我々はそれぞれ、実験支援のためにポーラコボス博士エヴロスバシリウに感謝し、マウスのマクロファージを提供。また、材料と指導と助けを私たちの研究室マネージャーリチャードCriasiaに感謝します。最後に、著者は、ビデオ制作のヘルプはRamanpreet Kaurさんに感謝します。

1。組織培養<ol><li>コレラ毒素の測定にハツカネズミマクロファージ （BALB / cマウス）を使用します。当初は文化M.のすべてベンダーの指示に従って筋細胞 。 </li><li> 10％ウシ胎児血清で完了L-グルタミンを有するダルベッコ変法イーグル培地中でBALB / cマウス細胞を伝播して、1％の抗生物質/抗真菌剤またはRPMI 1640ベース媒体は、10％ウシ胎児血清、5％L-グルタミン、および1で完了し％抗生物質/抗真菌剤試薬。 </li><li> 37℃でベントキャップ付き75cm 2のコーニングフラスコ中の細胞を増殖°C、95％空気および5％CO 2。 </li><li>脂肪酸の決定の決定のため、メーカーの指示に従って配信の24時間以内にヒト腸上皮細胞を持ち出す。 </li><li> 37℃、10％FBSおよび30 ngの/ mlのヒトEGFで完了HybriCareメディアベントキャップ付75cm 2のコーニングフラスコ、℃で人間の腸管上皮細胞を伝播抗生物質を使用しないでくださいメーカーの指示に従って、/抗真菌薬。 </li><li>密集度約70％で、すべてのセル（1:3）を分割します。 </li><li>フリーズと標準凍結プロトコルを使用して研究を通して、すべてのセルを持ち出す。 </li><li> 注：脂肪酸のより大規模な濃度決定のために、最初は速く成長し、保守が容易にマウスマクロファージを使用しています。細かいスケール脂肪酸濃度の決定のために、ヒト上皮細胞を使用しています。すべてのコレラ毒素治療（説明を参照してください）​​、マウスマクロファージを使用してください。 </li></ol> 2。脂肪酸とコレラ毒素トリートメント<ol><li>オレイン酸は、同社が提供するガラスアンプルから滅菌ガラスバイアルにリノール酸とリノレン酸を転送します。 </li><li>個別滅菌エッペンドルフチュー​​ブに各脂肪酸を転送し、1:6希釈で100％エタノールで最初にそれを溶解した後、10μgの/μlの最終濃度のためにRPMI 1640不完全なメディアである。 </li><li>ボルテックス各ソリューションとTRANSFえーそれは冷凍庫内のストレージ用ガラスバイアル（-20°C）へ。 </li><li> 96ウェル組織培養プレートでよく2,500細胞/皿で細胞。 </li><li> MTTアッセイの準備のために脂肪酸と細胞の治療のために200μlに合計うまくボリュームを持って適切な完全なメディアを追加します。 </li><li> 24時間増殖期間の後、メディアを取り出し、新鮮な培地と交換してください。 </li><li> （結果を参照）ウェルにテストする各脂肪酸の適切な濃度を追加します。 200μlに合計うまくボリュームを持って完全なメディアを追加します。 </li><li> MTTアッセイを開始する前に24時間処理プレートをインキュベートする。 </li><li>クライオバイアルに1 mg / mlのアリコートと溶液の濃度でPBSでコレラ毒素を溶解し、2-8でクライオバイアルを保管℃に</li><li>細胞治療のため、1 ngの/μLの最終作業濃度のどちら滅菌PBS（pH7.4）に、または不完全なDMEM中で毒素1:100に希釈する。 </li><li>におけるコレラ毒素を持つ細胞を治療する上述したようにMTTアッセイプレート電池用製剤。 </li><li> 24時間増殖期間の後、脂肪酸アプリケーション（上）で使用される手順に従って、ウェルにテストする（我々の濃度について、結果を参照）コレラ毒素の適切な濃度を追加します。 </li><li> MTTアッセイを開始する前に24時間処理プレートをインキュベートする。 </li><li>それぞれの脂肪酸及びコレラ毒素治療の両方のための正（紙全体に正c）および陰性対照（負c）は、完全培地および70％エタノールでインキュベートした細胞、など。 </li><li>すべてのサンプルとトリートメントにはポジティブコントロールのためのより多くの複製（本研究ではn = 6）で、3反復（n = 3）での最小値を使用しています。 </li></ol> 3。 MTTアッセイ<ol><li> 2.5 mg / mlの濃度にMTT原液を作る。 </li><li>テストする、96ウェルプレートの各ウェル中の溶液を取り除き、新鮮な完全なメディア溶液200μlと交換してください。 </li><li>広告各ウェルにMTT溶液を10μlのD。 </li><li> 37℃で3-4時間、プレートをインキュベートする。 </li><li>メディアソリューションを捨て、各ウェルにイソプロパノールで0.04 M HClを100μlのを追加します。 </li><li> 5分間室温でプレートをインキュベートする。 </li><li>各ウェルから新しい遠心管にソリューションを移す。 </li><li> 1分間、またはペレットが形成されるまで、室温で、20,000×gで遠心分離します。 </li><li>各サンプルの20〜40μlの間にマイクロプレートリーダーへの転送。 </li><li>分光光度計を用いて570nmで吸光度を読み取る。 </li></ol>

<ol>
