나는에 도움을 실험 설계를 개념화 및 재료 지원, 치료 요법 및 부검을 확립 도움 A. Pessier 분석, MWH 채트, MJ Robak, K. 테스 커 및 D. Lenger 도움을 CL 리차드 - Zawacki에게 감사의 말씀을 전합니다 실험실과 촬영 기간 동안 자신의 공간의 사용에 대한 지원 및 타 롱가 동물원에서 M. 맥파든과 P. 할로. 이 프로젝트는 부분적으로 LA Brannelly에게 수여 루이지애나 환경 교육위원회의 승인을 통해 생활 기술에 의해 야생 동물과 수산의 루이지애나 부에 의해 투자되었다.

윤리 승인 :이 연구와 그 방법은 툴레 인 대학의 동물 사용 및 관리위원회 (. 프로토콜없이 0407)에 의해 승인되었다. 1. BD 감염에 대한 테스트 <ol><li> BD에 대한 보라고 <ol><li> 멸균 면봉 면봉 동물 ventrum에 다섯 번, 각 허벅 다리, 양쪽, 45 스트로크의 총 각 다리에 다섯 번. 보라고는 양서류의 피부에 BD에 대한 샘플링의 비 침습적 인 방법입니다. </li><li> 부드럽게 DNA의 가장 큰 금액을 면봉에 수집하기 위해 스트로크시 사이에 면봉을 회전. </li><li> -20 ℃에서 1.5 ml의 마이크로 튜브에 보관 면봉 </li></ol></li><li> DNA 추출 및 qPCR의 분석 <ol><li> 동물 조직에 대한 지침에 따라, 시판되는 DNA 분리 키트를 이용하여 면봉으로부터 게놈 DNA를 추출한다. </li><li> 보일 다음 정량 실시간 PCR (qPCR에)를 사용하여 추출 된 DNA를 분석 <eM&gt; 등 17, 다음과 같이 수정과. : </li><li> 이중 탈 이온수로 DNA 추출 샘플보기 1:10 희석. </li><li> PCR 억제를 방지하기 위하여 소 혈청 알부민 (BSA)의 0.7 μl를 추가. </li><li> PCR 억제가 결과에 영향을 미치는되지 보장하기 위해 각 샘플의 내부 양성 대조군을 포함합니다. </li><li> 긍정적이고 부정적인 제어 및 유주자 (감염) 부하를 추정하기 위해 희석 기준의 일련의 실행 샘플. </li></ol></li></ol> 2. 접종 <ol><li> 문화 BD <ol><li> (40 분 동안 121 ° C) 멸균 및 냉각, 1 L의 역삼 투 또는 탈 이온수에 10g의 트립 톤을 추가하여 트립 톤 국물을 준비한다. 일단 23 ° C로 냉각, 1 ㎖ 페니실린 - 스트렙토 마이신을 추가합니다. </li><li> 23 ° C에서 이레 동안 트립 톤 국물에 (Phyllomedusa 여우 원숭이에서 Guabal, 파나마에서 격리 J. Longcore에서 JEL411) BD 변형 성장 </li></ol></li><li> B로 이동로스는 문화 트립 톤 한천 플레이트합니다. <ol><li> 판을 만들려면 1 L 역삼 투 또는 탈 이온수와 오토 클레이브 (40 분 동안 121 ° C)에 10g의 트립 톤과 10g의 한천을 추가합니다. </li><li> 멸균 혼합물을 터치 할 정도로 멋지다하지만 응결 전에, 층류 후드, 페트리 접시에 가득 1/4-1/3을 부어 한 번. </li><li> 한천이 완전히 응고 및 냉각되면, 각각의 판에 0.5 ㎖ BD 국물을 추가하고 접시 주위에 균일하게 확산. 건조하도록 허용합니다. </li><li> 플레이트는 BD의 zoospores, 파라 필름과 인감 플레이트에 접종하면. </li><li> 판 5-7 일 동안 23 ° C에서 배양 할 수 있습니다. 