저자는 우리에게 모델을 가르치고 돕기 위해 박사 제이 Pepose 감사드립니다. 이 작품은 건강 보조금 EY11885 (PMS), EY21247 (PMS) 및 안과 부서에 실명을 방지하기 위해 연구에서 제한 교부금의 국립 연구소에 의해 지원되었다.

동물 케어 참고 :이 항 염증이며 따라서 모델을 무효화하는 것처럼 진통제는이 모델에 사용되지 않습니다. 1. 바이러스의 작성 <ol><li> 3-4일에 감염 0.01 다중성의 시작 희석을 (T-150 술병에 약 5 × 10 5 상패 형성 단위)를 사용하여 Vero 세포 (80 % 합류)에 HSV-1을 성장하는 것은 세포의 모든 때까지 반올림 업 쉽게 플라스크를 클릭하여 dislodged 수 있습니다. </li><li> 4에 멸균 원심 분리기 튜브 및 Sorvall 전설 RT에서 원심 분리기로 미디어를 제거 ° C와 15 분에 3,500 RPM. 미디어 총 5 ML 각 50 ML 튜브의 표면에 뜨는 (1.3 참조), resuspend 큰 펠릿을 제거 후. 이 30 초 폭발에서 1 분에 Sonicate. </li><li> 4 Beckman에서 ° C J2-21 원심 분리기에서 1 시간 동안 표면에 뜨는 및 9,000 rpm으로 50 ML 멸균 오크 릿지 튜브와 원심 분리기에 넣어 제거합니다. 이 작은 바이러스 펠릿을 생산하는 것입니다. 표면에 뜨는 폐기하십시오. </li><li> 1.2 지혜에서 sonicate 결합H 1.3에서 펠렛 45 초에 다시 sonicate. 4에 5 분에 1,500 rpm으로 Sorvall 전설 RT의 원심 분리기 ° C. 표면에 뜨는를 저장합니다. 이 -70에서 aliquoted와 저장되어있는 바이러스 재고 ° C.를 구성 </li><li> Vero 세포에 Titer 바이러스. 에서 10 -8 희석 (각각 10 배 희석되는) 2 개의 희석 세트에서 2 배까지 나는 보통 판 10 0. 바이러스의 적정은 12도 무균 접시에 수행됩니다. </li></ol> 2. 마우스 감염 <ol><li> 마취 cocktail/20 g 마우스 몸무게의 0.1 ML 각 마우스를 주사 (케타민, 60 밀리그램 / kg + Xylazine 5 MG / HBSS에 희석되어 kg). 이 적절한 복용량를 사용하고이 약의 효과가 매우 일시적인 있습니다 마취의 가장 안전한 방법 중 하나이기 때문에 칵테일이 사용됩니다. </li><li> 쥐가 anesthetized 때하면 각막이 천공하지 않도록 해부 현미경을 사용하여 30 게이지 바늘로 오른쪽 눈의 각막을 긁어. </li><li> 각 마우스 rece주요 감염 동안 손상 각막을 보호하기 위해, 풀링 된 인간의 혈청 (1:800의 50 % 바이러스 중화위한 효과적인 복용량과 안티 - HSV의 반응성 시그마, Temecula, 캘리포니아) 1 ML의 intraperitoneal 주사를 ived. </li><li> 귀 클립 넣거나 귀를 펀치로 번호 쥐를. </li><li> 마우스의 모든 번호를 한 후, 20 μl pipettor를 사용하여, 우리는 10 6 PFU (5 μl)와 각 눈을 감염. 우리는 한쪽 눈을 일반적으로 오른쪽 눈을 감염. 우리는 또한 그들이 anesthetized있는 동안은 촉촉한 유지하기 위해 다른 눈 HBSS의 5 μl를 놓습니다. 5 UL은 절차 미만 10 분에서 수행되며, 동물 따라 마취와 함께 복용되어 있기 때문에 눈의 보습을 유지하기에 충분한 것입니다. 감염은 Vero 미디어 1 ML에 수화했다 멸균면 작은 주걱으로 마우스에게 삼일 후 감염을 보라고하여 확인합니다. 그런 다음 100 μl를 48 잘 접시에서 자란 Vero 세포에 추가됩니다. 이 번호판은 cytopathic 효과를 매일 모니터링<html> (CPE). </li></ol> 3. 지연에서 마우스의 재 활성화 <ol><li> 마우스는 기본 감염 후 적어도 오주 anesthetized 있습니다. 그것은 우리가 일년 다음과 같은 주요 감염에 마우스를 재 활성화하고 응답에 큰 차이를 관찰하지 않은 것을인지해야합니다. </li><li> 일단 anesthetized 그들은 단지 하나의 눈이 UV-B 빛에 노출되는 등 302 nm의 피크 파장에서 UV-B 방출 TM20 Chromato-VU의 transilluminator를 배치하고 있습니다. 각 마우스는 250 UV-B의 MJ 빛 cm 2로 노출되어있다. </li><li> 재가입 먼저 1-7 후 UV-B 조사 일에 다음 날 0에 Vero 미디어 1 ML에 위치 (자연 shedders에 대해 제어 할 수 UV-B 노출 전)되고 멸균면 작은 주걱으로 눈을 보라고에 의해 결정됩니다. 2.5에 설명 된대로 면봉 소재)가 평가됩니다. 