저자는 트레이시 J. 어린 양에게 수여 면역학 및 면역 주사와 NIH 새로운 혁신 상 (1DP2AI112242-01)에 대한 아동 센터를 통해 자금을 인정합니다. 저자는 또한 rad1 S.의 관대 한 기여 나탈리아 P. Degtyareva 감사합니다 cerevisiae의.

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저자는 공개 아무것도 없어.

함께, 프로토콜은 여기에 장에 이종 치료 단백질의 전달을위한 개발과 영양 요 바이오 틱 효모 균주의 시험에 필요한 필수 단계를 설명합니다. 이 조작 및 재조합 바이오 틱 효모의 테스트는 개별 실험실은 익숙하지 않을 수있는 기술과 자원을 필요로한다. 많은 선행 연구가 여러 효모와 마우스 균주에 대해 위의 프로토콜을 설명하고 있지만 따라서, 이러한 방법은 저자의 지식이 상세한, 통합 형...

프로 바이오 틱 미생물을 효​​율적이고 경제적으로 위장관에 이종 단백질을 전달하는 흥미로운 잠재적 인 수단이다. 이 생물은 아직 1 식민지화하지 않는 위장관 통과 생존 제어 투약이 가능하고 약물 표현에 노출을 제한 할 수있다. 또한, 쉽게 대규모 이종 단백질을 생산하는 이들 미생물을 설계하는 능력은 그들을 합성 전달 입자 경제적 인 대안을 렌더링한다. 최근 2 boulardii에 생균제 효모 사카의 영양 요 구성 균주를 사용하여 입증하지만, 이러한 접근법의 개발은, 효모 및 분자 생물학에서 동물 처리 기술 및 면역 학적 방법에 이르기까지 전통적으로 특정 연구에서 결합되지 실험 기법에 대한 지식을 필요로한다. 본원에 기재된 각각의 방법은 그 자체로 신규 아니다 따라서 비록실험 프로토콜이 원고의 목적은 쥐 위장관에 약물 전달 비히클로서 생균제 효모 실험 테스트에 필요한 기술을 통합 도입을 제공하는 것이다. 제공에 필수적인 프로토콜의 편집이다 : 쉽게 유 전적으로 조작 할 수있는 효모의 영양 요 구성 돌연변이 균주의 1) 생성; 효모 배양 2) 변환은 이종 단백질을 발현하는 단계; 구강 투여를 통해 장에 재조합 효모의 3) 관리; 자신의 이종 단백질 발현의 쥐 소장 및 평가에서 가능한 재조합 바이오 틱 효모의 4) 복구. 다수의 양성 및 음성 선택 방법 효모 종의 조작에 존재하지만 첫째, 이러한 영양 요 구성 마커를 사용함으로써 부정적 선택 효율 효모 형질 전환 및 선택 될 수있는 용이성을 모두 증가시킨다. 공동 항생제를 사용하여 형질 전환 체의 긍정적 인 선택,ntrast 크게 효모 조작의 비용을 증가시킨다. 또한, 항생제 함유 고체 배지에 효모의 선택은 밖으로 합성 드롭 고체 배지 (게시되지 않은 관찰)에 영양 요 효모의 선택에 대해 변형되지 않은 배경 식민지의 증가 성장을 할 수 있습니다. 영양 요 구성 효모가 필수 아미노산 또는 우라실의 합성에 중요한 효소가 결여 균주이다. 효모가 필수 대사 물질이 부족 미디어 밖으로 합성 드롭에 도금 할 때 따라서 음의 선택이 가능 누락 된 대사 또는 대사 유전자로 보충하면 효모는 증가 할 수 있습니다. 많은 일반적으로 사용되는 사카로 마이 세스 세레 비지에 실험실 균주는 이미 사실 영양 요 구성 돌연변이 3입니다. 공업, 임상, 바이오 틱 및 효모 그러나, 일반적으로 필요한 모든 영양소를 합성하는 능력 prototrophic이다. 효모를보다 효율적으로 유전자 조작을 사용하려면, 영양 요 구성 유전자를 선택적으로 타겟팅 할 수 있습니다항생제없이 선택 될 수있는 균주를 생성한다. 6 - 영양 요 구성 마커 유전자의 구체적인 타겟팅 CRISPR / Cas9 4 타겟으로 최근에 상동 재조합에 의존 PCR 매개 유전자 파괴를 통해 달성 될 수있다. 또한, UV 돌연변이 유발 빠르게도 7 기술적으로 어려운 여러 플라스미드와 함께하는 형질 전환 효모 균주의 영양 요 구성 돌연변이 체를 생성 할 수 있습니다. PCR 타겟팅 및 CRISPR / Cas9 광범위 다른 설명되었지만,이 원고 한 음성 선별보다는 효모 형질 전환 체의 양성 항생제 선택할 수 영양 요 구성 균주를 제작하는 UV 돌연변이 유발 접근법을 설명하는 상세한 프로토콜 부분에 제시 하였다. 이종 단백질의 경구 전달을 위해 같은 영양 요 구성 균주의 사용에 필요한 다음 단계는 플라스미드 DNA로 형질 전환 효모이다. 찬성의 첫 번째 성공적인 변환 이후1978 사카로 마이 세스 세레 비지에 (8)에 대해보고 된 spheroplasts t는 수많은 변형 효율을 증가 효모 종은 유 전적으로 변형 될 수있는 것을 특징으로 완화되었다. S.로 DNA의 성공적인 전환을위한 전기 사용 cerevisiae를 먼저 1985 9에서 설명하고, 이후지지 삼투 셀 (10)에 1 M 소르비톨 배양 첨가를 통해 개선되었다. 전기 효율은 또한 효모 종 및 균주, 세포 수와 성장 단계, 전기 체적, 전계 강도, 및 특정 버퍼 (11)에 의존하는 것으로 나타났다. 이 특별한 장비를 필요로하지 않기 때문에 원래 이토 외. (12)에 의해 기재된 리튬 아세테이트 (LiOAc) 변환은, 가장 일반적으로 사용되는 프로토콜 사이에서 변환된다. 