Name: trans-vivo delayed type hypersensitivity assay for antigen specific regulation
Uploaded: 2013-05-02T12:00:00
Description: Watch this Scientific Journal Video about Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation at JoVE.com

Forfatterne ønsker å erkjenne bidrag fra AM VanBuskirk til utviklingen av vår forståelse av det regulerte DTH respons i resipienter. Dette arbeidet ble støttet NIH tilskudd PO1AI084853 og R01AI066219-06, og av EU-sponset en studie.

En. Utarbeidelse av lymfocytter <ol><li> Samle blod inn ACD (syresitratdekstrose) rør. </li><li> Isoler PBMC fra friskt humant perifert blod ved hjelp Lymfocytt Separation Medium i henhold til standard metoder. </li><li> Vask PBMC tre ganger med PBS for å fjerne kontaminerende blodplater. Blodplater ble funnet å påvirke trans-vivo DTH analysen. Maximal tillatte blodplater kontaminering av PBMC forberedelse er ≤ 1x10 7 / injeksjon. </li><li> Hvis det er en merkbar røde blodlegemer forurensning, utfør...

<ol>
	<li>Carrodeguas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans+vivo+analysis+of+human+delayed-type+hypersensitivity+reactivity.">Trans vivo analysis of human delayed-type hypersensitivity reactivity.</a> Hum. Immunol. 60, 640-651 (1999).</li><li>VanBuskirk, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+allograft+acceptance+is+associated+with+immune+regulation.">Human allograft acceptance is associated with immune regulation.</a> J. Clin. Invest. 106, 145-155 (2000).</li><li>Burlingham, W. J., Jankowska-Gan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+and+injection+site+are+crucial+for+detecting+linked+suppression+in+transplant+recipients+by+trans-vivo+DTH+assay.">Mouse strain and injection site are crucial for detecting linked suppression in transplant recipients by trans-vivo DTH assay.</a> Am. J. Transplant. 7, 466-470 (2007).</li><li>Burlingham, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+tolerance+to+a+maternal+kidney+transplant+is+selective+for+HLA+class+II:+Evidence+from+trans-vivo+DTH+and+alloantibody+analysis.">Loss of tolerance to a maternal kidney transplant is selective for HLA class II: Evidence from trans-vivo DTH and alloantibody analysis.</a> Human Immunology. 61, 1395-1402 (2000).</li><li>Geissler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+allograft+acceptance+and+the+'tolerance+assay':+In+vitro+anti-donor+T+cell+assays+show+hyporeactivity+to+donor+cells+but,+unlike+DTH,+fail+to+detect+linked+suppression.">Human liver allograft acceptance and the 'tolerance assay': In vitro anti-donor T cell assays show hyporeactivity to donor cells but, unlike DTH, fail to detect linked suppression.</a> Transplantation. 72, 571-580 (2001).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+allograft+acceptance+and+the+'tolerance+assay'.+II.+donor+HLA-A,+-B+but+not+DR+antigens+are+able+to+trigger+regulation+of+DTH.">Human liver allograft acceptance and the 'tolerance assay'. II. donor HLA-A, -B but not DR antigens are able to trigger regulation of DTH.</a> Hum. Immunol. 63, 862 (2002).</li><li>Cai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+H+Antigen+HA-1-specific+Regulator+and+Effector+CD8++T+Cells,+and+HA-1+Microchimerism,+in+Allograft+Tolerance.">Minor H Antigen HA-1-specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance.</a> J. Exp. Med. 199, 1017-1023 (2004).</li><li>Rodriguez, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+and+graft+survival+in+kidney+transplant+recipients+are+both+enhanced+by+human+leukocyte+antigen+matching.">Immune regulation and graft survival in kidney transplant recipients are both enhanced by human leukocyte antigen matching.</a> Am. J. Transplant. 4, 537-543 (2004).</li><li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD4+CD25low+adaptive+T+regulatory+cells+suppress+delayed-type+hypersensitivity+during+transplant+tolerance.">Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance.</a> J. Immunol. 178, 3983-3995 (2007).</li><li>Derks, R. A., Jankowska-Gan, E., Xu, Q., Burlingham, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+type+determines+the+mechanism+of+bystander+suppression+by+adaptive+T+regulatory+cells+specific+for+the+minor+antigen+HA-1.">Dendritic cell type determines the mechanism of bystander suppression by adaptive T regulatory cells specific for the minor antigen HA-1.</a> J. Immunol. 179, 3443-3451 (2007).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+reduction+of+immunosuppression+in+older+renal+transplant+recipients+who+exhibit+donor-specific+regulation.">Successful reduction of immunosuppression in older renal transplant recipients who exhibit donor-specific regulation.</a> Transplantation. 88, 533-541 (2009).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretransplant+immune+regulation+predicts+allograft+outcome:+bidirectional+regulation+correlates+with+excellent+renal+transplant+function+in+living-related+donor-recipient+pairs.">Pretransplant immune regulation predicts allograft outcome: bidirectional regulation correlates with excellent renal transplant function in living-related donor-recipient pairs.</a> Transplantation. 93, 283-290 (2012).</li><li>Knechtle, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+and+limited+use+of+tacrolimus+to+avoid+rejection+in+an+alemtuzumab+and+sirolimus+regimen+for+kidney+transplantation:+clinical+results+and+immune+monitoring.">Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.</a> Am. J. Transplant. 9, 1087-1098 (2009).</li><li>Haynes, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-specific+indirect+pathway+analysis+reveals+a+B-cell-independent+signature+which+reflects+outcomes+in+kidney+transplant+recipients.">Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.</a> Am. J. Transplant. 12, 640-648 (2012).</li><li>Burlingham, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17-dependent+cellular+immunity+to+collagen+type+V+predisposes+to+obliterative+bronchiolitis+in+human+lung+transplants.">IL-17-dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants.</a> J. Clin. Invest. 117, 3498-3506 (2007).</li><li>Bobadilla, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH-17,+Monokines,+Collagen+Type+V,+and+Primary+Graft+Dysfunction+in+Lung+Transplantation.">TH-17, Monokines, Collagen Type V, and Primary Graft Dysfunction in Lung Transplantation.</a> Am. J. Respir. Crit. Care Med. 177 (6), (2008).</li><li>Olson, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prostate+tumor+antigen-specific+CD8++regulatory+T+cells+are+inhibited+by+CTLA-4+or+IL-35+blockade.">Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade.</a> J. Immunol. 189, 5590-5601 (2012).</li></ol>

