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14.2.1: Argitektuur van die immuunstelsel - Biologie

14.2.1: Argitektuur van die immuunstelsel - Biologie


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Leerdoelwitte

  • Definieer geheue, primêre reaksie, sekondêre reaksie en spesifisiteit
  • Onderskei tussen humorale en sellulêre immuniteit
  • Onderskei tussen antigene, epitope en haptens
  • Beskryf die struktuur en funksie van teenliggaampies en onderskei tussen die verskillende klasse teenliggaampies

Kliniese fokus: Deel 1

Olivia, 'n eenjarige baba, word deur haar ouers na die noodkamer gebring, wat haar simptome rapporteer: oormatige gehuil, prikkelbaarheid, sensitiwiteit vir lig, ongewone lusteloosheid en braking. 'N Dokter voel geswelde limfkliere in Olivia se keel en oksels. Daarbenewens is die area van die buik oor die milt geswel en sag.

Oefening ( PageIndex {1} )

  1. Wat dui hierdie simptome aan?
  2. Watter toetse kan bestel word om die probleem te diagnoseer?

Adaptiewe immuniteit word gedefinieer deur twee belangrike eienskappe: spesifisiteit en geheue. Spesifisiteit verwys na die vermoë van die adaptiewe immuunstelsel om spesifieke patogene te teiken, en geheue verwys na die vermoë om vinnig te reageer op patogene waaraan dit voorheen blootgestel is. Byvoorbeeld, as 'n individu herstel van waterpokkies, ontwikkel die liggaam a geheue van die infeksie wat sal spesifiek beskerm dit teen die veroorsakende middel, die varicella-zoster-virus, as dit later weer aan die virus blootgestel word.

Spesifisiteit en geheue word verkry deur in wese sekere selle wat betrokke is by die immuunrespons te programmeer om vinnig te reageer op daaropvolgende blootstelling van die patogeen. Hierdie programmering vind plaas as gevolg van die eerste blootstelling aan 'n patogeen of entstof, wat 'n primêre reaksie veroorsaak. Latere blootstellings lei tot 'n sekondêre reaksie wat vinniger en sterker is as gevolg van die liggaam se geheue van die eerste blootstelling (Figuur ( PageIndex {1} )). Hierdie sekondêre reaksie is egter spesifiek vir die betrokke patogeen. Byvoorbeeld, blootstelling aan een virus (bv. varicella-zoster-virus) sal nie beskerming teen ander virussiektes (bv. masels, pampoentjies of polio) bied nie.

Adaptiewe spesifieke immuniteit behels die werking van twee verskillende seltipes: B -limfosiete (B -selle) en T -limfosiete (T -selle). Alhoewel B-selle en T-selle voortspruit uit 'n algemene hematopoietiese stamseldifferensiasie-weg, is hul plekke van rypwording en hul rolle in adaptiewe immuniteit baie verskillend.

B-selle word volwasse in die beenmurg en is verantwoordelik vir die produksie van glikoproteïene wat teenliggaampies of immunoglobuliene genoem word. Teenliggaampies is betrokke by die liggaam se verdediging teen patogene en gifstowwe in die ekstrasellulêre omgewing. Meganismes van adaptiewe spesifieke immuniteit wat B -selle en teenliggaamproduksie behels, word humorale immuniteit genoem. Die rypwording van T-selle vind in die timus plaas. T -selle funksioneer as die sentrale orkestreerder van beide aangebore en aanpasbare immuunreaksies. Hulle is ook verantwoordelik vir die vernietiging van selle wat met intrasellulêre patogene besmet is. Die doelwit en vernietiging van intrasellulêre patogene deur T-selle word sel-gemedieerde immuniteit, of sellulêre immuniteit, genoem.

Oefening ( PageIndex {2} )

  1. Noem die twee kenmerkende eienskappe van adaptiewe immuniteit.
  2. Verduidelik die verskil tussen 'n primêre en sekondêre immuunrespons.
  3. Hoe verskil humorale en sellulêre immuniteit?

Antigene

Aktivering van die adaptiewe immuunverdediging word veroorsaak deur patogeen-spesifieke molekulêre strukture wat antigene genoem word. Antigene is soortgelyk aan die patogeen-geassosieerde molekulêre patrone (PAMP's) wat in patogeenherkenning en fagositose bespreek word; Alhoewel PAMPs molekulêre strukture is wat op talle patogene voorkom, is antigene uniek aan 'n spesifieke patogeen. Die antigene wat byvoorbeeld aanpasbare immuniteit teen waterpokkies stimuleer, is uniek aan die varicella-zoster-virus, maar verskil aansienlik van die antigene wat met ander virale patogene geassosieer word.

Die term antigeen is aanvanklik gebruik om molekules te beskryf wat die produksie van teenliggaampies stimuleer; die term kom eintlik uit 'n kombinasie van die woorde antiliggaam en genlDaar word gesê dat 'n molekule wat die produksie van teenliggaampies stimuleer, antigene is. Die rol van antigene is egter nie beperk tot humorale immuniteit en die produksie van teenliggaampies nie; antigene speel ook 'n noodsaaklike rol in die stimulering van sellulêre immuniteit, en om hierdie rede word antigene soms meer akkuraat na verwys as immunogene. In hierdie teks sal ons egter tipies na hulle verwys as antigene.

Patogene beskik oor 'n verskeidenheid strukture wat antigene kan bevat. Antigene van bakteriese selle kan byvoorbeeld geassosieer word met hul kapsules, selwande, fimbriae, flagella of pili. Bakteriële antigene kan ook geassosieer word met ekstrasellulêre gifstowwe en ensieme wat dit afskei. Virusse beskik oor 'n verskeidenheid antigene wat verband hou met hul kapsiede, omhulsels en die spykestrukture wat hulle gebruik om aan selle vas te maak.

Antigene kan aan enige aantal molekulêre klasse behoort, insluitend koolhidrate, lipiede, nukleïensure, proteïene en kombinasies van hierdie molekules. Antigene van verskillende klasse verskil in hul vermoë om aanpasbare immuunverdediging te stimuleer sowel as in die tipe reaksie wat hulle stimuleer (humoraal of sellulêr). Die strukturele kompleksiteit van 'n antigene molekule is 'n belangrike faktor in die antigeniese potensiaal daarvan. Oor die algemeen is meer komplekse molekules meer effektief as antigene. Die driedimensionele komplekse struktuur van proteïene maak hulle byvoorbeeld die mees effektiewe en kragtige antigene, wat beide humorale en sellulêre immuniteit kan stimuleer. In vergelyking hiermee is koolhidrate minder kompleks in struktuur en dus minder effektief as antigene; hulle kan slegs humorale immuunverdediging stimuleer. Lipiede en nukleïensure is die minste antigene molekules, en kan in sommige gevalle slegs antigene word as dit met proteïene of koolhidrate gekombineer word om glikolipiede, lipoproteïene of nukleoproteïene te vorm.

Een rede waarom die driedimensionele kompleksiteit van antigene so belangrik is, is dat teenliggaampies en T-selle nie 'n hele antigeen herken en daarmee in wisselwerking tree nie, maar met kleiner blootgestelde streke op die oppervlak van antigene wat epitope genoem word. 'n Enkele antigeen kan verskeie verskillende epitope besit (Figuur (PageIndex{2})), en verskillende teenliggaampies kan aan verskillende epitope op dieselfde antigeen bind (Figuur (PageIndex{3})). Byvoorbeeld, die bakteriële flagellum is 'n groot, komplekse proteïenstruktuur wat honderde of selfs duisende epitope met unieke driedimensionele strukture kan besit. Boonop bevat flagella van verskillende bakteriese spesies (of selfs stamme van dieselfde spesie) unieke epitope wat slegs deur spesifieke teenliggaampies gebind kan word.