전염성 바이러스를 가진 면봉을 위해 바이러스의 양이 12도 접시를 사용하여 표준 상패 분석​​에 의해 tittered 있습니다. </li></ol>ove_title &quot;&gt; 4. 임상 평가 <ol><li> 마우스는 쌍안 해부 현미경을 사용하여 가면 관찰자에 의해 임상 안과 질환에 대한 평가입니다. </li><li> 각막 불투명도가 0 명확한 기질을 나타냅니다 0-4의 규모에 평가되며, 1 가벼운 stromal opacification을 나타냅니다, 2 알아볼 수 창포 기능과 함께 적당한 투명도를 나타냅니다, 3 나타냅니다 학생 여백, 4를 제외하고 정의 아이리스 세부의 손실과 밀도 투명도를 나타냅니다 더 뒤쪽 전경을 조망할 수있는 총 불투명도. </li><li> Neovascularization은 4 동일한 당장 자리로 각막을 나누어 이러한 당장 자리의 각 vascularization의 범위를 결정하여 8-1의 규모에 평가하고 있습니다. </li><li> Blepharitis은 다음과 같이 측정되지 않습니다 : 0, 더 병변, 1, 최소한의 눈꺼풀이 붓기, 2, 적당한 붓기와 피 각질의 안구 방전, 3, 심각한 부종, 중간 periocular 머리 손실, 피부 병변, 그리고 4, 눈을 겉 껍질이있는 다물고있는 심각한 부종, 심각한 periocular 헤어 손실 및 피부 병변. </li><li> 뇌염 inclu의 증거ded가 다시 뻗치고 머리, 그리고 명백한 neurologic 이상을 hunched. 그들은 뇌염의 중요한 징후를 표시 할 때 마우스는 euthanized 있습니다. </li></ol> 5. 대표 결과 위에서 설명한 된 재발 모델이 처음 Shimeld, 외 발행되었습니다. 1-3. 그들은 테스트 및 실험실 년 4-15을 통해 많은 원고를 게시하는이 모델의 수정 된 버전을 사용하고 있습니다. 모델은 역할을 정의하는 데 사용 된이 재발 HSV-1 병 10 시토 킨은 인터페론-γ 재생. 로 그림 3에 도시 된 바와 같이,보다 IFNγ 표시 악화 각막 질환을 부족한 마우스는 야생 형 생쥐에게 14 했어요. 또한 여러 가지 백신 6,10,11을 구성의 치료 가치를 정의하는 데 성공적으로 사용되었다. 하나의 사건에서 테스트 백신 prophylactically 잘 작동하지만, 6 therapeutically 효과가 증명하지 않은 있습니다. 그러나,복제 - 무능 바이러스 백신을 테스트했을 때이 prophylactically 만 효과가 아니라, therapeutically도 10,11 매우 효과적이었고, 참조 10 그림 5를 참조하십시오. 최근 우리는 성별 차이가 재발 HSK를 겪고 생쥐에서 감지 된 여부를 결정하는이 모델을 사용했습니다. 그림 1은 설명으로 남성 C57BL / 6 마우스는 여성 C57BL / 6 쥐를보다 훨씬 적은 각막 투명도를 표시합니다. 마찬가지로, 각막 neovascularization이 평가되었을 때, 남성은 여성 마우스 마우스 (그림 2)보다 각막에 훨씬 적은 새로운 혈관의 성장을 보여 주었다. 우리는 현재이 표현형 쥐의 다른 변종 유사한 분석을 수행하여 특정 변형 여부를 테스트하고 있습니다. 우리는 또한 차이가 남성 호르몬의 부족이나 에스트로겐의 존재로 인해 여부를 결정합니다. <img alt="그림 1" src="/files/ftp_upload/4276/4276fig1.jpg" /> 그림 1. UV-B 유발 재 활성화에 따라 남성과 여성의 C57BL / 6 마우스 종자의 각막 질병. Latently 감염 마우스는 UV-B 조사 및 마우스 35일에 대한 각막 투명도에 대한 모니터링 된을 다시 활성화 할 수 있도록 유도했다. 다음과 같이이 연구에 사용 마우스의 번호는 있었다 : 남성, N = 15, 여성, N = 15. 결과는 평균 ± SEM을 나타냅니다. * 상당히 큰 바이러스 유발 질환은 일 14-35 (P = 0.001-0.01)에 대한 여성의 C57BL / 6 마우스에서 발생했습니다. <img alt="그림 2" src="/files/ftp_upload/4276/4276fig2.jpg" /> 그림 2. UV-B 유발 재 활성화에 따라 남성과 여성의 C57BL / 6 마우스 종자의 각막 질병. Latently 감염 마우스는 UV-B 조사 및 마우스 35일에 대한 각막 neovascularization에 대한 모니터링 된을 다시 활성화 할 수 있도록 유도했다. 남성, N : 이러한 연구에 사용 마우스의 번호는 다음과 같이있었습니다= 15, 여성, N = 15. 결과는 평균 ± SEM을 나타냅니다. * 상당히 큰 바이러스 유발 질환은 일 14-28 (P = 0.001-0.01)에 대한 여성의 C57BL / 6 마우스에서 발생했습니다.