접종 전에 생존을 보장하기 위해 운동을 유주자 확인합니다. </li></ol></li><li> 유주자 접종 솔루션에 대한 홍수 판 <ol><li> BD 배양과 성장의 5~7일 후, 세 수돗물 5 ㎖의 각 판 홍수와 zoospores 10 분 동안 미디어를 입력 할 수 있습니다. </li><li> 유주자의 suspe을 붓고휴대용 용기에 nsion. </li><li> 혈구와 유주자 농도를 추정한다. </li><li> 세 수돗물로 1 × 10 6 3 당 ML의 농도로 혼합물을 희석. </li></ol></li><li> 동물을 접종 <ol><li> 각각 50 ㎖ 용기에, 그 ventrum에 3 ㎖ 접종 혼합물을 부어 각각의 동물에 접종. 추가 접종은 접종 용기의 바닥에 떨어지는 것을 허용한다. </li><li> 각 동물은 감염을 확인하기 위해 24 시간 동안 접종 용기에 남아있을 것입니다. </li><li> 접종 24 시간 후, 개별 소독 terraria에 동물을 반환합니다. </li><li> 감염 2 주에 구축 할 수 있습니다. 면봉과의 qPCR 결과 감염을 확인하고 치료를 시작합니다. </li><li> BD-음성 대조군의 경우, 5 ㎖와 홍수 BD없는 한천 플레이트는 상기와 같은 방법을 사용하여 수돗물 및 모의 접종 동물을 세. </li></ol></li></ol> 3. 치료식이 요법 <ol><li> Prepa처리 욕 재 <ol><li> 1 L 양서류 링거액 (- 가장 효과적인 치료 농도 0.0025 %의 이트라코나졸)에 2.50 ML에게 수성 이트라코나졸 (1 % Sporanox 구강 솔루션)를 추가합니다. <ol><li> 양서류 링거액 제조법 : 1 L 역삼 투 또는 탈 이온수, 6.6 g의 소듐 클로라이드, 0.15 g의 염화 칼륨, 0.15 g의 염화 칼슘, 및 0.2 g의 중탄산 나트륨. 오토 클레이브 솔루션 (40 분, 121 ℃) 및 실내 온도까지 냉각 할 수 있습니다. </li><li> 0.01 % 이트라코나졸 치료 농도의 경우, 1 L 양서류 링거액에 10.00 ml의 수성 이트라코나졸 (1 % Sporanox 구강 솔루션)를 추가 </li><li> 0.005 % 이트라코나졸 치료 농도의 경우, 1 L 양서류 링거 용액에 5.00 ML에게 수성 이트라코나졸 (1 % Sporanox 구강 솔루션)를 추가합니다. </li></ol></li><li> BD-양성 및 BD-음성 대조군의 경우, 이트라코나졸과 양서류 링거액 동물 목욕. </li></ol></li><li> 치료 &lt;올&gt; <li> 처리 액 35 mL를 소독 50 ㎖ 용기에 개별 양서류를 놓습니다. </li><li> 확인 동물은 완전히 부드럽게 용기를 소용돌이로 5 분 동안 적용됩니다. </li><li> 소독 terraria에 동물을 반환합니다. 6 일 동안 반복합니다. </li></ol></li><li> 치료의 성공을 보장합니다 <ol><li> 일 처리에 BD 감염에 대한 시험 동물은 시작됩니다. </li><li> 감염을 보장하기 위해 4 주 동안 일주일에 해제 한 BD 감염에 대한 실험 동물 일주 처리가 완료되면, 한 번. </li></ol></li></ol> 생물 등 4. BD <ol><li> 개인 terraria 매주 소독. <ol><li> 10 % 표백제 해결책 목욕 terraria 및 치료 컨테이너 목욕. </li><li> 흐르는 물에 두 번 표백 용기를 내십시오. </li><li> 용기는 다시 사용하기 전에 24 시간 동안 건조 할 수 있습니다. </li></ol></li><li> 교차 오염을 방지하기 위해 동물을 처리 할 때 깨끗한 니트릴 장갑을 착용BD의 병원체. 각각의 동물을 처리하는 사이에 장갑을 변경합니다. </li><li> 10 % 표백제에 솔루션을 가져 와서 폐기 전에 모든 액체 폐기물을 소독. </li><li> 잠재적으로 폐기 전에 BD에 노출되는 모든 고체 폐기물을 압력솥. </li></ol>