Die grootte van 'n antigeen is nog 'n belangrike faktor in sy antigeniese potensiaal. Terwyl groot antigene strukture soos flagella verskeie epitope besit, is sommige molekules te klein om self antigene te wees. Sulke molekules, haptens genoem, is in wese vrye epitope wat nie deel uitmaak van die komplekse driedimensionele struktuur van 'n groter antigeen nie. Om 'n hapten antigene te word, moet dit eers aan 'n groter draermolekule (gewoonlik 'n proteïen) heg om 'n gekonjugeerde antigeen te produseer. Die hapten-spesifieke teenliggaampies wat geproduseer word in reaksie op die gekonjugeerde antigeen, kan dan met onkonjugeerde vrye haptenmolekules in wisselwerking tree. Dit is nie bekend dat Haptens met enige spesifieke patogene geassosieer word nie, maar hulle is verantwoordelik vir sommige allergiese reaksies. Byvoorbeeld, die hapten urushiol, 'n molekule wat in die olie van plante voorkom wat giftige klimop veroorsaak, veroorsaak 'n immuunrespons wat kan lei tot ernstige uitslag (kontakdermatitis genoem). Net so kan die hapten penisillien allergiese reaksies veroorsaak vir middels in die penisillienklas.

Oefening ( PageIndex {3} )

  1. Wat is die verskil tussen 'n antigeen en 'n epitoop?
  2. Watter faktore beïnvloed 'n antigeen se antigeenpotensiaal?
  3. Waarom is haptens tipies nie antigene nie, en hoe word dit antigene?

Teenliggaampies

Teenliggaampies (ook genoem immunoglobuliene) is glikoproteïene wat in beide die bloed en weefselvloeistowwe teenwoordig is. Die basiese struktuur van 'n teenliggaam -monomeer bestaan ​​uit vier proteïenkettings wat deur disulfiedbindings aan mekaar gehou word (Figuur ( PageIndex {4} )). 'N Disulfiedbinding is 'n kovalente binding tussen die sulfhidriel R groepe wat op twee sistien aminosure voorkom. Die twee grootste kettings is identies aan mekaar en word die swaar kettings genoem. Die twee kleiner kettings is ook identies aan mekaar en word die ligte kettings genoem. Saamgevoeg vorm die swaar en ligte kettings 'n basiese Y-vormige struktuur.

Die twee 'arms' van die Y-vormige teenliggaammolekule staan ​​bekend as die Fab-streek, vir "fragment van antigeenbinding." Die verste punt van die Fab -streek is die veranderlike gebied, wat dien as die plek van antigeenbinding. Die aminosuurvolgorde in die veranderlike gebied dikteer die driedimensionele struktuur, en dus die spesifieke driedimensionele epitoop waaraan die Fab-streek in staat is om te bind. Alhoewel die epitoop-spesifisiteit van die Fab-streke identies is vir elke arm van 'n enkele teenliggaammolekule, vertoon hierdie streek 'n hoë mate van variasie tussen teenliggaampies met verskillende epitoop-spesifisiteite. Binding aan die Fab-streek is nodig vir die neutralisering van patogene, agglutinasie of samevoeging van patogene en teenliggaamafhanklike selgemedieerde sitotoksisiteit.

Die konstante gebied van die teenliggaammolekule sluit die stam van die Y en onderste gedeelte van elke arm van die Y in. Die stam van die Y word ook die Fc-streek genoem, vir "fragment van kristallisasie," en is die plek van komplementfaktorbinding en binding aan fagositiese selle tydens teenliggaam-gemedieerde opsonisering.

Oefening ( PageIndex {4} )

Beskryf die verskillende funksies van die Fab -streek en die Fc -streek.

Klasse teenliggaampies

Die konstante gebied van 'n teenliggaammolekule bepaal sy klas, of isotipe. Die vyf klasse teenliggaampies is IgG, IgM, IgA, IgD en IgE. Elke klas beskik oor unieke swaar kettings wat onderskeidelik met die Griekse letters γ, μ, α, δ en ε aangedui word. Teenliggaamklasse toon ook belangrike verskille in oorvloed in serum, rangskikking, liggaamsplekke van aksie, funksionele rolle en grootte (Figuur (PageIndex{5})).

IgG is 'n monomeer wat verreweg die grootste teenliggaam in menslike bloed is, en is verantwoordelik vir ongeveer 80% van die totale serum teenliggaam. IgG dring doeltreffend in weefselruimtes binne, en is die enigste teenliggaamplas met die vermoë om die plasentale versperring oor te steek, wat passiewe immuniteit bied aan die ontwikkelende fetus tydens swangerskap. IgG is ook die mees veelsydige teenliggaamplas in terme van sy rol in die liggaam se verdediging teen patogene.

IgM word aanvanklik geproduseer in 'n monomere membraangebonde vorm wat dien as 'n antigeenbindende reseptor op B-selle. Die afgeskeide vorm van IgM vorm 'n pentameer met vyf monomere van IgM wat saamgebind word deur 'n proteïenstruktuur wat die J-ketting genoem word. Alhoewel die ligging van die J -ketting relatief tot die Fc -streke van die vyf monomere verhoed dat IgM sommige van die funksies van IgG kan verrig, maak die tien beskikbare Fab -terreine wat verband hou met 'n pentamere IgM dit 'n belangrike teenliggaam in die liggaam se arsenaal van verdediging. IgM is die eerste teenliggaam wat deur B-selle geproduseer en afgeskei word tydens die primêre en sekondêre immuunrespons, wat patogeen-spesifieke IgM 'n waardevolle diagnostiese merker maak tydens aktiewe of onlangse infeksies.

IgA is verantwoordelik vir ongeveer 13% van die totale serum teenliggaam, en sekretoriese IgA is die algemeenste en mees voorkomende teenliggaamplas wat voorkom in die slymafskeidings wat die slymvliese beskerm. IgA kan ook gevind word in ander afskeidings soos borsmelk, trane en speeksel. Sekretoriese IgA word in 'n dimere vorm saamgevoeg met twee monomere wat verbind is deur 'n proteïenstruktuur wat die sekretoriese komponent genoem word. Een van die belangrikste funksies van sekretoriese IgA is om patogene in slym vas te vang, sodat hulle later uit die liggaam verwyder kan word.

Soortgelyk aan IgM, is IgD 'n membraangebonde monomeer wat op die oppervlak van B-selle voorkom, waar dit dien as 'n antigeenbindende reseptor. IgD word egter nie deur B-selle afgeskei nie, en slegs spoorhoeveelhede word in serum opgespoor. Hierdie spoorhoeveelhede kom waarskynlik uit die agteruitgang van ou B -selle en die vrystelling van IgD -molekules uit hul sitoplasmiese membrane.

IgE is die minste volop teenliggaamplas in serum. Net soos IgG word dit as 'n monomeer afgeskei, maar sy rol in adaptiewe immuniteit is beperk tot anti-parasitiese verdediging. Die Fc -streek van IgE bind aan basofiele en mastselle. Die Fab-gebied van die gebonde IgE reageer dan met spesifieke antigeenepitope, wat veroorsaak dat die selle kragtige pro-inflammatoriese bemiddelaars vrystel. Die inflammatoriese reaksie wat voortspruit uit die aktivering van mastselle en basofiele help in die verdediging teen parasiete, maar hierdie reaksie is ook sentraal tot allergiese reaksies (sien Siektes van die immuunstelsel.)

Oefening (PageIndex{5})

  1. Watter deel van 'n teenliggaammolekule bepaal sy klas?
  2. Watter klas teenliggaampies is betrokke by beskerming teen parasiete?
  3. Beskryf die verskil in struktuur tussen IgM en IgG.

Antigeen-teenliggaam interaksies

Verskillende klasse teenliggaampies speel belangrike rolle in die liggaam se verdediging teen patogene. Hierdie funksies sluit in neutralisering van patogene, opsonisering vir fagositose, agglutinasie, komplementaktivering en teenliggaamafhanklike selgemedieerde sitotoksisiteit. Vir die meeste van hierdie funksies bied teenliggaampies ook 'n belangrike skakel tussen adaptiewe spesifieke immuniteit en aangebore nie -spesifieke immuniteit.

