著者は、私たちにモデルを教えて助けるために博士ジェイPeposeに感謝したいと思います。この作品は、国民健康補助EY11885（PMS）の研究所、EY21247（PMS）と眼科に失明を防ぐための研究からの無制限の助成金によって支えられている。

動物のケアに注意してください：彼らは、抗炎症であり、したがってモデルをinvalidateするような鎮痛剤は、このモデルでは使用されません。 1。ウイルスの作製<ol><li>細胞のすべてを持っている丸みを帯びたアップまで3-4日間感染の多重度0.01（T-150フラスコ、約5×10 5プラーク形成単位）の開始希釈を用いてVero細胞（80％コンフルエント）に、HSV-1を育てると簡単にフラスコを押すことで外れている。 </li><li> ℃、15分間3500 rpmで4時ソーバル伝説RTに滅菌遠心管と遠心分離機にメディアを取り出します。メディア合計5mlで各50mlチューブから上清（1.3を参照）、再懸大型ペレットを除去した後。 2 30秒のバーストで1分間超音波洗浄します。 </li><li>上清を除去し、9,000 rpmで50ミリリットル滅菌オークリッジ管や遠心分離機に入れ、4℃で1時間°ベックマンJ2-21遠心分離機でC用。これは、小さなウイルスのペレットを製造することである。上清を捨てる。 </li><li> 1.2ウィットから超音波処理組み合わせるhを1.3からペレットと45秒のために再度超音波洗浄します。 4℃で5分間1,500 rpmでソーバル伝説RTで遠心℃、上清を保存します。これは-70℃で小分けして保存されているウイルスストック℃を構成する</li><li> Vero細胞で力価ウイルス。 10 -8希釈（それぞれ10倍希釈である）2つの独立した希釈セットから2倍へ私は通常板10 0。ウイルスの力価測定は12ウェル滅菌プレート上で行われる。 </li></ol> 2。マウスへの感染<ol><li>麻酔cocktail/20グラムのマウスの体重（ケタミン、HBSSで希釈し60 mg / kgの+キシラジン5 mg / kg）を0.1mlの各マウスを注入します。それは適切な用量が使用され、この薬剤の効果は非常に短命であるアール麻酔の安全な手段の一つであるため、このカクテルに使用されます。 </li><li>マウスは、角膜に穴がないことを確認するために、解剖顕微鏡を用いて、30ゲージの針で右眼の角膜麻酔スクラッチあるとき。 </li><li>各マウスRECE初感染時の損傷から角膜を保護するために、プールしたヒト血清（1:800の50％ウイルス中和のために有効な用量と抗HSV反応性シグマ、テメキュラ、カリフォルニア州）の1ミリリットルの腹腔内注射をived。 </li><li>耳のクリップのいずれかで、または耳パンチで数匹のマウスを。 </li><li>すべてのマウス番された後、20μlのピペッターを使用して、我々は10 6 PFU（5μl）を使用して各眼に感染します。我々は、片目だけ、通常は右眼に感染します。我々はまた、彼らは麻酔している間に潤いを保つために、他の目にHBSSの5μlを置く。 5 ulは手続きが未満10分間で行われ、動物がそれに応じて麻酔薬を投与されているので目の水和を維持するのに十分である。感染症は、ベロメディア1mlに水和させた滅菌綿棒でマウスを3日後に感染を拭き取りにより確認されている。その後、100μlを48ウェルプレート中で増殖させたVero細胞に添加される。これらのプレートは、細胞変性効果のために毎日監視している<html> （CPE）。 </li></ol> 3。レイテンシからのマウスの再活性化<ol><li>マウスは一次感染後少なくとも5週間麻酔する。それは、我々は1年以下の初感染にマウスを再活性化しているし、それに応答して有意な差は認められていないことに留意すべきである。 </li><li>一度麻酔それらはただ一つ目は、UV-B光にさらされるように302 nmのピーク波長でのUV-Bを発するTM20クロマト-VUトランスイルミネーターを配置されます。各マウスは250 mJのUV-B光cm 2にさらされている。 </li><li>再活性化は、まず1から7までのポストのUV-B照射の日に次に0日にベロメディアの1ミリリットルに配置（自発sheddersために制御するために、UV-B曝露前）とされている滅菌綿棒で目を拭き取りによって決定されます。 