저자는 이식 규제 DTH 반응에 대한 우리의 이해의 발전에 AM VanBuskirk의 기여를 인정하고 싶습니다. 이 작품은 NIH 보조금에게 PO1AI084853 및 R01AI066219-06을 지원하고, EU가 후원 한 연구가 있었다.

1. 림프구의 준비 <ol><li> ACD (산성 구연산 포도당) 튜브에 혈액을 수집합니다. </li><li> 표준 방법에 따라 림프구 분리 매체를 사용하여 신선한 인간의 말초 혈액에서 PBMC를 분리합니다. </li><li> 오염 혈소판을 제거하는 PBS로 PBMC 세 번 씻으십시오. 혈소판은 트랜스 생체 DTH 분석을 방해 발견되었다. PBMC 준비의 최대 허용 혈소판 오염 ≤ 1X10 7 / 주입이다. </li><li> 눈에 띄는 적혈구 오염이있는 경우, 첫 번째 세척 후 ACK 용해 버퍼를 사용하여 적혈구의 용해를 수행합니다. PBS로 2 회 세척하여 ACK 버퍼를 제거합니다. </li></ol> 2. Alloantigen의 준비 <ol><li> 위와 같이 프로 시저를 사용하여 기증자의 말초 혈액에서 PBMC를 분리합니다. </li><li> 120x10 6 세포 / ㎖의 농도 (4X10 6 세포 / 30 μL)에서 PBS에서를 Resuspend 기증자 PBMC. </li><li> 단백질을 방지하기 위해 혼합물에 1 μM PMSF 추가분해. </li><li> 2 MM-프로브 sonicator로 일곱 1 초 펄스를 사용하여 세포 현탁액을 초음파 처리. (주 : 물자 감기를 유지하고 과도한 거품을 방지 거품이 발생하면, 세포 현탁액은 2 ~ 3 분 동안 얼음에 앉아 보자..) </li><li> 혈구를 사용하여 세포의&gt; 90 %의 중단을 확인합니다. </li><li> 냉장의 microfuge에서 20 분 동안 4 ° C에서 14,000 rpm으로 혼합물을 원심 분리기. </li><li> 새로운 2.0 ML 안전 잠금 튜브에 뜨는을 전송하고 단백질 농도를 결정한다. </li></ol> 3. 주사에 대한 세포의 준비 <ol><li> 2.0 ML 안전 잠금 튜브에 각각 주입 나누어지는 7X10 6 PBMC합니다. </li><li> 의 microfuge에서 분 동안 6,000 rpm으로 원심 분리하여 그 상등액을 제거합니다. </li><li> PBS에서 함께 또는 항원이없는 세포를 resuspend을. PBS로 35 μL를 주입 볼륨을 조정합니다. 다음 계획은 주사에 사용됩니다 : 부정적인 제어 : PBMC + PBS 긍정적 인 제어 : PBMC + TT / DT (25 &amp; 무; G / 주입) 실험 항원 특정 응답 : PBMC + 시험 자세 (4-8 ㎍ / 주입) 실험 항원 특정 규정 : PBMC + 시험 자세 + TT / DT </li></ol> 4. 사전 측정, 주입 및 사후 측정 <ol><li> isoflurane을 함께 CB17 SCID 마우스를 마취. 스프링 캘리퍼스를 사용하여 후면 발바닥의 두께를 측정한다. 측정 부위의 일관성을 유지하기 위해 벤치 마크를 제공하기 위해, 하나의 가장자리 다리의 마지막 산책 패드를 만지고, 발바닥의 중심에서 캘리퍼를 넣어. 게이지 판독 값이 안정 될 때 발바닥의 두께가 기록됩니다. </li><li> 천천히 28 게이지 바늘 ½ CC 인슐린 주사기를 사용하여 쥐의 발바닥에 피하 세포 현탁액을 주입. 바늘 발가락 향하게하고, 경사가 위를 향하도록 주입을 수행합니다. (참고 : 누수가 없는지 확인). </li><li> 주사 후 18 ~ 24 시간은, 발바닥 부종의 isoflurane을 반복 측정에 마우스를 마취. </li><li> Subtr발바닥 부종 값을 가져 포스트 분사 값에서 주입 전 각 발바닥의 두께 행동, 10 -4 인치의 단위로 데이터를 표현한다. 치료 (PBMC + 기증자 alloantigen, TT / DT, 또는 기증자 alloantigen + TT / DT)에서 얻은 발바닥 부종 값에서 제어 발바닥 부종 (PBMC + PBS)를 뺀 항원 특이 그물 붓기를 계산합니다. PBS에 배경 응답을 통해 ≥ 25X10 -4 인치의 항원 TT / DT를 호출하는 긍정적 인 제어 응답이 유효한 것으로 간주되는 테스트가 필요합니다. </li><li> 다음 공식을 사용하여 각 주사의 순 붓기를 비교하여 기증자의 항원의 존재 리콜 반응의 억제를 확인합니다 : <img alt="식 1" fo:src="/files/ftp_upload/4454/4454eq1highres.jpg" src="/files/ftp_upload/4454/4454eq1.jpg" /></li></ol>

<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>

이 논문의 저자가 공개하는 관심 없음 충돌이 없습니다.

