우리는 숙련 된 지원 커스틴 Thim (아스트라 제네카, 룬드, 스웨덴), 베니 타 달 버그 박사 앤더스 에크 룬드 (카롤린스카 연구소, 스톡홀름, 스웨덴)뿐만 아니라 닥터 마틴 Stampfli (맥 매스터 대학, 해밀턴, ON, 캐나다)에 감사드립니다 및 전문가의 조언.

동물 연구는 북부 스톡홀름 동물 복지 윤리위원회에 의해 승인되었다. 실험 절차는 스웨덴어 법을 준수 하였다. 1. LPS 에어로졸을 생성 <ol><li> P. 정제 0.5 g을 녹여 부드러운 교반과 함께 50 ml의 멸균 식염수에 aeruginosa의 LPS 및 용해를 확인합니다. 1 mg / ml의 최종 농도로, 9 ml의 멸균 식염수에 용해하여 LPS를 1 mL로 희석. -20 ° C에서 알루미늄 호일과 가게와 빛으로부터 보호합니다. </li><li> 실온에서 어두운 가용화 LPS를 사용하기 전에 해동하고 즉시 잘 혼합한다. </li><li> 환기 수준 II 생물 학적 후드에서 분무기에 붉은 입구를 삽입하고, 제조업체 (그림 1의 실험 장치를 제시 검토 방식)에 의해 제공되는 튜브를 통해 가압 실내 공기에 분무기를 연결합니다. 주의 : 적절한 개인 보호 장비, 반 얼굴 조각 재사용 respirat 포함또는 미립자 필터, 고글, 장갑, 보호 의복 노광의 과정 동안 사용되어야한다. </li></ol><img alt="그림 1" src="/files/ftp_upload/51470/51470fig1highres.jpg" /> 도 1 :. 에어로졸을 생성하기 위해 사용되는 실험 장치의 개략도 표현은 에어로졸 생성기의 입구는 공기 공급 장치에 연결된다. 분무기의 출구는 제 15.9 mm 튜브와 공기 필터 및 공기 공급을 통해 유량계에 연결되어 2 kbar 압력 5.0 L / m으로 조절된다. 출구 이동식 뚜껑과 압력 증가를 방지하기 위해 5mm 구멍에 끼워 플렉시 박스 옆으로 접속된다. <ol start="4"><li> 에어 필터를 통해 질량 유량계에 분무기의 출구를 연결합니다. 전기 공급 장치에 질량 유량계를 연결합니다. </li><li> 1.0 ~ 2.0 바에서 남은 압력, 5 L / min으로 공기 공급을 조정합니다. </li><li> 질량 유량계를 제거하고공기 공급 장치를 분리합니다. </li><li> 150 X 163 X 205mm, 이동식 뚜껑 장착 : 분기합니다 및 치수 두 플렉시 유리 상자에 연결하는 15.9 mm 튜브에 분무기의 출구를 연결합니다. 각 박스에는 압력 증가를 방지하기 위해 입구 대향 측 5mm 구멍을 가져야한다. </li><li> 각 플렉시 유리 상자에 5 마우스까지 올려 놓고 뚜껑을 닫습니다. </li><li> 분무기를 열고 단독으로 멸균 식염수 또는 차량에 용해 최소 4 ML의 LPS로 삽입을 채우기 (멸균 식염수를, 볼륨 8 ml의를 초과 할 수 없습니다). 공기 공급 장치의 입구를 다시하는 것은 연결합니다. </li><li> 에어로졸은 10 분 동안 폐쇄 플렉시 글라스 상자에 흐르게. 지속적으로 동물을 모니터링합니다. 반드시 공기 공급이 분무기의 입구에 단단히 고정되어있는 상태 확인합니다. </li><li> 공기 공급 장치를 분리합니다. 뚜껑이 닫힌 상​​태에서, 동물이 2 분 동안 플렉시 유리 상자에 남아있을 수 있습니다. </li><li> 뚜껑을 열고 에어로졸 분산 할 수 있도록하고, T로 동물을 반환그는 케이지. 동물 젖은 나타날 경우 저체온증을 방지하기 위해, 저 발열로 설정 가열 패드에 새장을 배치합니다. </li><li> 처음 30 분 동안 연속적으로 동물을 모니터링하고, 상기 제 6 시간 동안, 그 후 매 2 시간. 동물 중 및 분무 과정을 거친 후 정상적인 호흡 패턴 및 활동을 전시한다. </li></ol> 2. 