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Chapters study guide about complement, chapters 3 and 4
Typology: Study notes
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Exam 2 StudyGuide Chpt. 3 and Chapter 3 1 ) Whenthefollowingreceptorsareengagedwhatmechanism isinitiatedinaphagocyte? Mannosereceptor,CR 3 andCR 4 ,orScavengerreceptor. Thesereceptorsaremacrophagephagocyticreceptors.Whenengaged,theyinducephagocytosisbymeans ofopsonization. MannoseReceptor–PhagocyticPRR,targetsbacteria.Ligands:LPSonGram(-)bacteria CR 3 &CR 4 – BindtoiC 3 bfragmentsaswellasLPS ScavengerReceptor–Preferencefornegativelychargedmolecules 2 ) Whenthefollowingreceptorsareengagedwhatmechanism isinitiatedinamacrophage? TLR’s. TLR(TollLikeReceptor)–cytokinesignalingreceptors,NOTphagocytic.Theyengageinproductionand secretionofcytokines. 3 ) WhatdoTLR3, 4 , 5 and7bindto? Location Cell Ligand Organism Recognized TLR3 Endosome NKCells Doublestrand RNA RNAVirus TLR4 PlasmaMembrane Macrophage,Mast Cell,Dendritic Cell,Eosinophil LPS Gram(-)Bacteria TLR5 PlasmaMembrane Intestinal Epithelium Flagellin Bacteria TLR7 Endosome Dendriticcells,B- Cells,Basophiles, Eosinphil SinglestrandRNA RNAVirus 4 ) KnowtheTLR 4 signalingmechanism.AlloftheextracellularincludingLBPand intracellular,MyD 88 - TIRdomainandIKK-NFkB- signaling. BacterialLPSfromGram(-)BacteriaisbroughtbyLBP(LPSBindingProtein)tothecomplexofTLR 4 , MD 2 ,andCD 14 onthesurfaceofthemacrophage MyD 88 adaptorproteinbindstoTIRdomain,theTLR 4 componentinsidetheplasmamembrane Thissetsofachainreaction.BindstoproteinkinaseIRAK 4 whichphosphorylates,thenphosphorylates TRAF 6 adapterprotein. ThisleadstophosphorylationandactivationofkinasecomplexcalledIKK,whichactivatestranscription factorNFkB.
NFkBisheldincytoplasmwithinhibitorIkBininactiveform.IKKphosphorylatesIkB,releasingNFkB, whichtravelstothenucleusandbeginstranscriptionofcytokines. ThesearesecretedviaEndoplasmicReticulum 5 ) Inthepreviousquestion,what 5 genes(thatyoulearnedabout)aretranscribedbecauseof theTLR- 4 recognition? IL- 1 andTNF-a–vasodilationandvascularpermeability IL- 6 – metabolizesfat+musclecellstoraisetemperature CXCL 8 – recruitsneutrophils(chemokine) IL- 12 – recruitsNKcells(chemokine) 6 ) Whatisthegeneralterm giventothese 5 geneproducts?(Hint; 2 partnameincludingwhat theyresultinalongwithwhattypeofmoleculetheyare) InflammatoryCytokines,responsibleforinflammation 7 ) Knowwhatneutrophilsdoandwhatlineagetheycomefrom duringhematopoiesis. Neutrophilsare Granulocytes(granulesincytoplasm) Pyogenic–formpus Leukocytes–whitebloodcells Polymorphonuclear–multi-lobednucleus Primaryphagocyticcellsoftheinnateimmunesystem.Mostcommonincirculation. Lineage:Hematopoieticstemcellinbonemarrow myeloidprecursor granulocytesneutrophil 8 ) Whatisanothernameforneutrophils. Polymorphonucleargranulocytes 9 ) Knowthe 4 classesofadhesionmoleculesandwhichadhesionmoleculetheybindto.Ex; LFA- 1 bindstoICAM- 1 andvascularaddressin(CD- 34 bindstoL-selectin) 4 Classesofleukocyteadhesionmolecules–determinemovementofleukocytesfrombloodtotissue