Neutralisasie behels die binding van sekere teenliggaampies (IgG, IgM of IgA) aan epitope op die oppervlak van patogene of toksiene, wat hul aanhegting aan selle verhoed. Sekretoriese IgA kan byvoorbeeld aan spesifieke patogene bind en die aanvanklike aanhegting aan dermslijmvliesselle blokkeer. Spesifieke teenliggaampies kan op dieselfde manier aan sekere gifstowwe bind, wat hulle verhinder om aan die teikenselle te heg en sodoende hul toksiese effekte te neutraliseer. Virusse kan deur dieselfde meganisme geneutraliseer word en verhoed word om 'n sel te besmet (Figuur ( PageIndex {6} )).

Soos beskryf in Chemical Defenses, is opsonisering die bedekking van 'n patogeen met molekules, soos komplementfaktore, C-reaktiewe proteïen en serumamyloïde A, om te help met fagosietbinding om fagositose te vergemaklik. IgG-teenliggaampies dien ook as uitstekende opsoniene, wat hul Fab-plekke aan spesifieke epitope op die oppervlak van patogene bind. Fagositiese selle soos makrofage, dendritiese selle en neutrofiele het reseptore op hul oppervlaktes wat die Fc -gedeelte van die IgG -molekules herken en bind; dus help IgG sulke fagosiete om die patogene wat hulle gebind het, aan te sluit en te verswelg (Figuur ( PageIndex {7} )).

Agglutinasie of aggregasie behels die kruisverbinding van patogene deur teenliggaampies om groot aggregate te skep (Figuur ( PageIndex {8} )). IgG het twee Fab-antigeen-bindingsplekke, wat aan twee afsonderlike patogeenselle kan bind, wat hulle saamklonter. Wanneer veelvuldige IgG-teenliggaampies betrokke is, kan groot aggregate ontwikkel; hierdie aggregate is makliker vir die niere en milt om uit die bloed te filter en makliker vir fagosiete om dit te vernietig. Die pentamere struktuur van IgM bied tien Fab -bindingsplekke per molekule, wat dit die doeltreffendste teenliggaam vir agglutinasie maak.

Nog 'n belangrike funksie van teenliggaampies is aktivering van die komplementkaskade. Soos in die vorige hoofstuk bespreek, is die komplementstelsel 'n belangrike komponent van die aangebore verdediging, wat die inflammatoriese reaksie bevorder, fagosiete werf na die plek van infeksie, die verbetering van fagositose deur opsonisering en die dood van gram-negatiewe bakteriese patogene met die membraanaanvalkompleks (MAC ). Komplementaktivering kan deur drie verskillende weë plaasvind (sien [skakel]), maar die mees doeltreffende is die klassieke roete, wat die aanvanklike binding van IgG- of IgM-teenliggaampies aan die oppervlak van 'n patogeensel vereis, wat die werwing en aktivering van die C1 moontlik maak. kompleks.

Nog 'n belangrike funksie van teenliggaampies is teenliggaamafhanklike selgemedieerde sitotoksisiteit (ADCC), wat die dood van patogene wat te groot is om te fagositiseer, verbeter. Hierdie proses word die beste gekenmerk vir natuurlike moordenaarselle (NK -selle), soos getoon in Figuur ( PageIndex {9} ), maar dit kan ook makrofage en eosinofiele insluit. ADCC vind plaas wanneer die Fab-streek van 'n IgG-teenliggaam aan 'n groot patogeen bind; Fc-reseptore op effektorselle (bv. NK-selle) bind dan aan die Fc-gebied van die teenliggaam, wat hulle in die nabyheid van die teikenpatogeen bring. Die effektorsel skei dan kragtige sitotoksiene af (bv. perforien en gransieme) wat die patogeen doodmaak.

Oefening (PageIndex{6})

  1. Waar word IgA normaalweg gevind?
  2. Watter klas teenliggaam kruis die plasenta en bied beskerming aan die fetus?
  3. Vergelyk die meganismes van opsonisering en teenliggaamafhanklike selgemedieerde sitotoksisiteit.

Belangrike konsepte en opsomming

  • Adaptiewe immuniteit is 'n verworwe verdediging teen vreemde patogene wat gekenmerk word deur spesifisiteit en geheue. Die eerste blootstelling aan 'n antigeen stimuleer a primêre reaksie, en daaropvolgende blootstelling stimuleer 'n vinniger en sterk sekondêre reaksie.
  • Adaptiewe immuniteit is 'n dubbele stelsel humorale immuniteit (teenliggaampies wat deur B -selle vervaardig word) en sellulêre immuniteit (T -selle gerig teen intrasellulêre patogene).
  • Antigene, ook genoem immunogene, is molekules wat aanpasbare immuniteit aktiveer. 'N Enkele antigeen beskik oor kleiner epitope, wat elkeen in staat is om 'n spesifieke aanpasbare immuunrespons te veroorsaak.
  • Die vermoë van 'n antigeen om 'n immuunrespons te stimuleer, hang af van verskeie faktore, insluitend sy molekulêre klas, molekulêre kompleksiteit en grootte.
  • Teenliggaampies (immunoglobuliene) is Y-vormige glikoproteïene met twee Fab-plekke vir bindende antigene en 'n Fc-gedeelte wat betrokke is by komplementaktivering en opsonisering.
  • Die vyf klasse teenliggaampies is IgM, IgG, IgA, IgE, en IgD, wat elkeen verskil in grootte, rangskikking, ligging binne die liggaam en funksie. Die vyf primêre funksies van teenliggaampies is neutralisasie, opsonisering, agglutinasie, komplementaktivering en teenliggaamafhanklike sel-gemedieerde sitotoksisiteit (ADCC).

Meervoudige keuse

Teenliggaampies word deur ________ vervaardig.

A. plasmaselle
B. T -selle
C. beenmurg
D. B -selle

A

Sellulêre aanpasbare immuniteit word uitgevoer deur ________.

A. B-selle
B. neutrofiele

B

'N Enkele antigeenmolekule kan bestaan ​​uit baie individuele ________.

A. T-selreseptore
B. B-sel reseptore
C. MHC II
D. epitope

D

Watter klas molekules is die meeste antigene?

A. polisakkariede
B. lipiede
C. proteïene
D. koolhidrate

C

Bypassend

Pas die teenliggaamplas by sy beskrywing.

___IgAA. Hierdie klas teenliggaampies is die enigste wat die plasenta kan oorsteek.
___IgDB. Hierdie klas teenliggaampies verskyn die eerste na die aktivering van B -selle.
___IgEC. Hierdie klas teenliggaampies is betrokke by die verdediging teen parasitiese infeksies en is betrokke by allergiese reaksies.
___IgGD. Hierdie klas teenliggaam word in baie groot hoeveelhede in slymafskeidings aangetref.
___IgME. Hierdie klas teenliggaampies word nie deur B-selle afgeskei nie, maar word op die oppervlak van naïewe B-selle uitgedruk.

d, e, c, a, b

Vul die spasie in

Daar is twee krities belangrike aspekte van adaptiewe immuniteit. Die eerste is spesifisiteit, terwyl die tweede ________.

geheue

________ immuniteit behels die produksie van teenliggaammolekules wat aan spesifieke antigene bind.

Humoraal

Die swaar kettings van 'n teenliggaammolekule bevat ________ -streeksegmente, wat help om die klas of isotipe daarvan te bepaal.

konstant

Die veranderlike streke van die swaar en ligte kettings vorm die ________ plekke van 'n teenliggaam.

antigeenbinding

Kort antwoord

Wat is die verskil tussen humorale en sellulêre adaptiewe immuniteit?

Wat is die verskil tussen 'n antigeen en 'n hapten?

Beskryf die meganisme van teenliggaamafhanklike selgemedieerde sitotoksisiteit.


Immuunrespons in COVID-19: 'n oorsig

Die immuunstelsel beskerm teen virusse en siektes en produseer teenliggaampies om patogene dood te maak. Hierdie oorsig bied 'n kort oorsig van die immuunstelsel rakende die beskerming van die menslike liggaam teen COVID-19, illustreer die proses van die immuunstelsel, hoe dit werk, en sy meganisme om virusse te beveg en bied inligting oor die mees onlangse COVID-19-behandelings aan. en eksperimentele data. Verskeie tipes potensiële uitdagings vir die immuunstelsel word ook bespreek. Aan die einde van die artikel word voedsel voorgestel en vermy, en fisiese oefening word aangemoedig. Hierdie artikel kan op hierdie kritieke oomblik wêreldwyd as die nuutste tegnologie gebruik word vir belowende alternatiewe oplossings wat verband hou met die oorlewing van die koronavirus.