기관지 폐포 세척 (BAL) <ol><li> 교육 기관의 수의 직원에서 권장하는 실험 종료 시점에서, 깊이 효과에 이소 플루 란과 동물을 마취. 큰 수술을 실시 마취의 충분한 깊이를 보장하기 위해 철수 반사를 확인하기 위해 뒷발을 꼬집어. 70 % 에탄올과 동물의 모피를 스프레이. </li><li> 가위를 사용하여 복부를 열고 동물에게서 피를 뽑다하는 대동맥을 절단. 혈액을 흡수하기 위해 복부를 통해 조직의 조각을 놓습니다. </li><li> 안락사를 follwing을하기에 가위 하나의 전후방 컷을 사용가슴 노출. 흉골의 앞쪽 끝으로 흉곽을 들어 올려 모든 폐 엽으로 절단하지 않고, 대부분의 복부 지점에서 다이아 프램에 구멍에 가위를 사용합니다. 전후방 방향으로 두 컷 (턱 아래 회의)하여 흉곽을 엽니 다. </li><li> 부드럽게 집게를 사용하여 흉곽 떨어져 당겨, 그리고 후두 아래의 기관을 잘라. </li><li> 집게로기도를 들어 올려 흉강에 로브를 연결하는 인대를 절단하고 부드럽게 지방과 심장 조직에 의해 당겨 폐를 제거합니다. </li><li> 폴리에틸렌 관 (: 0.58 mm, 외경, 내경 0.965 mm)를 넣 기관에 및 실크 스레드의 문자열로 튜브를 고정합니다. </li><li> 실크 스레드와 multilobe (우측 폐의 네 로브)를 묶어. </li><li> 폴리에틸렌 튜브에 23 게이지 바늘을 삽입하고 천천히 1 ML의 주사기와 단일 로브에 얼음처럼 차가운 멸균 PBS의 250 μl를 주입. </li><li> 조심스럽게 폐를 누릅니다30 시간과 주사기를 통해 액체를 수집합니다. 200 μL PBS와 절차를 반복합니다 (약 300 μl의 PBS 450 μL의 총 주입 볼륨에서 하나의 로브에서 복구해야합니다). </li><li> 얼음에 기관지 세척액 (BALF)을 유지, 또는 즉시 BAL 세포를 열거합니다. 전지 (11)를 염색 터크 용액을 사용하여 세포 heamocytometer 카운트. 염색 액의 희석 배수와 heamocytometer의 카운트 필드 내의 부피 세포 수를 곱하여 총 세포 수를 계산한다. </li><li> 다른 11 설명 된대로 cytocentrifuged 세포에서 차동 세포 수를 준비합니다. </li></ol> 조직 학적 평가를위한 폐 조직의 3 포르말린 고정 <ol><li> multilobe를 제거하고 스냅인 동결을 드라이 아이스에. -80 ° C에서 보관하십시오. </li><li> 금속 지지대에 제거 플런저와 60 ML의 주사기를 탑재합니다. 10 % 포르말린에와 주사기를 채우일정한 압력의 20cm를 나타내는 실험실 벤치 위에 20cm의 높이. </li><li> 포르말린의 흐름을 제어하는​​ 밸브가 플라스틱 튜브를 통해 60 ml의 시린지에 고정 기관지 23 게이지 바늘을 연결한다. </li><li> 5 분 동안 포르말린 폐 엽 Insufflate. 바늘을 분리하고 실크 스레드에서 당기는 기관을 닫고 폐의 압력을 유지하면서 기관에서 폴리에틸렌 튜브를 함께 제거합니다. </li><li> 포르말린 폐 엽 잠수함 4 ° C에서 24 시간 동안 수정. </li><li> 70 % 에탄올로 적어도 20 분 동안 고정 된 조직을 세 번 씻은 다음, 다음 처방 (1 시간마다)를 통해 탈수 크실렌 : <ul><li> 3X 70 % 에탄올 </li><li> 3X 95 % 에탄올 </li><li> 3X 100 % 에탄올 </li><li> 3 배 크실렌 </li><li> 1 배 (액체) 파라핀 </li></ul></li><li> 헤 마톡 실린 및 에오신에 포함 탈수 4-5 μm의 섹션으로 잘라 파라핀 조직, 얼룩은 조직 학적 평가를 허용합니다.</li>
</ol>