Selectins(LSelectin)bindtoaddressins(CD 34 ) Vascularaddressins(CD 34 )bindtoselectins(LSelectin) Integrin(LFA- 1 )bindstoIgsuperfamilyproteins(ICAM) IgSuperfamily(ICAM- 1 )bindstointegrins(LFA- 1 ) 10 )Knowthe 4 stepsofextravasation. Extravasation–neutrophilsleavebloodintotissuebysqueezingthroughgapsinepithelialcells Step 1 :RollingAdhesion Cytokines/inflammatorymediatorsinduceselectinexpressiononvascularendotheliumtobindneutrophils Rollingadhesionsslowneutrophilsdown
Step 2 :TightBinding Inducedby: LFA-ICAM interaction(leukocyteadhesionmolecules) CXCL 8 toCXCL 8 R(chemokinesandreceptor) Step 3 :Diapedesis Neutrophilpenetratesandsqueezesbetweenendothelialcellsintoconnectivetissue Step 4 :Migration CXCL 8 guidestheneutrophiltoinfectedarea 11 )Howcanyoutellthedifferencebetweenageneralcytokinevs.achemokine? Chemokinesareatypeofcytokinethatdirectscelltraffic. 12 )Neutrophilgranulesandthenamesofthem. Primary–Azurophilic Secondary–Specific Tertiary–Gelatinate 13 )Respiratoryburstassociatedwithneutrophils. Respiratorybursts–Poweringneutrophilattack,increaseinoxygenconsumptiontoraisepHofthe phagosomesothegranulecontentscanbecomeactivetokillpathogen.Thisproducestoxicoxygenspecies thatdiffuseoutanddamagehostcells.Tolimitthisdamage,enzymesaresynthesizedthatinactivatethese molecules. 14 )Whatarethe 2 acutephaseproteinsyoulearnedaboutandwhatcantheydo?Iam excludingserum amyloidAfrom this.Hint:Wetalkedabouteachbeingabletodotwo things. C-ReactiveProtein(CRP)–activatesclassicalpathwayofcomplement,alsoactsasopsonin MannoseBindingLectin(MBL)–activateslectinpathwayofcomplement,alsoactsasopsonin Thesearecreatedinthehepatocytes(livercells) 15 )Knowall 3 complementpathways.Whathastobepresentforthem tostart,theinitiating enzymeandallthefollowingcomponentscutinorder. AlternativePathway C 3 proteinisturnedintoiC 3 (C 3 H 2 O)throughhydrolysis.Thisinitiatesthepathway. FactorBbindstoiC 3 FactorDarrivesandcleavesFactorBintoBbandBa.Baleaves,andBbstaysattachedtoiC 3 toform iC 3 Bb.ThisisaC 3 Convertase,soitsplitsotherC 3 proteins iC 3 BbcleavesC 3 proteinsintoC 3 aandC 3 b.
C 3 bbindstosurfaceofpathogen.Itcanfunctionasanopsonin,orcontinuethepathwaytobecomeaC 3 convertase. FactorBbindstoC 3 b,andiscleavedbyFactorD,creatingC 3 bBb.ThisisthealternativeC 3 convertase.It cancleaveotherC 3 proteins. ItsplitsevenmoreC 3 proteinsintoC 3 aandC 3 b. IfaC 3 bfragmentbindstotheC 3 bBb,itbecomesC 3 b 2 Bb,whichisthealternativeC 5 convertase.Thiscan splitC 5 proteins. LectinPathway MBLattachestopathogensurface MASP- 2 cleavesC 4 intoC 4 aandC 4 b C 4 bbindstopathogen MASP- 2 cleavesC 2 intoC 2 aandC 2 b.C 2 aisthelargerfragment. C 2 abindstoC 4 btomakeC 4 b 2 a,theclassicalandlectinC 3 convertase.ThiscancleaveC 3 proteinsinto C 3 bandC 3 a. C 3 bcanbindtoC 4 b 2 atomakeC 4 b 2 a 3 b,theC 5 convertaseofthelectinandclassicalpathways ClassicalPathway C-ReactiveProtein-OR-oneIgM -OR-twoIgGbindstosurfaceofpathogen.C 1 bindstotheantibody. C 1 scleavesC 4 andC 2 tocreateC 4 b 2 a,theC 3 convertase. PathwaycontinuesthesamewayasLectin. 