We thank Peter Staeheli for providing B6.A2G-Mx1 mice and Semra &#214;zcelik for technical advice.
This work was supported by the German Research Foundation through SFB829 and KN536/16, and the K&#246;ln Fortune Program/Faculty of Medicine, University of Cologne.

윤리 문. 희생 동물의 표피 시트의 제조는 Landesamt 대 투르, UMWELT 및 Verbraucherschutz, 노스 린 - 웨스트 팔리 아 (독일)의 가이드의 권고 사항을 엄격히 준수하여 수행됩니다. 이 연구는 LANUV 노르 트라 인 베스트 팔렌 (수 8.84-02.05.20.13.018)에 의해 승인되었다. 악기와 문화 미디어 1. 준비 <ol><li> 둘 베코 변성 이글 배지 (DMEM) / 고 포도당 글루타민 디 펩티드, 10 % FCS, 100 IU / ㎖ 페니실린을 함유하고 / ㎖ 스트렙토 마이신 100 μg의이 (이하, 「DMEM」이라 함)에서 상피 시트를 배양. </li><li> 상피 시트의 제조를위한, 심하게 가열 PBS (최대 37 ° C)에서 디스 파제 II에게 분말 (5 ㎎ / ㎖)에 녹인다. 0.22 μm의 필터를 통해 솔루션을 필터링 및 치료에 직접 사용합니다. </li><li> 표피 전체의 준비 (13) 마운트를 들어, 광을 차단 준비0.5 % 분유, 냉수 물고기 피부와 TBS에서 0.5 % 트리톤 X-100에서 0.25 % 젤라틴과 어. 물질 회전 믹서에 실온에서 적어도 2 시간 동안 혼합물을 배양하여 용해하도록 허용합니다. 또한 세척 버퍼로 PBS에 트윈 20 0.2 %를 준비합니다. 고정 용, 3.4 %와 PBS에서 4 % 포름 알데히드를 준비합니다. </li><li> 저온부의 면역 염색을 위해, 5 % 정상 염소 혈청과 0.2 % 트윈 20에서 PBS 버퍼로 차단 제조. 고정 용, PBS에 0.​​5 %의 포름 알데히드를 준비합니다. </li></ol> 쥐 피부에서 표피 시트 2. 준비 <ol><li> 감염 연구를위한 신생아 다시 피부 표피의 준비 <ol><li> 해부 접시에 해고 마우스 (1~3일 출생 후) 놓고 사지를 잘라 몸통에 가까운 꼬리는 전체 몸통으로부터 피부를 효율적으로 제거 할 수. 사지 제거하는 동안, 직경이 가능한 한 작은 상처를 유지하려고. </li><li> 곡선 미세 집게와 몸통을 수정부드럽게 몸통으로부터 피부를 분리하기 위해 뒷면을 따라 꼬리 지느러미에 두개골에서 피부 아래에 무딘 끝이 위를 이동 ND. 백을 따라 피부를 슬릿. </li><li> FACS 분석, 각질 세포 또는 RNA의 준비의 격리, 들어 몸통을 밀어 조직과 뭉툭한 팁 가위를 해결하기 위해 집게를 사용하여 몸통에서 완벽한 피부를 벗겨. 저온부 또는 전체 마운트의 준비를 위해 다시 피부의 조각을. </li><li> 약 10 × 10mm의 1-2 조각으로 메스와 함께 다시 피부를 잘라. ~ 1 ml의 멸균 PBS로 6 웰 플레이트 잘 하나에 조각을 넣어. 피부면이 바닥을 향하도록해야합니다. </li><li> 1.5 ml의 디스 파제 II (5 ㎎ / ㎖ PBS)에 의해 PBS를 교체하고 4 ° C에서 / 30 (전체 마운트에 대한) 37 ° C에서 분 또는 O 중 하나는 N 품어. PBS로 3 회 반복한다. 