이 작품은 Wellcome 트러스트에서 비테 Kampmann (056608, GRO77273) 개인 장학금에 의해 투자되었다.

1. BCG 럭스 기자 mycobacteria 재고 준비 <ol><li> BCG 성장 럭스 미들 7H9 국물이 0.2 % 글리세롤, 0.05 % 십대 초반 80와 10 %의 ADC 농축을 포함하는 중간에 로그 단계로 200rpm에서 잡고 (셔틀 플라스미드 pSMT1로 변환 M. bovis BCG 몬트리올 변형). </li><li> 중복에 luminometer 튜브 (900μl PBS + 100μl BCG 룩스 문화)에서 멸균 PBS에서 BCG 럭스 문화의 1시 10분 희석의 1ml를 준비합니다. </li><li> 컴퓨터의 뒷면에 N - decyl 알데히드 (루시페라제 효소 기판)이있는 튜브를로드하고 뚜껑이 자리에 안전하고 튜브 있는지 확인하십시오. </li><li> luminometer에 배치 3 빈 마중물 튜브의 각으로 100 ucl X 3 주입으로 설정 주요 프로그램을 통해 기판과 수상 luminometer 주입기. </li><li> luminometer로 2 튜브를로드하고 각 튜브에 기판의 100 ucl을 주입에서 설정 프로그램을 통해 신호를 1 초 간격 20 초위한 읽어보십시오. </li><li> 주식 1x10 8 RLU / ML에 2x10 8 RLU / ML을 (이것은 성장의 3~4일에 대한 소요)에 도달되면, 50ml 팔콘 튜브의 문화 멸균 30 % 글리세롤의 동일한 볼륨을 추가하고 부드럽게 섞는다. </li><li> -80에서 레이블 2ml 나사 캡 microtube하고 저장 ° C.으로 나누어지는 1.5ml 볼륨을 </li></ol> 2. 결정 재고 RLU / CFU 상관 관계 및 aliquots의 내용 <ol><li> 통풍 모자와 200ml Erlenmeyer 플라스크 10 % ADC의 보완과 함께 미들 7H9 문화 매체 15ml를 추가합니다. </li><li> 20 %의 십대 초반의 hygromycin와 30μl의 15μl를 추가합니다. </li><li> 안전 캐비닛에 실내 온도 (RT)에서 냉동고와 서리를 없애다에서 BCG 럭스의 병을 제거합니다. 매체에 유리병의 내용을 추가하고 뚜껑을 조입니다. </li><li> 37 200rpm에서 흔들어 ° C를 4 일간 함​​께 품어. </li><li> 매일, CFU 결정을위한 일련 10 배 dilutions을 (1시 10분, 1:100, 1:1000, 1:10,000과 1:100,000)까지합니다. </li><li> 병렬 같은 dilutions을 설정하고 (일부 1.2-1.6 참조) 발광을 결정하기 위해 중복. </li><li> 미들 7H11 한천 배지 (0.5 % 글리세롤 10 % OADC 보완, 20 % 십대 초반과 hygromycin 1 ML 1 ML을 포함하는 액체 매질 1 L)의 두 3 구획 접시를 준비합니다. </li><li> 각 챔버에 걸쳐 균등하게 액체를 배포 별도 밧침대를 사용하여 각 접시의 세그먼트에 각 희석의 100μl, 나가 플레이트. </li><li> parafilm 각 접시, 멸균 플라스틱 봉투에 장소, 압력솥 테이프와 인감 도장, 37에서 품어 ° C를 2 주 동안 다운 향하게 뚜껑과 함께. </li><li> CFUs이 (2-3 주) 나타날 때까지 정기적으로 검사합니다. </li><li> 식민지를 계산하려면, 보육과 식민지 카운터에 장소에서 접시를 제거합니다. 중복 접시에서 각 희석에 대한 의미를 계산합니다. </li><li> 각각의 희석에 대한 동등한 RLU 및 CFU 카운트를 사용하여 RLU / CFU 비율을 계산합니다. 비율은 3과 5 RLU / CFU 사이 여야합니다. </li></ol> 3. 