Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

No conflicts of interest to disclose.

여기에서 우리는 L.의 EGD 균주가 남성 또는 여성 C57BL / 6J 마우스에서 25 모노 사이토 기본 실험 감염을 수행하는 방법에 대한 프로토콜을 설명합니다. 이 프로토콜은 생체 (31)에 타고난 및 적응 림프구에 의한 IFN-γ 생산에 새로운 작은 분자 IS001의 효과를 연구하기위한 목적으로 설립되었다. 세균 통관과 생존 후 감염을 모니터링함으로써, 통찰력 IFN-γ의 이러한...

IFN-γ는 세포 내 병원체에 대한 면역 매개 종양 성장을 제어하기위한 5 중요 사이토킨이다. 박테리아 내성이 사이토 카인의 중요성은 IFN-γ의 신호 전달 경로에서의 돌연변이는 마이코 박테리아 및 살모넬라 6 감염에 매우 민감하여 인간 관찰에 분명하다. 마찬가지로, 마우스, IFN-γ 또는 L.는 10, 11 모노 사이토 겐을 포함 마이코 박테리아 7-9 및 기타 세포 내 병원균에 저항의 IFN-γ 수용체 전시 결함 중 하나에 슈만 편모충 주요 12, 살모넬라 티피 무리 움 (13), 특정 바이러스 (11) 결핍. 맞서 싸우는 병원균뿐만 아니라, IFN-γ 종양 (14)에 대한 호스트 방어에 중요한 역할을한다. IFN-γ의 높은 생산은 감염 또는 암의 맥락에서 유용하지만, 이러한 사이토 카인의 생산을 장기간 t 연계 된비 - 비만 당뇨 마우스 모델 (18) 전신성자가 면역 15-17의 발전 형 가속도 O I 당뇨병. IFN-γ의 주요 소스는 NK 세포, NKT 세포, γδ T 세포, T 헬퍼 1 (받은 Th1) 세포 및 세포 독성 T 림프구 (CTL)를 포함 5,19,20. IFN-γ에 의해 선천성 및 후천성 면역을 모두 향상 (1)를 조절 (3) 강화 식세포, (2) 항원 제시 세포상의 공동 - 자극 분자의 발현을 증가시키는 주요 조직 적합성 복합체 (MHC) 클래스 I 및 II 발현 식세포 작용 및 염증성 사이토 카인의 생산 및 살균 요인 (예를 들면, 일산화 질소 및 활성 산소 종), (4) (5) (면역 글로불린 항체 클래스 스위칭을 촉진하는 Th1 효과기 세포로의 나이브 CD4 + T 세포의 분화를 촉진 IG) 2a 및 마우스에서 IgG3 ()는 (6) 케모카인의 생성을 유도하는 난 부위로 면역 세포를 모집nfection 및 (7) NK 세포 및 CTL 반응을 향상 5,19. 병원균과 종양 호스트 응답 IFN-γ의 결정적인 중요성을 감안할 때, 재조합 IFN-γ는 (19에서 검토) 다양한 감염과 악성 종양에 대한 치료로 테스트되었습니다. IFN-γ 또는받은 Th1 촉진 사이토킨 인터루킨 -12 (IL-12)의 전신 투여는 부작용과 연관 투여 량 관련 독성 (19, 21)가되기 때문에, 의한 IFN-γ 생산을 증가시키기 위해 대안적인 전략의 개발에 대한 관심이있다 면역 세포. 새로운 바이오 작은 분자의 개발은 에이전트가 면역 반응 중 IFN-γ의 생산을 증가 여부를 테스트하기 위해 생체 검사 도구의 필요 여부이는 동물의 생존의 증가로 의미있는 생물학적 효과로 변환합니다. 그램 양성 세균 리스테리아 모노 사이토 겐 쥐의 실험 감염에 대한 쓸모있는 모델이었다세포 내 병원균 1,22에 대한 호스트 면역 IFN-γ의 역할을 해독. 병원균 정맥 또는 복강 (IP)와 쥐의 감염은이 일 포스트 (3) 사이에 4를 발생하는 비장의 피크 세균 부하 주민 대식 세포 및 간세포에 의해 내면화 될 비장과 간,에 박테리아의 급속한 보급에 이르게 감염 1,3,22. NK 세포에 의한 IFN-γ의 생산은 대 식세포의 활성화 및 병원체 3에 대한 초기 저항 중요하다; 그러나 높은 전염성 투여 량, IFN-γ의 생산은 병원균 통관 (23)에 유해 할 수 있습니다. NKT 세포는 병원균 2,24 초기 제어시 비장, 간에서 IFN-γ의 공급원이며, 이러한 제조는 NK 세포를 포함하는 다른 2 종류의 세포에 의한 IFN-γ 생산을 증폭하는 것으로 나타났다. 이상적 상대있는 반면, 적응 형 T 림프구 이상 작용, CD8 + T 세포큘라은 병원균의 간극을 매개하고 다시 감염 1,4,22에 대한 보호를 제공하기위한 중요하다. 이 감염 모델 (1 검토) 이유에서 연구자 매력적인왔다. 우선, 병원체 감염이 높은 재현성과 강한받은 Th1 세포 성 면역 반응을 유도한다. 둘째, 치사 감염시 세균 부하가 쉽게 측정 될 수있는 간 및 비장에서 농축된다. 셋째, 병원체 안전하게 바이오 레벨 2 (BSL2) 조건 하에서 처리 될 수있다. 넷째, 생물 및 면역 반응이 광범위하게 특성화되었다 생성있다. 마지막으로, 돌연변이와 유전자 변형 종자의 다양한 사용할 수 있습니다 개발되었다. L.의 EGD 균주와 C57BL / 6J 마우스의 접종을위한 기본 프로토콜은 여기에 25 모노 사이토 겐입니다 설명 및 IFN 측정 - γ 다시NK, NKT, 적응 림프구 후 감염에 의해 sponses. 또한 설명 치사 감염 후에 박테리아 비장, 간 부하를 측정하고, 병원체의 변형 LD 50 복용량 감염 후 생존 연구를 수행하는 방법이다. 마지막으로, 대표 데이터는이 프로토콜은 감염 L. 모노 사이토 겐에서 IFN-γ 반응과 마우스의 생존에 대한 새로운 치료법의 효과를 스크리닝하기 위해 사용될 수 있는지 나타낸다.