<ol>
	<li>Shimeld, C., Hill, T. J., Blyth, B., Easty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+model+of+recurrent+herpetic+eye+disease+in+mice.">An improved model of recurrent herpetic eye disease in mice.</a> Curr. Eye Res. 8, 1193-1205 (1989).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+infection+and+induction+of+recurrent+herpetic+eye+disease+in+mice.">Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.</a> J. Gen. Virol. 71, 397-404 (1990).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+protects+the+mouse+eye+from+damage+after+herpes+simplex+virus+infection+by+limiting+spread+of+virus+in+the+nervous+system.">Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.</a> J. Gen. Virol. 71, 681-687 (1990).</li><li>Laycock, K. A., Lee, S. F., Brady, R. H., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+model+of+recurrent+herpes+simplex+viral+keratitis+induced+by+ultraviolet+B+radiation.">Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.</a> Invest. Ophthalmol. Vis. Sci. 32, 2741-2746 (1991).</li><li>Keadle, T. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+ameliorates+corneal+disease+in+a+mouse+model+of+recurrent+herpetic+keratitis.">IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis.</a> Microbial Path. 38, 13-21 (2005).</li><li>Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+recombinant+glycoprotein+D+subunit+vaccine+on+the+development+of+primary+and+recurrent+ocular+infection+with+herpes+simplex+virus+type+1+in+mice.">Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.</a> J. Inf. Dis. 176, 331-338 (1997).</li><li>Keadle, T. L., Laycock, K. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+cytokines+IL-1+and+TNF-a+are+required+for+recurrent+herpetic+keratitis+in+NIH+mice.">Proinflammatory cytokines IL-1 and TNF-a are required for recurrent herpetic keratitis in NIH mice.</a> Invest. Ophth. Vis. Sci. 41, 96-102 (2000).</li><li>Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+cytokine+expression+in+a+murine+model+recurrent+herpetic+stromal+keratitis.">Corneal cytokine expression in a murine model recurrent herpetic stromal keratitis.</a> Ocular Immunol. Inflam. 9, 193-205 (2001).</li><li>Keadle, T. L., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++and+CD8++cells+are+key+participants+in+the+development+of+recurrent+herpetic+stromal+keratitis+in+mice.">CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice.</a> Microbial Path. 32, 255-262 (2002).</li><li>Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+immunization+with+a+virion+host+shutoff-defective,+replication-incompetent+herpes+simplex+virus+type+1+strain+limits+recurrent+herpetic+ocular+infection.">Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection.</a> J. Virol. 76, 3615-3625 (2002).</li><li>Keadle, T. L., Laycock, K. A., Morris, J. L., Leib, D. A., Morrison, L. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+vaccination+with+vhs(-)+herpes+simplex+virus+reduces+the+severity+of+recurrent+herpetic+stromal+keratitis+in+mice.">Therapeutic vaccination with vhs(-) herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice.</a> J. Gen. Virol. 83, 2361-2365 (2002).</li><li>Keadle, T. L., Morris, J. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aminoguanidine+on+primary+and+recurrent+ocular+herpes+simplex+virus+infection.">The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection.</a> Nitric Oxide. 13, 247-253 (2005).</li><li>Stuart, P. M., Morris, J. E., Sidhu, M., Keadle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL3+protects+mice+from+corneal+pathology+during+recurrent+HSV-1+infection.">CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.</a> Front. Biosci. 13, 4407-4415 (2008).</li><li>Keadle, T. L., Alexander, D. E., Leib, D. A., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+is+not+required+for+recurrent+herpetic+stromal+keratitis.">Interferon gamma is not required for recurrent herpetic stromal keratitis.</a> Virology. 380, 46-51 (2008).</li><li>Carr, D. J., Austin, B. A., Halford, W. P., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Interferon-gamma+by+an+adenovirus+vector+blocks+herpes+simplex+virus+Type+1+reactivation+in+vitro+and+in+vivo+independent+of+RNase+L+and+double-stranded+RNA-dependent+protein+kinase+pathways.">Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.</a> J. Neuroimmunol. 206, 39-43 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

