuteswereallowedforhaemolysisinsteadof30minutes.Theresultsareexpressedasthereciprocalofthehighestdilutiongiving2plus(50percent)haemolysisestimatedvisually.Ariseintitreoftwotubes(2log2units)wasacceptedassignificant.Thedilutionsofthetestantigens,intermsofwetweightoftissue,wererabbitliver,rabbitkidneyandratliver1/80,rabbitspleen,heartandmuscle1/40-1/50,rabbitbrain1/200-1/400,chloroformmethanolextractsofliverandkidney1/10,andofbrain1/20.DifcoKolmercardiolipinantigendiluted1/20wasusedasWassermannantigen.ABSORPTIONA1/10homogenateofrabbittissuein0'25Msucrosewhichhadbeenstoredat-180wasmixedwith4volumesofveronal-bufferedcalciummagnesiumsalineandleftat370for15minutesand4°for90minutes.Itwascentrifugedat144,700gfor30minutesandthedepositresuspendedinavolumeofbufferequaltothatoftheoriginalhomogenate.Thissuspensionwasmixedwithanequalvolumeofa1/4dilutionofserumtaken4weeksafterprimaryimmunization,leftat370for30minutesand40for30minutesandcentri-fugedat44,000gfor30minutes.Thesupernatantserumwasstoredat-18°andin-activatedbeforeuse.Asacontrol,unabsorbedserumwashandledinparallel.GELDIFFUSIONThishasbeendescribed(AshersonandDumonde,1962).Intheexperimentsonserotaken1monthafterprimaryimmunization,theantigenwellscontaineda1/3extracta20
Organ-SpecificAutoantibodiesrabbittissueinmolarglycinesaline.InthelaterexperimentstheantigenwellscontainedrabbittissueinmolarglycinesalinebufferedtopH7-2withsodiumbarbiturateand001percentethylenediaminetetraacetatewasaddedtotheextractandtheagar.SUCROSEGRADIENTCENTRIFUGATIONThemethodofCharlwoodwasemployed.Ten,20and30percentsucrosewaslayeredina12ml.'lusteroid'tubeandleftovernight.Onemillilitreofundilutedserumorserumdilutedwithanequalvolumeofsalinewasmixedwith1311-labelledhumanyglobulin(forwhichweareindebtedtoDr.P.A.Charlwood)andwascarefullylayeredontopandthetubescentrifugedat56,500gfor14hours.Thebottomofthetubeswaspuncturedbyaneedleandsuccessiveequalfractionseachslightlygreaterthan1ml.werecollected.Theradioactivitywasmeasuredandthefluidwasdialysedagainstcalciummagnesiumsalinebufferandtestedforantibodyactivitybycomplementfixation.Thelocationofthelowmolecularweightglobulinwasdeterminedbythe131I-labelledmarker,thatofthemacroglobulinbythedistributionofthenaturallyoccurringrabbitsheepcellhaemolysinwhichisamacroglobulin.CHLOROFORMMETHANOLEXTRACTIONOnegrammeoffreshrabbittissuewashomogenizedwithsalinetogiveafinalvolumeof10ml.,andaddedto100ml.ofa1:2chloroformmethanolmixture,leftatroomtemperaturefor24hours,andfiltered.Thefiltratewasevaporatedunderreducedpressureandthedepositdissolvedin5ml.oftoluene.Analiquotofthetoluenesolutionwastaken,thetolueneevaporatedunderreducedpressureandtheresiduetakenupinmethanol.Themilkyfluidproducedbyadding9volumesofbufferwasusedasantigen.Activitywasexpressedintermsoftheweightofrabbittissuefromwhichtheextractwasobtained.SOLUBLEANTIGENRabbittissuewashomogenizedin9volumesofsucroseusingaPotterhomogenizerandthenucleiremovedbycentrifugationat500gfor10minutes.Thedepositobtainedbycentrifugationat15,000gfor17minuteswaswashedinsucrose,resuspendedinM/2glycinebufferedwithveronal(pH7.2)anddisintegratedbyultrasonicvibrationat20,000cyclespersecondfor21minutes.Thesupernatantwasobtainedbycentrifugationat144,700gfor30minutes.RESULTSAUTOANTIBODYFORMATIONFOLLOWINGASINGLEINJECTIONOFRATTISSUEEighteenrabbitswereinjectedwithratliver,kidney,brain,heart,muscleorspleeninFreund'scompleteadjuvant.Beforeimmunization,thetitreofcomplement-fixingactivityoftheseraheatedat560againstrabbitkidneyrangedfrom16to364.Whentheserawereheatedat650,thetitrefellto<4inseventeenoftheeighteenrabbits.21
22G.L.AshersonandD.C.DumondeTable1showsthatafterimmunizationtherewasasignificantriseinthetitreofcomplement-fixingactivityagainstrabbitkidneyinatleasttwelveoftheeighteenrabbitswhentheserawereheatedat560andinfifteenoftheeighteenrabbitswhentheserawereTABLE1TITREOFCOMPLEMENT-FIXINGACTIVITYAGAINSTRABBITANDRATTISSUEHOMOGENATESINTHESERAOFRABBITSAFTERONEINJECTIONOFRATTISSUEINFREUNDSCOMPLETEADJUVANTImmunizingratantigenBleedTestantigenLiverKidneyBrainMuscleHeartSpleen17f1819121314910111234567815OaRatliver560b5446455<345536665641560>7>7>7>7>7>77>77656>7>7>7>77>74560>87>8878777667>78888>80Ratliver650<2<2<2<2<2<2<2<2<2<2<2<2<2<2<2<23<2165067743334322243424246508678664664366775780Correspondingrabbit560<33<35d>4d4d5<3<3<3<3<35556641organc56076>76d7d>7d>7764<35>776>77645605556d5d6d98>85<467667860Correspondingrabbit650<2<2<2<2e<2e<2e<2<2<2<2<2<2<2<2<2<22<21organc6503332e<2e<2e42<22<22<22<2<23<246505542e<2e<2e5642<25<22<2<23<2ORabbitkidney560544656>6556646>66>6651560>7>7>7>7>7>7>7>7>7766>7>7>7>77>74560>867888>878777>88788>80Rabbitkidney650<2<2<2<2<2<2<2<2<2<2<2<2<2<2<2<23<216503344333222<22232<23<24650756656543223343548aTimeinweeksafterimmunization.bTemperatureofinactivationofserum.cRabbitorgancorrespondingtoratorganusedforimmunization.dTestantigen-rabbitheart.eTestantigen-rabbitliver.fRabbitNo.Theresultsareexpressedasthelogarithmtothebase2ofthereciprocalofthehighestdilutionofserumgivingsignificantcomplement-fixation.heatedat65°.Asignificantriseinthetitreofcomplement-fixingactivityagainstthecorrespondingrabbitorgan(thatistherabbitorgancorrespondingtotheratorganusedforimmunization)occurredinallrabbitsinjectedwithratliver,kidney,brainandheart,intwooftherabbitsinjectedwithratmuscleandintwooftherabbitsinjectedwithratspleen.AUTOANTIBODYFORMATIONFOLLOWINGREPEATEDINJECTIONSOFRATTISSUEAfterthefourthinjectionofrattissueinFreund'scompleteadjuvanttherewasasignificantriseintitreofheat-stablecomplement-fixingactivityagainstrabbitkidneyin
Organ-SpecificAutoantibodies23tenoftheelevenrabbitstested.Table2showsthatineightrabbitstherewasasignificantriseintitreoftheheat-stablecomplement-fixingantibodyagainstthecorrespondingrabbitorgan.TABLE2TITREOFCOMPLEMENT-FIXINGAUTOANTIBODYINRABBITSBEFOREANDAFTERFOURTHINJECTIONOFRATORGANS(Seruminactivatedat650)TestrabbitantigenSerumImmunizingCorrespond-Homologousorganratantigeningauto-logousorgan*LiverKidneyBrainMustcleHeartSpleenPretPost+PrePostPrePostPrePostPrePostPrePostPrePost2Muscle<48<4<4<48<48<48<44<4<44Heart<416<4<4<416<4<4<44<416<4<45Heart<416<4<4<416<4<4<4<4<48<4<46Spleen<4<4<4<4<4<4<4<4<4<4<4<4<4<47Spleen<48<44<416<48<4<4<4<4<488Spleen<432<416<432<416<4<4<48<41615Spleen<4<4<4<4<416168<4<4<4<4<4<411Brain832<4<4<48832<4<4<4<4<4<412Kidney8128<416464<48<4<4<44<41617Liver<4<4<4<4<48<4<4<4<4<4<4<4<418Liver<432<432<4128<44<4<4<4<4<44*Rabbitorgancorrespondingtoratorganusedforimmunization.tSerumtaken10weeksafterprimaryimmunization.