我々は、技術的な助言をB6.A2G-Mx1のマウスとSemraÖzcelikを提供するためのピーターStaeheliに感謝します。 この作品は、SFB829とKN536 / 16を通じてドイツ研究財団、医学のケルンフォーチュンプログラム/学部、ケルン大学によってサポートされていました。

倫理ステートメント。 屠殺した動物からの表皮シートの製造はLandesamtエリーゼナチュール、環世界とVerbraucherschutz、ノルトライン=ヴェストファーレン（ドイツ）のガイドの勧告に厳密に従って実施されます。研究はLANUV NRW（番号8.84-02.05.20.13.018）によって承認されました。 楽器と培養培地の調製<ol><li>グルタミンジペプチド、10％FCS、100 IU / mlペニシリン、および100μg/ mlストレプトマイシン（以下「DMEM」と称する）を含むダ​​ルベッコ改変イーグル培地（DMEM）/高グルコースに表皮シートを養います。 </li><li>表皮シートの製造のために、慎重に加熱したPBSでディスパーゼII粉末（5 mg / mlの）を溶解（最大37℃）。 0.22μmのフィルターを通して溶液を濾過し、治療のためにそれを直接使用しています。 </li><li>表皮全体の製造は13をマウントするために、バフを遮断準備0.5％の粉乳、冷水魚皮から0.25％のゼラチン及びTBS中0.5％のTriton X-100とER。物質は、回転ミキサー上で室温で少なくとも2時間混合物をインキュベートすることによって溶解させます。また、洗浄用緩衝液としてPBS中の0.2％のTween 20を準備します。固定のために、3.4％、PBS中4％ホルムアルデヒドを準備します。 </li><li>凍結切片の免疫染色のために、PBS中の5％正常ヤギ血清および0.2％Tween 20でブロッキングバッファーを準備します。固定のため、PBS中の0.5％ホルムアルデヒドを準備します。 </li></ol>マウスの皮膚から表皮シートの作製<ol><li>感染研究のための新生児背中の皮膚からの表皮の調製<ol><li>解剖皿に断頭マウス（生後1〜3日）を配置し、四肢と完全な胴体から皮膚の効率的な除去を可能にする胴体に近い尾をカット。四肢の除去中に、直径が可能な限り小さくカットを維持しようとします。 </li><li>湾曲した微細な鉗子Aと胴体を修正優しく胴体から皮膚を分離するために、バックに沿って尾側する頭蓋から皮膚の下に鈍い先端のはさみを動かすND。背面に沿って皮膚をスリット。 </li><li> FACS分析、ケラチノサイトまたはRNAの準備を単離するために、胴体をオフにプッシュする組織と鈍い先端のはさみを修正するために鉗子を使用して、胴体から完全に皮をはがし。凍結切片またはホールマウントの調製のために背中の皮膚の部分だけを取ります。 </li><li>約10×10mmの1-2個にメスで背部皮膚をみじん切りに。 〜1ミリリットル滅菌PBSで6ウェルプレートの1ウェルに作品を配置。真皮側が下を向いていることを確認します。 </li><li> 1.5ミリリットルのディスパーゼIIでPBSを交換してください（5 mg / mlのPBS）と（ホールマウント用）、37℃で、またはO / N 4℃で30分間のいずれかでインキュベートします。 PBSで3回洗浄します。静かに表皮を持ち上げるために、基礎となる真皮および他の鉗子を修正するために、1つの鉗子を使用して、完全なシートとして表皮を除去します。このステップEASを作るために双眼鏡を使用して、Y。下部にその基底側を含むDMEMに表皮シートを転送します。感染実験のためにすぐにシートを使用してください。 注：主にFACS分析、ケラチノサイトまたはRNAの準備を単離するための新生児の皮膚からの表皮の準備を使用してください。理由は、その脆弱性のため、凍結切片またはホールマウントのための感染シートの製造は、あまり適していません。 </li></ol></li><li>感染研究のための大人の尾の皮膚から表皮の調製<ol><li>頚椎脱臼により1〜3ヶ月の年齢でマウスを安楽死させます。背中の長い埋め込み毛包が無傷の表皮シートの分離を妨げるので、代わりに背中の皮膚の尾を取ります。屠殺したマウスから尾をカットし、メスで長さ方向にスリット。 </li><li>骨から皮をはがしし、約5×5mmの小片にメスでそ​​れを切ります。 1つのウェルに4枚まで入れて、2.1.5で説明したようにディスパーゼII中でのインキュベーションを進めます 注：使用大人太極拳リットルの皮膚は、凍結切片またはホールマウントを調製しました。あるいはすぐに使用する、尾部は、かなりの感染効率を損なうことなく記憶または4℃で約24時間、輸送することができます。尾部は、4​​8時間まで維持される場合、表皮シートがほとんどHSV-1に感染したことはできませんでした。 </li></ol></li><li> FACS分析のために表皮の解離 注：FACS分析による表面タンパク質の発現の検出は、細胞表面と目的のタンパク質を損なわない適切な細胞分離技術を必要とします。 <ol><li> RTで15分間、1.5ミリリットル/ウェルの組換えトリプシンに基づく細胞解離溶液に、37℃で15分間基底側を6ウェルプレート中の新生表皮シートをインキュベートします。 </li><li> 3ミリリットルのDMEMを追加することにより、インキュベーションを停止します。ケラチノサイトが溶解を得るように穏やかに6ウェルプレートに表皮を移動させるように湾曲した微細なピンセットを使用して、ケラチノサイトを解離します。細胞懸濁液を収集し、このステップを3回繰り返します常に新鮮なDMEMを使用。 注：この解離手法は、ケラチノサイトの表面にネクチン1を検出することが適当です。 </li><li>あるいは、RTで15分間、37℃で15分間、酵素を含まない細胞解離溶液に表皮シート（CDS）をインキュベートします。優しく上下CDSをピペットでケラチノサイトをフラッシュするために、室温でさらに15分間インキュベートします。 </li><li>細胞懸濁液を収集し、新鮮なDMEMを使用して、フラッシング工程を3回繰り返します。 注：この解離がHVEMの表面発現を検出するのに適しています。 CDS溶液の欠点は、より少ない細胞が、組換えトリプシンに基づく細胞解離溶液よりも解離することです。表面発現に加えて、ICP0式として、核または細胞質発現は、組換えトリプシン基づく細胞解離溶液で感染表皮シートの解離後にFACSによって分析することができます。 </li></ol></li><li>ケラチノサイトまたはexを単離するための表皮の解離道のRNA <ol><li> 1.5ミリリットル/ウェルの組換えトリプシン基づく細胞解離溶液を含んでいる皿の底部に向かって基底側で6ウェルプレートに新生児表皮シートを置きます。 RTで30分間インキュベートします。 3ミリリットルのDMEMを加え、穏やかに皿に表皮を移動するために、湾曲細かい鉗子を使用して、ケラチノサイトを解離。 </li><li>細胞懸濁液を収集し、新鮮なDMEMを使用して、この手順を3回繰り返します。この細胞懸濁液は、RNAを抽出したり、文化の一次マウスケラチノサイトにするために使用します。 </li></ol></li></ol> HSV-1と表皮シートの3感染<ol><li> 500μlのDMEM以上にHSV-1 WT株17 11の溶液を調製し、表皮シートはフローティングされたウイルス液で培地を交換してください。この時点は、0時間12として定義される。37℃で24ウェルプレートの1ウェルに表皮シートの感染を行っ。ウイルス力価の計算は、推定された数に基づいています表皮シート（5×5 mmのシートあたり約2×10 5細胞 ）あたりの基底層での細胞。 </li><li> HSV-1で約100プラーク形成単位（P​​FU）/細胞で感染し、1時間のパイでDMEMによるウイルス含有培地を交換 注：新生児の皮膚から表皮シートの場合、シートはインキュベーション中に解離し始めることに注意してください。 </li></ol>感染した細胞の可視化4. <ol><li>表皮ホールマウント13 <ol><li> RTまたはOで2時間/ N 4℃のいずれかの3.4％ホルムアルデヒドで表皮シートを修正。 PBSで2回洗浄し、0.5％粉乳、冷水魚皮、室温14で1時間、TBS中の0.5％のTriton X-100 0.25％ゼラチンでブロックします。 </li><li> 15を室温でブロッキング緩衝液中で1:60に希釈したマウス抗ICP0（モノクローナル抗体11060）とシートO / Nインキュベートします。中間径フィラメントネットワークを可視化するために、ウサギポリクローナルと同時にインキュベート抗マウスケラチン14（100ng / mlの）。 </li><li>室温で4時間の間に、PBS-0.2％Tween 20で4〜5回洗浄します。 AF488結合抗マウスIgG（1 / mlの）、AF555結合抗ウサギIgG（1 / mlの）、およびDAPI（4 &#39;、6-ジアミジノ-2-フェニルインドール）（100 ngのでO / Nインキュベート/ ml）を、室温でブロッキング緩衝液中。 </li><li>室温で4時間、PBS-0.2％Tween 20で洗浄します。試料スライドの上にその基底側マウントの表皮シート、蛍光マウンティング培地中に埋め込んで、カバーガラスでカバーしています。 </li></ol></li><li>表皮凍結切片<ol><li>成人表皮シートを室温で20分間、PBS中の4％ホルムアルデヒドで固定し、PBSで2回洗浄しました。液体窒素中の培地と凍結凍結組織で固定シートを埋め込みます。クライオミクロトームで8-10ミクロンのセクションをカット。 </li><li> RTで10分間PBS中0.5％ホルムアルデヒドで再びセクションを修正、PBSで2回洗浄し、5％正常ヤギ血清でブロックし、0.2％Tween 20をPBS中で室温で30分間、次いで、Mで60分間染色ウーズ抗ICP0（モノクローナル抗体11060）15は、ブロッキング緩衝液で3回洗浄し、続いて、ブロッキング緩衝液中で1:60に希釈しました。 </li><li>その後、RTで45分間ブロッキング緩衝液中にAF488結合抗マウスIgG（1μg/ ml）およびDAPI（100ng / mlの）でインキュベートし、3回洗浄します。蛍光マウンティング培地中のセクションを埋め込み、カバーガラスでカバーしています。 </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+adhesion+molecule+nectin-1+is+critical+for+normal+enamel+formation+in+mice.">The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.</a> Hum. Mol. Genet. 17, 3509-3520 (2008).</li><li>Wang, Y., Subudhi, S. K., Anders, R. A., Lo, J., Sun, Y., Blink, S., Wang, Y., Liu, X., Mink, K., Degrandi, D., Pfeffer, K., Fu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+herpesvirus+entry+mediator+as+a+negative+regulator+of+T+cell-mediated+responses.">The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.</a> J. Clin. Invest. 115, 711-717 (2005).</li></ol>

