專業(yè)英語 Unit 3教案學(xué)習(xí)資料

UnitThreeULTRASTRUCTURALANDIMMUNOHISTOCHEMICALFINDINGSINORALHAIRYLEUKOPLAKIAVirchowsArchivAPatholAnatHistopathol(1988)412:533-542Summary.ThreecasesofHLfromthelateralborderofthetongueofmalehomosexualAIDSpatientswereinvestigatedbythinsectionelectronmicroscopy.Keratinocytescontainedcondensedchromatinintheirpyknoticnucleiandafeworganellesintheedematouscytoplasm.Chromatinwasincloseassociationtothenuclearmembraneandshowedapunched-outappearance.Particletypicaloftheherpesvirusgroupwasabundantintheuppertwothirdsoftheepitheliuminallthreecases.Virusparticleswereseenfrequentlyinthenucleioftheballoonedkeratinocytes,butrarelyincellscontainingCandidaalbicans.Viralnucleocapsidswereobservedbuddingattheinner-nuclearmembrane,therebyacquiringtheprospectiveviralenvelope.Complete,envelopedvirionswerefoundintheendoplasmicreticulumandintheextracellularspace.ThesevirionswereidentifiedimmunohistochemicallyasEpstein-Barrvirus(EBV)usingtwomonoclonalantibodiesdirectedagainstEBVcapsidandmembraneantigen,respectively.Candidaalbicanswasobservedinthestratumcorneumandintheupperlayerofthestratumspinosum.Specialcytoplasmictubularstructuresarrangedinparallelbundleswerefoundinkoilocytoticcellsinadditiontocharacteristicmembranestructurescomposedofundulatingconvolutedmembranes.Epithelialbasementmembraneswerealwaysintact.Keywords:Hairyleukoplakia,Electronmicroscopy,Epstein-Barrvirus,CandidaalblcansIntroductionOralhairyleukoplakia(HL),anewclinicalentity,wasfirstdescribedbyGreeenspanetal.(1984)aspathognomonicfortheHIV-infection.HLischaracterizedbywhitishplaquesatthelateralborderofthetongue,whichcannotberubbedoff.Thelesion,mainlyobservedinHIV-infectedhomosexualmales,wasalsoreportedinheterosexualHIV-infectedindividuals,hemophiliacs,atransfusionrecipient(Greenspanetal.1986;Rindumetal.1987),andintravenous(iv)drugabusers(Reichartetal.1986,L987).AsurvivalanalysisshowedthattheprobabilityofdevelopingAIDSinpatientswithHLwas40%during16monthsand83%during31months(Greenspanetal.1987)Lightmicroscopicinvestigations(Greenspanetal.1984;Greenspanetal.1985;Eversoleetal.1986)indicated,thatthelesionischaracterizedbyparakeratosis,acanthosis,koilocytosisandlackofinflammationinthesubepithelialtissue.Usingelectronmicroscopy(EM)particlesoftheherpesvirusgroupwerefoundinHL(Greenspanetal.1984,1985;BeltonandEversole1986;Konrad1986).ImmunofluorescencestudiesdemonstratedEpstein-Barrvirus(EBV)antigen,andanucleicacid-hybridizationprocedurerevealedEBVviralDNAinahighcopynumber(Greenspanetal.1985;Loningetal.1987)PurposeofthepresentstudywastodescribeultrastructuralfeaturesofHLandtoidentifythevirionparticlesusingmonoclonalantibodiesagainstEBVcoreandmembraneantigens.MaterialsandmethodsFromJuly1984toJuly1987among195HIVseropositiveindividualsatotalof61patients(malen=59,femalen=2;homo-/bisexualn=55,i.v.drugabusetsn=5,bloodtransfusionrecipientn=l)withtheclinicaldiagnosisofHLhavebeenobserved.TheaverageageofHLpatientswas34.2years.AtthetimeofserodiagnosisfourHIVpositiveindividualswereclinicallysymptomless,18patientsshowedARC,and39patientsAIDSsymptoms.ForthepresentstudybiopsiesofHLwereobtainedfrom3malehomosexualpatients(age39,46and83years),showingdifferentAIDSmanifestations(multipleKaposisarcoma:n=l,opportunisticinfections:n=2).Allthreepatientsdiedwithin2.8monthsafterbiopsy.Biopsiesweretakenfromthelateralmarginofthetongueunderlocalanesthesia(UltracainDSR).Priortobiopsyallthreepatientshadbeentreatedfortwoweekswithtopical(Mikonazole),twopatientsadditionallywithsystemicantimycotictherapy(Ketokonamle).Tissuesweredividedandonepartwasfixedin2.5%glutaraldehydeinPBSfor3hours.AfterwashingwithPBSspecimenswerepostfixedinl%OSO4forlhat4?C,andafterwardstreatedforlhatroomtemperaturewithinl%uranylacetate.Afterdehydrationinagradedseriesofethanol,specimenswereinfiltratedby3changesofpropyleneoxideandembeddedinEpon812followingroutinetechniques(Gelderblometal.1974).Semithinsections(0.5-1minthicknessofeachspecimenswerecut,stainedwithtoluidineblue,andevaluatedlightmicroscopicallyforpathognomonicchanges.Afterfindingsuspectiveareasinlightmicroscopy,ultrathinsections(40-60nm)werecut.Theseweremountedonbaregrids,poststainedwithleadcitrate,stabilizedwithcarbonandexaminedusingaZeissEM10Aat60kV.Theotherpartofthebiopsywasfixedindimethylsuberimidate(DMS,HasselandHand,1974)for2h,washedinPBSandinfiltratedstepwisewith5%,l.0,l.2Mand2.3MsucroseinPBS.Biopsiesweredeepfrozeninliquidnitrogen.Semithincryosectionsof0.5mwerecutusingtheReichertFC4cryoattachment(ReichertAG,Wien).SectionswereincubatedwithmonoclonalantibodiesdirectedagainstEBVcapsid(VCA)andmembraneantigen(MA)(dilutionl:1000inPBS).Immunobindingwasvisualizedusingamodificationofthealkaline-phosphatase-mouse-anti-alkaline-phosphatasetechnique(APAAP;Beckeretal.1987).Controlsincludedtheuseofsecondandthirdstepantibodies,aswellasnormaltissuesofHIVseronegativepersons.ResultCommonfeaturesinallcaseswerehyperkeratosis,parakeratonsisandacanthosisofepithelium.Abovethebasalcelllayeranincreaseddegreeofintracellularedemaofspinouscellswasnoted.Theballoonedkeratinocytesoftenlosttheirchromatinpatternandsometimesbasophilicintranuclearinclusionswereobserved.Candidaalbicanswasfoundwithinepithelialcellofthesuperficiallayers.Infiltrationbyinflammatorycellswasnotapparent,neitherinthelaminapropriaofthemucosanorintheepithelium.Inallcases,balloonedkeratinocyteswereseeninclusterswithinthespinouslayeroftheepitheliumonelectronmicroscopy.Thesecellswereincreasedinsizeandcontainedonlyfeworganelles,includingdegeneratedmitochondriaandabundantintermediatesizedkeratinfibrils.Fociofcondensedchromatinwithapunched-outappearancewerefoundinpyknoticnucleimainlyincloseassociationwiththenuclearmembrane.Candidaalbicanswasfrequentlyobservedinthestratumcorneumaswellasintheupperlayersofthestratumspinosum.Virusparticlesoftheherpesvirusgroupwerepresentintheupperspinouslayermainlyinandaroundkoilocytoticcells,andintheintercellularspaceofflattenedcellsofthesuperficiallayers.ViruswasalsoobservedoccasionallyincellsinfectedbyCandidaalbicans.Thenucleiofinfectedcellscontainedvaryingamountsofviralnucleocapsidswithadiameterof100nm;whilesomecapsidsalreadycontainedtheelectron-densetoroidaldesoxyribonucleoproteinportiontypicalforherpesviruses,othersrepresentedemptyproteincapsids.Occasionally,theegressofherpesviruscapsidsfromthenucleiwasobserved.Duringegressthenucleocapsidacquiresitsfutureviralenvelopemostlyfromtheinnernuclearmembraneinabuddingprocess.Themembranewasthickenedinregionsofnucleocapsidenvelopment.Virusparticleswerealsofoundinthecytoplasm,eitherfreeasnucleocapsidsorascompleteenvelopedparticlescontainedwithintheendoplasmicreticulum.Virionswerefinallyreleasedinabundantnumbersintotheextracellularspace.Enveloped,matureherpestypevirionswithinthecytoplasmandintheextracellularspaceshowedidenticaldiametersof150nm.Twocharacteristicstructuresoccurredwithinthecytoplasmofballoonedkeratinocytesinfectedbythevirus.Oneconsistedoftubules,35nmindiameter,arrangedinparallelbundlesaboutlpminlength.Anotherinclusionbodywascomposedofhighlyundulating,convolutedmembranes.Thesewereobservedclosetothenucleiofkoilocytoticcellsconsistingoftwo,partlyfusedlipidbilayers.Theinnerfusedpartofthisstructurewaselectrondenseand25nminwidth.Thesubepithelialbasementmembranedidnotrevealanyirregularities.LangerhanscellswerenotobservedwithintheHLlesions.AspecificimmunostainingwasnotedintheuppertwothirdsoforalepitheliausingsemithincryosectionsandEBV-specificmonoclonalantibodies.AntibodiesagainstEBVcapsid(VCA)andmembraneantigens(MA)revealedacompletelydifferentstainingpattern.WhileVCAwaspredominantinandaroundthenucleiofkeratinocytes,MAwasobservedinhighconcentrationintheinterepithelialspacesinthesametissueareas,whereEBVVCAwasobservedinconsecutivesections.DiscussionHL,anexclusivelyorallyoccuringlesion,wasreportedtobeanearlyindicatorofHIV-associatedimmunosuppression(Eversoleetal.1986;Silvermannetal.1986).TheoccurrenceofHLindifferentriskgroups,asobservedinthepresentstudy,haswellbeendocumentedintheliterature(Greenspanetal.1984,1986;Reichartetal.1987;Rindumetal.1987).TheaetiologyofHLhaslongbeensupposedtobevirus-relatedandinmostinvestigationsaherpestypeviruswasdetected(Greenspanetal.1984.1985;Konradetal.1986).Furthermore,insomeofthesestudiespapillomavirus-likeparticleswereobservedinadditiontotheherpestypevirus(Greenspanetal.1984,1985).WhileCreenspanetal.(1984,1985)reporteddoubleinfectionsinHLbyEBV(usinginsituhybridization)andHPV(EM,immunohistochemistry),wehavenotbeenabletodetectpapillomavirusinHLontheEMlevelinthisinvestigation.InafurtherinsituhybridizationstudyofsevenHLallspecimenswerenegativewiththeappliedHPVtype6,11,13,16,18probeswhilefiveofthesespecimenswereEBVpositive(Loningetal.1987)Fromtheobservationthatpapillomavirus-likeparticleswerepresentinall23casesofHLattheEMlevelGreenspanetal.(1985)consideredwhetherthecoexistenceofEBVandHPVinHLisfortuitous.SinceHPVwasnotdemonstratedinourthreecasesorinafurther7cases(Loningetal.1987)ourfindingsdonotsupporttheconceptofsimultaneousinfectionofHL.However,negativefindingsregardingHPVdonotruleoutapossibleinvolvementofasecondvirus.IndifferenttissuesfrompatientswithARCorAIDSintracytoplasmicinclusionshavebeendescribed,appearingasvesicularrosettes,testtubeandring-shapedforms(TRF),ortubuloreticularstructures(TRS)(Onerheimetal.1984;Kuntzetal.1987).WithinthecytoplasmofEBVproducingcells,weobservedonlyonetypeofparalleltubularstructures,whichresembledinbothsizeandappearancetheinclusionsdescribedbyBeltonandEversole(1986).Theoriginandnatureoftheseaggregatesisasyetunknown.Whiletheoccurrenceoftheseparallelarranged,intracytoplasmictubularstructureshasonlybeendescribedinHL.TRFinclusionshavealsobeenfoundinassociationwithdifferentimmunologicalandneurodegenerativediseasesaswellasinviralinfections.Thesemembranederivedinclusionsappearedtobeassociatedwiththeproductionofalpha-andbeta-interferon,butnotofgamma-interferon(Onerheimetal.L984).Theoccurrenceoftheseaggregatesmaybeamorphologicalindicatorofinterferonproduction,inducedbyviralinfection,andmayreflecttheself-perpetuatingimmunologicaldysfunction.Inaddition,undulatingmembranestructureswereobservedwithinthecytoplasmofkoilocytoticcells,consistingofpartlyfusedmembranessimilartotheabovementionedTRF/TRS.ItisprobablethatthesemembranestructuresareindeedprecursorsofTRF/TRS.WedidnotobserveLangerhanscellsorothersignsofcellularimmunereactionintheepithelium.ClearanceofCandidaalbicansbycellularimmunemechanismswasnotfoundinourstudynorinotherreports(BeltonandEversole1986).ThisisprobablyduetodefectivemonocytemigratoryfunctioninAIDS-patientsandgivesreasontoexpectfurtherfunctionaldisturbancesinantigenprocessingandpresentationbymonocytesandLangerhanscells(Danielsetal.1987;Beckeretal.1987).RegardingtheetiologyofHL,ourEMandimmunohistochemicalfindingscorroboratethecloseassociationofEBVwiththislesion.Fromclinicalobservationsthevirusseemstobeinvolvedinthepathogenesis.ofHL,sinceduringtreatmentwithhighdosesofacyclovir(4x800mg/dayduring14days)clinicalregressionofthelesionhasbeenfound(Freidman-Kien1986).TheinvolvementofEBVinHLmaybeimportantforthefurtherprogressoftheundelyingHIVdisease,becauseEBVisknowntobecapableoftransformingB-lymphocytessothatHIV(Montagnieretal.1984)caninfectthem.TherecruitmentofsuchtransformedB-cellsinadditiontotheCD4+T-helperlymphocytes,monocytes/macrophagesandLangerhanscellsmayenhanceHIVproductionconsiderably.ThisthendirectlyleadstoahigherlevelofinfectedcellsengagedinHIVreplication,andconcomitantlytoacontinuouslyincreasinglossofCD4+-lymphocytesbydirectcytotoxicityofHIVand/orbythemanifoldHIVinducedimmunopathogenicmechanisms(Gelderbolometal.1985;KlatzmannandGluckman1986).Finally,themassesofEBVproducingcellsinHLareindicativeofasevereimmunodeficiencyobservedinthesepatients.VOCABULARYl.hairyleukoplakia毛狀白斑2.homosexual同性交的n．同性戀者3.AIDS(AcquiredImmunodeficiencySyndrome)艾滋病4.thinsection超薄切片的別稱(電鏡技術(shù)用語)5.semithinsection半薄切片(電鏡技術(shù)用語)6.condensedchromatin“濃縮的”染色質(zhì)7.punched-outappearance穿孔樣表現(xiàn)(改變)8.pyknoticnuclei“皺縮的”細(xì)胞核9.herpesvirusgroup皰疹病毒族10.balloonedkeratinocyte“汽球狀”角質(zhì)細(xì)胞11.Candidaalbicans白色念珠菌12.nucleocapsid核殼，外殼13.bud芽，vi．芽生14.viralenvelop病毒包膜15.endoplasmicreticulum內(nèi)質(zhì)網(wǎng)16.extracellularspace細(xì)胞間隙17.Epstein-BarrvirusEB病毒(γ組皰疹病毒)18.monoclonalantibody單克隆抗體19.against抗…20.stratumcorneum角質(zhì)細(xì)胞層21.stratumspinosum棘細(xì)胞層22.koilocytoticcell“凹狀”細(xì)胞23.undulatingconvoluted波狀的，折迭狀的24.basementmembrane基膜25.pathognomonic特定的癥狀，有確診意義的癥狀、表現(xiàn)26.HIV-infectionHIV感染27.plaquen．斑，溶菌斑；血小板28.ruboff擦掉(撕脫)29.heterosexual異性戀的n異性戀者30.hemophiliac血友病人31.intra-venousdrugabuser靜脈吸毒者32.survival生存，成活33.parakeratosis不全角化癥34.acanthosis棘層增生癥35.subepithelialtissue上皮下組織36.nucleicacid-hybridization核酸雜交37.EBVcoreEB病毒的“核”38.seropositive血清陰性的39.homo-/bisexual同性／同性、異性戀的40.serodiagnosis血清學(xué)診斷41.ARC(AIDSRelatedComplex)艾滋相關(guān)癥候群42.biopsy活體組織的病理檢查43.multiple多重的，多倍的44.KaposiSacoma卡波濟(jì)肉瘤45.opportunisticinfection機(jī)遇性感染46.prior在前面的47.glutaraldehyde戊二醛48.washing刷洗49.postfixed后固定的50.uranylacetate醋酸雙氧鈾51.dehydration脫水52.gradedseriesofethanol梯度酒精53.infiltrate浸潤54.propyleneoxide氧化丙烯55.Epon812包埋電鏡標(biāo)本的一種環(huán)氧樹脂56.toluidineblue甲苯胺藍(lán)57.ultrathinsection超薄切片58.nm(nanometre)納米，毫微米59.mounted裱的60.baregrid裸(銅)網(wǎng)，超薄切片的戴網(wǎng)61.leadcitrate檸檬酸鉛62.stabilizedwithcarbon(噴)鍍碳膜63.dimethylsuberimidate(DMS)類似于二甲基亞礬的一種冷凍保護(hù)劑64.stepwise逐步的65.sucrose蔗糖66.cryosection冷凍切片67.cryoattachment作冷凍切片的附加設(shè)備68.incubate孵育69.immunobinding免疫結(jié)合(粘合)70.visualize顯現(xiàn)71.modification改良72.control對照73.secondantibody第二抗體74.seronegative血清陰性的75.adhering粘附76.hyperkeratosis過度角化癥77.basophilic嗜堿性的78.intranuclearinclusion核內(nèi)包涵體79.superficiallayer淺層80.laminapropria