This work was supported by "Spanish Ministry of Economy and Competitiveness" [grant number BIO2014-5258P], "European Commission" by the H2020 programs [grant numbers TBVAC2020 643381].

모든 마우스는 통제 된 조건 하에서 유지와 질병의 징후가 관찰되었다. 실험 연구는 실험 동물의 보호와 관할 지역 윤리위원회의 승인과 유럽 국가 지침과 일치 하였다. BCG 덴마크어의 정량화 글리세롤 주식 및 결핵균의 H37Rv 1. 준비 주 : 설명 된 모든 프로토콜은 BSL3 조건 하에서 수행 하였다. <ol><li> BCG 덴마크어 또는 H37Rv 균주의 해동 냉동 글리세롤 주식 및 트윈 80 0.05 % (v / v)로 및 ADC (알부민, 포도당, 카탈라제) 10 % (v / v)의 보충 7H9 매체 (10) 10ml에 100 μl를 접종. </li><li> 배양 로그 상에 성장 될 때까지 37 ℃에서 일주일 동안 정적 조건에서 인큐베이션. </li><li> 신선한 배지 200㎖에 10 ml의 문화를 전송하여 세균 배양을 확장 할 수 있습니다. 20 추가 품다정적 조건에서 일. </li><li> g X 2500에서 15 분 동안 50 ml의 원심 분리기 튜브와 원심 분리기에 전송합니다. </li><li> 상층 액을 버리고 잔류 볼륨에서 세균 펠렛을 재현 탁. (- 튜브 당 15 10) 각각의 튜브 멸균 3 mm 직경의 유리 구슬에 추가합니다. </li><li> 소용돌이 적극적 위해 1 분 동안 세균 덩어리를 떼어 놓다합니다. 트윈 80 0.05 %와 PBS 10 ml의 각 펠렛을 재현 탁 (v / v)로. </li><li> 가장 큰 세균 단위 및 유리 구슬이 정착 할 수 있도록 10 분 동안 튜브를 남겨주세요. </li><li> 하나의 신선한 50 ml의 원심 분리기 튜브 (최종 복구 된 볼륨 38 ㎖)에 작은 덩어​​리와 하나의 박테리아 및 전송 볼륨을 포함하는 4 튜브의 각에서 상층 액 9.5 mL로 복구 할 수 있습니다. 400 × g으로 10 분 동안 원심 분리기. </li><li> 15 %의 최종 글리세롤 농도를 수득 한 신선한 50 mL의 원심 분리 튜브 (주로 단일 박테리아를 함유 함) 상등액 35 mL를 복구 및 글리세롤로 50 % (V / V) PBS 15 mL로 추가 (V / V). </li><li> 부드럽게 믹스냉동에 적합한 튜브에 1 ㎖의 분량 씩 배포 할 수 있습니다. -80 ° C에서 보관 (한 많은 약 50 분취 글리세롤 주식을 제공합니다). </li><li> 글리세롤 주식을 정량화하기 위해, 동결 후 글리세롤에게 일을 해동하고 PBS 900 μl를 포함하는 별도의 1.5 ml의 튜브에 일곱 10 배 연속 희석을 수행합니다. </li><li> 플레이트 55 mm 직경 배양 접시에서 제조 된 ADC 10 % (v / v)의 보충 7H10 고체 한천 배지 (10)의 각 희석액 100 ㎕. </li><li> 조심스럽게 3mm 직경의 유리 구슬 (접시 당 약. 10)를 추가하고 부드럽게 동등하게 한천 접시 위에 볼륨을 배포 판을 흔들어. 구슬을 취소하고 파라 필름으로 한천 플레이트를 밀봉. </li><li> 37 ° C 21 일 동안 품어 단일 콜로니를 정확하게 구별 할 수있는 희석에 집락 형성 단위를 (CFU)를 계산하여 세균 농도를 결정한다. </li></ol> 2. 마우스 예방 접종 <ol><li> 사전 정량 BCG 글리세롤을 해동하고 사전에 PBS에 희석이 현탁액이다. 동물 당 투여 량을 고려하여 최종 부피를 계산하는 것은 피하 투여 100 μL (피하 접종 ml의 당 107 CFU) 및 비강 내 투여 (비강 예방 접종에 대해 2.5 × 10 7 CFU) 40 μL이다. </li><li> 이소 플루 란과 산소의 혼합물로 쥐를 마취하는 설치류에 대한 전문 마취 워크 스테이션을 사용합니다. 마취 챔버 내에 마우스를 유지하는 마취 2 % 이소 플루 란을 유도하는 5 %를 사용한다. (기타 수락 마취 방법이 사용될 수있다). </li><li> 피하 백신 투여에 세균 현탁액 (107 CFU / ㎖) 1 ㎖의 26 G 바늘을 1 mL를 주사기를 채운다. 공기 방울을 제거합니다. </li><li> 층류 후드 내부에 발생하기 쉬운 위치에 평평한 표면에 마취 마우스를 놓고 마우스의 측면에 피하에게 백신 100 ㎕ (106 CFU / 복용량을) 다시 접종. 