The authors declare that they have no competing financial interests.

C57BL / 6から、成人の皮膚の表皮シートは、HSV-1に感染している場合で約100 PFU /細胞より低いウイルス用量が少なく、感染細胞と相関し、遅いながら、私たちは、毛包間表皮内の基底層のほぼ全ての細胞に感染を観察感染の進行。一般的には、毛包には、感染した細胞の可変数を示します。開発毛包の内側を覆うかなり小さいケラチノサイトのほとんどが感染している間、大人の毛包の唯一の?...

単純ヘルペスウイルス（HSV）は、生命を脅かす感染症に軽度の合併症のない皮膚粘膜病変から、ヒトでの疾患の範囲を引き起こす可能性があります。 HSV 1型（HSV-1）が支配的に可能性が高い生殖器感染症を引き起こす1 HSV 2型（HSV-2）、一方、口腔顔面感染症および脳炎に関連しています。 HSVは、培養中の細胞に入り、感染を開始し、ウイルス子孫を生産する方法を理解する上で重要な進展があるが、我々は細胞レベル2での組織へのウイルスの侵入経路（複数可）について少し知っています。 HSV皮膚または感染、マウス、ウサギ、モルモット、粘膜の研究のための動物モデルとして使用されています。皮膚感染は皮内注射によって、またはウイルスの存在下で肌を掻くことによって設立され、病気の発症は、ウイルス産生と相関していました。これらの方法は、疾患の病因の様々な側面を理解することを助け、及び抗ウイルス薬を評価するために使用されます。 TISSUでHSV感染を研究するために、Eレベルは、器官型ヒト皮膚モデルが適用されています。感染率は、これらのいかだ培養で制限されているように、感染症、ウイルスの拡散および抗ウイルス成分の影響を調査した研究の限られた数しか3-6を公開されています。 無傷の上皮におけるHSV-1感染の間に役割を果たしている細胞の決定を特徴づけるために、我々は、マウスの表皮7のex vivo感染研究のためのプロトコルを確立しました。皮膚は、新生児からか、成体マウスの尾から調製しました。 HSV-1は、ウイルス含有培地中に浸漬した完全な皮膚サンプルを、感染することができませんでしたので、私たちは、ディスパーゼII処理によって真皮から表皮を分離しました。ウイルス含有培地上で表皮シートの浮上した後、感染した細胞を様々な時間感染後（PI）7に表皮基底層に可視化することができます。ウイルス複製Aの​​前に、個々の細胞における感染の開始を可視化しますNDウイルス産生は、我々は、HSV-1感染中に発現最初のタンパク質のうちの1つである感染した細胞の蛋白質0（ICP0）に対する抗体で染色しました。 ICP0の細胞内局在は、初期感染時に明確な相を通過します。 ICP0は、ウイルス遺伝子発現の初期段階の間に、核病巣に存在するが、細胞質へのICP0の再局在は、感染8の次の段階を示しています。 我々は、感染の間に、様々な細胞因子の潜在的な役割をテストするために別のマウスモデルから表皮シートの ex vivo感染アッセイを使用していました。アクチンダイナミクスの重要な調節因子としてのRac1の影響に対処するために、我々は、RAC1遺伝子9のケラチノサイト特異的欠失を有するマウスの表皮を感染させました。このモデルは、表皮角化細胞層におけるHSV-1感染の効率に欠損のRac1の影響を研究するために私達を可能にしました。再対照同腹子の感染表皮との比較Rac1の不在は、表皮の基底層7における感染の開始に影響を及ぼさないことを示し、有意差はvealedありません。さらに、マウスモデルの使用は、私たちは細胞の受容体は、表皮内への進入を媒介する対処することができました。 HSV-1とネクチン1またはHVEM欠損マウスのいずれかから感染表皮シートは、組織への最初のウイルスの侵入を強くネクチン1 10の存在に依存することを明らかにしました。さらに、我々の結果は、HVEMはまた、ネクチン1 10未満の効率が、マウスの表皮における受容体として働くことができることを実証しています。 表皮層における感染細胞の空間分布に対処するために、我々は、組織切片および表皮ホールマウント（ 図1）にICP0発現を可視化します。完全な皮膚の凍結切片では、何のICP0発現細胞は、（ 図1）検出されません。対照的に、表皮シートの凍結切片は、細胞質を実証すでに3時間のPI（ 図1）での基底層でICP0式。後の時点で、基底層直上の層に拡散するウイルスを可視化することができます。基底層中の感染細胞の空間分布は、容易に表皮ホールマウント（ 図1）に従うことができます。 100 PFU /細胞でのHSV-1の感染の際に、毛包間表皮における基底ケラチノサイトの約50％が最も感染した細胞が核ICP0を表現するこの時点で1.5時間のパイでICP0の発現を示します。初期遺伝子発現の後の段階を示す細胞質へICP0の再局在は、3時間のPI（ 図1）で、ほぼすべての細胞に存在します。 ex vivoで HSV-1感染時に感染した細胞を可視化これらのモードは、阻害剤のまたは組織中のウイルスの侵入と拡散上/削除変異した細胞成分の効果を研究するための強力なアッセイを提供します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