	<li>Franceur, M. L., Golden, G. M., Potts, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+Acid:+Its+effects+on+stratum+corneum+in+relation+to+(trans)+dermal+drug+delivery.">Oleic Acid: Its effects on stratum corneum in relation to (trans) dermal drug delivery.</a> Pharm Res. 7 (6), 621-627 (1990).</li><li>Tandon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+diffraction+and+spectroscopic+studies+of+oleic+acid-sodium+acetate.">X-ray diffraction and spectroscopic studies of oleic acid-sodium acetate.</a> Chem. Phys. Lipids. 109 (1), 37-45 (1990).</li><li>Kris-Etherton, 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+debate+about+n-6+polyunsaturated+fatty+acid+recommendations+for+cardiovascular+health.">The debate about n-6 polyunsaturated fatty acid recommendations for cardiovascular health.</a> Journal of the American Diabetic Association. 110 (2), 201-204 (2010).</li><li>Rizzo, W. B., Phillips, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenoleukodystrophy:+dietary+oleic+acid+lowers+hexacosanoate+levels.">Adrenoleukodystrophy: dietary oleic acid lowers hexacosanoate levels.</a> Annals of Neurology. 21 (3), 232-239 (1987).</li><li>Bozan, B., Temelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+oxidative+stability+of+flax,+safflower+and+poppy+seed+and+seed+oil.">Chemical composition and oxidative stability of flax, safflower and poppy seed and seed oil.</a> Bioresource Tech. 99, 6354-6359 (2005).</li><li>Krein, S., Meldurm, H., Hawkins, S., Foy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+benefits+of+conjugated+linoleic+acid+to+3-dimensional+wrinkle+morphology.">Clinical benefits of conjugated linoleic acid to 3-dimensional wrinkle morphology.</a> J. American Academy of Dermatology. 60 (3), AB30 (2009).</li><li>Yu, Y., Correll, P. H., Heuvel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+linoleic+acid+decreases+production+of+pro-inflammatory+products+in+macrophages:+Evidence+for+a+PPARy+dependent+mechanism.">Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: Evidence for a PPARy dependent mechanism.</a> Biochimica et Biophysica Acta. 1581 (3), 89-99 (2002).</li><li>Palombo, J., Ganguly, A., Bistrian, B., Menard, 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+anti-proliferative+effects+of+biologically+active+isomers+of+conjugated+linoleic+acid+on+human+colorectal+and+prostatic+cancer+cells.">The anti-proliferative effects of biologically active isomers of conjugated linoleic acid on human colorectal and prostatic cancer cells.</a> Cancer Letters. 177, 163-172 (2002).