추가의 분석은 세포를 중간 로그 상에 수집되는 경우 LiOAc 효모 변환 효율을 크게 증가시키는 것으로 나타났다열 성장은 42 ° C에서 12 폴리에틸렌 글리콜 (PEG)과 DNA의 존재 하에서 충격된다. PEG와 전체 그대로 효모의 배양이 가능 세포막에 DNA의 첨부 파일을 개선을 통해뿐만 아니라 막 (13)에 다른 효과를 통해, 효율적인 변환을 위해 필수적이다. 리튬 자체도 손상 전지 (14)의 투과성을 증가시킨다. 대부분의 실험실 S. 있지만 세레 비지에 균주 쉽게 LiOAc 변형 3을 이용하여 변환 될 수 있고, 다른 효모 종은 더욱 효율적으로 다른 프로토콜을 사용하여 변환 될 수있다. 피 키아 파스 토리스, 예를 들면, 가장 효율적으로 변환 오히려 LiOAc 변환 (13)보다 전기를 통한 것이다. 이것은 중요하다, 따라서, 복수의 테스트 유 전적으로지지 않은 효모 균주를 수정을 시도 할 때와 변화의 방법은 배양 기간과 시약의 농도를 최적화 할 수 있습니다. 이 원고 따라서 LiOAc 그럴 모두 설명영양 요구 돌연변이와 야생 유형 S.의 변환을위한 기술로 ansformation 및 전기 boulardii에. 관심있는 독자는 효모 변형, 다른 프로토콜 및 행동 13, 15의 가능한 메커니즘의 추가 논의의 진화의 철저한 설명은 최근 리뷰에 관한 것이다. 플라스미드가 쉽게 검출 가능한 단백질을 코딩하는 효모로 형질 전환은 적절한 발현 이종 단백질의 기능을 보장하기 위해 다운 스트림 테스트 또한 필수적이다. 무수한 다른 단백질 치료 연구의 궁극적 인 목적 및 면역, ELISA, 및 다른 기술에 의해 단백질이 검출 가능한 항체에 따라 선택 될 수있다. 이러한 기술에 대한 프로토콜은 완전히 다른 16,17 설명되었지만, 표준 곡선과 비교하여 형질 전환 효모에서 이종 단백질 생산의 수준을 결정하기 위해 사용될 수있다. 목적 데모 및 표시 할효모 생물학 매우 일반적으로 사용되는 단백질의 성공적인 제조 방법이 원고는 형광 현미경을 사용하여 연속적인 검출이 가능 플라스미드 인코딩 녹색 형광 단백질 (GFP)로 형질 전환을 제공한다. 부품 셋, 넷에 설명 된대로 이종 단백질, 적절한 관리 및 위장관 조직 내에서 이러한 미생물의 검출 인 표현 바이오 틱 미생물의 생산에 똑같이 중요. 구강 투여를 통해 재조합 효모의 관리를 직접 C57BL / 6 마우스 자연스럽게 18 구토 능력이있는에서 위,에 효모의 제어 수량의 전달을 위해 수 있습니다. 그러나, 부적절한 동물 처리 및 위관은 식도 손상 및 천공, 위 천공, 기관 관리, 흡인 성 폐렴 (19, 20)로 이어질 수 있습니다. 빈약 한 기술과 경험 부족은 또한 쥐의 면역 반응 및 실험 resu의 변동성을 증가시킬 수있다구강 투여에 21,22 동물 응력에 기인 한 LTS. 적절한 기술의 연습 따라서뿐만 아니라 동물의 불편을 감쇠 할 수 있습니다뿐만 아니라, 실험 결과의 정밀도를 높일 수 있습니다. 이 원고는 설명하고 재조합 효모의 제어 용량의 관리를 위해 동물 처리 및 구강 투여를 보여줍니다. 마지막으로, 효모 및 이종 단백질의 존재를 림프 조직을 분석하여 재조합 효모의 성공적인 전달을 확인하는 것이 중요하다. 가장 용이하고 예측 효모의 존재를 검사 할 수 위장관 면역 조직 파이어 패치이다. 파이어 패치는 점막 면역 반응의 유도 (23)의 키 사이트입니다 소장을 따라 차 림프 기관입니다. 내강에서 항원 microfold 통해 transcellularly 상피 (M) 세포를 전달하고 묶 노출 시킴 따라서 파이어 패치로 출시제시 세포 D 항원 내강 내용 장내한다. 장 상피를 가로 지르는 입자 흡수도 술잔 세포에 의해 달성 될 수 있지만, 이러한 셀은 차지 입자 직경이 24 미만 0.02 ㎛ 인 것으로 나타났다. Transepithelial 수상 돌기는 CD103 + 수지상 세포 (DC)도 25 루멘 장에서 작은 입자를 차지 연장; 그러나, CD103 + DC가 세균보다 큰 입자를 가지고 있음을 보여주는보고는 현재 존재하지 않는다. 따라서, 그대로 생균제 효모 직경 3-6 μm의 평균 크기 사이의, M 세포에 의해 용해 및 파이어 패치로 전송 될 가능성이 가장 높은. 여기에 설명 된 절차가 쉽게 바이오 틱 균의 흡수를 평가하기 위해 적응 될 수 있지만, 수집 및 생존 재조합 효모 파이어 패치 선별 프로토콜이다. 요약하면, 배달 재조합 효모 생균제 평가소장에 rapeutic 단백질은 동물의 취급 및 면역학에 분자 생물학에 걸친 실험 기술의 능력을 필요로한다. 1 여기를 제시 프로토콜) 쉽게 부정적인 항생제없이 선택할 수있는 세대 및 영양 요 효모 균주의 선별, 2) 다른 프로토콜은 효모를 변환 및 이종 단백질의 발현을 활성화, 3) 적절한 동물 처리 기술과 경구 위관의 시위 재조합 효모의 위장 납품 및 파이어 판 절제술 4) 프로토콜과 가능한 재조합 효모 및 기능 이종 단백질을 스크리닝. 결합하여, 이러한 프로토콜은 생성 및 위장관에 이종 단백질 치료제를 제공 할 수있는 생균제 효모의 테스트를 허용한다.