そのヒト宿主を入力するには、単純ヘルペスウイルス1型（HSV-1）は、粘膜表面、皮膚、または角膜の障壁を克服しなければなりません。 HSV-1の目標は、最初の入力時にケラチノサイトおよび神経細胞の潜伏感染が続いている上皮における一次感染を確立します。再活性化した後、ウイル​​スは皮膚の小胞または粘膜潰瘍として現れる皮膚粘膜のサイトで明らかになることができます。 HSV-1は、皮膚や粘膜に侵入し、到達したか、その受容体は、あまり理解されていません。細胞レベルでの上皮組織へのHSV-1の侵入経路を調べるために、我々は、皮膚中の原発性および再発性感染症の部位を表すマウス表皮の ex vivo感染モデルを、確立しました。アッセイは、マウスの皮膚の準備が含まれています。表皮は、ディスパーゼII処理により真皮から分離されています。ウイルス含有培地に表皮シートを浮遊した後、組織が固定され、感染による種々の時間の感染後に視覚化することができますHSV-1前初期タンパク質ICP0に対する抗体を感染させた細胞を染色します。 ICP0発現細胞は、1.5時間の感染後に既に基底ケラチノサイト層で観察することができます。より長い感染時間で、感染した細胞は、感染は、基底ケラチノサイトに限定されないことを示す、基底上層において検出されるが、ウイルスが組織内の他の層に広がります。様々なマウスモデルの表皮シートを使用して、感染のプロトコールは、組織内へのHSV-1の侵入に寄与する細胞成分の関与を決定することができます。さらに、アッセイは、初期エントリ段階、細胞から細胞への広がりおよびウイルス産生を妨害する組織で阻害剤を試験するのに適しています。ここでは、詳細にex vivoでの感染プロトコルを記述し、ネクチン1またはHVEM欠損マウスを用いて、我々の結果を提示します。

この方法の課題は、HSV-1は、基底層から浸透することが可能に表皮シートを調製することです。重要なステップは、マウスの系統に応じて、適合させる必要があり、ディスパーゼII処理によって真皮から表皮を分離することです。ディスパーゼIIの濃度は、2.5から5 mg / mlの、そして20〜45分間のインキュベーション時間の範囲であることができます。中間フィラメントタンパク質のケラチン14の?...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

単純ヘルペスウイルス1型を持つマウス表皮の ex vivo 感染

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.1K ビュー.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

ビデオ: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

免疫・感染学

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

単純ヘルペスウイルスの待ち時間と再活性化の研究の主ニューロン培養システム

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

抗原特異的規制用のトランス生体内遅延型過敏アッセイ

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

遺伝子または薬物療法で使用するためのHIV-1産生を標的RNAの有効性および毒性の評価

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivoひと免疫不全ウイルス 1 における抗癌剤感受性との人間のリンパ組織と女性の性器粘膜の感染症

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

伝染性ファブリキウス嚢病ウイルスの病原性を研究するためのEx Vivo鶏ファブリキウス嚢細胞培養モデル

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

蛍光共焦点顕微鏡を用いた単純ヘルペス ウイルス 1 蛋白質の核-細胞質転流の時空間解析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

2型糖尿病と関連した非アルコール性脂肪性肝炎のための高度なマウスモデル

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

狂犬病ワクチンの効力を評価するインビトロELISA試験

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...