부드럽게 표피를 들어 올릴 기본 진피와 다른 집게를 해결하기 위해 하나의 집게를 사용하여 그대로 시트로 표피를 제거합니다. 이 단계 EAS를 만들기 위해 쌍안경을 사용하여Y. 아래쪽으로는 기초면 DMEM에 표피 시트를 전송합니다. 감염 실험에 즉시 시트를 사용합니다. 주 : 주로 FACS 분석, 각질 세포 또는 RNA의 제조를 위해 절연 신생아 피부 표피의 제제를 사용한다. 때문에 그 취약성의, 저온부 또는 전체 마운트에 대한 감염 시트의 준비는 덜 적합하다. </li></ol></li><li> 감염 연구를위한 성인 꼬리 피부에서 표피의 준비 <ol><li> 자궁 전위에 의해 1~3개월 세의 나이에 쥐를 안락사. 뒷면에 긴 포함 된 모낭이 손상 표피 시트의 분리를 방해하기 때문에 대신 다시 피부의 꼬리를 가져 가라. 희생 생쥐의 꼬리를 잘라 메스와 길이를 슬릿. </li><li> 뼈에서 피부를 벗겨 약 5 × 5mm의 조각으로 메스로 들어온다. 잘 하나에서 4 개까지 넣고 2.1.5에서 설명한대로 디스 파제 II에서 배양 진행 참고 : 사용 성인 타이L 피부 저온부 또는 전체 마운트를 제조 하였다. 또한 즉시 사용에, 꼬리는 저장 또는 중요한 감염 효율을 잃지 않고 4 ℃에서 약 24 시간 동안 전송 될 수있다. 꼬리가 48 시간으로 유지하는 경우, 표피 시트는 거의 HSV-1에 감염되지 않을 수 있습니다. </li></ol></li><li> FACS 분석을위한 표피의 해리 주 : FACS 분석에 의해 표면 단백질의 발현의 검출은 세포 표면 및 관심 단백질을 손상하지 않는 적절한 세포 분리 기술을 필요로한다. <ol><li> RT에서 15 분 동안 1.5 ㎖ / 웰 재조합 트립신 계 세포 해리 용액에 37 ℃에서 15 분 동안 기부 쪽과 6 웰 플레이트에서 태어난 표피 시트를 배양한다. </li><li> 3 ml의 DMEM을 추가하여 부화를 중지합니다. 각질 용해 얻을 수 있도록 부드럽게 6 잘 판에 표피를 이동 곡선 미세 집게를 사용하여 각질을 떼어 놓다. 세포 현탁액을 수집하고이 과정을 3 회 반복항상 신선한 DMEM을 사용. 참고 :이 해리 절차는 각질 세포의 표면에 nectin-1을 감지하는 것이 적절하다. </li><li> 대안 적으로, RT에서 15 분, 37 ° C에서 15 분 동안 효소를 무 세포 해리 용액에 표피 시트 (CDS)를 배양한다. 부드럽게 위아래로 CD를 피펫 팅하여 각질을 플러시 실온에서 또 다른 15 분을 품어. </li><li> 세포 현탁액을 수집하고 신선한 DMEM을 사용하여 세척 단계를 3 회 반복한다. 참고 :이 해리는 HVEM의 표면 발현을 감지하기에 적합합니다. CDS 용액의 단점은 더 적은 세포가 재조합 트립신 계 세포 해리 용액보다 해리된다는 점이다. 또한 재조합 트립신 계 세포 해리 용액 감염된 상피 시트의 해리 후에 발현 등 ICP0 식 핵 또는 세포질 발현을 FACS로 분석 될 수있다 표면. </li></ol></li><li> 표피의 해리는 각질이나 전직을 분리요로 RNA <ol><li> 1.5 ㎖ / 웰 재조합 트립신 계 세포 해리 용액을 포함하는 접시의 바닥쪽으로 기부 쪽과 6 웰 플레이트에서 태어난 표피 시트를 넣는다. 