</li><li>Granda, M., Sinclair, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acids+and+obesity.">Fatty acids and obesity.</a> Current Pharm. Design. 15 (36), 4117-4125 (2009).</li><li>Rosenstein, E., Kushner, L., Kramer, N., Kazandjian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+dietary+fatty+acid+supplementation+in+the+treatment+of+adult+periodontitis.">Pilot study of dietary fatty acid supplementation in the treatment of adult periodontitis.</a> Prostaglandins, Leukotrienes and Essential F.A. 68 (3), 213-218 (2003).</li><li>Ferretti, A., Flanagan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithromboxane+activity+of+dietary+alpha-linolenic+acid:+a+pilot+study.">Antithromboxane activity of dietary alpha-linolenic acid: a pilot study.</a> Prostaglandins Leukot. Essent. Fatty Acids. 54 (6), 451-455 (1995).</li><li>Arpita, C., Pradeep, K. D., Chowdhury, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Fatty+Acids+and+Cholesterol+Present+in+Bile+on+Expression+of+Virulence+Factors+and+Motility+of+Vibrio+cholera.">Effect of Fatty Acids and Cholesterol Present in Bile on Expression of Virulence Factors and Motility of Vibrio cholera.</a> Infection and Immunity. 75 (4), 1946-1953 (2007).</li><li>Yamasaki, S., Asakura, M., Neogi, S. B., Hinenoya, A., Iwaoka, E., Aoki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+virulence+potential+of+Vibrio+cholerae+by+natural+compounds.">Inhibition of virulence potential of Vibrio cholerae by natural compounds.</a> Indian J. Med. Res. 133 (2), 232-239 (2011).</li><li>Hendriksen, R. S., Price, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+Genetics+of+Vibrio+cholerae+from+Nepal+in+2010:+Evidence+on+the+origin+of+the+Haitian+outbreak.">Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the origin of the Haitian outbreak.</a> mBio. 2 (4), 1-6 (2011).</li><li>Schaeffler, A., Gross, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-induced+induction+of+Toll-like+receptor-4/nuclear+factor-kappa+B+pathway+in+adipocytes+links+nutritional+signaling+with+innate+immunity.">Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-kappa B pathway in adipocytes links nutritional signaling with innate immunity.</a> Immunology. 126 (2), 233-245 (2009).</li></ol>

この研究の著者らは、この研究の結果からの経済的利害関係を持っていません。この研究のための資金はキーン大学がFTに提供されたが、FTは、現在Kingsboroughコミュニティカレッジの従業員です。

脂肪酸とコレラ毒素の濃度の提案脂肪酸は粘膜免疫を高める方法の正確なメカニズムは不明であるが、いくつかの研究は、それらの有益な効果を調べることを試みた。我々の研究は、細胞が細胞は細胞の生存に重要な影響を及ぼすことなく耐えることができるだけでなく、コレラ毒素の最大濃度に耐えることができる脂肪酸の​​最大濃度を決定する方法を提供することを...