전체 혈액에 대한 BCG 접종 럭스 문화의 준비 <ol><li> 통풍 모자와 200ml Erlenmeyer 플라스크 10 % ADC의 보완과 함께 미들 7H9 문화 매체 15ml를 추가합니다. </li><li> 20 %의 십대 초반의 hygromycin와 30μl의 15μl를 추가합니다. </li><li> 안전 캐비닛에 RT에서 냉장고와 서리를 없애다에서 BCG 럭스의 병을 제거합니다. 매체에 유리병의 내용을 추가하고 뚜껑을 조입니다. </li><li> 37 200rpm에서 흔들어 ° C를 2-4일 위해 함께 품어. </li><li> 중복에 luminometer 튜브 (900μl PBS + 100μl BCG 룩스 문화)에서 멸균 PBS에서 BCG 럭스 문화의 1시 10분 희석의 1ml를 준비합니다. </li><li> 기판과 프라임 luminometer (위에서 설명한 것처럼), 부드럽게 2 튜브 소용돌이와 luminometer로로드하고 판독을 (위에서 설명한 것처럼) 가져가라. </li><li> 7x10 6 RLU의 등가를 (이것은의 단핵구 수를 가정, 1시 1분 약 monocytes에 2x10 약 5 CFU / ML 혈액과 BCG의 비율 inoculum를 제공합니다 제공하는 PBS의 문화를 희석이 읽기를 사용하여 혈액 ML 당 4x10 5 2x10 약 5 monocytes). </li></ol> 4. 전체 혈액의 준비 <ol><li> 방부제없는 헤파린이 들어있는 튜브에 정맥 혈액 5ml 3을 가져가라. </li><li> 50ml 팔콘 튜브에 혈액을 전송 및 글루타민과 25mM HEPES를 (없음 펜 / strep)이 포함된 RPMI 1640의 동일한 볼륨 희석. </li><li> . 0hrs 3과 96hrs 3 필요한 경우, 추가 timepoints에 대한 추가 튜브를 설정합니다. 때마다 포인트 살균 보석 튜브 (총 6 튜브에 세중의에 희석 혈액 나누어지는의 900μl </li><li> 에서 10 % ADC 보충 + 50μg/ml hygromycin (BCG 럭스 문화 사용된 같은 ​​매체)와 미들 브룩 7H9 문화 매체 나누어지는에 900μl의 성장 제어를위한 중복. </li><li> 혈액의 각 튜브에 희석 BCG 럭스의 100μl를 추가하고 2 성장 제어튜브. </li><li> 잘 믹스 37의 락을 인큐베이터에서 쪽에서 시간 - 포인트 96hr과 2 성장 컨트롤 3 bijous을 배치 ° C, 20 레브 / 분 (CO 2는 필요하지 않습니다.) </li></ol> 5. 측정 발광 (0hr 및 96hrs에서) <ol><li> 10 분 2,000그램에서 3 bijous을 원심 분리기. </li><li> 조심스럽게 -80시 동결에 2ml 나사 캡 microtubes의 펠렛과 장소를 방해하지 않고 뜨는의 300μl 제거 ° C 후속 시토킨 분석. </li><li> 삭제 뜨는의 볼륨을 교체하기 위해 각 보석에 PBS의 300μl를 추가합니다. </li><li> 해당 50ml 팔콘 튜브에 각 보석의 내용을 대기음 각 튜브에 8ml 증류수를 추가합니다. 10 분 타이머를 시작합니다. 주의 사항 : 이것은 시간에 민감한 단계이며,이 부화는 세포가 먼저 물과 접촉했을 때부터 시작, 더 이상보다 10 분해서는 안됩니다. </li><li> 해당 팔콘 튜브에 팁 전에 5 초 동안 증류수와 소용돌이 2ml 각 보석을 씻어. </li><li> 10 분 부화 끝에, 10 분 동안 2천그램에서 팔콘 튜브를 원심 분리기. </li><li> Surfanios의 살균제에 가만히 따르다에 뜨는. </li><li> 각 펠렛과 와동에 몇 유리 구슬을 추가합니다. </li><li> 각각의 튜브와 와동에 멸균 PBS의 1ml을 추가합니다. </li><li> luminometer 튜브에서 PBS (PBS 900μl가 + 100μl 샘플)와 중복의 시간과 포인트 96hr 각 샘플에 대한 1시 10분 dilutions합니다. 