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아브라함 로스는 베링거 인 겔 하임, 아스트라 제네카 R &amp; D와 아스트라 제네카 R &amp; D에서 연구 보조금에서 여행 보조금에서 강사 수수료를 받고있다. 매그너스 노르 아스트라 제네카 R &amp; D의 풀 타임 직원과 아스트라 제네카 R &amp; D에서 연구 보조금을 받았다. 요한 Grunewald는과 토베 버그는 아스트라 제네카 R &amp; D 자금 지원 관련없는 연구 프로젝트에 공동 연구자이다.

에어로졸은 LPS 상피 점막하 호중구 특징으로기도의 염증 반응을 발생시키고, 전도성기도뿐만 아니라 폐포를 둘러싼 공간. 이것은 함께 혈장 누출, 급성 폐 손상의 병리 대표 나타내는 BALF에서 증가 된 총 단백질 함량으로한다. LPS는 무균 염증을 유도 같이, 반응은 적응성 면역 반응에 독립적이며, 세균 감염에 대한 관련성에 한계가있다. 기술은 그러나 적응 면역 반응을 제외하여 염증 기전을 해?...

리포 폴리 사카 라이드 (LPS)는 그람 음성 세균 (1)의 세포벽 성분이다. LPS에 도전 급성 폐 손상, 급성 염증 세포의 침윤 염증과 부종이 특징으로하는 증후군의 잘 문서화 된 모델입니다. 또한, 폐 호중구 또한 COPD 악화 4를 모델링하는데 사용되어 만성 폐쇄성 폐 질환 (COPD) 3, 및 인간에서 LPS 챌린지의 특징이다. 따라서, LPS 노출의 실험 모델은 임상 적으로 인간의 병리를 이해하기 위해 적절하고 가치있는 도구입니다. 여기에 설명 된 에어로졸 LPS의 폐 전달의 목적은 전신 개입 없이도 도통 및 호흡기도에 호중구 염증 반응을 생성하는 것이다. LPS 도전의 여러 기술 이전에 기술되었다. LPS의 정맥 내 주입 투여 가장 일반적으로 사용되는 경로이다. 이 기술에 쉽게 접근 할 수 있지만, t그는 차 손상은 폐에 호중구 마이그레이션 다음 폐 상피 세포의 보조 파괴, 내피이다. 인트라 정맥 투여는 동물 모델에서 임상 사진을 복잡하게 할 수 있습니다 전신 염증이를 유도한다. 전신 염증은 내 기관 관리와 관찰되지 대조적이다. 이 기술은, 그러나, 노동 집약적이며, 마취제뿐만 아니라 상당한 훈련 5, 6을 필요로한다. 또한, 행정이 경로를 통한 폐 침착은 호흡 7에 따라 달라집니다. 따라서, 폐 침착은기도에 내 기관 관리 및 변수 침착이 관찰 될 수 있습니다에 필요한 마취의 깊이에 의해 영향을 받는다. 대조적으로, 에어로졸 화 된 LPS와 폐 전달은 최소한의 훈련을 필요로하며, 쉽게 작은 가진 동물의 다수 또는 개인 5, 8 사이에 편차가없이 달성 될 수있다.최근의 연구는 에어로졸 배달 증착에 관한 내부 기관 노선 우수하다는 및 LPS의 관련성 용량이 모델 8 호중구 염증을 유도 확인합니다. 에어로졸 Psuedomonas aeruginosa의 LPS가 폐포 (9), (10)를 포함하여기도 루멘과 폐 실질, 눈에 띄게 염증 반응을 생성에 이전의 연구는 그 도전을 증명하고있다. 염증은 호중구 폐부종의 존재를 특징으로 우세, 따라서 급성 폐 손상의 기전을 해결하고 질병 병리에 기여하는 메커니즘을 더 지식을 얻기 위해 사용될 수있다.