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Brain Heart Infusion Broth, Modified</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">299070</td>
			<td style="width:273px;">any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>BD</td>
			<td style="width:107px;">214010</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:107px;">X100</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1xPBS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:107px;">D8537</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">TissueLyser II</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:107px;">85300</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ammonium Chloride (NH3Cl)</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">KHCO3</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Na2EDTA</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">RPMI 1640</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">22400089</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fetal Bovine Serum&#160;</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">12483</td>
			<td>Before use, heat-inactivate at 56 &#176;C for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">L-glutamine</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">25030</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Non-essential amino acids</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">11140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Penicillin/Streptomycin</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">15140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">GolgiStop Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554724</td>
			<td>Use 4 &#956;l in 6 ml cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">16% Paraformaledehye</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">15710</td>
			<td>Dilute to 4% PFA in ddH20 or 1xPBS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">10 x Perm/Wash buffer</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554723</td>
			<td>Dilute 10x in ddH20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fc block, Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">14-0161</td>
			<td>Dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fixable Viability Dye eFluor 506</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">65-0866</td>
			<td>Dilute 1:1000 (we have also used viability dyes from Molecular Probes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD4-PE-Cy5 (GK1.5)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">15-0041</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD8-FITC (53-6.7)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">11-0081</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PBS57/mCD1d tetramer-APC</td>
			<td style="width:191px;">NIH Tetramer Core Facility</td>
			<td style="width:107px;">N/A</td>
			<td>Obtained as a gift from the facility</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse TCR&#946;-PE-Cy7 (H56-597)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-5961</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">47-3351</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD45 PE-Cyanine7 (30-F11)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-0451</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse IFN gamma-PE (XMG1.2)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">12-7311</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">OneComp eBeads</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">01-1111</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Mouse IFN gamma ELISA kit</td>
			<td style="width:191px;">eBioscience</td>
			<td>88-7314</td>
			<td>Used for measuring the interferon gamma in the culture supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 mL vented tubes for culture</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for culturing the bacteria, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">bacterial petri dishes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2 ml cyrovials</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">UV spectrometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">safety engineered needles</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">C57BL6/J</td>
			<td style="width:191px;">Jackson laboratories</td>
			<td style="width:107px;">Stock#000664</td>
			<td>Order for arrival at 7 wks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Bleach</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70% Ethanol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Glass beads</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Centrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>rotor, buckets, bucket covers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microcentrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Sterile Glycerol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette Tips</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Surgical instruments</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70 micron strainers</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">3 ml syringe</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette gun</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Filtration Units</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Trypan Blue</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Dilute 1 to 9 in ddH20, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Hemocytometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Round bottomed plates</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">FACs tubes</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">BD LSR II</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></td>
			<td>Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Flowjo software</td>
			<td style="width:191px;">Treestar</td>
			<td style="width:107px;"></td>
			<td>Used for data analysis. Other types of data analysis software will also be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Multichannel pipettor (0-300 &#181;l)</td>
			<td style="width:191px;">Eppendorf</td>
			<td style="width:107px;"></td>
			<td>Used for washing cells and adding antibodies during flow cytometry staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Acetic Acid</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for washing glass beads, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microbank Bacterial Preservation System</td>
			<td style="width:191px;">Pro-lab Diagnositics</td>
			<td style="width:107px;"></td>
			<td>Used as an alternative to glycerol stocks for long-term storage of bacteria</td>
		</tr>
	</tbody>
</table>

experimental infection with listeria monocytogenes as a model for studying host interferon-&#947; responses