성장 컨트롤 각각에 대해 동일한 dilutions하십시오. </li><li> 부드럽게 튜브 소용돌이와 luminometer로로드하고 판독을 (위에서 설명한 것처럼) 걸릴 주의 : 동일한 샘플 triplicates 사이의 수치가 서로의 15 % 이내에 있어야합니다. </li></ol> 6. 대표 결과 : 그것은 대사 활동이 suboptimal 한, 즉 이전에 샘플을 inoculating하는 48-72시간 이상 성장, 전체 혈액 럭스 assays를위한 성장 단계에 로그 박테리아를 사용하는 것이 중요합니다 냉동고의 직선 밖으로 하나 경우 또는 고정 단계 인치 이전 성장 다양성을 피하기 위해 48 또는 72시간 중 하나에 일련의 실험을 위해 표준화되어야합니다. 그림 1은 BCG 럭스의 대표 문화의 성장 곡선을 보여줍니다. 고정 단계에 도달할 때까지 두 배로 시간은 24 시간 정도입니다. <img alt="그림 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> BCG의 그림 1. 대표 성장 곡선 럭스 7H9 매체와 RLU와 CFU의 상관 관계에 96시간 이상. RLU 값이 항상 CFU와 연관 있지만, 하나의 CFU에 해당 RLU의 숫자는 감염의 일관된 다중성을 보장하기 위해 일련의 실험을 통해 같은 냉동 주식을 사용하는 것이 좋습니다 이유있는가 (즉, 주식에 따라 달라질 수 입니다만). 표 1은 접종 시간 (T 0)에서, 96시간에서 원료 날짜의 예를 보여줍니다. 성장 비율은 공식 T 96 / T 0을 사용하여 계산하지만, 다른 시간 간격은 물론도 측정할 수 있습니다. 중요한 세포 사망이 발생하는대로이 단, 권장, 96시간 넘어 문화를 사용하지 않습니다. <img alt="표 1" src="/files/ftp_upload/3332/3332table1.jpg" /> 표 1. 접종 (T0)의 시간과 96시간 (T96) 3 성인 기증자로부터 계산의 성장 비율에 원시 데이터의 예. 사용할 수있는 항원 - 특정 메모리 반응과 가능성 또한 호중구 카운트에 따라 성장 비율과 함께 아이들의 혈액에서 그림 2와 HIV 감염없이 여기 그림과 같이, 개인 간의 차이가 있습니다. 평균, 어린 아이들이 성인보다 높은 성장 비율을 가지고, 투베르쿨린 피부 반응 검사 (TST) +했습니다 개인이 TST - 봤어요 개인보다 낮은 성장 비율을 갖고 있으며 HIV에 감염된 환자는 CD4 T 세포 인구의 결핍으로 인해 성장 비율을 가지고 mycobacteria에 세포 면역 반응의 핵심 중재자 중 하나. <img alt="그림 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> 그림 2 인터 기증자 다양성 :. 기본 HIV 상태에 따라 환자의 집합에 대한 T 0 대 96시간의 성장 비율. 일정 기간 동안 성장 비율의 재현성은 12 개월 이상 단일 기증자 제어 실험 반복 출혈에서 출혈이 64 회 기증자의 결과를 요약한 그림 3에 표시됩니다. 다양성의 잠재적인 원인은 많은 bioassays에서 관찰 호스트 크린 시토킨의 수준 이내 mycobacterial sensitization이나 다양성의 변화 수 있습니다. <img alt="그림 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> 그림 3. 12 개월 동안 12 차례에 (A) 단일 기증자. 12m 이상의 두 경우에 (B) 복합 (64) 기증자onths.