실온에서 30 분 동안 품어. 3 ml의 DMEM을 추가하고 부드럽게 접시에 표피를 이동 곡선 미세 집게를 사용하여 각질을 떼어 놓다. </li><li> 세포 현탁액을 수집하고 신선한 DMEM을 사용하여이 단계를 3 회 반복한다. RNA 또는 문화 차 쥐의 각질 세포에 압축을 풉니이 세포 현탁액을 사용합니다. </li></ol></li></ol> HSV-1과 표피 시트 3. 감염 <ol><li> 500 ㎕의 DMEM 이상에서 HSV-1 중량 균주 17 (11)의 용액을 제조하고 표피 시트 플로팅되는 바이러스 용액으로 교환 매체. 이 시점은 0~12 시간으로 정의한다. 37 ℃에서 24 웰 플레이트의 한 웰에 표피 시트의 감염을 수행한다. 바이러스 역가의 계산은 추정 된 개수에 기초표피 시트 당 기저층에서 세포의 (약 5 × 5mm 시트 당 2 × 10 5 세포). </li><li> HSV-1에서 약 100 플라크 형성 단위 (PFU) / 세포로 감염 1 시간 PI DMEM에 의해 바이러스 함유 배지를 대체 주 : 신생아 피부의 표피 시트의 경우, 시트 배양 동안 해리 시작 있음을 알아 두셔야합니다. </li></ol> 감염된 세포 4. 시각화 <ol><li> 표피 전체 마운트 (13) <ol><li> 3.4 %의 포름 알데히드 중 4 ℃에서 RT 또는 O / N에서 2 시간 동안과 표피 시트를 고정합니다. PBS로 두 번 세척하고 0.5 % 분유, 냉수 물고기 피부와 RT (14)에서 1 시간 동안 TBS에서 0.5 % 트리톤 X-100에서 0.25 % 젤라틴으로 차단합니다. </li><li> 15 실온에서 버퍼를 차단에서 1:60으로 희석 마우스 항 - ICP0 (단클론 항체 11060)와 시트 O / N을 품어. 중간 필라멘트 네트워크 시각화 토끼 폴리 클로 날 항 - 부화 동시에마우스 케라틴 14 (NG 100 / ㎖). </li><li> 실온에서 4 시간 동안 PBS-0.2 % 트윈 20으로 4 ~ 5 회 반복한다. O / N이 함께 AF488 - 공액 안티 - 마우스 IgG (1 μg의 / ㎖), AF555 - 공액 안티 - 토끼 IgG의 (1 μg의 / ㎖) DAPI (4 &#39;, 6-diamidino -2- 페닐 인돌) (100 NG 부화 RT에서 블로킹 완충액에서 / ㎖). </li><li> 실온에서 4 시간 동안 PBS-0.2 % 트윈 20 씻으십시오. 표본 슬라이드의 상단에 자신의 기저면 산의 표피 시트, 형광등 설치 매체에 포함 된 커버와 커버. </li></ol></li><li> 표피 저온부 <ol><li> 성인 상피 시트를 실온에서 20 분 동안 PBS 중의 4 % 포름 알데히드로 고정하고 PBS로 2 회 세척 하였다. 조직에 포함 된 고정 시트는 액체 질소 중간 동결 동결. cryomicrotome으로 8 ~ 10 μm의 섹션을 잘라. </li><li> 실온에서 10 분 동안 PBS 0.5 %의 포름 알데히드 다시 부분 수정 PBS, 실온에서 30 분 동안 PBS에 5 % 정상 염소 혈청과 0.2 % 트윈 20 블록을 2 회 반복 한 다음 M 60 분간 얼룩안티 ICP0 (모노클로 날 항체 11,060) (15)가 차단 완충액으로 3 회 세척 한 후, 블로킹 완충액에서 1:60으로 희석을 여러개. </li><li> 이어서 실온에서 45 분 동안 블로킹 완충액에서 AF488 - 공액 안티 - 마우스 IgG (1 μg의 / mL) 및 DAPI (100 NG / ml)로 배양하고 3 회 반복한다. 형광 장착 매체 섹션을 포함하고 커버 슬립 커버. </li></ol></li></ol>

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G., 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=Cellular+cholesterol+efflux+mediated+by+cyclodextrins:+Demonstration+of+kinetic+pools+and+mechanism+of+efflux.">Cellular cholesterol efflux mediated by cyclodextrins: Demonstration of kinetic pools and mechanism of efflux.</a> J. Biol. Chem. 271, 16026-16034 (1996).</li><li>Montgomery, R. I., Warner, M. S., Lum, B. J., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus-1+entry+into+cells+mediated+by+a+novel+member+of+the+TNF/NGF+receptor+family.">Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.</a> Cell. 87, 427-436 (1996).</li><li>Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., Spear, P. 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The authors declare that they have no competing financial interests.

When epidermal sheets of adult skin from C57BL/6 are infected with HSV-1 at approximately 100 PFU/cell, we observe infection in nearly all cells of the basal layer in the interfollicular epidermis while lower virus doses correlate with less infected cells and a slower progress of infection. In general, hair follicles show a variable number of infected cells; while most of the rather small keratinocytes lining the developing hair follicles are infected, only the hair germ of the adult hair follicle is completely infected....

단순 포진 바이러스 (HSV)는 생명을 위협하는 감염에 약한 단순한 피부 점막 병변에서 인간의 질병의 범위가 발생할 수 있습니다. HSV 1 형 (HSV-1)이 지배적 가능성 생식기 감염 1 원인 HSV 2 형 (HSV-2), 반면, 구강 안면 뇌염 감염과 관련된다. HSV는, 문화에서 셀을 입력 감염을 시작하고 바이러스 자손을 생산하는 방법의 이해에 상당한 진전이있는 동안, 우리는 세포 수준 2에서 조직에 바이러스 침입 경로 (들)에 대해 거의 알고있다. HSV 피부 또는 점막 감염 마우스의 연구를 들어, 토끼, 기니아 피그 동물 모델로서 사용되어왔다. 피부 감염은 피내 주사 또는 바이러스의 존재에 의해 피부를 긁 확립시키고, 질병 발달은 바이러스 생산과 관련이 있었다. 이러한 방법은 질병 발병의 다양한 측면을 이해하는 데 도움이, 그리고 항 바이러스 약물을 평가하는 데 사용됩니다. tissu에서 HSV 감염을 연구하는E 수준은, 인간의 피부의 Organotypic 모델이 적용되어왔다. 감염률은 이들 뗏목 배양 제한되는 바와 같이, 감염, 바이러스 확산을 조사하는 연구와 항 바이러스 성분의 효과의 제한된 수는 3-6 발표되었다. 그대로 상피에서 HSV-1 감염 기간 동안 역할을 휴대 결정기를 특성화하기 위해, 우리는 뮤린 표피 (7)의 생체 감염 연구를위한 프로토콜을 확립. 신생아 피부 또는 성체 마우스의 꼬리로부터 제조 하였다. HSV-1 바이러스 함유 배지에서 침수 된 완벽한 피부 샘플을, 감염 없었기 때문에, 우리는 디스 파제 II 처리에 의해 진피에서 표피를 분리. 바이러스 - 함유 배지에서 표피 시트 부동 후 감염된 세포를 다양한 시간의 postinfection (PI) (7)에서 기저 표피 층으로 시각화 될 수있다. 바이러스 복제에 앞서 각 셀 감염의 개시를 시각화ND 바이러스 생산, 우리는 HSV-1 감염 기간 동안 발현되는 첫 단백질이다 감염된 세포 단백질 0 (ICP0)에 대한 항체로 염색. ICP0의 세포 현지화는 초기 감염시 가지 단계를 통과한다. ICP0는 바이러스 유전자 발현의 초기 단계에서의 핵 병소에 존재하지만, 세포질로의 ICP0 relocalization 감염 (8)의 후속 단계를 나타낸다. 우리는 감염 동안 다양한 세포 인자의 역할 가능성을 테스트하기 위해 다른 마우스 모델에서 상피 시트의 생체 감염 분석을 사용 하였다. 굴지 역학의 핵심 레귤레이터로 RAC1의 영향을 해결하기 위해, 우리는 RAC1 유전자 (9)의 각질 세포 고유의 삭제와 쥐의 표피를 감염. 이 모델은 표피의 각질 층에서 HSV-1 감염의 효율성에 결함이 RAC1의 결과를 연구하기 위해 우리를 허용했다. 다시 제어 한배 새끼의 감염 표피에 비교RAC1의 부재 (7)의 표피 기저층 감염의 개시에 아무런 영향을 미치지 않았다 나타내는 큰 차이를 vealed 없다. 상기 마우스 모델의 사용은 우리가 세포 수용체는 표피 진입을 중재 할 수있는 해결. HSV-1 nectin -1- HVEM 또는 - 결핍 마우스에서 표피 중 시트 감염은 조직으로 진입 초기 바이러스 강하게 nectin-1 (10)의 존재에 의존한다는 것을 밝혔다. 또한, 우리의 결과는 HVEM 또한 nectin-1 (10)보다 덜 효율적 있지만, 쥐의 표피에있는 수용체 역할을 할 수 있음을 보여줍니다. 표피 층에 감염된 세포의 공간 분포를 해결하기 위해, 우리는 조직 절편, 표피 및 전체 마운트 (도 1)의 발현을 ICP0 시각화. 전체 피부의 저온부에서 더 ICP0 발현 세포는 (그림 1) 검출되지 않습니다. 대조적으로, 상피 시트의 저온부가 세포질을 보여이미 3 시간 PI (그림 1)에서 기저층에서 ICP0 식입니다. 나중에 시간에, 바이러스가 가시화 될 수있다 층을 suprabasal 확산. 기저층에 감염된 세포의 공간 분포를 용이하게 표피 전체 마운트 (도 1)에 따라 될 수있다. HSV-1 100 PFU / 셀에 감염되면, interfollicular 표피의 기저 각질 세포의 약 50 %는 대부분의 감염된 세포 핵 ICP0을 표현하는이 시점에서 1.5 시간 파이에서 ICP0 식을 보여줍니다. 초기 유전자 발현의 후기 단계를 나타내는 세포질 ICP0의 relocalization 3 열연 PI (도 1)의 거의 모든 세포에 존재한다. 생체 HSV-1 감염에 감염된 세포를 시각화의이 모드는 억제제 또는 조직에서 바이러스 성 항목 및 확산에 / 삭제 돌연변이 세포 성분의 효과를 연구 할 수있는 강력한 분석을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