Immuunresponse op adenovirus en adeno-geassosieerde vektore wat gebruik word vir genterapie van breinsiektes: die rol van immunologiese sinapse om die selbiologie van neuro-immuuninteraksies te verstaan

Navorsers het talle pre-kliniese en kliniese geenoordragstudies uitgevoer met behulp van rekombinante virale vektore afgelei van 'n wye reeks patogeniese virusse soos adenovirus, adeno-geassosieerde virus en lentivirus. Aangesien virale vektore van patogeniese virusse afgelei is, het hulle 'n inherente vermoë om 'n vektorspesifieke immuunrespons te induseer wanneer dit in vivo gebruik word. Die rol van die immuunrespons teen die virale vektor is geïmpliseer in die inkonsekwente en onvoorspelbare vertaling van pre-kliniese sukses in terapeutiese doeltreffendheid in menslike kliniese proewe wat geenterapie gebruik om neurologiese afwykings te behandel. Hierin ondersoek ons ​​die uitwerking van die aangebore en aanpasbare immuunresponse deeglik op terapeutiese geenuitdrukking wat deur adenovirale, AAV- en lentivirale vektorsisteme bemiddel word in beide prekliniese en kliniese eksperimente. Verder word die immuunreaksies teen genterapie -vektore en die gevolglike verlies aan terapeutiese geenuitdrukking ondersoek in die konteks van die argitektuur en neuroanatomie van die brein se immuunstelsel. Die hoofstuk sluit af met 'n bespreking van die verwantskap tussen die eliminasie van transgeenuitdrukking en die in vivo immunologiese sinapse tussen immuunselle en teiken viraal geïnfekteerde breinselle. Alhoewel dit belangrik is dat sistemiese immuunrespons teen virusvektore wat sistemies ingespuit word in 'n aantal proewe skadelik is, die resultate van kliniese proewe in breingene -terapie nie hierdie algemene gevolgtrekking ondersteun nie, wat daarop dui dat breingene -terapie veiliger kan wees vanuit 'n immunologiese oogpunt.

Syfers

Fig. (1). Anti-adenovirale immuunrespons skakel heeltemal uit ...

Fig. (1). Anti-adenovirale immuunresponse skakel transgeen-uitdrukking van eerste generasie adenovirale vektore heeltemal uit

Fig. (2). Anti-adenovirale immuunresponse is nie in staat nie ...

Fig. (2). Anti-adenovirale immuunresponse is nie in staat om transgeenuitdrukking van HC-Ad vektore uit te skakel nie

Fig. (3). Vergelyking van reeds bestaande antwoorde teen ...

Fig. (3). Vergelyking van reeds bestaande reaksies teen eerste generasie adenovirus, hoë kapasiteit adenovirus en transgeen

Fig. (4). Reeds bestaande reaksies teen AAV-vektore

Fig. (4). Reeds bestaande reaksies teen AAV-vektore

Fig. (5). SMAC-vorming by immunologiese sinapse ...

Fig. (5). SMAC -vorming by immunologiese sinapse in vivo , tussen T-selle en besmette ...


Uitgelaai

Hoogtepunte

Verwante verhale

Toe Carl F. Nathan, mikrobiologie en immunologie, Weill Cornell Medicine, in Desember 1966 sy aanvaarding by die Harvard University Medical School ontvang het, het hy dit nie gevier nie. Vroeër die dag het hy gesien hoe sy ma aan kanker sterf.

"Ek het daardie dag 'n emosionele en intellektuele verbintenis tot die veld gemaak," sê Nathan. “Ek wou my dankbaarheid teenoor haar uitspreek en probeer om te laat terug te betaal deur ander mense in dieselfde situasie te help.”

Nathan het na die mediese skool, 'n koshuis en 'n onkologie-genootskap gegaan, maar hy het gou gefrustreerd geraak met chemoterapie as die verstek, en gewoonlik ondoeltreffende, behandeling destyds. Nathan, reeds geskeur tussen kliniese onkologie en fundamentele navorsing, het gesien dat die wortel van die probleem - om te verstaan ​​hoe die immuunstelsel van die liggaam werk - nuwe benaderings kan ontdek om nie net kanker nie, maar ook aansteeklike siektes te bestry.

Die immuunstelsel teenoor bakterieë

'Die immuunstelsel het 'n enorme vernietigende krag,' sê Nathan. 'Dit kan enige weefsel wat volgens hom besmet is, vernietig. Maar op daardie stadium het ons niks geweet waaruit die vuurkrag bestaan ​​en hoe dit gereguleer is nie.”

In 1977 begin Nathan voltyds met navorsing aan die Rockefeller Universiteit om uit te vind, en kyk spesifiek na die immuunrespons teen aansteeklike siektes. 'Ek het gedink ek kan vinniger vorder met behulp van aansteeklike siektes,' sê hy, 'want dit was die situasie waarin hierdie immuunvermoë ontwikkel het, terwyl kanker mense gewoonlik na hul voortplantingsouderdom aantas.'

Nathan het geweet dat neutrofiele en makrofage die selle van die immuunstelsel is wat patogene direk kan doodmaak, eerder as om besmette gasheerselle dood te maak. Oor die volgende dekade sou hy ontdek dat makrofage geaktiveer word deur 'n proteïen, genaamd interferon-gamma. Interferon-gamma word deur T-limfosiete geproduseer wanneer hulle bakterieë opspoor. Sy laboratorium het ook bevind dat, tot sy en ander se verbasing, hierdie aktivering die produksie van reaktiewe suurstofspesies soos superoksied en waterstofperoksied moontlik maak, wat die selle dan as wapens teen die bakterieë gebruik.

"Ons kon toe interferon-gamma bekendstel as 'n behandeling vir kinders wat gebrekkig was in hierdie stelsel, wat aan bakteriële of swaminfeksies sou gesterf het," sê Nathan. Die behandeling werk ook vir melaatsheid, wat veroorsaak word deur 'n mycobacterium. Hierdie pad verduidelik egter nie alles nie. 'Ons was deeglik bewus daarvan dat iets ontbreek,' sê Nathan.

Toe hy in 1986 na Cornell verhuis het, het hy die tweede groot moordpad ontdek. Die immuunreaksie op aansteeklike siektes het ook neutrofiel- en makrofaagproduksie van 'n ander proteïen, die ensiem iNOS (induseerbare stikstofoksiedsintase) ingesluit, wat reaktiewe stikstofspesies maak - 'n ander wapen.

Toe albei hierdie weë in muismodelle uitgeslaan is, was die resultaat nie versoenbaar met die lewe in die natuur nie. "Hulle het die normale aantal makrofage en neutrofiele en kon hulle selfs na besmette plekke mobiliseer, maar die selle kon die bakterieë nie doodmaak nie," verduidelik Nathan. 'Dit is die ernstigste immuungebrek -fenotipe vir bakteriële infeksies waarvan ek weet met 'n normale aantal gemobiliseerde fagosiete, en dit toon aan dat hierdie twee stelsels deels onderling oortollig is, maar gesamentlik onontbeerlik is vir die daaglikse lewe.'

Bestudeer 'n blywende aansteeklike siekte, tuberkulose

Nathan en sy span het begin toets om vas te stel watter siektes floreer toe die iNOS -pad geblokkeer is. Tuberkulose - veroorsaak deur Mycobacterium tuberculosis (Mtb)-die grootste bakteriële infeksie wat die dood veroorsaak in die wêreld, was boaan die lys.

“Ek het dus oor Mtb begin leer,” sê Nathan, “en ek het besef daar is ’n enorme hoeveelheid menslike biologie wat Mtb ons probeer leer as ons daarna sou luister.”

