Name: overcoming unresponsiveness in experimental autoimmune encephalomyelitis (eae) resistant mouse strains by adoptive transfer and antigenic challenge
Uploaded: 2012-04-09T13:00:00
Description: Watch this Scientific Journal Video about Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge at JoVE.com

Delvist understøttet af tilskud fra National Institutes of Health (R01 NS37253 og N01 NS055167) og National Scleroseforeningen (RG 3288-A6).

1. Fremstilling CFA-emulgeret Antigen <ol><li> Opløs marsvine MBP (fremstillet ifølge fremgangsmåden ifølge Swanborg1 og opbevaret i lyofiliseret form ved 4 ° C) i phosphatpufferopløsning (PBS) ved en koncentration på 2 mg / ml. Hvis syntetiserede MBP peptider (MBP 60-80, SHHAARTTHYGSLPQKSQR) 2 anvendes, fremstilles en 2 mg / ml opløsning. Udarbejde en passende mængde af antigen-opløsning (0,1 ml per mus kan immuniseres) i en glassprøjte med luer-lok. </li><li> Opstille en volumen af ?...

<ol>
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Undersøgelser af EAE i mus ofte benytte neuroantigens, basisk myelinprotein (MBP), proteolipidprotein (PLP) eller myelin oligodendrocyt protein (MOG). Tidligere undersøgelser anvendes oftest MBP. PLP stimulerer stærke og konsekvente svar fra SJL mus. For nylig, MOG er almindeligt anvendte neuroantigen fordi B6 mus er følsomme for sygdomsinduktion med dette antigen. Mange af genet målrettede musestammer er på B6 genetisk baggrund. Interessant B6 mus er modtagelige for EAE-induktion med MOG men er resistente over fo...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Marsvin rygmarv </td><td> Rockland Immuno-kemikalier, Inc. Gilbertsville, PA 19525 <a href="http://www.rockland-inc.com" target="_blank">www.rockland-inc.com</a> </td><td> GP-T065 </td><td> frosset </td></tr><tr><td> Freunds adjuvans, komplet H37Ra </td><td> Difco Laboratories Detroit, MI </td><td> 231131 </td><td></td></tr><tr><td> MultiFit udskiftelige kan sprøjte </td><td> BD <a href="http://www.bd.com/us" target="_blank">www.bd.com / os</a> </td><td> 512133 </td><td></td></tr><tr><td> RPMI 1640 </td><td> MediatechInc. Manassas, VA <a href="http://www.cellgro.com" target="_blank">www.cellgro.com</a> </td><td> 10-040-CM </td><td></td></tr><tr><td> OVA </td><td> Sigma-Aldrich <a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a> </td><td> A5503 </td><td></td></tr><tr><td> Mus </td><td> Jackson Laboratory Bar Harbor, Maine </td><td> B6 mus: 0000664 </td><td></td></tr></tbody></table>

overcoming unresponsiveness in experimental autoimmune encephalomyelitis (eae) resistant mouse strains by adoptive transfer and antigenic challenge

Eksperimentel autoimmun encephalomyelitis (EAE) er en inflammatorisk sygdom i centralnervesystemet (CNS) og er blevet anvendt som en dyremodel til undersøgelse af den humane demyeliniserende sygdom, multipel sclerose (MS). EAE er karakteriseret ved patologisk infiltration af mononukleære celler i CNS og kliniske manifestation af paralytisk sygdom. Svarende til MS, EAE er også under genetisk kontrol at visse musestammer er modtagelige for sygdomsinduktion, mens andre er resistente. Typisk C57BL / 6 (H-2b)-mus immuniseret med myelin basisk protein (MBP) ikke udvikler paralytiske symptomer. Dette unresponsiveness er bestemt ikke skyldes defekter i antigen-processering eller antigen præsentation af MBP, som en forsøgsprotokol beskrevet her blevet anvendt til at inducere kraftig EAE i C57BL / 6 mus såvel som andre anerkendte resistente musestammer. Desuden var encephalitogene T-cellekloner fra C57BL / 6 og Balb / c-mus reaktive med MBP er lykkedesfuldt isoleret og opformeret. Den eksperimentelle protokol indebærer anvendelse af en cellulær adoptiv overførsel i hvilket MBP-primet (200 ug / mus) C57BL / 6 donor lymfeknudeceller isoleres og dyrkes i fem dage med antigenet for at udvide pulje af MBP-specifikke T-celler. Ved afslutningen af ​​dyrkningsperioden, er 50 millioner levedygtige celler overføres til naive recipienter syngene gennem halevenen. Modtager mus, så der blev behandlet normalt ikke udvikler EAE, hvilket bekræfter deres resistente status, og de kan forblive normal ubestemt tid. Ti dage efter celleoverførsel, der modtager musene udfordret med komplet Freunds adjuvans (CFA)-emulgeret MBP i fire steder i flankerne. Svær EAE begynder at udvikle sig i disse mus ti til fjorten dage efter udfordring. Resultaterne viste, at induktionen af ​​sygdom var antigent specifikt som udfordring med irrelevante antigener ikke kliniske tegn på sygdom. Betydeligt, en titrering af antigendosis anvendes tiludfordre de modtagende mus viste, at det kunne være så lav som 5 mg / mus. Desuden viste en kinetisk undersøgelse af timingen af ​​antigenudfordring denne udfordring til at fremkalde sygdom var effektivt så tidligt som 5 dage efter antigenudfordring og så længe som over 445 dage efter antigenudfordring. Disse data kraftigt peger mod involvering af en &quot;langlivede&quot; T-cellepopulation til at opretholde passivitet. Inddragelsen af ​​regulatoriske T-celler (tregs) i dette system ikke er defineret.

Watch this Scientific Journal Video about Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge at JoVE.com

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Visse musestammer er i stand til at modstå induktion af eksperimentel autoimmun encephalomyelitis (EAE) med myelin basisk protein. Beskrevet her er en simpel immuniseringsprotokol, der vender unresponsiveness og inducerer paralytisk sygdom i flere typiske EAE resistente muse pletter.

Overvinde unresponsiveness i eksperimentel autoimmun encephalomyelitis (EAE) Resistente musestammer ved adoptiv overførsel og antigenudfordring

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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