+Serumtaken7daysafterthefourthimmunization.Theresultsareexpressedasthereciprocalofthehighestdilutionofserumgivingsignificantcomplement-fixation.AUTO-REACTIVENATUREOFANTIBODYTable2alsoshowsthattheseraoftheseelevenrabbitsreactedincomparabletitreswithautologousandhomologousrabbittissues.Table4showsthatthehighestdilutionsofautologousandhomologousrabbittissuegivingsignificantcomplementfixationwithafixedquantityofantibodywerecomparable.DISTRIBUTIONOFANTIGENACTIVITYINRABBITTISSUESTable3recordsthehighestdilutionofadultandfoetalrabbitliver,kidney,brain,heartandmusclegivingsignificantcomplementfixationwithelevenseraheatedat65°,obtained1monthafterasingleinjectionofrattissue.
24G.L.AshersonandD.C.DumondeTABLE3ANTIGENTITRATION.MINIMUMCONCENTRATIONOFRABBITORGANHOMOGENATEREACTINGWITHIMMUNESERA*RabbitImmuniziglTestrabbitantigenserumConcentrationratantigenLiverKidneyBrainHeartMuscleAdultFoetalAdultFoetalAdultFoetalAdultFoetalAdultFoetal31/8Muscle650t<80<80320<20<160<40<10<201608051/32Heart5606406401280320102403203203201601601/8650<80<80160<20<160<40<10<20<20<1091/64Brain56°8016025603201024016020<2020401/8650<80<80640401024040<10<20s2010101/32Brain5603201601280320204801280404040801/8650320160320402048080<10<20420<10111/64Brain560<80<80128016020480640-404040401/8650<80<80160<2010240<40<10<2020<10121/64Kidney560160160128016051208020<20<20401/16650<80<8064080<160<40<10<20<20<10131/64Kidney560<80<80640802560<4020<20<20201/8650<80<80320<20<160<40<10<20<20<10141/64Kidney56016016012801605120<402040<20401/16650<80<80640160<320<40<10<20<20<10171/64Liver560160320640805120<4020<20<20201/8650128016032040t320<40<10<20<20<10181/16Liver560640320320805120402040<20201/86501280160160<20<160<40<10<20<20<10191/32Liver56025603206401601024080<10<20A20201/166501280160160<20<160<40<10<20<20<10C71/64560Infectionwith2560128025606402560640640320640320Eimeriastiedae*Serataken28daysafterprimaryimmunization.tTemperatureofinactivationofserum.Theresultsareexpressedasthereciprocaloftheminimumconcentrationofrabbittissuegivingsignificantcom-plement-fixation.TheserumofarabbitexperimentallyinfectedwithEimeriastiedae,thecauseofhepaticcoccidiosis,wasalsoused(AshersonandRose,1963).Thenineserafromrabbitsthatreceivedratliver,kidneyandbrainrevealedthehighestcomplement-fixingantigenactivityinthecorrespondingrabbitorgan.Thecomplement-fixingantigenactivityoffoetaltissuewaslessthanthatofadulttissue.Aftertheserahadbeenheatedat560theactivityoftheantigensfellintheorderbrain,kidneyandliverwhetheranti-brainoranti-kidneyserawereusedfortesting.Theanti-liverserarevealedmorecomplement-fixingantigenactivityinbrainthaninliver.