Wetenskaplikes dink dat tuberkulose (TB) al minstens 70 000 jaar bestaan. Nathan sê: 'As ons ophou om daaraan te dink, het ons dit nie uitgeskakel nie, en dit het ons nie uitgeskakel nie. Daar is dus 'n soort ewewig. "

“Mtb kry die immuunstelsel om longweefsel te vernietig, wat ons laat hoes of nies. Dan ry dit op klein druppeltjies — vloeibare longweefsel. ”

Dit hou verband met die patogeen se lewensiklus en dat mense sy enigste natuurlike gasheer is. "Voordat ons in stede gewoon het, sou dit baie belangrik gewees het vir TB om nie almal in 'n dorp vinnig dood te maak voordat hulle kinders gehad het nie," verduidelik Nathan, "want dan sou daar geen nuwe gashere wees nie."

Mtb moet sy tyd neem om openlike siektes te veroorsaak. Dit moet ook deur die immuunstelsel erken word om nuwe slagoffers te besmet. "Mtb kry die immuunstelsel om longweefsel te vernietig, wat ons laat hoes of nies," verduidelik Nathan. "Dan vat dit 'n rit op klein druppels - vloeibare longweefsel. "Mtb moet hierdie stywe tou loop," gaan hy voort, "om ons immuniteit aan te blaas, maar dit ook te titreer, dit te oorleef en dit dan uit te buit om by die volgende gasheer uit te kom."

Hierdie verhouding laat baie vrae ontstaan: hoe oorleef Mtb, nadat dit deur die immuunstelsel erken is? Wat is die verdediging daarvan? "Vir my is dit 'n handboek wat ons vertel wat menslike immuniteit na die stryd bring," sê Nathan. In baie breë terme het hy en ander sewe strategieë gevind wat die bakterieë gebruik, van die aftakeling van die immuunstelsel se chemie, om skade te herstel, om skade te sekwestreer.

Hierdie werk bring Nathan ook 180 grade van die ondersoek na hoe die immuunstelsel bakterieë doodmaak tot hoe die bakterieë terugveg. "Die doel is om dit van beide kante te sien, en dan kan jy dalk die poppemeester wees," sê hy. "Dan het jy 'n kans om die immuunstelsel meer van die tyd meer suksesvol te maak."

Nathan het ensieme gevind wat Mtb help om baie van sy verdedigingsmeganismes uit te voer. Hy het ook remmers van die ensieme gevind. 'Maar dit beteken ongelukkig nie dat ons dwelms gevind het nie,' sê hy.

Gesoek: Nuwe middels vir tuberkulose

Namate Nathan meer oor Mtb geleer het, het hy bewus geword van 'n ander probleem, een wat groot gevolge vir die wêreldwye gesondheid kan hê - antimikrobiese weerstand. 'Ek het ontsteld geraak omdat die toename in weerstand saamgeval het met die terugtrekking van die meeste farmaseutiese industrie om nuwe antibiotika te probeer maak.'

Ekonomie het 'n rol gespeel in hierdie onttrekking, maar Nathan sê dit is ook omdat maatskappye sukkel om nuwe antibiotika te vind. "Die praktyke wat hulle in die 1950's en 60's gebruik het, was so doeltreffend dat dit 'n dogma geword het, 'n gedagtevries," sê hy. "Hier is waar mense wat uit 'n ander dissipline na die probleem kom, dalk nuwe maniere van dink kan bring."

Terwyl akademici dikwels navorsing en tegnologie vir die industrie verskaf het, was daar min geleenthede vir sy-aan-sy-samewerking. Nathan, saam met baie ander, werk daaraan om die grense af te breek. "Nou werk ons ​​van die begin af saam met bedryfsvennote," sê hy. 'Hierdie arm-in-arm-samewerking is ongelooflik doeltreffend, en as u by die onvermydelike probleme kom, het u 'n hele multidissiplinêre span om daaroor na te dink.'

Nathan is betrokke by drie projekte wat die samewerking van akademici en kollegas in die bedryf ondersteun: die Bill and Melinda Gates Foundation se TB Drug Accelerator-program, die Tri-Institutional Therapeutics Discovery Institute en Tres Cantos Open Lab. Nathan is ook hoofnavorser vir 'n toelae van sewe jaar van die National Institute of Allergy and Infectious Disease (NIAID), wat ses instellings, vyf Weill Cornell Medicine-laboratoriums en bedryfsvennote bymekaar bring in 'n TB-navorsingseenheid. Bydraes van die NIAID kan $ 46 miljoen beloop.

Vir hierdie pogings dra Nathan sy nuwe begrip van Mtb by, insluitend wat hy geleer het oor die verdediging daarvan en die betrokke ensieme. 'Maar daar is 'n groot leerkurwe,' sê hy. 'Ontdekking van dwelms is vol mislukking, en ek het geleer hoeveel maniere om te misluk.'

Verbindings waarvan Nathan groot hoop gehad het dat dit op een of ander manier giftig sou wees of om onverklaarbare redes nie werk nie. "Maar daar is enorme entoesiasme oor die volgende groep verbindings, so ons spring terug," sê hy. 'Ek dink nie u kan so lank in die wetenskap hou as u nie kan opstaan ​​as u platgeslaan is nie.'

Through the joys and disappointments, Nathan is always driving forward. “It’s a thrill to discover something that answers a question you didn’t even know you were asking,” he says. “It’s a thrill to see people in my lab bring their own insights and launch their own paths. But we haven’t succeeded nearly well enough yet. We have so much farther to go.”


Курс 2

Fundamentals of Immunology: T Cells and Signaling

Course 2 of a three course specialization called Fundamentals of Immunology. Each course in the specialization presents material that builds on the previous course's material.

This is the second half of the journey through the defenses your body uses to keep you healthy. In the first part we learned about innate immunity and B cell function. The second part covers T cell function and coordination of the immune response. Fundamentals of Immunology: T cells and Signaling builds on the first course to describe the functions of Complement, MHC presentation to T cells, T cell development and signaling. The early lectures survey cells, tissues and organs using metaphors, cartoons and models to improve understanding and retention. This course includes the structure of both MHC proteins and T cell receptors and the sources of variation. The course provides animations of gene rearrangement, developmental processes and signal cascades. Testing employs multiple choice questions testing facts, concepts, and application of principles. Questions may refer to diagrams, drawing and photographs used in lecture and reproduced in the outline. What You’ll Learn: How complement uses adaptive and innate triggers to target pathogens. The detailed structure and coding of MHC proteins and both alpha-beta and gamma delta receptors and how these proteins interact to initiate an adaptive immune response. The basics of signaling, and the varieties of external receipt and internal activation pathways. We bine the process of putting together how signals and crosstalk control the activity of the immune system.


Materiale en metodes

Animals and challenge infections

Female BALB/c mice were purchased from Harlan Olac (Bicester, UK). DO11.10 mice, with CD4 + T cells specific for the OVA323-339 peptide in the context of the MHC class II molecule I-A d recognized by the KJ1.26 clonotypic antibody [96] were obtained originally from N. Lycke, University of Göteborg, Sweden. MD4 mice containing HEL-specific B cells [51] were backcrossed onto the BALB/c background. All mice were maintained at Biological Services, University of Glasgow, under specific pathogen-free conditions and first used between 6 and 8 weeks of age in accordance with local and UK Home Office regulations.

To initiate a malaria infection, mice were inoculated with 1 × 10 6 P. chabaudi AS-infected erythrocytes intra-peritoneally. Parasitemia was monitored by thin blood smears stained with Giemsa's stain. Peak parasitemia occurred at 5-6 days post-infection, after which time parasite levels declined and remained at low but usually detectable levels for the remainder of the experiments (see Figure 1a), as previously described [97]. Infected mice were held in a reverse light/dark cycle so that parasites harvested at 08:00 h were at the late trophozoite stage. For studies in vitro, blood was collected when parasitemia was 30-40%. Infected blood was recovered into heparin (10 IU/ml) by cardiac puncture and diluted in phosphate-buffered saline (PBS Invitrogen, Paisley, UK) to the required concentration of pRBCs.

At various times following malaria infection, mice were immunized intravenously with 500 μg OVA (Sigma-Aldrich, Poole, UK), or a conjugate of OVA and HEL (Biozyme, Gwent, UK) [50], along with 50 ng LPS (from Salmonella equi-abortus Sigma-Aldrich).