포진 성 각막염 stromal의 재발 모델 (HSK)는 여기서 설명하는 것은 연구자가 인간의 질병 중에 관찰되는 그와 더 일관성있는 모델이 인간의 HSK 공부를 할 수 있습니다. 따라서, 모델의 장점은 질병 주요 질병 중에 자극에 의​​해 이미 면역 체계의 맥락에서 발생한다는 점입니다. 이 마우스가 이미 HSV-1에 면역 반응이 있기 때문에, 그 면역 반응을 활성화하는 요인은 원래를 생성 한 것과 가능성이 ?...

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-C1</td>
		</tr>
		<tr>
			<td>Pen/ Strep</td>
			<td>Sigma</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>52-21</td>
		</tr>
		<tr>
			<td>Transilluminatior</td>
			<td>UVP</td>
			<td>95-0452-02</td>
		</tr>
		<tr>
			<td>Sonicatior</td>
			<td>Branson</td>
			<td>Sonifier 450</td>
		</tr>
		<tr>
			<td>Oak Ridge Tubes</td>
			<td>Nalgene</td>
			<td>Z 720410</td>
		</tr>
		<tr>
			<td>Flasks- 7150</td>
			<td>Corning</td>
			<td>430823</td>
		</tr>
		<tr>
			<td>Plates- 12 well</td>
			<td>Corning</td>
			<td>CLS 3513</td>
		</tr>
		<tr>
			<td>Plates- 48 well</td>
			<td>Corning</td>
			<td>CLS 3548</td>
		</tr>
		<tr>
			<td>Sterile Conical Tubes- 30 ml</td>
			<td>Corning</td>
			<td>CLS 430828</td>
		</tr>
		<tr>
			<td>Sterile Cotton-tipped Applicators</td>
			<td>Fisher</td>
			<td>23-400-125</td>
		</tr>
		<tr>
			<td>30G Needles</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
		</tr>
		<tr>
			<td>25G Needles</td>
			<td>BD</td>
			<td>305122</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309604</td>
		</tr>
	</tbody>
</table>