케이지에 마우스를 반환하고 prope 수 있도록 그것을 모니터RLY는 마취에서 회복. </li><li> 각 마우스 행정부 사이의 주사기 바늘을 변경합니다. 상술 한 바와 같이 기포를 제거했다. </li><li> 비강 내 투여의 경우, 후드 안쪽에 누운 자세에서 마취 마우스를 놓습니다. </li><li> 마이크로 피펫으로 2.5 × 107 CFU / ㎖로 현탁액의 20 μl를 가져 가라. 마우스의 볼륨을 숨을 쉴 수 있도록 방울 사이의 시간을두고 접종 드롭하여 드롭 전체 볼륨이 입금 될 때까지 두 개의 콧 구멍 사이에 배치 참고 :. 마우스는이 절차를 수행하는 동안 호흡 곤란으로 고생 할 수 있습니다 그들이에 남아 있어야하므로 가급적으로 방지하기 위해 다음 중 하나를 관리하다 전에 각 드롭 동화. </li><li> 또 다른 20 μL와 마이크로 피펫을 재 작성하고 작업을 반복합니다. 마우스가 첫 번째 접종 후 마취에서 깨어 시작되면, 이전에 두 번째로 마취 실에 넣습니다. 케이지에 마우스를 반환하고 올바르게 복구되었는지 확인마취에서. </li></ol> H37Rv 스트레인 3. 마우스 비강 도전 <ol><li> 미리 정량 H37Rv 글리세롤을 해동하고 ㎖의 당 2,500 CFU의 세균 현탁액을 준비하기 위해 PBS에 희석. 동물 당 투여 량을 고려하여 최종 부피를 계산하면 40 μL이다. </li><li> 2.5에 설명 된대로 H37Rv는 비강 접종 - 2.7. 마우스 당 최종 도전 선량이 10 CFU / 40 μL이다. </li></ol> 폐에서 유도 된 면역 반응 4. 분석 <ol><li> 폐 세포 현탁액 주 : 폐 백신 유발 적응성 면역 반응의 평가는 단일 세포 현탁액의 생성을 필요로한다. 이를 위해, 우리는 GentleMACS 티슈 dissociator를 사용한다. <ol><li> 자궁 경부 전위 (또는 다른 승인 된 인도적인 방법)에 의해 마우스를 희생와 마우스가 점멸 반사의 존재를 확인하기 위해 안구에 접촉 죽은 확인합니다. </li><li> 층류 후드 내부에서 L을 추출멸균 가위와 집게를 사용하여 깨끗한 표면에 배치 ungs. 면도 폐 기관지 및 결합 조직을 제거하고 (처리 될 때까지 얼음 상에 유지) PBS로 1.5 ㎖의 튜브에 폐를 옮긴다. </li><li> pH가 7.4, 150 mM의 NaCl을, 5 밀리미터의 KCl, 1 mM의의 MgCl 2, 1.8 mM의 CaCl2를 10 mM의 HEPES-NaOH를 5 mL를 함유 기관 해리 (C-dissociator 튜브)에 대한 적절한 튜브에 폐를 배치, 휴대 정지를 생성하려면 완충기. </li><li> 콜라게나 D 100 ㎎ / ㎖ 100 ㎕ (2 ㎎ / ㎖ 최종 농도)과의 DNase I 20,000 IU / ㎖의 40 μl를 (160 IU / ml의 최종 농도를 추가합니다. 주 : 콜라게나 D 또는 DNaseI의 스톡 솔루션은 각각이 1 ㎜ (V / V), 20 mM 트리스 - 염산 pH를 7.5의 Y의 MgCl 동결 PBS 효소 또는 글리세롤 50 % 용해 제조된다. </li><li> 조직 dissociator에 튜브를 놓고 폐 조각에 대해 사전 정의 된 프로그램 m_lung_01를 사용합니다. 30 분 동안 수욕에서 37 ℃에서 인큐베이션 튜브. </li><li> 장소 일전자 상기 dissociator 튜브 및 단일 세포 현탁액에 완전한 폐 균질화에 대해 사전 정의 된 프로그램 m_lung_02를 사용합니다. </li><li> 50 mL의 원심 분리 튜브에 장착 된 70 ㎛의 나일론 세포 여과기를 통하여 세포 현탁액을 통과한다. RPMI 1640 배지 10 ㎖로 스트레이너 씻으십시오. </li><li> 원심 분리기 세포 400 XG에 5 분간 및 상층 액을 버린다. </li><li> 버퍼를 용균 적혈구의 1 ML을 추가 텍싱하여 혼합하고 실온에서 1 분 동안 품어. </li><li> 400 X g에서 5 분 동안 RPMI 1640 배지 및 원심 분리기 세포의 10 ML을 추가합니다. </li><li> 완전 RPMI 1640 배지에 0.5 ㎖ 중의 재현 탁 된 세포를 10 % 열 - 불 활성화 된 태아 송아지 혈청 (FCS), 2 mM의 L 글루타민, 100 μg의 / ㎖ 스트렙토 마이신 100 IU / ㎖ 페니실린, 50 μM 2- 머 캅토 에탄올을 첨가. 주 : FCS 30 분 동안 56 ℃에서 배양하여 비활성화된다. </li><li> 세포를 카운트 10 7 세포 / ml의 최종 세포 밀도에 도달하는 전체 RPMI에게 1640 배지를 추가합니다. 