2.5で説明したようにスワブ材料）が評価されます。感染性ウイルスを持っているそれらのスワブのウイルスの量は、12ウェルプレートを用いた標準的なプラークアッセイによりtitteredされています。 </li></ol>ove_title &quot;&gt; 4。臨床評価<ol><li>マウスは、双眼実体顕微鏡を用いてマスクされた観察者によって臨床眼疾患のために評価されます。 </li><li>角膜混濁が0クリアストロマを示す0〜4のスケールで評価され、1が軽度の間質混濁、2を示す識別可能な虹彩の機能と適度な不透明度を示し、3は瞳孔の余白を除いて定義された虹彩の細部が失われた状態で密な不透明度を意味し、4を示しノー後部を望む総不透明。 </li><li>血管新生は、4つの等しい象限に角膜を分割し、これらの各象限における血管新生の程度を決定することによって、1から8までのスケールで評価されています。 </li><li>眼瞼炎は、次のように測定されていない：0、全く病変; 1、最小限のまぶたが腫れ、2、中等度の腫れや無愛想な眼漏、3、重度の腫れ、適度な眼周囲の脱毛、皮膚病変、および4、目をまぶしたシャットダウンを伴う重度の腫れ、厳しい眼周囲の脱毛、皮膚病変。 </li><li>脳炎もの含めるの証拠DEDは、バックフリルの髪、そして明らかな神経学的異常を覆いかぶさる。彼らは脳炎の重要な兆候を表示するときに、マウスを安楽死させています。 </li></ol> 5。代表的な結果上述した再発モデルは最初Shimeld らによって発表された1-3。彼らはテストの結果、私たちの研究室では、年4月15日の上に多くの原稿を公開するために、このモデルの修正版を使用してきました。モデルは、再発性HSV-1の10疾患におけるサイトカインインターフェロン-γの果たす役割を定義するために使用された。 図3に示すように、よりIFNγの表示悪化角膜疾患を欠損したマウスは、野生型マウスに14をしました。それはまた、いくつかの異なるワクチン構築6,10,11の治療的価値を定義するために首尾よく使用されていました。 1ケースでテストワクチンが予防的にうまくいきましたが、6治療的に有効であることが判明しなかったことに注意してください。しかし、複製能力のないウイルスワクチンを試験したとき、それは予防的に唯一の効果はありませんでしたが、治療的にも10,11非常に有効であった、参考文献10の図5を参照してください。さらに最近では、我々は性差が再発HSKを受けたマウスで検出可能であったかどうかを判断するために、このモデルを使用している。 図1が示すように 、雄のC57BL / 6マウス雌のC57BL / 6マウスの場合よりも有意に低い角膜混濁を表示します。同様に、角膜血管新生を評価したところ、雄マウスでは有意に少ない雌マウス（ 図2）したより角膜の新しい血管の成長を示した。我々は現在、この表現型は、マウスの他の株で同様の分析を行うことにより、特定の菌株であるかどうかをテストしています。我々はまた、違いはテストステロンやエストロゲンの存在の欠如によるものであるかどうかを判断します。 <img alt="図1" src="/files/ftp_upload/4276/4276fig1.jpg" /> 図1のUV-B誘発再活性化後に雄と雌のC57BL / 6マウス系統における角膜疾患。潜伏感染マウスは35日間角膜混濁についてモニターしたUV-Bを照射し、マウスで復活させるように誘導した。これらの研究のために使用したマウスの数は以下の通りであった：男性は、n = 15、女性は、n = 15。結果は平均値±SEMを示す。 *かなり大きいウイルス誘発性の疾患は、14から35日間のために雌のC57BL / 6マウス（P = 0.001から0.01）であった。 <img alt="図2" src="/files/ftp_upload/4276/4276fig2.jpg" /> 図2 UV-Bの再活性化によって誘導される、以下の雄と雌のC57BL / 6マウス系統における角膜疾患。潜伏感染マウスは35日間角膜血管新生のために監視されたUV-Bを照射し、マウスで復活させるように誘導した。これらの研究のために使用したマウスの数は以下の通りであった：男性は、n= 15;女性は、n = 15。結果は平均値±SEMを示す。 *かなり大きいウイルス誘発性の疾患は、14から28日間のために雌のC57BL / 6マウス（P = 0.001から0.01）であった。