Organ-SpecificAutoantibodiesTable4presentsresultswithnineserainactivatedat650obtainedafterrepeatedinjectionsofrattissue.Theseraofrabbitswhichreceivedratliver,kidney,brainandheartrevealedthegreatestcomplement-fixingantigenactivityinthecorrespondingrabbitorganandrabbitkidney.TABLE4ANTIGENTITRATION.MINIMUMCONCENTRATIONOFRABBITORGANHOMOGENATEREACTINGWITHSERAOFRABBITIMMUNIZEDWITHFOURINJECTIONSOFRATORGANS(Seruminactivatedat65°)TestrabbitantigenImmunizingSerumratantigenCorrespondingHomologousorganautologousorgan*LiverKidneyBrainMuscleHeart,Spleen2tMuscle160<20320640640<20<204Heart64020320<40<20320<205Heart64020320<40<20320<207Spleen40160640640<20<20808Spleen16032012801280<208016011Brain2560<20320>5120<20<20<2012Kidney64012801280640<20408017Liver320640320160<20<20<2018Liver25602560640160<20<2080#Rabbitorgancorrespondingtoratorganusedforimmunization.tTheserawereusedatadilutionof1/4.ABSORP1'IONSTUDIESSerataken1monthafterprimaryimmunizationwereabsorbedwithrabbitkidneyandthecorrespondingrabbitorganhomogenates.Selectiveabsorptionwasobtainedwiththreeoutofthreeanti-liversera,twooutof'twoanti-kidneysera,threeoutofthreeanti-brainsera,oneoutoftwoanti-muscleseraandtwooutofthreeanti-spleensera.TypicalresultsaregiveninTable5.TheserumofrabbitNo.8,whichhadreceivedratspleen,hadatitreof64againsttheWassermannantigen.Thiswasremovedbyabsorptionwithrabbitkidneyandspleen.Theseresultsshowthattheserareactedwithantigenswithmuchhigheractivityinsomerabbitorgansthaninothers.GEL-DIFFUSIONSTUDIESTwoofthethreeanti-muscleseraandthreeofthethreeanti-brainserataken1monthafterimmunizationgaveprecipitationlineswiththecorrespondingrabbitorgan.Adoubtfullinewasobtainedwiththethreeanti-liversera.Oneoftheanti-spleenseragavealinewithrabbitserum.Theotherseraandseratakenbeforeimmunizationwereuniformlynegative.Thismadeitunlikelythatthelineswereartefactsofthetypedescribedby25
G.L.AshersonandD.C.DumondeTABLE5ABSORPTIONOFRABBITSERABYRABBITKIDNEYANDOTHERRABBITORGANSTestantigenRabbitImmunizingAbsorbed.-serumratantigenwithrabbitRatliverRabbitkidneyCorrespondingRatlivrRabbikidneyrabbitorgan19LiverNil1286432Kidney128<88Liver12864814KidneyNil25625664*Kidney1286416#Liver128256<8*10BrainNil64128256Kidney64S16256Brain6464<163MuscleNil128128128Kidney1283264Muscle128256325HeartNil256256128Kidney256161615SpleenNil25625664Kidney2563216Spleen2562568*Rabbitliverusedasantigen.Berenbaum,KitchandCope(1962).Theoneanti-muscleserum,thetwoanti-heartseraandthetwoanti-liverserapreparedbyrepeatedinjectionsofrattissuereactedwiththecorrespondingrabbitorganandotherwisefailedtoreactwithrabbitliver,kidney,brain,spleenora1/20dilutionoftheserumoftherabbitfromwhichtheorgansweretaken.Theserumpreparedbytherepeatedinjectionofratkidneygaveafaintlinewithrabbitheartandkidneyandoneofthefourserapreparedbytheinjectionofratspleengavelineswithrabbitliver,kidneyandheart.Thelinesproducedbytheanti-liverandanti-kidneyseradidnotstainforprotein.NATUREOFANTIBODYThesedimentationbehaviourofthecomplement-fixingactivityinsixserawasstudiedbysucrosegradientcentrifugation.Thehighestconcentrationofmacroglobulin,asshownbythetitrationofthenaturalrabbitsheepcellhaemolysin,occurredinfractions3and4.Thehighestconcentrationofthe131I-labelledhumanyglobulinoccurredinfractions7and8.Fraction1,takenfromthebottomofthetube,showednocomplement-fixingactivity.Thecomplement-fixingactivityoftwoserafromunimmunizedrabbits,containingnaturalantibody,andtwoserafromrabbitNo.18,taken7and28daysafterimmunizationwithratliver,appearedinfractions2-5.Thehighestconcentrationofactivityappearedinfractions3and4.Thesefractionscouldbediluted1/2to1/4beforelosingactivity.TheactivityintwoserafromrabbitNo.11,taken7and28daysafter26
immunizationwithratbrainappearedinfractions3-6and2-7.Thehighestconcentrationagainstrabbitbrainandkidneyappearedinfractions4and5.Thesefractionscouldbedilutedto1/8to1/16withoutlosingactivity.Itwasconcludedthatsomeofthecomple-ment-fixingactivityoftheseserahadthesedimentationbehaviourofmacroglobulin.Someofthecomplement-fixingactivityinthetwoserataken28daysaftertheinjectionofratliverandratbrainappearedinfractions7and8.Thisactivitywaslostondilution.Itwasconcludedthatthecomplement-fixingactivityofserataken28daysafterim-munizationhadthesedimentationbehaviourofboth7Sandmacroglobulin.Thecomplement-fixingactivityinthe7Sglobulinfractionswasunchangedbyheatingat650;thatofthemacroglobulinfractionwasdestroyedinmostsera.Theactivityinthemacroglobulinfractionofserumtaken7daysafterimmunizationshowedadropoftwotothreetubes,butcouldbedetectedintheundilutedfractions.Theseresultsshowedthatcomplement-fixingactivityassociatedwiththemacroglobulinfractionwasheatlabileat65°whileactivityassociatedwiththesmallmolecularweightyglobulinwasheatstable.NATUREOFANTIGENChloroformmethanolextractswerepreparedfromrabbitliver,kidneyandbrainandtestedforcomplement-fixingantigenactivitywithanti-liver,anti-kidneyandanti-brainsera.Theliverextractwasinactive.Table6showsthattwoanti-brainandtwoanti-TABLE6CHLOROFORMMETHANOLEXTRACTSOFRABBITTISSUE.ANTIBODYANDANTIGENTITRESTest(rabbit)antigenRabbitImmunizingExtractofExtractofserumratantigenrabbitkidneyrabbitbrainAntibodyAntigenAntibodyAntigentitretitretitretitre10Ratbrain168064204811Ratbrain81064102412Ratkidney3216032<2014Ratkidney32801620Theantibodytitreisthereciprocalofthehighestdilutionofserumgivingsignificantcomplementfixation.Theantigentitreisthereciprocalofthehighestdilutionofrabbitorganhomo-genategivingsignificantcomplementfixationwitha1/8dilutionofserum.kidneyserafixedcomplementwithkidneyandbrainextractsandthattheseserarevealedthehighestcomplement-fixingantigenactivityintheextractofthecorrespondingrabbitorgan.TheserumofrabbitNo.9taken1monthafterimmunizationwithratbrainwasinactive.Oneofthethreeanti-ratliverserareactedwiththerabbitbrainextract.Twooftheanti-brainserareactedinlowtitrewiththeWassermannantigen.Theanti-brainserareactedwithasolubleantigenpresentinthesupernatantpreparedbytheultrasonicdisintegrationoflowspeeddepositsofrabbitkidneyandbrain.Theanti-kidneyseraonlyreactedwiththekidneysupernatant.Liversupernatantwasin-active.Organ-SpecificAutoantibodies27