주 : 세포 수를 들어, 우리는 자동 세포 계수기를 사용한다. </li><li> U-바닥 96 웰 멸균 접시에 세포 현탁액을 100 μl를 배포 (10 6 세포 / 웰). </li><li> 상업 H37Rv 정제 된 단백질 유도체 15 ㎍ / ㎖ (PPD) (5 μg의 / ml의 최종 농도)로 전체 RPMI 1640 배지의 잘 50 μL /를 추가합니다. </li></ol></li><li> ELISA에 의해 사이토 카인 결정을위한 뜨는 수집 <ol><li> 4.1.14에서 37 ° C에서 48 시간 5 % CO 2 판을 품어. </li><li> 5 400 XG에 분, 상층 액 100 ㎕를 복구하기위한 원심 분리기 판. </li><li> -80 ° C에서 보관하십시오. </li><li> 각각 IFNγ에 대한 분석 버퍼와 IL17A 결정에 상층 액을 1/10 및 1/2 희석. 제조업체 지침 (ELISA 개발 키트)에 따라, 상업 EL​​ISA 키트와 사이토 카인 농도를 결정합니다. </li></ol></li><li> 사이토 카인의 결정에 대한 세포 내 얼룩유동 세포 계측법에 의해 CD4 + 세포 -producing <ol><li> 4.1.14에서 37 ° C에서 18 시간 5 % CO 2 판을 품어. </li><li> 각 웰 (10 μg의 / ml의 최종 농도)에 Brefeldin 1 ㎎ / ㎖ (DMSO에 용해 주)의 1.5 μl를 추가 여섯 추가 시간 동안 배양한다. </li><li> 3200 XG에서 1 분 동안 4.3.2에서 접시를 원심 분리기 및 상층 액을 버린다. </li><li> 10 % FCS (1/500)를 RPMI 1640에 희석 항 - 마우스 CD4-FITC의 50 μL와에 재현 탁 세포를 4 ℃에서 10 분 동안 품어. </li><li> , 3,200 XG에서 1 분 동안 4.3.4에서 접시를 원심 분리기 뜨는을 버리고 100 μL / 웰의 고정 솔루션을 추가 할 수 있습니다. 20 분 동안 4 ° C에서 품어. </li><li> permeabilization 용액 150 μL / 워시로 두 번 세포를 씻으십시오. 참고 : 10 배 농축 재고 1X 작업 용액을 제조 탈 이온수로 희석한다. </li><li> 안티 마우스 IFNγ-APC 및 항 - 마우스 IL17A - APC.Cy7의 50 μL에 재현 탁 세포는 permeabil에 희석화 용액 (1/250 모두 항체). </li><li> 실온에서 1 시간 동안 품어. </li><li> permeabilization 용액 150 μL / 워시로 두 번 세포를 씻으십시오. </li><li> 유동 세포 계측법 획득을위한 PBS 200 μL에 재현 탁 된 세포. </li></ol></li></ol> 기관지 폐포 세척에서 면역 글로불린 5. 분석 (BAL) <ol><li> BAL 샘플 획득 <ol><li> 자궁 경부 전위 (또는 다른 승인 된 인도적인 방법)에 의해 마우스를 희생와 마우스가 점멸 반사의 존재를 확인하기 위해 안구에 접촉 죽은 확인합니다. </li><li> 확인 완전히 절제하지 않도록하고, 적절한 정밀 멸균 집게와 가위를 사용하여 기관을 노출하고기도에 작은 상처를 수행합니다. BAL 샘플을 오염시킬 수있는 출혈을 일으킬 않도록주의하십시오. </li><li> 기관에 얼음처럼 차가운 PBS 800 μL와 멸균 1 ML의 주사기에 연결된 정맥을 소개합니다. </li><li> PBS를 폐하고 연구에 천천히 접종폐 붕괴를 방지하기 위해 매우 느리게 주사기의 피스톤을 후퇴 Ecover 주방 BAL 볼륨. 참고 : (500) 600 μL의 복구 만족입니다. </li><li> 1.5 ML 튜브에 BAL 볼륨을 전송하고 그 혈액 (처리까지 얼음에 유지)에 오염되지 않은 나타내는, BAL은 채색 유지 확인합니다. </li><li> 새로운 튜브에 5 400 XG에 분 및 전송 상등액을위한 원심 분리기 BAL 샘플. </li><li> -80 ° C에서 보관 상층 액. </li></ol></li><li> BAL 상층 액에서의 IgA의 결정 참고 : 사용 된 모든 시약은 RT에 있습니다. <ol><li> M. (10 μg의 / ㎖) PBS에 희석 PPD 100 ㎕와 코트 고 단백질 결합 폴리스티렌 평면 바닥 96 웰 플레이트 결핵 - 특정의 IgA 결정, 또는 전체의 IgA 결정을 위해 PBS에 10 배 희석 BAL의 상층 액 100 ㎕와. </li><li> 4 ℃에서 밤새 품어. </li><li> 뜨는을 취소하고 D에 종이 타월에 접시를 누릅니다공예. 