<ol>
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저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

이 프로토콜을 이용하여, 우리는 안전하고 효과적으로 양서류 BD에 대한 이트라코나졸의 치료 치료 복용량 및 시간을 절감 할 수 있었다. 가장 낮은 처리 용량과 시간 (6 일 동안 0.0025 %)이 subclinically 감염 I.에 대한 성공적인 치료했다 nebulifer. 낮은 투여 량 및 처리 시간 모두의 효과는 양서류 모든 종 및 나이 클래스에 사용될 제공된 안전하고 효과적인 치료법은 없기 때문에, <e...

수륙 양용 비행기는 현재 모든 척추 동물 분류군 1의 생물 다양성에 가장 큰 감소를 경험하고 있습니다. . Chytridiomycosis, chytrid 곰팡이 병원균 Batrachochytrium의 dendrobatidis (BD)에 의한 피부 질환, BD는 생물 다양성 손실의 원인에 대한 기록에 최악의 병원균이 극적으로 감소 2의 주요 원인 중 하나입니다 그것은 양서류 600 종 이상 감염되어있다 많은 200 종의 멸종 위기 또는 2,3 멸종있는 300 종의 감소를 일으키는 원인이되었다. 양서류 chytrid 균은 특정 호스트되지 않고 심각한 질병을 일으키는 진행하는 BD 감염을 허용하는 호스트의 어떤 자질 불분명하다. Chytridiomycosis 전해질 손실 및 심장 마비 4의 결과로, 피부에 이온 채널을 방해하여 죽음의 원인이됩니다. 또한, (된 BD zoospores을 포함하지 않는) BD 상층 액은 올챙이의 면역 반응 5 의원 원인G 연구원은 BD 부산물 6 독성을 생산하고 생각합니다. 많은 종은 빠르게 감소하고 있기 때문에, 포로 보증 식민지 사육 프로그램은 중요한 보존 조치하고, 어떤 종의 유일한 생명선이 될 수 있습니다. 구축 및 무병 식민지를 유지하는 것은 양서류 관리 시설에 대한 도전입니다. BD 감염을 치료하기위한 현재의 화학 요법의 방법은 효과적이지만, 치료되는 동물에 유해 할 수 있으며, 단일 치료법은 종 및 양서류 7의 연령 등급에 걸쳐 성공적인 없었다. BD 감염에 대한 치료의 몇 가지 임상 시험은 7을 수행하고 있습니다. 치료의 가장 일반적으로 사용되는 세 가지 방법은 열 치료, 클로람페니콜 및 이트라코나졸이다. 열 치료는 몇 가지 부작용, 여러 삶의 단계에서 사용할 수있을 것으로 보인다 nonchemotherapeutic 치료 옵션이며, gener입니다동맹 효과적이다. 치료 연구에서 채트와 리차드 - Zawacki 8은 처리 된 동물의 96 %가 10 일 동안 30 ° C에서 보관 후 감염을 취소 한 것으로 나타났습니다. 이 시험은 100 % 성공하지 않지만, 다른 사람들이 자신의 종족에 대한 열 치료를 평가하기위한 기준을 제공합니다. 열 치료는 많은 종에 효과적이지만, 장시간 고온으로 유지 할 수 많은 고산 종 비실용적이다. 클로람페니콜, 항균, 많은 종의 BD 치료에 성공적으로 사용하고, 체외에서 곰팡이 성장을 억제하고있다. 항균 같이, 클로람페니콜 구체적 균류 공격하지 않지만 진핵 세포 9에서 아폽토시스를 일으키는 것으로 알려져있다. 그것은 행동의 정확한 메커니즘은 알 수 있지만, 클로람페니콜 치료, 세포 내 포자낭을 죽이고, 피부의 세포 사멸을 일으키는 것으로 추측하고있다. 임상 시험 때문에 실시하지 않고,되어 있지만용량과 처리 시간, 평가되지 않은 현재의 치료 프로토콜은 일반적으로 지상파 양서류이 치료가 실용적이지, 2~4주 10, 11에 침수가 필요합니다. 인체 노출은 재생 불량성 빈혈 (12)의 드문 형태와 관련되어 있기 때문에 클로람페니콜은 동물 사용에 대한 일부 국가에서 제한되어 있습니다. 이트라코나졸, 아 졸계 항진균제, 가장 널리 포로 양서류 BD 감염의 치료에 사용된다. 가장 널리 사용되는 프로토콜 5 분 11 일 연속 하루 동안 0.01 % 이트라코나졸 솔루션 목욕 동물입니다. 이 프로토콜은 양서류에 효과적이지만, 양서류는 11, 13의 임상 시험을 기반으로하지 않는, 포유 동물의 관리에 사용되는 표준에서 파생되었다. 이 프로토콜은 특히 속라나와 올챙이 최근 metamorphs (11), 특정 종에 부작용을 일으킬 수 있습니다.올챙이 처리 이트라코나졸의 훨씬 더 낮은 농도 (0.0005 %)을 이용하여 성공을 거두었지만 탈색 (14)와 관련되었다. 이 같은 농도는 최근 metamorphs에 상용화에 박차를 가하고 감염 (15) 치료에 실패한 것으로 밝혀졌다. 이트라코나졸 치료와 부작용의 위험이 있기 때문에, 일부 실무자는 11 일 연속 절반 보통 농도 (0.005 %)를 사용하고이 BD 감염 16을 ​​치료에 성공을 발견했다. 본 연구는 BD 감염 (15)을 치료하는 이트라코나졸을 사용하여 임상 치료 시험을 요약 한 것입니다. 이 프로토콜은 이트라코나졸과 BD 감염된 동물의 치료에 안전하고 효과적인 모두 가장 낮은 용량과 처리 시간을 찾기 위해 시도에 대해 설명합니다. 본 연구에서 사용 된 동물은 걸프 코스트 두꺼비의 최근 metamorphs, Incilius nebulifer했다.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