Preparation of bone-marrow DCs

DCs were prepared from bone marrow as previously described [98]. Cell suspensions were obtained from femurs and tibias of female BALB/c mice. The bone-marrow cell concentration was adjusted to 5 × 10 5 cells/ml and cultured in six-well plates (Corning Costar, New York, USA) in complete RPMI (cRPMI: RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) (all from Invitrogen) and 10% fetal calf serum (FCS Labtech International, Ringmer, UK) containing 10% of culture supernatant from X63 myeloma cells transfected with mouse granulocyte-macrophage colony stimulating factor (GM-CSF) cDNA. Fresh medium was added to the cell cultures every 3 days. On day 6, DCs were harvested and cultured at the required concentration for each individual experimental procedure, as described below. This technique generated a large number of CD11c + DCs largely free from granulocyte and monocyte contamination, as previously described [98].

In vitroculture of DCs with fixed infected or uninfected erythrocytes

Blood from P. chabaudi AS-infected mice was washed twice in PBS before being resuspended in cRPMI for addition to DCs. For fixation, infected blood was washed three times in PBS and resuspended in 0.5% paraformaldehyde for 30 min at 4°C. Fixed erythrocytes were then washed in PBS, resuspended in 0.06% Gly-Gly (Sigma-Aldrich) for 5 min at 4°C and washed twice more in PBS before being resuspended in cRPMI for addition to DC. After 24 h culture, DCs were stimulated with 1 μg/ml LPS and the expression of cell-surface molecules was analyzed 18 h later by flow cytometry. To confirm complete fixation, we showed that 2 × 10 7 fixed, infected erythrocytes could not establish infection when injected intra-peritoneally into a female BALB/c mouse.

In vitroculture of CD40L-transfected fibroblasts with DCs

The cell lines 3T3-CD40L and 3T3-SAMEN [46] were kind gifts from P. Hwu (NCI, Bethesda, USA). Cells were grown in cRPMI in T75 tissue culture flasks (Helena Biosciences, Gateshead, UK) and, when confluent, harvested and distributed in six-well plates at 2.5 × 10 5 cells/ml of cRPMI. Bone-marrow-derived DCs were cultured with infected or uninfected erythrocytes at a ratio of 1:100. After 24 h, DCs were harvested, resuspended at 1 × 10 6 cells/ml and cultured in a 1:1 ratio with either 3T3-CD40L or 3T3-SAMEN cells for a further 24 h. The level of CD40 expression on DCs was analyzed by flow cytometry and culture supernatants collected for IL-12 cytokine analysis.

T-cell stimulation in vitro

Bone-marrow DCs were centrifuged at 450 × g, resuspended at 1 × 10 6 cells/ml and 500 μl aliquots were distributed into 24-well tissue culture plates (Corning Costar) with pRBCs or RBCs. After 24 h incubation at 37°C in 5% CO2, DCs were antigen-loaded for 6 h with 5 mg/ml OVA (Worthington Biochemical, Freehold, USA). OVA-specific T cells were isolated from the mesenteric and peripheral lymph nodes of DO11.10 transgenic mice [96] on the SCID background and cultured at a 1:1 ratio with DCs. T-cell proliferation was assessed after 48, 72, 96 and 120 h of culture and assessed by incorporation of [ 3 H]thymidine (0.5 μCi/well) for the last 24 h of culture. Cells were harvested using a Betaplate 96-well harvester (Wallac Oy, Turku, Finland) and [ 3 H]thymidine incorporation measured on a Betaplate liquid scintillation counter (Wallac).

Cytokine assay

For the detection of IL-12 (p40 and p70) and IL-10, OptEIA™ enzyme-linked immunosorbent assay (ELISA) kits (Becton Dickinson, Oxford, UK) were used according to the manufacturer's instructions. For T-cell cytokines, Mouse Th1/Th2 6-Plex kit (Biosource, Nivelles, Belgium) was used according to the manufacturer's instructions. For analysis of cytokine production ex vivo, single-cell suspensions of spleen cells were prepared by rubbing through Nitex mesh (Cadisch & Sons, London, UK) in RPMI 1640 medium. After washing, cells were resuspended at 4 × 10 6 cells/ml in cRPMI, either alone or with 1 mg/ml OVA or 5 μg/ml concanavalin A (ConA Sigma-Aldrich) and supernatants sampled after 48 h. These were stored at -20°C until analysis by standard sandwich ELISA protocol (antibodies used: for IFN-γ capture, R4-6A2 for IFN-γ detection, XMG1.2 for IL-5 capture, TRKF5 for IL-5 detection, TRKF4 Pharmingen, Oxford, UK) and the levels of cytokine in supernatants calculated by comparison with recombinant cytokine standards (R & D Systems, Abingdon, UK).

Vloeisitometrie

Aliquots of 1 × 10 6 cells in 12 × 75 mm polystyrene tubes (Falcon BD, Oxford, UK) were resuspended in 100 μl FACS buffer (PBS, 2% FCS and 0.05% NaN3) containing Fc Block (2.4G2 hybridoma supernatant) as well as the appropriate combinations of the following antibodies: anti-CD4-PerCP (clone RM4-5), anti-CD11c-PE (clone HL3), anti-CD40-FITC (clone 3/23), anti-CD69-PE (clone H1.2F3), anti-CD80-FITC (clone 16-10A1), anti-CD86-FITC (clone GL1), anti-MHC-II (clone 2G9), anti-B220-PE (clone RA3-6B2), PE-hamster IgG isotype control, FITC-rat IgG2a, κ isotype control and FITC-hamster IgG1, λ isotype control (anti-TNP) (all Pharmingen), biotinylated KJ1.26 antibody or biotinylated HEL. Biotinylated antibodies were detected by incubation with fluorochrome-conjugated streptavidin (Pharmingen). After washing, samples were analyzed using a FACSCalibur flow cytometer equipped with a 488 nm argon laser and a 635 nm red diode laser and analyzed using CellQuest software (both BD BioSciences, Oxford, UK).

Preparation of erythrocyte ghosts from infected and uninfected mouse blood

Ghosts from infected and uninfected erythrocytes were generated as previously described [67]. Briefly, blood was collected into heparin by cardiac puncture and washed three times in PBS. Infected and uninfected erythrocytes were concentrated in PBS supplemented with 113 mM glucose (Sigma-Aldrich) and 3% FCS. Infected erythrocytes were incubated in an equal volume of glycerol buffer (10% glycerol (Sigma-Aldrich) supplemented with 5% FCS in PBS) for 1 h at 4°C. Parasites and ghosts were separated in a continuous Percoll (Amersham Biosciences, Little Chalfont, UK) gradient (ρ: 1.02-1.10 g/cm 3 ) in intracellular medium buffer (IM: 20 mM NaCl, 120 mM KCl, 1 mM MgCl2, 10 mM glucose, 5 mM Hepes pH 6.7) by centrifugation at 5,000 × g vir 30 min. Ghosts were then washed in IM buffer and layered on a two-step Percoll gradient (ρ: 1.01+1.02 g/cm 3 ) to separate them from ghosts that might still contain parasites. Ghosts from uninfected erythrocytes were obtained by adding a 40-fold volume of phosphate buffer (5 mM NaH2PO4/Na2HPO4, 1 mM PMSF, 0.01% azide, pH 8.5). The suspension was centrifuged at 32,000 × g vir 30 min. Ghosts from infected and uninfected erythrocytes were then washed three times in PBS before being resuspended in cRPMI for addition to DCs at a ratio of 100:1.

Hemozoin preparation

HZ was isolated from supernatants obtained from cultures of P. falciparum gametocytes, kindly provided by Lisa Ranford-Cartwright, (Division of Infection and Immunity, University of Glasgow, UK). Endotoxin-free buffers and solutions were used throughout. Supernatants were centrifuged for 20 min at 450 × g. The pellet was washed three times in 2% SLS and resuspended in 6 M guanidine HCl. Following 5-7 washes in PBS, the pellet was resuspended in PBS and sonicated for 90 min using Soniprep 150 (Sanyo Scientific, Bensenville, USA) at an amplitude of 5-8 μm to minimize aggregation and maintain the HZ in suspension. Total heme content was determined as previously described [99] by depolymerizing heme polymer in 1 ml of 20 mM NaOH and 2% SDS, incubating the suspension at room temperature for 2 h and then reading the optical density at 400 nm using a UV-visible Helios spectrophotometer (Thermo Spectronic, Cambridge, UK). DCs were pulsed with 1-20 μM HZ - a concentration range similar to that seen when DCs were cultured at a 1:100 ratio with pRBCs.