16 )Nameofthecomplementconvertasesandtheircomponents. Alternative ClassicalandLectin C 3 Convertase iC 3 BbandC 3 bBb C 4 b 2 a C 5 Convertase C 3 b 2 Bb C 4 b 2 a 3 b 17 )TypeIinterferonsvs.TypeIIinterferons. Interferonsaresensorproteinsincytoplasmthatcandetectvirusnucleicacids.Resultsintheproductionof cytokinesandinterferons. TypeIInterferons AlphaandBeta Interfererswithviralreplication Alertsimmunesystem AllnucleatedcellscanproduceTypeIinterferons TypeIIInterferons OnlyNKCellsandCytotoxicT-Cells(CD 8 )andT-HelperCells(CD 4 )canmakeit
Gamma 18 )WhatcellscanproduceTypeIinterferons? Allnucleatedcells 19 )WhatisthemaininnateimmunecellactivatedbyTypeIinterferons? TypeIinterferonsactivateNKcells 20 )Knowthe 2 effectorfunctionsofNKcells.Hint;oneiscytotoxic Cytotoxicfunction–killinginfectedcellsviaapoptosis.Partofinnateimmunity ProduceTypeIIinterferons 21 )WhattypeofinfectiondoNKcellstypicallyfightagainst? Viralinfections 22 )WhattypeofresponseiscreatedinacellthatanNKcellhastargeted(virallyinfected)? Apoptosis 23 )WhatcytokinedoesanNKcellsecretetoactivatemacrophages? TypeIIinterferon 24 )WhathappenstovirallyinfecteddendriticcellswhenNKcellsoutnumberthem?What happenstovirallyinfecteddendriticcellswhentheyoutnumberNKcells? WhentherearemoreNKcellsthaninfecteddendriticcellstheNKcellsarecytotoxictothe dendriticcells WhentherearemoreinfecteddendriticcellsthanNKcellstheNKcellssignalthedendritic cellstomigratetothenearestsecondarylymphoidtissuetoactivatetheadaptiveimmune system Chapter 4 1 ) WhatdoesthegermlineconfigurationoftheIgvariableregiongeneslooklikeinallcells exceptmatureB-cells? WhatdoesthegermlineconfigurationoftheTCRvariableregion geneslooklikeinallcellsexceptmatureT-cells? Hasnotgonethroughsomaticrecombination.L,V,D,andJgenesegments.Uncut.ContainsDNAexons. 2 ) Whatgenesegmentsneedtocometogethertomakeafunctionalgene(forthevariable region)inB-cellsforheavyandlightchains? Heavychain:V,J,andDgenesegments LightChain:VandJgenesegments 3 ) Whatarethedifferentisotypesofheavychainsandlightchains?
HeavyChain:IgG( 1 , 2 , 3 ,and 4 )IgA,( 1 , 2 ),IgM,IgE,IgD CanberememberedasGAMED LightChain:Kappaandlambda 4 ) Whichchain(heavyorlight)conferstheantibody’sisotype,heavyorlight? Heavychainconfersisotype(typeofIg,GAMED) 5 ) Knowthemajorpointsforthedifferentantibodies. IgG 3 constantregions,hashinge 4 differentsubclasses( 1 , 2 , 3 ,and 4 )orderedbyconcentrationinblood Foundinvascularandextravascularspacesandsecretions MostabundantIginblood,providesbulkofimmunityforbloodbornepathogens. Small OnlyIgthatcancrossplacenta Caninitiateclassicalpathwayofcomplement Secretedasmonomer IgA 3 constantregions,hashinge 2 differentsubclasses( 1 , 2 ) MostcommonIg abundantinexternalsecretionsex.Saliva,milk Bivalent.HeldtogetherbyJ-Chain. Firstlineofdefenseagainstmicrobesenteringmucosalsurfacesex.Respiratorytract,urinarytract MonomericIgAfoundincirculationonsurfaceofB-Cell. DimericIgAissecretedintolumenandgut IgM 4 constantregions,nohinge