<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>

特別な利害関係は宣言されません。

ヘルペス性間質性角膜炎の再発モデル（HSK）は、ここで説明すると、研究者は、人間の病気の間に観察されているものとより一貫性のあるモデルで人間のHSK試験を勉強することができます。したがって、このモデルの強みは、病気は原発性疾患時の刺激によってすでに免疫システムのコンテキストで発生することがあります。これらのマウスはすでに、HSV-1に対する免疫応答を持っているの?...

<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

ヘルペス性眼疾患と呼ばれるヘルペス性間質性角膜炎（HSK）は、角膜の潜在的に失明に至る感染症であり、その治療のために30万人以上の通院各年の結果。臨床疾患を持つ患者の1〜2％が感染し、角膜の視力喪失を経験するでしょう。これらのケースの大半はヘルペスで潜伏感染の再活性化の結果は、私はウイルスではなく、急性疾患に起因する単純に入力されています。興味深いことに、急性感染症は、ほとんどの場合、この病気を研究するために使用されるモデルです。しかし、HSK試験の再発モデルは臨床疾患の間に発生するものをより反映であろうと感じました。 HSK試験のために再発の動物モデルは、ウサギとマウスの両方を採用してきた。ウサギの利点は、彼らは、任意の既知の刺激が存在しない場合の待ち時間から再活性化を経験するということです。それはウサギモデルがIMMUNを持っていないので、それは多くの免疫学的要因が再発HSK試験に果たす役割を探求することは困難である、と述べたマウスが持っているologicalと遺伝資源。再活性化は、UV-B光への曝露によって誘発されるので、我々はそれがそこには多くのリソースが利用可能であるという利点を持っており、また再活性化が発生するときに我々は知っているので、再発HSKのマウスモデルを使用することにしました。これまでのところ、このモデルでは、この病気に重要ないくつかの免疫学的要因を定義するためにそれを使用して、それらの研究室ができました。また、私たちは、治療およびワクチンの有効性の両方をテストすることができました。

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

ヘルペス性角膜疾患のほとんどの研究は、一次感染モデルを使用しています。しかし、HSV-1の初感染は、典型的には、ヒトの疾患につながるものではない。ここでは、より密接に人間の病気を模倣ヘルペス性角膜疾患の再発モデルについて述べる。

マウスにおける再発性ヘルペス性間質性角膜炎、人間のHSKを研究するためのモデル

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 ビュー.  Saint Louis University. ヘルペス性角膜疾患のほとんどの研究は、一次感染モデルを使用しています。しかし、HSV-1の初感染は、典型的には、ヒトの疾患につながるものではない。ここでは、より密接に人間の病気を模倣ヘルペス性角膜疾患の再発モデルについて述べる。

ビデオ: 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.

ザ<em&gt;シトロrodentium</em&gt;マウスモデル：感染性大腸炎に勉強病原体とホスト貢献

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

免疫・感染学

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.

不定冠詞<em&gt;インビトロ</emヒトマクロファージの分化と偏光の不均一性を研究するために&gt;モデル

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.

の4Dのマルチモダリティイメージング<em&gt;シトロバクターrodentium</emマウスにおける&gt;感染症

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.

ネズミリンパ節間質細胞の単離

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).

小説<em&gt;インビトロ</em&gt;ヒト全血と内皮の間の相互作用を研究するためのモデル

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.

と実験感染<em&gt;リステリア菌</em&gt;ホストインターフェロンγ応答を研究するためのモデルとして、

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.

研究のための方法論<em&gt; B。枯草菌</em小分子バイオフィルム阻害剤を特徴付けるためのモデルとしての&gt;バイオフィルム

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.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

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 イメージング勉強結核感染と非ひと霊長類における治療のためのツールとしての解析

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.

アチミックマウスにおけるヒト胸膜中皮腫の正形細胞モデルのPET/CTによる移植とモニタリング

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