Assessment of antigen-specific antibody responses

Peripheral blood was collected and the plasma was separated by centrifugation at 450 × g for 10 min and stored at -20°C until analysis. OVA-specific IgG was measured by standard sandwich ELISA using a peroxidase-conjugated anti-mouse total IgG (Sigma-Aldrich).

Adoptive transfer of antigen-specific lymphocytes

Lymph nodes and spleens were homogenized and the resulting cell suspensions washed twice by centrifugation at 400 × g for 5 min and resuspended in RPMI. The proportions of antigen-specific T cells were evaluated by flow cytometry, and syngeneic recipients received 3 × 10 6 antigen-specific cells. In some experiments, cells were labeled with the fluorescent dye CFSE (Molecular Probes, Oregon, USA) immediately before use [100]. The level of CFSE in cells was analyzed by flow cytometry and expressed as the mean proportion of antigen-specific T cells under each CFSE peak.

Immunhistochemie

Spleens were frozen in liquid nitrogen in OCT embedding medium (Miles, Elkart, USA) in cryomoulds (Miles) and stored at -70°C. Tissue sections (8 μm) were cut on a cryostat (ThermoShandon, Cheshire, UK) and stored at -20°C. Sections were blocked and stained as previously described [55], using B220-FITC to stain B-cell areas and biotinylated-KJ1.26 to detect OVA-specific DO11.10 cells, and visualized using Streptavidin-Alexa Fluor 647 (Molecular Probes). All photographs were taken at 20× magnification.

To visualize HZ deposition in DCs, cells were photographed using an Axiovert S-100 Zeiss microscope using a 63× oil-immersion lens by normal bright-field imaging. To image HZ in splenic DC, 8 μm sections were cut as described above and stained with biotinylated-CD11c followed by Streptavidin-HRP and finally tyramide-488 (PerkinElmer, Boston, USA). Images were then taken of bright-field and green fluorescence and the images merged by inverting and then false coloring the bright-field image such that deposited HZ appeared red and CD11c appeared green.

Laser-scanning cytometry

Sections were stained as described above. Sections were then scanned on a laser-scanning cytometer equipped with argon, helium, neon, and ultraviolet lasers (Compucyte, Cambridge, USA) and visualized with the Openlab imaging system (Improvision, Coventry, UK). The localization of transgenic T cells and B-cell follicles were plotted. Using these tissue maps the number of transgenic T cells in defined gates was calculated for three gates in periarteriolar lymphoid sheath (PALS) and three B-cell follicle gates per section. Data are plotted as the mean proportion of transgenic T cells in each gate relative to the number of transgenic T cells in the entire section and are the mean of triplicate readings from three mice per group.

Isolation of DCs from spleen

Spleens were excised and single-cell suspensions obtained as described above. In some experiments, cells were stimulated with 1 μg/ml LPS for 18 h before analysis by flow cytometry. To obtain purified DCs from spleens of mice, single-cell suspensions were labeled using a CD11c isolation kit (Miltenyi Biotec, Bisley, UK) according to the manufacturer's instructions. DCs were then purified using two MS magnetic columns (Miltenyi Biotec) and found to be 85-95% pure by flow cytometric analysis.

Statistiese analise

Results are expressed as mean ± standard error or standard deviation as indicated. Significance was determined by one-way ANOVA in conjunction with the Tukey test using Minitab. A bl-value of less than 0.05 was considered significant.


Toward synthetic detection platforms

Despite breakthroughs in our molecular understanding of NLR activation, knowledge of subsequent signaling steps and mechanisms remains weak. The pathways that connect NLR activation to outputs such as transcription of defense genes, changes in cell permeability, localized cell death, and systemic signaling remain poorly understood. Do activated, or dimerized, or oligomerized plant NLRs recruit new signaling proteins? How distinct are the signaling pathways controlled by the various N-terminal signaling domains recruited to the NLR chassis during evolution? Are integrated decoy domain NLRs modular? Can we engineer new or additional decoy domains into them to create or extend NLR function? As more structural and mechanistic information emerges on how plant and animal NLRs function, the engineering of novel, bespoke, and useful recognition capacities in plant and animal immune systems will become a more realistic goal.


Therapeutic options targeting immune deviation

Three major concepts emerge concerning therapeutic and preventive options in COVID-19: (1) targeting the virus and its cellular life cycle by antiviral drugs, (2) developing vaccines that can either prevent or mitigate disease symptoms after infection, and (3) targeting the deviation of the immune response to avoid or mitigate severe and fatal disease outcome ( Riva etਊl., 2020 Vabret etਊl., 2020 ). Targeting the innate immune system might play a role in all three areas.

The antivirals are divided in direct and indirect acting antivirals, targeting molecules and mechanisms of the virus itself or host cell proteins, respectively. Because SARS-CoV-2 antagonizes the type I IFN system, drugs strengthening this cellular defense system might improve early innate immune responses. Type I IFN therapy in patients with genetic deficits in the IFN system ( Zhang etਊl., 2020a ), but not in those with autoimmune phenocopies of these deficits ( Bastard etਊl., 2020 ), might be beneficial if provided sufficiently early ( Hadjadj etਊl., 2020 Wang etਊl., 2020b ). More than 20 clinical trials are currently evaluating the efficacy of type I IFN treatment (https://www.clinicaltrials.gov, accessed 2021/01/19) ( Wang etਊl., 2020b Zhou etਊl., 2020a ), the best time window, and benefits versus risks of IFN therapy. Alternatives such as IFNλ (type III IFN) only targeting receptors on epithelial cells without the broader effects of type I IFNs ( Prokunina-Olsson etਊl., 2020 ) are also under clinical evaluation. Other indirect antivirals blocking viral cell entry (e.g., by targeting proteases such as TMPRSS2) have been suggested as potential therapies ( Kaur etਊl., 2021 ), yet definitive proof for clinical efficacy is still lacking. Antiviral protein interaction mapping revealed further promising sets of antivirals: those that affect translation and those that modulate the sigma-1 and sigma-2 receptors, the cellular interaction partners of SARS-CoV-2 NSP6 and ORF9c ( Gordon etਊl., 2020 ).

Based on results from phase-III vaccine trials utilizing mRNA vaccines ( Anderson etਊl., 2020 Polack etਊl., 2020 ), two vaccines have been approved in the United States, United Kingdom, Israel, and the European Union, and several million individuals have been vaccinated since late 2020. A most surprising finding in light of age-related changes in the adaptive and the innate immune system is the similarly high efficiency of these vaccines in the elderly population. This unexpected success requires further mechanistic and molecular evaluations about the elicited immune response because this might give insights how age-related alterations of the immune system are overcome by this type of vaccination.

The third strategy is targeting the deviation of the immune response to avoid or mitigate severe and fatal disease outcomes. A starting point for many therapeutic strategies has been the hyperinflammatory state in severe disease ( Tang etਊl., 2020 Wang etਊl., 2020a ). Not surprisingly, trials targeting cytokines such as IL-6, IL-1, IFNγ, IL-1R, TNF, CXCL8, GM-CSF, GM-CSF receptor, or IL-37 have been reported, as well as strategies attempting to achieve disruption of chemokine signaling (e.g., via CCR1, CCR2, and CCR5) to prevent overt innate immune cell recruitment into the lung ( Wang etਊl., 2020a ). Because COVID-19 presents with such heterogeneity, a certain drug might be beneficial in one setting, while having no effect in another. For example, inconsistent results have been reported from large clinical trials when trying to inhibit IL-6 associated with hyperinflammation ( Huang and Jordan, 2020 ). A randomized clinical trial assessing tocilizumab, an anti-IL-6 antibody ( Stone etਊl., 2020 ), showed no benefit for moderately ill patients concerning effectiveness of preventing intubation or death. In contrast, among critically ill patients, the risk of in-hospital mortality was lower in patients treated with tocilizumab in the first 2ꃚys of ICU admission ( Gupta etਊl., 2021 ), which has also been reported in earlier trials ( Huang and Jordan, 2020 ). It cannot yet be ruled out that targeting IL-6 might be beneficial for a group of patients in specific clinical settings. This might be similarly true for targeting IL-1 with anakinra for which several smaller studies were reporting beneficial effects ( Iglesias-Julián etਊl., 2020 Kooistra etਊl., 2020 ), yet results from larger randomized trials are still missing.