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:107px;">170-2501</td>
			<td style="width:275px;">Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick Roller Drum</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:107px;">M1053-4004</td>
			<td style="width:275px;">Example of roller drum for yeast culture incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Stratalinker 2400</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:107px;">400075-03</td>
			<td style="width:275px;">Example stratalinker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stuart Colony Counter SC6PLUS</td>
			<td style="width:107px;">11983044</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:275px;">Plate stand with magnification records colony count upon sensing pressure from pen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scienceware Colony Counter&#160;</td>
			<td style="width:107px;">F378620002</td>
			<td style="width:107px;">Bel-Art Scienceware</td>
			<td style="width:275px;">Hand held colony counter pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica plating device</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-1</td>
			<td rowspan="2" style="width:275px;">Example of replica plating stand and pads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen squares</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;L shaped sterile cell spreaders&#160;</td>
			<td style="width:107px;">Fisherbrand</td>
			<td style="width:107px;">14665230</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deoxyribonucleic acid, single stranded from salmon testes</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D7656-1ML</td>
			<td style="width:275px;">Example carrier DNA for yeast LiOAc transformation</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gavage needles</td>
			<td style="width:107px;">Braintree Scientific</td>
			<td style="width:107px;">N-PK&#160;002</td>
			<td style="width:275px;">For mice 15-20 g, the suggested needle is a 22 gauge (1.25 mm ball), 1 in long, straight reusable gavage needle. For mice weighing greater than 20 g, 20 gauge or larger straight or curved gavage needles may be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1mL sterile slip-tip disposable tuberculin syringe&#160;</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:107px;">BD 309659</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Blunt forceps such as Electron Microscopy Sciences 7&#34; (178 mm) serrated tip, broad grip forceps</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">77937-28</td>
			<td style="width:275px;">Example of blunt forceps needed for dissection&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Straight and curved dissection scissors&#160;</td>
			<td style="width:107px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:107px;">72966-02 and 72966-03</td>
			<td style="width:275px;">Examples of scissors needed for dissection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IMDM</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:107px;">12440053</td>
			<td style="width:275px;"></td>
		</tr>
	</tbody>
</table>