200 μL / PBS 잘 씻으시오. </li><li> 200 μL / 웰의 소 혈청 알부민 (BSA) PBS-트윈 80, 0.05 % (부피 / 부피) (블로킹 완충액) (V / w) 1 %로 차단. </li><li> 실온에서 1 시간을 품어. </li><li> 200 μL / PBS-트윈 80 0.05 % (v / v)의 (세척 버퍼) 잘 한 번 씻으십시오. </li><li> PPD를 코팅 우물에 희석 BAL의 상층 액 100 μl를 추가합니다. 실온에서 2 시간 동안 인큐베이션. 참고 : 전체의 IgA 결정은 PPD 별의 IgA와 병렬로 수행 할 경우,이 배양 동안 100 μL 세척 버퍼 전체의 IgA 우물을 둡니다. </li><li> 세척 buffer.0170004 200 μL / 웰로 3 회 세척 </li><li> 고추 냉이 퍼 옥시 다제 (HRP)의 100 μl를 추가 버퍼 (1 / 10,000) 차단에 희석 항 - 마우스의 IgA를 π 공역 계. </li><li> 1 시간 RT를 품어. </li><li> 세척 완충액 200 μL / 웰로 5 회를 씻으십시오. </li><li> 3,3 100 ㎕ / 웰 추가 &#39;, 5,5&#39;- 테트라 메틸 벤지딘 (TMB). t 실온에서 20 분 인큐베이션그는 어두운. </li><li> 100 μL / 웰의 H 2 SO 4 0.5N를 추가합니다. 파장 450 nm에서 분광 광도계 플레이트를 읽었다. </li></ol></li></ol> 폐 6. 세균 부하 결정 <ol><li> 폐 균질화 및 도금 <ol><li> 네 주 공격 후, 자궁 경부 전위 (또는 다른 승인 된 인도적인 방법)에 의해 마우스를 희생와 마우스가 점멸 반사의 존재를 확인하기 위해 안구에 접촉 죽은 확인합니다. </li><li> 층류 후드 내부의 평면 및 소독 표면에 고정 부정사 위치에 마우스를 놓습니다. </li><li> 절개를 확인하고 흉부 영역을 볼 수 떠날 가슴 영역에서 피부를 제거합니다. </li><li> 갈비뼈를 잘라 멸균 가위와 집게를 사용하여 폐와 심장을 수확. 깨끗한 표면에 장기를 놓습니다. </li><li> 심장, 기관지 및 결합 조직을 제거하여 폐를 청소하고 1 ㎖ 탈 이온수에 1.5 ㎖의 튜브에 폐를 전송할(처리 될 때까지 얼음에 유지). </li><li> 탈 이온수 1 ml를 포함, - (dissociator 튜브 M) 장기 균질화에 대한 적절한 튜브에 각 마우스의 폐를 전송하는 멸균 집게를 사용합니다. </li><li> 조직 dissociator에 튜브를 놓고 폐를 균질화하는 미리 정의 된 프로그램 RNA_1를 사용합니다. </li><li> 900 μL에 희석 폐 균질 100 ㎕를 시작으로 1.5 ml의 튜브에 5 ~ 10 배 연속 희석을 확인합니다. </li><li> 플레이트 희석은 1.11-1.14에 설명 된대로. </li></ol></li><li> PCR에 의해 BCG와 H37Rv 사이의 차별화 참고 : 비강 내 경로에 의해 BCG 접종 그룹에 해당하는 6.1.6에서 판에서, 우리는 CFU가 백신 수여 예방 효과가 맞습니다 평가로 간주하도록 BCG와 H37Rv 사이의 안목 PCR에 의해 식민지의 대표 번호를 분석 고유 H37Rv합니다. RD9이 PCR 표적 증폭, BCG와 M. 너와 다른 게놈 영역berculosis, H37Rv에 대한 2618 bp의 및 BCG (11)에 대한 465 염기쌍의 단편을 제공. <ol><li> PCR 튜브에 (9 μL / 샘플)을 배포 한 후 표시로 샘플 당 볼륨이있는 모든 샘플에 대한 마스터 믹스 재고를 준비합니다. 참고 : 버퍼 5 배 : 2 μL. 앞으로 프라이머 25 μM (GTGTAGGTCAGCCCCATCC) : Taq 중합 효소에게, μl의 5 단위 / 0.32 μL : 0.32 μL는 프라이머 25 μM (GCCCAACAGCTCGACATC)를 역 0.04 μL, 탈 H 2 O : 6.32 μl를. </li><li> 멸균 된 이쑤시개로 콜로니를 터치 마스터 믹스를 함유하는 PCR 튜브에 샘플 이쑤시개 젖어. </li><li> 다음 프로그램으로 PCR을 실행 : 10 분 ~ 95 ° C (이 단계는 박테리아 중단을 유발하는 것입니다), 10 분, 95 ° C, 1 분, 95 ° C에서 35주기 (30 초 (이 단계는 박테리아 혼란을 유발하는 것입니다) 58 ° C와 4 분 72 ° C에서)에서. </li><li> 단편을 시각화 티듐 브로마이드 실행 샘플을 1 % 아가 로스 겔에서 PCR 샘플을로드. </l난&gt; </ol></li></ol>