Whether targeting single effector molecules will be efficient in disrupting the COVID-19-associated immune deviation awaits the report of ongoing clinical trials. Targeting several of these pathways simultaneously (e.g., by inhibiting Janus kinases) might overcome such limitations ( McCreary and Pogue, 2020 Wu and Yang, 2020 ). In early clinical trials, the use of the JAK inhibitor baricitinib showed a reduction in serum levels of IL-6, IL-1β, and TNF ( Bronte etਊl., 2020 ), suppressed the production of proinflammatory cytokines in lung macrophages, and the recruitment of neutrophils to the lung ( Hoang etਊl., 2021 ). Randomized clinical trials have to be initiated to validate these promising results. Along these lines, therapies targeting the kallikrein-kinin system with icatibant ( van de Veerdonk etਊl., 2020 ), or the coagulation system with antibodies against C5a and C3a ( Mastellos etਊl., 2020 ), have been introduced as additional options to ameliorate clinical symptoms and reduce mortality rates. Further, these therapeutic strategies might be considered in combination with immunotherapies such as anti-IL-6 or anti-IL-1 in severe COVID-19.

Reverse transcriptomics of blood immune cells derived from COVID-19 patients predicted the broadly anti-inflammatory drug dexamethasone and other corticosteroids as potentially beneficial for a subgroup of severely ill COVID-19 patients ( Aschenbrenner etਊl., 2021 ). Indeed, dexamethasone was shown to be beneficial, particularly in patients with severe disease courses where it reduced 28-day mortality in randomized clinical trials ( RECOVERY Collaborative Group etਊl., 2021 Tomazini etਊl., 2020 WHO Rapid Evidence Appraisal for COVID-19 Therapies (REACT) Working Group etਊl., 2020 ). Further, molecular prediction of drug responses may guide the way for precision medicine approaches following patient stratification.


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How the surfaces of silicone breast implants affect the immune system

Every year, about 400,000 people receive silicone breast implants in the United States. According to data from the U.S. Food and Drug Administration, a majority of those implants needs to be replaced within 10 years due to the buildup of scar tissue and other complications.

A team led by MIT researchers has now systematically analyzed how the varying surface architecture found in these implants influences the development of adverse effects, which in rare cases can include an unusual type of lymphoma.

"The surface topography of an implant can drastically affect how the immune response perceives it, and this has important ramifications for the [implants'] design," says Omid Veiseh, a former MIT postdoc. "We hope this paper provides a foundation for plastic surgeons to evaluate and better understand how implant choice can affect the patient experience."

The findings could also help scientists to design more biocompatible implants in the future, the researchers say.

"We are pleased that we were able to bring new materials science approaches to better understand issues of biocompatibility in the area of breast implants. We also hope the studies that we conducted will be broadly useful in understanding how to design safer and more effective implants of any type," says Robert Langer, the David H. Koch Institute Professor at MIT and the senior author of the study.

Veiseh, who is now an assistant professor at Rice University, and Joshua Doloff, a former MIT postdoc who is now an assistant professor at Johns Hopkins University, are the lead authors of the paper, which appears today in Nature Biomedical Engineering. The research team also includes scientists from Rice University, Johns Hopkins, Establishment Labs, and MD Anderson Cancer Center, among other institutions.

Surface analysis

Silicone breast implants have been in use since the 1960s, and the earliest versions had smooth surfaces. However, with these implants, patients often experienced a complication called capsular contracture, in which scar tissue forms around the implant and squeezes it, creating pain or discomfort as well as visible deformation of the implant. These implants could also flip after implantation, requiring them to be surgically adjusted or removed.

In the late 1980s, some companies began making implants with rougher surfaces, with the hopes of reducing capsular contracture rates and making them "stick" better to the tissue and stay in place. They did this by creating a surface with peaks extending up to hundreds of microns above the surface.

However, in 2019, the FDA requested a breast implant manufacturer to recall all highly textured breast implants (about 80 microns) marketed in the United States due to risk of breast implant-associated anaplastic large cell lymphoma, a cancer of the immune system.

A new generation of breast implants that dates back a decade, having a unique and patented surface architecture that includes not only a slight degree of surface roughness, with an average of about 4 microns, but also other specific surface characteristics including skewness and the number, distribution, and size of contact points optimized to cellular dimensions, was designed to prevent those complications.

In 2015, Doloff, Veiseh, and researchers from Establishment Labs teamed up to explore how this unique surface, as well as others commonly used, interact with the surrounding tissue and the immune system. They began by testing five commercially available implants with different topographies, including degree of roughness. These included the highly textured one that had been previously recalled, one that is completely smooth, and three that are somewhere in between. Two of these implants had the aforementioned novel surface architecture, one with a 4-micron roughness and one with a 15-micron roughness, manufactured by Establishment Labs.

In a study of rabbits, the researchers found that tissue exposed to the roughest implant surfaces showed signs of increased activity from macrophages -- immune cells that normally clear out foreign cells and debris.

All of the implants stimulated immune cells called T cells, but in different ways. Implants with rougher surfaces stimulated more pro-inflammatory T cell responses, while implants with the unique surface topography, including 4-micron average roughness, stimulated T cells that appear to inhibit tissue inflammation.

The researchers' findings suggest that rougher implants rub against the surrounding tissue and cause more irritation. This may offer an explanation for why the rougher implants can lead to lymphoma: The hypothesis is that some of the texture sloughs off and gets trapped in nearby tissue, where it provokes chronic inflammation that can eventually lead to cancer.

The researchers also tested miniaturized versions of these implants in mice. They manufactured these implants using the same techniques used to manufacture the human-sized versions, and showed that more highly textured implants provoked more macrophage activity, more scar tissue formation, and higher levels of inflammatory T cells. The researchers also performed single-cell RNA sequencing of immune cells from these tissues to confirm that the cells were expressing pro-inflammatory genes.

"While completely smooth surface implants also had higher levels of macrophage response and fibrosis, it was very clear in mice that individual cells were more stressed and were expressing more of a pro-inflammatory phenotype in response to the highest surface roughness," Doloff says.

On the other hand, implants with the unique surface architecture, including an optimized degree or "sweet spot" of surface roughness, at about 4 microns on average, and other specific characteristics, appeared to significantly reduce the amount of scarring and inflammation, compared to either the implants with higher roughness or a completely smooth surface.

"We believe that this is due to such surface architecture existing on the scale of individual cells of the body, allowing the cells to perceive them in a different way," Doloff says.

Toward safer implants

After performing their animal studies, the researchers analyzed samples from a large bank of cancer tissue samples at MD Anderson to study how human patients respond to different types of silicone breast implants.

In those samples, the researchers found evidence for the same types of immune responses that they had seen in the animal studies. Among their findings, they observed that tissue samples that had been host to highly textured implants for many years showed signs of a chronic, long-term immune response. They also found that scar tissue was thicker in patients who had more highly textured implants.

"Doing across-the-board comparisons in mice, rabbits, and then in human [tissue samples] really provides a much more robust and substantial body of evidence about how these compare to one another," Veiseh says.

The authors hope that their datasets will help other researchers optimize the design of silicone breast implants and other types of medical silicone implants for better safety.

"The importance of science-based design that can provide patients with safer breast implants was confirmed in this study," says Roberto de Mezerville, an author of the paper and head of R&D at Establishment Labs. "By demonstrating for the first time that an optimal surface architecture allows for the least possible inflammation and foreign-body response, this work is a significant contribution to the entire medical device industry."


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