트랜스 생체 DTH 분석은 이식, 암,자가 면역 질환 환자에서 세포 매개 반응을 평가하는 잠재적 인 임상 응용 프로그램과 새로운 진단 테스트입니다. 이 리콜 T 이펙터 반응을 모니터링하는 데 유용뿐만 아니라 때문에 가치가있다,뿐만 아니라 그것은 T 규제 응답을 검색 할 수 있습니다. 인간의 DTH 규제를 감지하는 신뢰할 수있는 방법은 단독 요법 또는 내성 시험에 대한 후보자 환자에서 면역 억제 ?...

<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

지연 형 과민 반응 (DTH)은 숙주 면역 시스템은 최근 과거에 경험있는 외국 항원 (자세)에 대한 T 세포 의존 면역 반응의 생체 징후의 급격한입니다. DTH 반응은 종종 일반적으로 감작 후 몇 일 뒤에 초기 항원 경험과 도전 단계를 참조 감작 단계로 구분됩니다. 피부 테스트에 의해 입증 리콜 AG의 지연 형 과민 반응의 부족은 종종 anergy의 증거로 간주됩니다. 기존의 DTH 분석을 효과적으로 많은 미생물 감염을 진단하는 데 사용되었습니다. 이러한 림프구 침윤, 부종 및 조직 괴사와 유사한 면역 기능을 공유에도 불구하고, 직접 D​​TH 때문에 기증자의 항원 및 이식 손실 감작의 결과로 직접 분사의 가능성 이식 환자에서 가능한 진단 기술이 아니다. 이 문제를 방지하려면, 험투 마우스 &quot;트랜스 생체&quot;DTH 분석은 1,2 개발되었다. 이 테스트는 인간의 말초 혈액 단핵 세포 (말초 혈액) 및 특정 항원 pinnae에 피하 주사 또는 순진 마우스와 DTH와 같은 부종의 노상 18-24 시간 후 3 측정 된에 기본적으로 전송 DTH 분석이다. 식세포 나 높은 혈관 마우스 조직에서 T 세포 수지상 인간 항원 제시 세포에 의해 항원 프레 젠 테이션 염증 폭포를 트리거하고 붓기 반응의 결과로 마우스 면역 세포를 끈다. 반응은 항원 특이하고 사전 항원 감작이 필요합니다. TV-DTH 분석에 긍정적 인 기증자 반응 DTH 반응은 이식 환자는 이식 alloantigens 향해 염증성 면역 처리를 개발했다고 반영합니다. 이 분석의 가장 중요한 기능은 또한 방관자 억제를 일으키는 원인이 규제 T 세포를 감지하는 데 사용할 수 있다는 것입니다. 의 방관자 억제기증자의 항원의 존재 DTH 리콜 응답 허용 이식 2,4-14와 이식의 특징입니다. TV-DTH에 의해 alloreactivity 및 규제에 대한 이식의 모니터링은 거부 또는 악화 신장 기능의 높은 위험없이 면역 억제제의 감소 혜택을 누릴 수있는 환자의 하위 집합을 식별 할 수 있습니다. 유망한 지역은자가 면역 15,16의 모니터링 또한 종양 면역학 17 TV-DTH 분석의 응용 프로그램입니다.

1. TV-DTH 분석을 사용하여 기증자의 항원에 특정한 응답 신장 이식의 평가 신장 이식 기증자 반응 세포 성 면역을 테스트하기 위해 우리는 기증자의 항원 단독으로 또는 리콜 항원 파상풍 톡소이드 (TT)를 가진 환자의 PBMC를 주입했다. 양성 대조군 세포는 TT 형과 주입으로. 우리는 이식 지연 형 과민 반응의 세 가지 패턴 (그림 1)을 관찰합니다. 모든 환자는 자세 (TT)를...

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

우리는 잠재적으로 이식 거부의 높은 위험없이 이식 후 면역 억제제의 철수를 결정하는 데 사용할 수있는 유용한 진단 분석을 설명합니다. 분석은 지연 형 과민 반응의 원리를 사용하여받는 사람이 탑재 된 두 기증자 특정 이펙터 및 규제 면역 반응의 정확한 평가를 제공합니다.

항원 특정 규제 트랜스 생체 지연 형 과민 분석

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 ...

trans-vivo-delayed-type-hypersensitivity-assay-for-antigen-specific

Research

JoVE Journal

Immunology and Infection

16.0K 조회수.  University of Wisconsin-Madison, School of Medicine and Public Health. 우리는 잠재적으로 이식 거부의 높은 위험없이 이식 후 면역 억제제의 철수를 결정하는 데 사용할 수있는 유용한 진단 분석을 설명합니다. 분석은 지연 형 과민 반응의 원리를 사용하여받는 사람이 탑재 된 두 기증자 특정 이펙터 및 규제 면역 반응의 정확한 평가를 제공합니다.

비디오: Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

항원 특정 VIVO에서</emCFSE 라벨 대상 세포를 사용하여> 죽이는 분석

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

연구

면역학 및 감염병학

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA 전달을위한 셀 타입의 특정 안티 HIV gp120 aptamers의 개발

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

약물 과민성의 IGE - 중재 메커니즘을 조사 Basophil 인증 시험

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

표현형 및 기능 분석에 대한 검색 및 희귀의 농축 항원 특이 적 B 세포

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

항 원 특정 항 체 응답 다음 Clostridium 남과 어울리지 않 는 감염의 혈 청 학적인 결정에 대 한 단백질 Microarray 분석 결과

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

예방 접종 된 마우스에서 항원 특정 CD8 T 세포의 세포 주기 분석을위한 DNA / Ki67 기반 유동 세포 분석

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...