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The authors have nothing to disclose.

Although current vaccine against tuberculosis, BCG, is the most widely administered vaccine in history, tuberculosis remains one of the leading causes of death and morbidity from infectious diseases worldwide. This paradox is explained by the lack of protection of this vaccine against pulmonary tuberculosis, the responsible form of transmission. New vaccination approaches effective against pulmonary forms of the disease are urgently needed, as they would have the greatest impact on disease transmission globally.
<p c...

결핵 (TB)는 TB 놀라운 세계 보건 문제 일 수 있습니다 세계에서 HIV보다 더 많은 관련 사망의 원인이되는 주요 감염 질환 중 하나 다 약제 내성 균주의 증가 증가와 결합이다. 새로운 진단 도구,보다 효과적이고 독성이 적은 약물, 새로운 안전하고 효과적인 결핵 백신은 특히 개발 도상국에서, 긴급한 필요하다. 감쇠 Bacille 칼 메트 - GUERIN은 (BCG)은 현재 전 세계적으로 1970 년대 이후 출생시 피내 투여 된 TB, 대한 전용 허가 백신 살고있다. BCG는 아이들의 질환 (수막염 및 속 립성 결핵)의 심한 형태의 예방에 효과적인 것으로 간주되지만, 질병 전파 (2)의 책임 폐결핵에 대한 일관성 효과를 보여 주었다. TB 감염의 자연 경로를 모방 폐 예방 접종은 로컬 호스트 면역 반응을 프라이밍위한 매력적인 접근 방식을 나타냅니다에스. 피하 또는 피내 경로 3-6에 비해 이와 관련하여, 다른 관련 TB의 동물 모델에서 다양한 전임상 작품 폐 면역화 다음 큰 백신 효능을 보여 주었다. 그럼에도 불구하고, 폐 예방 접종에 의해 유발 보호 메커니즘은 잘 이해되지 않습니다. 지난 몇 년 동안, 여러 작품 IL17 점막 백신에 의한 예방 효과에 대한 결핍 된 마우스 모델에서와 같이, TB-특정 점막 면역 반응의 중요한 요소로 IL17 중재 응답으로 지적은 7,8를 손상된다. 최근에 우리는 BCG 관리는 DBA / 2 마우스, 피하 BCG 예방 접종 9 후 보호의 부족을 특징으로하는 마우스 변형을 보호 비강 처음으로 보여 주었다. 이러한 결과는 호흡기 결핵 예방 접종은 피내 BCG가 pulmon에 대한 효과로 간주됩니다 발병 국가에서 TB의 비율을 감소 시키는데 더 효과적 일 수 있다고 제안진 TB.