FirstantibodyproducedbyB-cells.ProducedbyimmatureB-cells,stilldevelopinginbone marrow. MonomericIgM onsurfaceofB-cellasantigenreceptor. PentamericIgM secretedasantibody Lowaffinity,highavidity Caninitiateclassicalpathwayofcomplement IgE 4 constantregions,nohinge Playsroleininflammationandallergicreactions Bindstomastcellstotriggerreleaseofhistamines Secretedasmonomer IgD 3 constantregions,hashinge SameantigenbindingsiteasIgM PerformsasanantigenreceptoronsurfaceofB-cell IgM andIgDassurfaceBCR’sinteractwithantigentotriggerBcellstoproliferateand differentiate(activationofB-cells)
Secretedasmonomer 6 ) WhatdotheconstantdomainsdofortheIg’s?Dotheybindtotheantigen,confereffector functionorhavenofunctionatall? Constantdomainsdetermineeffectorfunction. 7 ) WhatdoesCDRstandforandwhyaretheseimportant?WhatdoesHVstandfor?Where specificallyaretheseintheBCR/Ab? CDRstandsforComplementarityDeterminingRegion.HVstandsforHypervariableRegion.Thesemean thesamething. ThesearelocatedattheverytipofthevariableregionsintheFabportionoftheantibody,ontheheavyand lightchains(theantigenbindingsite).Thereare 3 HV/CDRsoneachregion,foratotalof 6 perantibody. Theseareimportantfordeterminingthespecificityanddiversityofantigenbindingsites. 8 ) WhichgenesegmentsprovidediversitytoCDR 1 and 2 ?WheredoesthediversityforCDR comefrom (whichgenesegment)? Diversitycomesfrom… HeavyChain LightChain CDR 1 Vsegments Vgenesegments CDR 2 Vsegments Vgenesegments CDR 3 Dgenesegments V/DJunction D/JJunction V/Jjunction 9 ) Knowthemajorcharacteristicsofthedifferentantibodies(Ab’s).Ex.bivalent,highest concentrationAbinblood,allowsforphagocytosis,cancrosstheplacenta,small(goodin extravascularareas) Referto# 5 fordetailedlist. Ex. Bivalent–IgA Highestconcentrationinblood–IgG Allowsforphagocytosis–IgG Crossplacenta–IgG Small-IgG 10 )Whatarethewayswecangetdiversityintheantigenbindingsites.(combinatorial,multiple V,JandDsegmentscanbeusedandNandPnucleotideadditions((junctionaldiversity)) Waystogetdiversityinantigenbindingsites: Somaticrecombination Somatichypermutation Junctionaldiversity(inclusionofPandNnucleotides)
CombinatorialV-D-Jjoining Multiplegermlinegenesegments 11 )Somaticrecombinationingreaterdetail.Raggenesandgeneproducts. a. RAG 1 andRAG 2 - whichtwocelltypesaretheseactiveinandwhatdotheydo? b. RSS–whataretheseandwhattwocelltypeswouldthesebeuseful? c. 1 2 / 23 or 1 turn- 2 turnrule d. TdT e. PandNnucleotideadditions Somaticrecombination–duringthedevelopmentoftheimmatureB-cellinthebonemarrow(primary lymphoidtissue),thearraysofVDJgenesegmentsarecutandreslicedbyDNArecombination.Thisbrings togetherasinglegenesegmentofeachtypetoformaDNAsequenceencodingthevariableregionoftheIg. Forlightregions,asinglerecombinationoccursbetweentheVandJsegments. Forheavyregions, 2 recombinationsareneeded. o FirsttojoinDandJ o SecondtojoinDJtoV RAG- 1 andRAG- 2 – Thesearerecombinationactivationgenes.Theyareonlyfoundin lymphocytes,TandBcells,thatarematuringinthethymusandbonemarrowrespectively.These initiatetheprocessofrecombinationbybindingtheRAGcomplextotheRSS RSS–theseareenzymesthatdirecttherecombinationofV,J,andDgenesegments.Theymake suretheDNAarebroughttogetherintherightorder.TheyareusefulforimmatureB-cellsandT- cells.OneRSSislocatedonthe 3 ’sideofeachVsegment, 5 ’sideofJsegement,andbothsides ofDsegment. 