Den trans-vivo DTH-analysen er en ny diagnostisk test med en potensiell klinisk anvendelse i vurderingen av celle-medierte responser i transplantasjon, kreft og autoimmune pasienter. Det er verdifullt fordi det er ikke bare nyttig for å overvåke tilbakekalling T effektor responser, men også den kan påvise T-regulerende responser. En sikker måte å oppdage menneskelig DTH regulering kan forutsi sikkerheten til immunsuppresjon tilbaketrekking hos pasienter som er kandidater for monoterapi eller toleranse studier. </p...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>ACD tube for blood collection</td>
			<td>BD</td>
			<td>02-684-26</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lymphocyte Separation Medium</td>
			<td>Cellgro</td>
			<td>25-072-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-Buffered Saline</td>
			<td>Cellgro</td>
			<td>21-031-CM</td>
			<td>Without calcium &amp; magnesium</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>BioWhittaker</td>
			<td>10-548E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TT/DT or EBV</td>
			<td>Sanofi Pasteur Inc./ Meridian Life Science, Inc.</td>
			<td>&nbsp;</td>
			<td>TT/DT 25 &mu;g/injection EBV 8 &mu;g/injection</td>
		</tr>
		<tr>
			<td>Protease inhibitor PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eosine for cell count</td>
			<td>Sigma-Aldrich</td>
			<td>E-6003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alloantigen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Purified HLA antigens, synthetic allopeptides can be used instead of donor cell-free lysates</td>
		</tr>
		<tr>
			<td>50 ml sterile centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-18</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml pipettes and pipettor</td>
			<td>BD Falcon</td>
			<td>13-675-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml safe-lock tubes</td>
			<td>Costar</td>
			<td>3213</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1000 &mu;l, 100 &mu;l , 10 &mu;l pipettes with sterile tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Full size centrifuge and microfuge</td>
			<td>Beckman Coulter/Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1/2cc or 1cc insulin syringes</td>
			<td>Becton Dickinson</td>
			<td>14-826-79</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Vibracell sonicator</td>
			<td>Divtech Equipment Co. Sonocs&amp; Materials Inc</td>
			<td>&nbsp;</td>
			<td>2 mm probe</td>
		</tr>
		<tr>
			<td>Dial thickness gauge</td>
			<td>Mitutoyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SCID mice</td>
			<td>Harlan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>&nbsp;</td>
			<td>Inhalant anesthesia</td>
		</tr>
	</tbody>
</table>