<table border="0" cellpadding="0" cellspacing="0" width="895">
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		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H9 broth</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">271310</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook ADC Enrichment</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">211887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Tween 80</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">TW00800250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3-mm diameter Glass Beads</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">038-138003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H10 Agar</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">262710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">1-ml syringe 26GA 0.45x10 mm</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">301358</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GentleMACS dissociator</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">C tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">M tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Collagenase D</td>
			<td style="width:176px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">DNaseI</td>
			<td style="width:176px;">Applichem</td>
			<td style="width:119px;">A3778,0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:176px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">RPMI 1640</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R0883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Red Blood Cell Lysing Buffer</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GlutaMAX Supplement</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:119px;">35050-061</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Penicillin-Streptomycin Solution</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Fetal Calf Serum</td>
			<td style="width:176px;">Biological Industries</td>
			<td style="width:119px;">04-001-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">2-Mercaptoethanol</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">M3148-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter 2.0 Handheld Automated Cell Counter</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC20040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter Cell Counter Sensors, 40 &#181;m</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC40050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:409px;">Mycobacterium Tuberculosis - Tuberculin PPD</td>
			<td style="width:176px;">Statens Serum Institut (SSI)</td>
			<td style="width:119px;">2390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IFN-&#947; ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3321-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IL17A ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3521-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Brefeldin A</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">553047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">BD Cytofix/Cytoperm Kit</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">555028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC-Cy7 Rat Anti-mouse IL-17A</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">560821</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC Mouse Anti-mouse IFNg</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">554413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&#8221;</td>
			<td style="width:176px;">UNIMED</td>
			<td style="width:119px;">27.134</td>
			<td>Used as trachea cannula for BAL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td>
			<td style="width:176px;">NUNC</td>
			<td style="width:119px;">430341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Albumin, from bovine serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Goat Anti-Mouse IgA (&#945;-chain specific)&#8722;Peroxidase antibody</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB)&#160;</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">T0440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">MyTaq DNA Polymerase</td>
			<td style="width:176px;">Bioline</td>
			<td style="width:119px;">BIO-21107</td>
			<td>The kit Includes Buffer 5x</td>
		</tr>
	</tbody>
</table>