수륙 양용 비행기는 척추 클래스의 가장 큰 감소를 경험하고 이러한 감소의 주요 원인은 질병 chytridiomycosis을 일으키는 곰팡이 병원균, Batrachochytrium의 dendrobatidis (BD)입니다. 포로 보증 식민지 멸종 위기 종의 양서류 세계적 중요하고 멸종의 중요한 위협이있는 사람들을위한 유일한 생명선이 될 수 있습니다. 무병 식민지를 유지하는 것이 포로 관리자의 우선 순위, 모든 종과 삶의 단계에서 아직 안전하고 효과적인 치료법이 발견되지 않았습니다. 일반적으로 사용되는 투여 량은 일부 개인과 종에 해가 될 수 있지만 가장 널리 사용되는 화학 요법 치료, 이트라코나졸입니다. 우리는 이트라코나졸의 낮은 및 안전하지만 유효 선량은 BD 감염을 치료하기 위해 찾을 수 있는지 여부를 평가하기 위해 임상 치료 실험을 수행 하였다. 우리는 발견 0.01-0.0025 %에서 치료 농도를 감소시키고 (11)로부터 치료 기간을 감소시킴으로써5 분 화장실 -6 일, 개구리 적은 부작용 적은 치료 관련 사망률과 BD 감염으로 치료 될 수 있습니다.

모든 동물 사주 종료 치료 후 BD-음성 인 경우 이트라코나졸 치료가 성공한 것으로 간주되었다. 치료를 시작하기 전에 치료가 끝난 사주 후, BD-양성 대조군 동물 16,765.12의 평균 유주자 부하가 있었을 때 2 주 접종 후, 모든 BD-접종 동물 ((17.34 ±) 1,121.7의 평균 유주자 부하, 감염 ) 14643.08를 ±. Incilius nebulifer의 치료 (N = 15) 6 일 동안 다섯 분 동안 0.0025 % ?...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

양서류 Batrachochytrium의 dendrobatidis 감염은 감소 된 농도 (0.0025 %) 및 일반적으로 사용되는 것보다 이트라코나졸의 짧은 기간 (6 일 동안 오분 화장실)을 사용하여 제거 할 수있다. 이하 사망률과 적은 부작용이 변경됨 치료 프로토콜을 사용하여 관찰 하였다.

감소 이트라코나졸의 농도와 지속 시간은 치료의 성공<em&gt; Batrachochytrium의 dendrobatidis</em&gt; 수륙 양용 비행기에 감염

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K 조회수.  James Cook University.  양서류 Batrachochytrium의 dendrobatidis 감염은 감소 된 농도 (0.0025 %) 및 일반적으로 사용되는 것보다 이트라코나졸의 짧은 기간 (6 일 동안 오분 화장실)을 사용하여 제거 할 수있다. 이하 사망률과 적은 부작용이 변경됨 치료 프로토콜을 사용하여 관찰 하였다. 

비디오: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

연구

면역학 및 감염병학

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용<em&gt; 녹농균</em&gt; 낭성 섬유증의 폐 관련성이 높은 조건에서

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

신생아와 성인 중추 신경계에서 배양 소교 세포

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

총 단백질 추출 및 2-D 겔 전기 영동 방법에 대한<em&gt; Burkholderia의</em&gt; 종

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

작은 인도 몽구스(헤르페스 오로 punctatus)에서이오페녹산 유사체의 분석은 구강 광견병 예방 접종 생물학적 마커로 사용하기위한 세라

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

재발 성 에스케리치아 콜라 이 요로 감염 Gardnerella 질 방광 노출에 의해 유발 마우스에서

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

감염성 단핵구증이 있는 어린이의 말초 혈액 샘플에서 면역 세포 하위 집단 분리

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

일본 뇌염 예방 접종 아동의 림프구 검출을 위한 유세포 분석기 간 실험실 간 이동 표준화

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...