12 / 23 Rule/ 1 - Turn- 2 - TurnRule–TheRSShastwonucleotidesequences.Onehas 23 basepairs( 2 turnsofDNAhelix)theotherhas 12 basepairs( 1 turnofDNAhelix) Youcanonlybringtogethera 12 witha 23 .Intheheavychain,youcannotputtogetheraVandJ directly,youneedaDsegmenttointervene. TdT–lymphocytespecificenzymepresentduringlymphocytematuration.ItaddsNNucleotides, generatingfurtherdiversity PandNnucleotideadditionsarereferredtoasjunctionaldiversity.PNucleotidesarePalindromes andNNucleotidesareNotencodedingermline. 12 )Whatissomatichypermutation?Wheredoesittakeplace?Whichcellsdoesitaffect?When doesithappen,beforeorafteractivation)?Isthereaspecificareathatismutatedmorethan otherareas? SomatichypermutationintroducespointmutationsthroughoutrearrangedvariableregionsusingAID enzyme.Thisaffectsthehypervariable/CDRregionontheantibody.ThisoccursonmatureB-cellsthat havebeenactivatedbyantigenpresentation,locatedinsecondarylymphoidtissuessuchaslymphnodes,but doesnotaffectplasmacells.Thisresultsinaffinitymaturation. 13 )Switchregions.Wherearetheylocated?Whatdotheyswitch?Howdoesitswitch?Which cellsdoesthishappeninandwhendoesithappen?Whatbenefitdoesitprovide?Whatcan betheAbisotypebeswitchedtoandwhichisotypescan’tbeswitchedto?
Switchregionschangetheconstantregionwhilemaintainingthesamevariableregion.Thishappens througharecombinationofwithinaclusterofCgenes,excisingthepreciousCgenes,andattachingnewC genetopreviouslyassembledVregion.Thisresultsinthesameantigenbindingsite,butdifferenteffector mechanisms.ThisisdonewiththeAIDenzyme.Thisoccursinactivated,matureB-cellsthatare proliferatinganddifferentiatinginresponsetoantigenpresentation,andthereforeoccursinsecondary lymphoidtissuesandorgans.ThiscanswitchIgM andIgDtoIgG,IgE,andIgA.IgM cannotbeswitched toIgDbecausetheDgenedoesnothaveswitchregions.Thisprocessisirreversible. Switchregionsflankthe 5 ’sideoftheCgene,withtheexceptionofthedeltaorDgene. 14 )Affinitymaturation.Whatdoesthisresultin? Affinitymaturationisaresultofsomatichypermutation.Itcanbeviewedasanevolutionofantibody specificity.Itresultsinincreasedaffinity. SomatichypermutationsresultinmutatedIg’sonsurfaceofB-cells.SomeoftheseIg’shavehigheraffinity forantigens.TheseIg’swillbepreferablyselected. 15 )Lookatfigure 4. 37 (thechangesintheimmunoglobulingenesthatoccuroveraBcell’s lifetime)Understandthatchart.Knowwhichchangesarepermanent(changestoDNA)vs. thosethatarenotpermanent(alternativesplicingofRNA).Youwillmostlikelyhavetogo backintothetexttogetagoodunderstandingofwhatisgoingoninthischart. ChangestoDNA,suchasinvariableregionassemblyfromgenefragments,somaticrecombination,somatic hypermutation,andisotypeswitching,arepermanentandirreversible. ChangestoRNA,suchasbetweensecretedandsurfaceantibodies,andbetweenIgM andIgDexpression. arereversible.