그 인간 숙주를 입력, 단순 헤르페스 바이러스 1 형 (HSV-1) 점막 표면, 피부 또는 각막의 장벽을 극복해야한다. HSV-1 대상은 초기 진입시 각화 및 신경 세포의 잠재 감염 뒤에 상피에 차 감염을 설정합니다. 재 활성화 한 후, 바이러스는 피부 소포 또는 점막 궤양으로 표시 피부 점막 부위에서 분명하게 할 수 있습니다. HSV-1은 피부 나 점막을 침범하고 도달 방법의 수용체는 제대로 이해된다. 세포 수준에서 표피 조직에 HSV-1의 침입 경로를 조사하기 위해, 우리는 피부의 기본 및 재발 성 감염 부위를 나타내는 쥐의 표피의 생체 감염 모델을 설립했다. 분석은 쥐의 피부의 준비를 포함한다. 표피는 디스 파제 II 처리하여 진피로부터 분리된다. 바이러스 - 함유 배지에서 상피 시트를 플로팅 한 후, 조직은 고정이고 감염에 의해 여러 번 postinfection에서 시각화 될 수있는HSV-1 즉각적인 초기 ICP0 단백질에 대한 항체로 감염된 세포를 염색. ICP0는 발현 세포 것은 1.5 열연 postinfection 이미 기저 각질 세포 층에서 관찰 될 수있다. 더 이상 감염 시간으로, 감염된 세포는 감염이 기초 각질 세포로 제한하지만, 바이러스가 조직에서 다른 층으로 확산되어 있지 않은 것을 나타내는, suprabasal 층에서 발견된다. 다양한 마우스 모델의 표피 시트를 사용하면, 감염 프로토콜은 조직으로 HSV-1 침입에 기여 세포 성분의 참여를 결정하는 허용한다. 또한, 분석은 초기 진입 단계에 지장 조직 억제제, 세포 간 확산 및 바이러스 생산을 테스트하기에 적합하다. 여기, 우리는 세부 사항에 생체 감염 프로토콜을 설명하고 nectin-1 또는 HVEM 결핍 마우스를 사용하여 우리의 결과를 제시한다.

The challenge of the method is to prepare epidermal sheets into which HSV-1 can penetrate from the basal layer. The critical step is the separation of the epidermis from the dermis by dispase II treatment, which, depending on the mouse strain, needs to be adapted. The concentration of dispase II can range from 2.5 to 5 mg/ml, and the time of incubation from 20 to 45 min. The staining of the intermediate filament protein keratin 14 readily allows predicting whether the basal epidermal layer can be infected or whether it w...

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Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

단순 포진 바이러스 1 형과 쥐 표피의 생체 감염

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

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Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.1K 조회수.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

비디오: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin의 1/Ionomycin에 의해 종양 반응 T 세포의 생체내 전 확장 및 일반 감마 체인 크린 시토킨 제제

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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면역학 및 감염병학

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

위드 필드와 공촛점 현미경과 Murine 림프절의 조각에서 T 세포의 생체내 이미징 전

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

헤르페스의 연구를위한 예비 신경 세포 문화 시스템은 바이러스 지연 및 재가입을 단순

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

전 비보 인간 면역 결핍 바이러스 1와 Histoculture 인간 림프 조직 및 여성 생식 기 점 막의 감염

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Bursal 전염병 바이러스 병 인을 공부 하는 Ex Vivo 닭 기본 Bursal 세포 배양 모델

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Immunofluorescent Confocal 현미경 검사 법에 의해 포진 심 플렉스 바이러스 1 단백질의 핵-세포질 전 좌의 시간적 분석

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

타입-2 당뇨병과 관련 하 여 무 알콜 수 막 염을 위한 진보 된 뮤 린 모형

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...