recurrent herpetic stromal keratitis in mice, a model for studying human hsk

포진 성 안구 질환, 포진 성이라고한다 stromal 각막염 (HSK)는 각막의 가능성이 눈부신 감염입니다 치료를위한 300,000 이상 임상 방문 각 연도에 발생합니다. 임상 질환을 가진 환자의 1 ~ 2 %에 감염된 각막의 비전 손실을 경험하게 될 것입니다. 이러한 경우의 대부분은 헤르페스에 의한 잠재 감염의 재 활성화의 결과는 급성 질환으로 인해 않는다고에게 바이러스를 입력하고 단순. 흥미롭게도, 급성 감염 대부분이 질병을 연구하는 데 사용되는 모델입니다. 그러나, HSK의 재발 모델은 임상 적 질환 중 발생하는 일이 더 반영 될 것이라고 생각했다. HSK에 대한 재발 동물 모델은 토끼와 쥐를 모두 고용했다. 토끼의 장점들은 지연의 재 활성화는 알려진 자극을 결석 경험한다는 것입니다. 그것은 토끼 모델이 immun가 없습니다 때문에 많은 면역 요인이 재발 HSK에서 재생하는 역할을 탐험하기가 어렵습니다, 말마우스가 있다고 ological 및 유전 자원. 재 활성화는 UV-B 빛에 노출에 의해 유도되기 때문에 우리는 거기에 많은 자원을 사용할 수있는 그런 이점을 가지고 있으며 또한 재 활성화가 발생합니다 때 알고 있기 때문에 재발 HSK의 마우스 모델을 사용하기로 결정했습니다. 지금까지이 모델이 질병에 중요한 여러 가지 면역 요소를 정의하는 데 사용하는 실험실 수있었습니다. 또한 우리는 치료 및 백신 효능을 모두 테스트 할 수 있습니다.

Watch this Scientific Journal Video about Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK at JoVE.com

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

포진 성 각막 질환의 대부분의 연구는 주요 감염 모델을 사용합니다. 그러나, HSV-1 기본 감염은 일반적으로 인간의 질병으로 이어질하지 않습니다. 여기보다 밀접하게 인간의 질병을 모방 포진 성 각막 질환의 재발 모델을 설명합니다.

마우스의 재발 포진 성 각막염 Stromal, 인간 HSK 유학을위한 모델

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

recurrent-herpetic-stromal-keratitis-mice-model-for-studying-human

Research

JoVE Journal

Immunology and Infection

11.8K 조회수.  Saint Louis University. 포진 성 각막 질환의 대부분의 연구는 주요 감염 모델을 사용합니다. 그러나, HSV-1 기본 감염은 일반적으로 인간의 질병으로 이어질하지 않습니다. 여기보다 밀접하게 인간의 질병을 모방 포진 성 각막 질환의 재발 모델을 설명합니다.

비디오: Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

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 ...

연구

면역학 및 감염병학

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

의 4D 좌석 Multimodality 이미징<em&gt; Citrobacter rodentium</em마우스에서&gt; 감염

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

쥐과 림프절 기질 세포의 분리

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

소설<em&gt; 시험관</em&gt; 인간의 전체 혈액과 내피 세포 사이의 상호 작용 유학을위한 모델

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

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

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 highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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 ...

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

심질 마우스에 있는 인간 흉막 중피종의 정형외 모형의 PET/CT에 의한 이식 및 모니터링

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...