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

Støttet delvis med tilskudd fra National Institutes of Health (R01 NS37253 og N01 NS055167) og Nasjonal Multippel sklerose Society (RG 3288-A6).

1. Forbereder CFA-emulgert Antigen <ol><li> Løs marsvin MBP (utarbeidet etter den metoden for Swanborg1 og lagret i lyofilisert skjema ved 4 ° C) i fosfatbuffer løsning (PBS) i en konsentrasjon på 2 mg / ml. Hvis syntetiserte MBP peptider (MBP 60-80, SHHAARTTHYGSLPQKSQR) 2 benyttes, utarbeide en 2 mg / ml. Tegn opp en passende mengde antigen løsning (0,1 ml per mus som skal vaksineres) i et glass sprøyte med Luer-Lok. </li><li> Tegn opp et volum på komplett Freund s adjuvans H37Ra (0.1WT%)...

<ol>
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Studier av EAE hos mus utnytter ofte de neuroantigens og myelin basic protein (MBP), proteolipid protein (PLP) eller myelin oligodendrocyte protein (Mog). Tidligere studier meste brukt MBP. PLP stimulerer sterke og konsistente svar fra SJL mus. Flere nylig, er Mog felles neuroantigen brukt fordi B6 mus er mottakelige for sykdom induksjon med dette antigenet. Mange av genet målrettede musestammer er på B6 genetisk bakgrunn. Interessant, B6 mus er utsatt for EAE induksjon med Mog men er motstandsdyktig mot sykdom induks...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Marsvin ryggmargen </td><td> Rockland Immuno-kjemikalier, Inc. Gilbertsville, 19525 PA <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> Freund er Adjuvant, komplett H37Ra </td><td> Difco Laboratories Detroit, MI </td><td> 231131 </td><td></td></tr><tr><td> Multifit utveksling-stand sprøyte </td><td> BD <a href="http://www.bd.com/us" target="_blank">www.bd.com / oss</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

Eksperimentell autoimmun encefalomyelitt (EAE) er en inflammatorisk sykdom i sentralnervesystemet (CNS) og har blitt brukt som en dyremodell for studier av det menneskelige demyeliniserende sykdom, multippel sklerose (MS). EAE er preget av patologisk infiltrasjon av mononukleære celler i CNS og ved klinisk manifestasjon av paralytisk sykdom. I likhet med MS, er EAE også under genetisk kontroll i at visse musestammer er mottakelige for sykdom induksjon mens andre er resistente. Vanligvis C57BL / 6 (H-2 b) mus immunisert med myelin basic protein (MBP) klarer å utvikle lammelse tegn. Denne manglende respons er absolutt ikke skyldes feil i antigen prosessering eller antigen presentasjon av MBP, som en eksperimentell protokoll som beskrives her hadde blitt brukt til å indusere alvorlig EAE i C57BL / 6 mus samt andre anerkjente resistente musestammer. I tillegg hadde encephalitogenic T celle kloner fra C57BL / 6 og BALB / c mus reaktive til MBP vært suksessfullisolert og forplantet seg. Forsøksprotokollen innebærer å bruke en mobil adoptiv overføring system der MBP-fylt (200 mikrogram / mus) C57BL / 6 donor lymfeknute cellene er isolert og dyrket i fem dager med antigenet å utvide pool av MBP-spesifikke T-celler. På slutten av kultur perioden, er 50 millioner levedyktige celler overført til naive syngeneic mottakerne gjennom halen blodåre. Mottaker mus så behandlet normalt ikke utvikler EAE, og dermed bekrefter sin motstandsdyktig status, og de kan forbli normal ubestemt tid. Ti dager etter celle overføring, er mottaker mus utfordret med komplett Freund adjuvans (CFA)-emulgert MBP i fire steder i flankene. Alvorlig EAE begynner å utvikle seg i disse musene ti til fjorten dager etter smitte. Resultatene viste at induksjon av sykdommen var antigene spesifikk som utfordring med irrelevante antigener ikke overtale kliniske tegn på sykdom. Betydelig, en titrering av antigen dosen som brukes tilutfordre mottakeren mus viste at det kunne være så lav som 5 mikrogram / mus. I tillegg viste en kinetisk studie av tidspunktet for antigene utfordring som utfordring å indusere sykdom var effektiv så tidlig som fem dager etter antigene utfordring og så lenge som over 445 dager etter antigen utfordring. Disse dataene sterkt peker mot involvering av en &quot;lang levetid&quot; T celle befolkning i å opprettholde manglende respons. Involvering av regulatoriske T-celler (Tregs) i dette systemet er ikke definert.

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 å motstå induksjon av eksperimentell autoimmun encefalomyelitt (EAE) med myelin basic protein. Beskrevet her er en enkel immunisering protokoll som reverserer den fungere bedre og induserer paralytisk sykdom i flere typiske EAE resistente mus flekker.

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