28G.L.AshersonandD.C.DumondeDISCUSSIONTheseresultsshowthattheinjectionofseveralratorgansinFreund'scompleteadjuvantcausedtheformationoforgan-specificcomplement-fixingactivityintherabbit.Thecomplement-fixingactivityintheserawasattributedtoantibodybecauseitbehavedondiethylaminoethylcellulosechromatographylikeamixtureofmacroglobulinandsmallmolecularweightyglobulin(AshersonandDumonde,1962)andonsucrosegradientcentrifugationlikemacroglobulinoramixtureofmacroglobulinandsmallmolecularweightyglobulin.Thetitresofantibodyactivityatconstantantigenconcentrationandofantigenactivityatconstantantibodyconcentration,measuredwithserainactivatedat65°,suggestedthattheanti-liver,kidney,brain,muscleandheartserashowedrelativeorganspecificity.Theabsorptionstudiesshowedthatsomeoftheanti-liver,kidney,brain,muscleandspleenserareactedwithatleasttworabbitantigens,whiletheOuchterlonyplateprovidedevidencefororgan-specificantibodiesinserapreparedagainstliver,brain,muscleandheart.Thetitrationofthecomplement-fixingactivityofchloroformmethanolextractsofrabbitbrainandkidneysuggestedthattheanti-brainandanti-kidneyserareactedwithtwodistinctantigenssolubleinfatsolvents,oneoccurringinhighconcentrationinrabbitbrainandtheotherinrabbitkidney.BrainantigenofthistypewasdescribedbyWitebskyandSteinfeld(1928).Thetitrationofantigenactivityusingserainactivatedat650providedevidenceoforgan-specificautoantibody.However,whentheserawereinactivatedat560,theantigenactivityofrabbitbrainwasgreaterthanrabbitliverorrabbitkidneywhethertheserumwaspreparedbytheinjectionofratliver,kidney,brainorheart.Itwasconcludedthattheseseracontainedarelativelyheatlabileautoantibodywhichreactedwithawidelydistributedantigenandaheat-stableantibodywithanorgan-specificcomponent.Thesimilarityofthetitresobtainedwithautologousandhomologousrabbittissueshowedthattheseracontainedtrueautoantibodiescapableoffixingcomplementwiththetissuesoftherabbitinwhichtheyoccurred.Theseresultsdonot,however,excludethepossibilitythattheseraalsocontainedisoantibodiesresemblingthosedescribedbyRose,MetzgarandWitebsky(1960).Thefindingofautoantibodiesabletoreactwiththetissueoftherabbitinwhichtheyoccurredindicatedthatatleastsomeoftheautoantigenswerenotfreelyaccessibletocirculatingautoantibody.Theautoantibodyfoundintheserabeforeimmunizationresemblednaturalauto-antibody.Thishasbeenshowntobedestroyedat65°andtolackorganspecificity(KiddandFriedewald,1942).However,agroupofeightunimmunizedanimalspurchasedfromadealercontainedcomplement-fixingautoantibodieswhichwereheatstableat650.Thetitresagainstrabbitkidneyafterheatingtheseraat56°rangedfrom32to128.Afterheatingat650thetitresrangedfrom8to32.Themeandropwas2-25log2units.Neverthelessthefindingofheat-stableandorgan-specificautoantibodiesfollowingtheinjectionofrattissueshowsthattheriseinautoantibodytitreswasnotmerelyduetoanincreaseinthelevelofnaturalautoantibody.AshersonandDumonde(1962)showedthatcertainautoantibodieslabileat65°behavedchromatographicallylikemacroglobulins.Wehavenowshownbysucrosegradientcentrifugationthatmacroglobulinantibody-isheatlabile,whilesmallmolecularweightantibodyisheatstable.Becauseofthelowantibodytitresinthefractionsobtainedbygradientcentrifugationthepresenceofsmallamountsoflowmolecularweight28
Organ-SpecificAutoantibodies29antibodyinseracouldnotbeexcluded.WiththisprovisothenaturalautoantibodyofKiddandFriedewaldandtheautoantibodiesformed1weekaftertheinjectionofrattissuewereapparentlymacroglobulinswhilethoseantibodiesformed1monthafterimmunizationcontainedboth7Sandmacroglobulinantibody.ThisisinkeepingwiththefindingofBauerandStavitsky(1961)andBenedict,BrownandAyengar(1962)thattheearlyantibodyresponsetoproteinantigenscontainsalargermacroglobulincomponentthanthelaterresponse.ACKNOWLEDGMENTSWeareindebtedtoProfessorE.G.L.BywatersandDr.L.E.GlynnfortheirencouragementandadviceandtoMrs.C.M.WalterandMr.G.Kennyforexperttechnicalassistance.WeareespeciallygratefultoDr.P.A.Charlwoodwhointroducedustothetechniqueofsucrosegradientcentrifugation.REFERENCESASHERSON,G.L.andDUMONDE,D.C.(1962).'Char-acterizationofautoantibodyproducedintherabbitbytheinjectionofratliver.'Brit.j.exp.Path.,43,12-20.AsHERSON,G.L.andDUMONDE,D.C.Tobesub-mittedforpublication.ASHERSON,G.L.andROSE,M.E.(1963).'Autoanti-bodyproductonintherabbit.III.TheeffectofinfectionwithEimeriastiedaeanditsrelationtonaturalantibody.'Immunology(Inpress).BAUER,D.C.andSTAVrrSKY,A.B.(1961).'Onthedifferentmolecularformsofantibodysynthesizedbyrabbitsduringtheearlyresponsetoasingleinjectionofproteinandcellularantigens.'Proc.nat.Acad.Sci.Wash.,47,1667-80.BENEDICT,A.A.,BROWN,R.J.andAYENGAR,R.(1962).'Physicalpropertiesofantibodytobovineserumalbuminasdemonstratedbyhaemagglutina-tion.'J.exp.Med.,115,195-208.BERENBAUM,M.C.,KITCH,G.M.andCOPE,W.A.(1962).'Spurious"auto-immune"reactionsingel-diffusionplates.'Nature(Lond.)193,81-2.CHARLWOOD,P.A.Personalcommunication.KIDD,J.G.andFRIEDEWALD,W.F.(1942).'Anaturalantibodythatreactsinvitrowithasedimentableconstituentofnormaltissuecells.I.Demonstrationofthephenomenon.'J.exp.Med.,76,543-56.KIDD,J.G.andFRIEDEWALD,W.F.(1942).'Anaturalantibodythatreactsinvitrowithasedimentableconstituentofnormaltissuecells.II.Specificityofthephenomenon:generaldiscussion.'J.exp.Med.,76,557-78.ROSE,N.R.,METZGAR,R.S.andWITEBSKY,E.(1960).'Studiesonorganspecificity.XI.Isoantigensofrabbitpancreas.J.Immunol.,85,575-87.WITEBSKY,E.andSTEINFELD,J.(1928).'Unter-suchungenuberspezifischeAntigenfunktionenvonOrganen.'Z.Immun.-Forsch.,58,271-96.BIMMUN.