L. 모노 사이토 인간 식품 매개 질환의 원인이되는 그람 양성 세균이다. 이 병원체와 쥐의 실험 감염은 세포 내 병원체에 대한 숙주의 면역 타고난 및 적응 면역 세포와 특정 사이토 카인의 역할에 매우 유익하고있다. 리스테리아 모노 사이토 겐과 치사 감염시 타고난 세포에 의한 IFN-γ의 생산은 대 식세포 및 병원균 1-3의 초기 제어 활성화를위한 중요합니다. 또한, 적응 메모리 림프구에 의한 IFN-γ 생산에 재감염 4 선천성 세포의 활성화를 프라이밍 중요하다. L. 따라서 감염 모델 모노 사이토 겐 IFN-γ 생산을 증가시키기 위해 설계된 새로운 치료법은 생체 내 IFN-γ 반응에 영향을 미치는 등과 같은 세균 간극을 증가 또는 마우스의 생존을 개선 생산적 생물학적 효과가 있는지 여부를 조사하기위한 훌륭한 도구 역할 ...에서감염. 리스테리아 모노 사이토 겐의 EGD 균주 C57BL / 6J 마우스의 복강 내 감염을 수행하고, 유동 세포 계측법에 의한 NK 세포, NKT 세포, 및 적응 림프구에 의한 IFN-γ 생산을 측정하는 방법의 기본 프로토콜이 여기에서 설명한다. (1) 성장하고 접종에 대한 박테리아를 준비 (2) 엔드 포인트로 비장과 간, 그리고 (3) 측정 동물의 생존에 박테리아 부하를 측정 : 또한, 절차에 설명되어 있습니다. 대표 데이터는이 감염 모델 L. 모노 사이토 겐이 감염 마우스의 생존에 대한 IFN-γ 반응의 특정 제제의 효과를 테스트 할 수있는 방법을 예시하기 위해 제공된다.

그림 3은 병원균의 10 5 CFU와 24 시간 후 감염에 비장 NK와 NKT 세포에서 IFN-γ의 염색 세포 계측법 몇 가지 일반적인 흐름을 제공합니다. 이 도면은 또한 표 2에 기재된 염색 패널 게이팅 전략을 예시한다. 그림 4는 105 CFU 리스테리아 모노 사이토 겐에 감염된 수컷 마우스는 PPARα 길항제 IS001 또는 차량 제어로 처리 ?...

Watch this Scientific Journal Video about Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃŽÂ³ Responses at JoVE.com

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

이 프로토콜은, 리스테리아의 EGD 균주 (L. 모노 사이토 겐)을 C57BL / 6J 마우스에 접종하고, 자연 살해 (NK) 세포, 자연 살해 T (NKT) 세포에 의한 인터페론 - γ (IFN-γ) 응답을 측정하는 방법을 설명 적응 T 포스트 감염 림프구. 이 프로토콜은 또한 병원균의 수정 된 LD (50) 투여로 감염 후 쥐에서 생존 연구를 수행하는 방법에 대해 설명합니다.

와 실험 감염<em&gt; 리스테리아</em&gt; 호스트 인터페론-γ 응답 유학을위한 모델로

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

experimental-infection-with-listeria-monocytogenes-as-model-for

Research

JoVE Journal

Immunology and Infection

13.5K 조회수.  University of Toronto.  이 프로토콜은, 리스테리아의 EGD 균주 (L. 모노 사이토 겐)을 C57BL / 6J 마우스에 접종하고, 자연 살해 (NK) 세포, 자연 살해 T (NKT) 세포에 의한 인터페론 - γ (IFN-γ) 응답을 측정하는 방법을 설명 적응 T 포스트 감염 림프구. 이 프로토콜은 또한 병원균의 수정 된 LD (50) 투여로 감염 후 쥐에서 생존 연구를 수행하는 방법에 대해 설명합니다. 

비디오: Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

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 ...

연구

면역학 및 감염병학

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

<em&gt; Citrobacter rodentium</em&gt; 마우스 모델 : 감염성 대장염에 대한 공부 병원체 및 호스트 기부

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

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 ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징<em&gt; 리스테리아</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

숙주 세포와 혐기성 세균의 상호 작용을 평가하기위한 모델 생물로 포피 진지 발리

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

유학을위한 방법론<em&gt; B. 서브 틸리</em작은 분자 바이오 필름 억제제를 특성화 모델로&gt; 바이오 필름

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes 뇌의 감염

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.