16 )AnAbismadeupof 4 chains( 2 heavyand2light)whatholdsthem together? Disulfidebonds 17 )InagivenAb,arethe 2 heavychainsexactlythesameandarethe 2 lightchainsexactlythe same? Yes 18 )Whatisallelicexclusionandhowdoesitaffectyouradaptiveimmunecellreceptors? Allelicexclusionmeansthatonlyoneheavychainandonlyonelightchainareproducedandexpressedina singleB-cell.ThisensuresthatIgM andIgDhaveasinglespecificity. 19 )From IgGtoIgGwheredoyouexpecttofindthegreatestaminoaciddifferences?Constant regionsorvariableregionsandiftheansweristhevariableregions,whichspecificregionsof thevariableregions? Youwouldfindthegreatestaminoaciddifferencesconcentratedinthehypervariable(HV)/CDRregionof thevariableregionoftheantibody. 20 )Dolightchainshaveisotypes?Ifso,howmanydifferentisotypesarethereandwhatare they?CanoneAbhavemultiplelightchainisotypes? Lightchainshavetwoisotypes:Kappaandlambda.OnlycanbepresentonaparticularB-cell. 21 )Whathappensduringsomaticrecombinationandwhathappensduringsomatic hypermutation?Whereandwhendoeachhappen? Somaticrecombination–duringthedevelopmentoftheimmatureB-cellinthebonemarrow(primary lymphoidtissue),thearraysofVDJgenesegmentsarecutandreslicedbyDNArecombination.Thisbrings togetherasinglegenesegmentofeachtypetoformaDNAsequenceencodingthevariableregionoftheIg, determiningitsspecificity. Somatichypermutation–introducespointmutationsthroughoutrearrangedvariableregionsusingAID enzyme.Thisaffectsthehypervariable/CDRregionontheantibody.ThisoccursonmatureB-cellsthat havebeenactivatedbyantigenpresentation,locatedinsecondarylymphoidtissuessuchaslymphnodes,but doesnotaffectplasmacells.Thisresultsinaffinitymaturation 22 )Whatenzymeisinvolvedinbothsomatichypermutationandisotypeswitching? AIDenzyme 23 )Lookatthedifferentepitopes.(ex.linearvs.discontinuous) EpitopesaretheregiononanantigenthatBCRcanbindto.Indiscontinuousepitopes,differentpartsofthe epitopearefoldedtogether. 24 )WhatdoesaB-celldotochangeamembraneBCRintoasecretedantibody? AllmembraneboundBCRscanbesecretedasantibodiesbyplasmacells,whicharemature,activatedB cellsthathavedifferentiated.AlternativesplicingofRNAcanremovethemembranebindingdomain. DifferencesinmembraneandsecretedantibodiesareinthecarboxyterminusofH-chains.Thesecreted antibodyhasahydrophilicsequence,whichallowsittobesoluble.
25 )IgDandIgM membraneimmunoglubulins.Aretheantigenbindingsitesthesame?Whatis differentbetweenthetwoimmunoglobulins?Howaretheybothonthesurfaceatthesame time?IftheycuttheDNAtochangeisotype,couldtheybothbeexpressedatthesametime? CirculatingB-cellsthathavenotbeenactivatedyetarecalledNaïveBCells.TheyexpressbothIgM and IgDontheirsurfaces.ThisistheonlyscenariowheretwoIg’scanbeexpressedconcurrentlyonthesameB cell.ThisoccursbecauseofdifferentialsplicingofsameprimaryRNAtranscript,whichdoesnotinvolve rearrangementofDNA.Theyhaveidenticalbindingsites.IgM isthefirstantibodycreatedbyaBcell.If theyalteredtheirDNAtoswitchisotypes,theycouldnotbeexpressedatthesametime. 26 )Lookatfig. 4. 21 and4. 28 .Haveanideaofthelinearityofconstantregiongenesandknow whichconstantregiongeneshaveswitchregionsinfrontofthem andwhichconstantregion genesdon’thaveaswitchregion.Whydoesn’tCdeltaneedaswitchregioninfrontofit?If youswitchedfrom IgM toIgG 1 couldyouevergobacktoIgG 3 orIgD? ThelinearityofthisheavychainbeginswiththeL-gene,followedbythecompletedVDJgene,followedby theconstantCgenes. Delta(D)constantregionsaretheonlyonesthatdonotneedaswitchregioninfront,becausetheyare highlyrepetitivesequencesthatmediaterecombination.
IfyouswitchfromIgM toIgG,youcannotreversethisprocess,becauseDNAcomponentswereslicedand removed.YoucannotswitchtoorfromanIgDbecauseitlacksswitchregions. 27 )HowmanysubclassesofIgGandIgAarethere?Fig. 4. 33 IgGhas 4 sub-classes.IgG 1 ,IgG 2 ,IgG 3 ,IgG 4 IgAhas 2 sub-classes.IgA 1 ,IgA 2 ,