trans-vivo delayed type hypersensitivity assay for antigen specific regulation

Forsinket hypersensitivitet respons (DTH) er en rask in vivo manifestasjon av T-celle-avhengige immunrespons til et fremmed antigen (Ag) at verten immunsystem har opplevd i den siste tiden. DTH reaksjoner er ofte delt i en sensibilisering fase, med henvisning til den initielle antigen erfaring, og en utfordring fase, som vanligvis følger flere dager etter sensibilisering. Mangelen på en forsinket hypersensitivitet respons til en tilbakekalling Ag demonstrert av huden testing er ofte sett på som et bevis på anergy. Den tradisjonelle DTH analysen har vært effektivt brukt i diagnostisering mange mikrobielle infeksjoner. Til tross for å dele lignende immun funksjoner som lymfocyttinfiltrasjon, ødem, og vevsnekrose, er den direkte DTH ikke et mulig diagnostisk teknikk hos transplanterte pasienter på grunn av muligheten for direkte innsprøytning som resulterer i overfølsomhet mot donor antigener og pode tap. For å unngå dette problemet, summingenen-to-mus &quot;trans-vivo&quot; DTH analysen ble utviklet 1,2. Denne testen er i hovedsak en overføring DTH assay, hvori humane perifere blodmononukleære celler (PBMC) og spesifikke antigener ble injisert subkutant inn i pinnae eller fotpute av et naivt mus og DTH-liknende hevelse måles etter 18-24 timers 3.. Antigen presentasjon av menneskelige antigenpresenterende celler som makrofager eller DCS-til T-celler i svært vaskulære mus vev utløser inflammatorisk kaskade og tiltrekker seg mus immunceller som resulterer i hevelse svar. Responsen er antigen-spesifikk og krever forutgående antigen sensibilisering. En positiv donor-reaktivt DTH respons i Tv-DTH analysen reflekterer at transplantasjon pasienten har utviklet en pro-inflammatorisk immune disposisjon mot pode alloantigener. Det viktigste trekk ved denne analyse er at den også kan brukes til å detektere regulatoriske T-celler, som forårsaker bystander suppresjon. Tilskuer undertrykkelse av enDTH tilbakekalling respons i nærvær av donor antigen er karakteristisk for resipienter med aksepterte allografts 2,4-14. Overvåkingen av resipienter for alloreactivity og regulering av Tv-DTH kan identifisere en undergruppe av pasienter som kan ha nytte av reduksjon av immunsuppresjon uten forhøyet risiko for avvisning eller forverret nyrefunksjon. En lovende området er anvendelsen av Tv-DTH analysen i overvåking av autoimmunitet 15,16 og også i tumor immunologi 17.

En. Evaluering av nyretransplanterte for donor antigen-spesifikk respons ved hjelp av Tv-DTH analysen For å teste donor-reaktivt cellulær immunitet hos nyretransplanterte vi injisert PBMC fra disse pasientene med donor antigener alene eller sammen med en tilbakekalling antigen tetanustoksoid (TT). Som en positiv kontroll-celler ble injisert med TT alene. Vi observerer tre mønstre av forsinket hypersensitivitet i resipienter (figur 1). Alle pasienter reagert sterkt til Recall...

Watch this Scientific Journal Video about Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation at JoVE.com

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Vi beskriver en verdifull diagnostisk analyse som potensielt kan brukes til å bestemme uttak av immunsuppresjon etter transplantasjon uten forhøyet risiko for pode avvisning. Analysen bruker prinsippene om Forsinket hypersensitivitet og gir nøyaktig vurdering av både donor spesifikke effektor og regulatoriske immunresponser montert mottakere.

Trans-vivo Forsinket hypersensitivitet analyse for antigenspesifikk forordning

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

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