<ol>
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관심 없음 충돌 선언하지 않습니다.

그들이 잠재적으로 유망한 백신 후보를 화면에 적용할 어디 결핵 백신 연구의 맥락에서 Bactericidal assays 잘, 동물 모델에 설립되었습니다. immunogenicity의 읽기 밖의 주요 등 다양한 장기의 기능에 세균 부하 감소. 인간 bactericidal assays의 첫 번째 세대 M. 감염, 혼자 macrophages을 사용하여 설계되었다 결핵. CFU 3 주 최소 시간이 지나면 mycobacterial 생존의 읽기 밖의 주요 ?...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 고양이 없음 </td></tr><tr><td> BCG 럭스 </td><td> 여러 </td><td> N / A </td></tr><tr><td> 미들 7H11 한천 </td><td> Becton 디킨슨 </td><td> 283810 </td></tr><tr><td> 미들 7H9 국물 </td><td> BD Biosciences </td><td> 271310 (500g) </td></tr><tr><td> 미들 ADC의 농축 </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> 미들 OADC 보충 </td><td> Becton 디킨슨 </td><td> 212240 </td></tr><tr><td> 십대 초반 80 </td><td> 시그마 알드리치 </td><td> 274364 </td></tr><tr><td> 글리세린 </td><td> 시그마 알드리치 </td><td> G5150 </td></tr><tr><td> Hygromycin B </td><td> 호슈 </td><td> 10843555001 (20ml) </td></tr><tr><td> N - decyl의 알데히드 </td><td> 시그마 </td><td> D7384 - 100G </td></tr><tr><td> 에탄올 (&gt; 99.7 %) </td><td> VWR </td><td> 101077 </td></tr><tr><td> 멸균 PBS </td><td> 집에서 </td><td> N / A </td></tr><tr><td> L - 글루타민, 25mM HEPES와 RPMI - 1640 </td><td> 시그마 </td><td> R0883 </td></tr><tr><td> 멸균 증류수 </td><td> 집에서 </td><td> N / A </td></tr><tr><td colspan="3"> 장비 </td></tr><tr><td> 튜브 luminometer (주사용 포트 필수) </td><td> Berthold AutoLumat LB953 또는 단일 튜브 luminometer 시리우스 </td><td></td></tr><tr><td> Luminometer 튜브, 12 X 75mm, pyrex 무테 </td><td> VWR의 코닝 </td><td> 99445-12 </td></tr><tr><td> 통풍 모자와 Erlenmeyer flasks의, 멸균 폴리 카보 네이트 250ml </td><td> 코닝 통해 피셔 과학 </td><td> 2,150,329 </td></tr><tr><td> BD Vacutainer 혈액 수집 튜브 (방부제없는 헤파린) </td><td> BD </td><td> 367676 </td></tr><tr><td> 50ml 팔콘 튜브, 원뿔 </td><td> BD </td><td> 352077 </td></tr><tr><td> 7ml Sterilin 표본 폴리 프로필렌 보석 튜브 </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin 유니버설 용기 (30 ML) </td><td> 실험실 분석 LTD </td><td> 128C </td></tr><tr><td> 유리 비즈 2mm 직경 </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10ml pipettes 및 피펫 소년 </td><td> 어떤 </td><td></td></tr><tr><td> 1000μl와 200μl pipettes 및 필터 팁 </td><td> 어떤 </td><td></td></tr><tr><td colspan="3"> 일반 실험실 장비 : </td></tr><tr><td> 클래스 II 안전 캐비닛 캐비닛 </td><td></td><td></td></tr><tr><td> 냉장고 (4 ° C) </td><td></td><td></td></tr><tr><td> 원심 분리기 </td><td></td><td></td></tr><tr><td> 튜브를 섞어 신나게 흔드는 플랫폼 (180 0) </td><td></td><td></td></tr><tr><td> 궤도 흔드는 인큐베이터는 박테리아 문화를 성장을위한 37 ° C에서 설정 </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