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.

피하 및 비강 :이 작품은 BCG의 관리의 두 경로의 비교에 대해 설명합니다. 피하 전세계 BCG 현재 임상 경로 인 피내, 비슷하다. 백신의 비내 경로 M.의 감염 천연 경로를 모방하는 것을 목표 목적으로 결핵은 폐에 직접 병원체의 주요 표적 장기를 면역 반응을 유도한다. 그림 1은 다음 워크 플로를 설명?...

Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

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

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 ...

protective-efficacy-pulmonary-immune-response-following-subcutaneous

Research

JoVE Journal

Immunology and Infection

10.5K 조회수.  University of Zaragoza. 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.

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

붉은 털원숭이 짧은 꼬리 원숭이의 세포 면역 반응의 여러 가지 빛깔의의 유동세포계측법의 분석

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

연구

면역학 및 감염병학

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

의 기생충 말벌에 대한 소개<em&gt; Drosophila</em&gt; 그리고 Antiparasite 면역 반응

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

면역 반응과 수면에서의 양적 측정<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

폐 마우스의 면역 조직 샘플의 수확과 함께 오른쪽 심실 수축기 압력 측정

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

맹장 결찰 및 마우스에서 autophagy에 공부를 모델로 패혈증 관통 유도

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

마우스 대 식세포의 타고난 면역 반응을 조사하기 위해 RNA 간섭을 사용하여

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

마우스의 호스트 면역 반응을 평가하기 위해 구인두 기관 내 PAMP 관리 및 폐포의 활용

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

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

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