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.

UV 조사 다음 생존 곡선의 생성은 별개 집락 형성 단위 (CFU)를 형성 할 수 있도록 희석하여 효모 세포의 도금을 필요로한다. 상기와 같이 수집 된 각 500 μL 샘플을 약 5 × 106 세포를 포함 그러나, 접시 당 100보다 큰 식민지를 정확하게 구별하기가 어렵다. 희석되지 않은 샘플과 조사 셀 직렬 1시 10분 희석 따라서도 1에서 설명한 바와 같이 CFU는 각각 UV 조?...

Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

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

개발 변환 관리 및 생균제 효모 사카 boulardii에의 외래 단백질 발현을 테스트 할 수있는 기술의 통일 된 설명은 여기에 제시 하였다.

마우스의 구강 투여에 따라 생균제 효모의 변환 및 위장 면역 조직에서 그들의 복구

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

transformation-probiotic-yeast-their-recovery-from-gastrointestinal

Research

JoVE Journal

Immunology and Infection

12.3K 조회수.  Emory University School of Medicine.  개발 변환 관리 및 생균제 효모 사카 boulardii에의 외래 단백질 발현을 테스트 할 수있는 기술의 통일 된 설명은 여기에 제시 하였다. 

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

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 ...

연구

면역학 및 감염병학

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

경구 전송<em&gt; 리스테리아</em&gt; 오염 된 음식의 섭취를 통해 마우스의

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNA의 타겟팅 디곡신 표지 DNA 프로브를 사용하여 파라핀 조직 내에서 박테리아의 제자리 검출

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

다양한 조직에서 면역 세포의 질병에 의존하는 유통의 마우스 및 평가에 실험자가 면역 뇌척수염의 유도

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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

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

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

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

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

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

Here, we present a protocol to perform a simple cellular fractionation for the subcellular separation of cytoplasmic and nuclear proteins in human fresh and frozen intestinal biopsies.

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Gavage를 사용 하 여 신생아 쥐에 있는 Probiotic 연구

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

마우스의 구강 투여에 따라 생균제 효모의 변환 및 위장 면역 조직에서 그들의 복구

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