<table border="0" cellpadding="0" cellspacing="0" style="width:931px;" width="929">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

급성 폐 손상 (ALI)는 제한된 치료 옵션과 높은 사망률과 폐포 호중구을 특징으로하는 심각한 질병이다. ALI의 실험 모델은 질병의 발병 기전에 대한 우리의 이해를 향상의 열쇠입니다. 그람 양성 세균에서 파생 된 리포 폴리 사카 라이드 (LPS)는 호중구기도의 염증과 쥐의 폐 실질을 유도한다. 이러한 LPS와 같은 화합물의 효율적인 폐 전달하지만, 달성하기가 어렵다. 여기에 설명 된 방법에서, 마우스의 폐 전달이 에어로졸 녹농균 LPS 챌린지에 의해 달성된다. 용존 LPS는 압축 공기에 연결되어 에어로졸 분무기로 하였다. 생쥐는 에어로졸이 중단 된 후 2 분 컨디셔닝 한 후, 10 분간 플렉시 유리 상자에 LPS 에어로졸의 연속 흐름 노출시켰다. 포르말린 관류 다음에 기관 삽관 이후 기관지 세척은, 다음 멸균 페이지의 특성을 허용하는 수행 하였다ulmonary 염증. 에어로졸 LPS는 기관지 폐포와 조직 학적 평가에 의해 검출 된 폐포 호중구을 특징으로하는 폐 염증을 생성합니다. 이 기술은 몇 가지 제품으로 작은 비용으로 설정하고, 최소한의 교육과 전문 지식을 필요로 할 수 있습니다. 노광 시스템은 따라서 통상적 폐 병변에 대한 이해를 향상시키기 위해 잠재적으로, 임의의 실험실에서 수행 될 수있다.

에어로졸 P.에 도전 aeruginosa의 LPS는 보통 초기와 후기 시점에서 모두 우위 호중구 특징으로기도 내강 및 치조 공간에서 표시된 염증 반응을 산출한다. 에어로졸 LPS는 폐 호중구을 유도 C57BL / 6by 및 BALB / C 마우스에게 에어로졸 화 된 P. 노출시켰다 aeruginosa의 LPS 또는 단독으로 차량과 호중구가 폐포 세척액에 열거 하였다. C57BL 차?...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

우리는 급성 폐 손상을 모델링하기 위해, 분무에 의해 에어로졸 리포 폴리 사카 라이드에 도전하여 호중구 폐 염증을 유도하는 방법을 설명합니다. 또한, 폐 격리, 기관 삽관 및 기관지 세척액에 대한 기본적인 수술 기법도 설명되어 있습니다.

에어로졸 리포 폴리 사카 라이드를 사용하여 생성 폐 호중구하는 방법

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

11.6K 조회수.  Karolinska Institutet.  우리는 급성 폐 손상을 모델링하기 위해, 분무에 의해 에어로졸 리포 폴리 사카 라이드에 도전하여 호중구 폐 염증을 유도하는 방법을 설명합니다. 또한, 폐 격리, 기관 삽관 및 기관지 세척액에 대한 기본적인 수술 기법도 설명되어 있습니다. 

비디오: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

배아 마우스에 Vasculature을 라벨링하는 방법

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

연구

면역학 및 감염병학

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Xpert MTB / 리프 시험과 폐 결핵 진단

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Lipopolysaccharide 취급 마우스 간에 면역 반응 동안 백혈구의 2 광자 Intravital 이미징

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

CRISPR/Cas9와 재조합 조류 Herpesvirus 벡터 생성 편집 진

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

수용 체와 효소 간의 기능 상호 작용을 공부에 대 한 분석 결과 Co-immunoprecipitation

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

생리 적 면역 원 성 수지상 세포 생성을 위한 새로운 프로토콜

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

형광 항생제 프로브를 사용한 세균 저항성 시각화

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

전신 흡입 시스템을 사용하여 쥐의 담배 연기 노출

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

미세주입에 의한 스트 롱실로이드 종에서 형질전환 및 녹아웃 생성

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

진공 안정화 이미징 시스템을 이용한 실험적 급성 폐 손상에서의 폐 미세순환의 생체내 광역 형광 현미경 검사

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...