기능성 assays 오래 주어진 백신 immunogenicity의 측정에 중요한 역할을했습니다. 이것은 통상 혈청 bactericidal titers으로 표시됩니다. 어린 시절 백신에 대한 응답으로 혈청 bactericidal titers의 연구는 일상적인 사용에 우리가 보호 면역 반응과 같은 컷 오프에 대한 수준을 개발하고 차단 확인할 수있게했습니다. 그런 assays는 그러한 TB 백신으로 효과기 T 세포 응답의 형태로 주로 세포 매개 면역을 유발 백신의 일상적인 평가에 앞으로 이동되지 않았습니다. 동물 모델에서 특정 백신 후보의 성능은 정기적으로 표준 bactericidal assays에서 평가되며, 현재의 모든 소설 TB - 백신 후보는 1 인체에 실험을 위상하기 전에 자신의 평가에서이 단계를 받게되었습니다. immunogenicity 따라서 소설 안티 - TB 백신의 보호 효능 가능성의 평가는 이상적으로 bactericidal assays의 측정을 포함하여, 지금은 인간의 모델과 유사한 단계 현명한 평가를 받아야한다. 그들이 잠재적으로 유망한 백신 후보를 화면에 적용할 어디 결핵 백신 연구의 맥락에서 Bactericidal assays은 이미 잘, 동물 모델에 설립되었습니다. immunogenicity의 읽기 밖의 주요 등 다양한 장기의 기능에 세균 부하 감소. 그러나, 그러한 assays 데이트 소설 안티 - 결핵 백신에 대한 임상 실험에 포함되지 않았습니다. 인간의 macrophages 내부 mycobacteria의 살인으로 이어지는 정확한 메커니즘에 대한 불확실성이 여전히 있지만, mycobacteria과 macrophages 및 T 세포의 상호 작용은 명확하게 필요합니다. 이 문서에서 설명하는 분석들은 상대적으로 개인의 기여에 대한 가정을하지 않고 전체 피에 다른 모든 세포와 체액성 요소와 같은 주요 세포 구성 요소의 연구를 가능하게 bactericidal assays의 소설 세대를 나타냅니다. 우리의 그룹에 의해 설명하는 분석은 전체 혈액의 작은 볼륨을 사용하여 이미 TB - 지방병 설정에서 성인과 어린이의 연구에 채용되었습니다. 우리는 BCG 백신의 immunogenicity를 볼 HIV 양성 환자에서 mycobacteria의 성장뿐만 아니라 체외에서 mycobacterial 생존에 방지 retroviral 요법과 비타민 D의 효과를 증가하고있다. 여기서 우리는 방법론을 요약하고이 비교적 간단하고 저렴한 비용 및 현장 친화적인 모델을 사용하여 우리의 재현성 데이터를 제시한다. 참고 : 정의 / 약어 BCG 럭스 = M. bovis BCG, 몬트리올 변형은 mycobacterial GroEL (hsp60) 발기인의 통제하에, 장염 harveyi에서 luxAB 유전자를 나르는 셔틀 플라스미드 pSMT1로 바꾸었다. CFU =의 콜로니 형성 단위 (mycobacterial 생존의 측정).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

우리는 mycobacteria의 열거에 대한 대안적인 접근 방식을 설명<em&gt; 체외에서</em기자 - 유전자를 사용하여&gt;는 대신 식민지 - 형성 단위 (CFU)의 mycobacteria 태그. 생물의 &quot;생존&quot;뿐만 아니라 호스트 반응 마커가 결핵의 맥락에서 호스트 / 병원체 상호 작용 연구를위한 저비용, 다목적 및 기능 시스템을 제공하는 동시에 측정됩니다.

Mycobacteria의 가능성을 측정하는 기능 전체 혈액 분석은 리포터 - 진 BCG 또는 M. TB (BCG 태그 사용 럭스 / M. TB 럭스)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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Research

JoVE Journal

Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

13.8K 조회수.  Imperial College London. 우리는 mycobacteria의 열거에 대한 대안적인 접근 방식을 설명 체외에서는 대신 식민지 - 형성 단위 (CFU)의 mycobacteria 태그. 생물의 "생존"뿐만 아니라 호스트 반응 마커가 결핵의 맥락에서 호스트 / 병원체 상호 작용 연구를위한 저비용, 다목적 및 기능 시스템을 제공하는 동시에 측정됩니다.

비디오: A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

연구

면역학 및 감염병학

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

오버레이 분석을 사용하여 성적으로 폐렴 구균 박테리오신에 대한 세균의 생산 및 감도를 측정하는

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

간단한 유세포 방법은 전체 혈액에서 단핵구 부분 집단에 대한 당 흡수 및 포도당 운송업자 발현을 측정하는

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

보호 효능 및 폐 면역 반응은 마우스에 피하 및 비강 BCG 관리에 따라

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

외 인 항 원 비 유전 표면 장식 Avidin 비타민 b 복합체 시스템을 통해 진 균 bovis BCG 백신에의 표현

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

예방 접종 후 기니 돼지 모델에서 측정 T 세포 응답에 최적화 된 인터페론-감마 ELISpot 분석 결과

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...