オレイン酸、リノール酸やリノレン酸などの脂肪酸の健康上の利点は、き、文書化され続けてきた。例えば、オレイン酸は、ボディ1,2における親油性薬物の浸透を促進するのに役立ち飽和脂肪酸3に代入するとき24％冠状動脈性心臓病を低減し、X-連結されるよう副腎4などの代謝性疾患を治療するために使用される脂肪酸代謝の遺伝性疾患。 。哺乳類におけるアラキドン酸、リノール酸（オレイン酸とは異なります）のために必要な前駆体は、体内で合成されず、そのような亜麻の種子の消費などによって外部ソースを介して取得する必要がありますが5つの研究は、次のようなリノール酸のいくつかの有益な健康への影響を示しています。肌のためのアンチエイジングの特性、6抗炎症作用、大腸と前立腺癌細胞の増殖が減少7、8、肥満と戦うための能力と昇進Ofの心臓血管の健康。9 ​​リノレン酸が歯周炎症、10および変調トロンボキサンとプロスタサイクリン合成を減少させる役割を果たしています11 Arpita 12 V.に胆汁脂肪酸とコレステロールの影響を検討病原因子と運動性のコレラ菌の表現。山崎13は、赤唐辛子、およびその他の天然抽出物、メタノール抽出物から、潜在的にコレラ毒素産生を減少させることができることが示された。これは、粘膜免疫を提供する介して、例えばコレラなどの感染症の予防及び緩和上記脂肪酸（例えば、亜麻種子など）に富んだ食品の使用を検討することも考えられる。我々は、細胞増殖アッセイ、ヒト腸管上皮細胞は、セリウムに有害な影響を与えることなく許容できる脂肪酸の​​最大濃度を用いて、脂肪酸を可溶化し、決定するために検討を重ねLL生存能力。我々は、オレイン酸、リノール酸及びリノレン酸は、低濃度で細胞生存度に有益な効果を提供すると仮定したが、より高い濃度では、それらは細胞に対して毒性であること。我々はまた、コレラ毒素を可溶化し、BALB / Cマウスのマクロファージ細胞生存率の有意な減少なしに耐えることができるコレラ毒素の最大濃度を決定した。私たちも、非常に低レベルでの細胞生存率にコレラ毒素の毒性効果を仮定する。コレラ毒素を可溶化し、細胞の生存性を大幅に低下させることなく耐えることができる毒素の最大量を決定するためにそれを使用する方法は、将来の研究のための利点を提供する。例えば、上記の方法の組み合わせが脂肪酸がコレラ感染に対する粘膜免疫細胞を提供するかどうかを決定するために使用することができる。我々の知る限り、これは合理的手法が検討されていない。 私たちは、ジ我々の予備的データはオレインかどうかを判断するために後で調査で使用することができますscuss方法、リノール酸とリノレン酸がコレラ感染に対する粘膜免疫で細胞を提供する。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells/Reagent</td>
		 </tr>
		 <tr>
			<td>Mus musculus macrophages</td>
			<td>ATCC</td>
			<td>ATCC RAW 264.7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle's Medium</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal bovine serum </td>
			<td>Bio-west</td>
			<td>S0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotic/antimycotic </td>
			<td>Hyclone</td>
			<td>SV3007901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human intestinal epithelial cells </td>
			<td>ATCC</td>
			<td>ATTC CCL-241</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HybriCare media </td>
			<td>ATCC</td>
			<td>46-X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-1376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-2376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>BD Falcon 96-Well Cell Culture Plates</td>
			<td>BD Biosciences </td>
			<td>351172</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer with Dynex Revelations 4.22 software</td>
			<td>Dynex</td>
			<td>91000101</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages

非ヒトとヒトの疾患の予防や緩和に脂肪酸の積極的な役割があっと広範囲に文書化され続けてきた。これらの役割は、炎症の予防だけでなく、感染症に対する粘膜免疫を含む感染と非感染性疾患への影響が含まれています。コレラは細菌コレラ菌によって引き起こされる急性の腸の病気です。それは発展途上国で発生し、放置すれば、死に至ることができます。コレラのためのワクチンは存在するが、それらは必ずしも有効と他の予防方法が必要とされているではありません。我々はそれぞれ、マウスBALB / Cマクロファージおよびヒト腸上皮細胞を使用して、3つの脂肪酸（オレインリノール酸およびリノレン酸）とコレラ毒素の許容濃度を決定するために設定してください。我々は、濃度範囲であると無脂肪酸の特定の濃度を決定するために、上記の脂肪酸および使用される細胞増殖アッセイを可溶化ヒト腸管上皮細胞の生存率に有害トン。我々は、コレラ毒素を可溶化し、統計的にBALB / Cマクロファージにおける細胞生存率を低下させない濃度範囲およびコレラ毒素の特定の濃度を決定するためのアッセイでそれを使用した。 我々は1-5 ngの/μlの間になるように最適な脂肪酸濃度を発見し、そして、それは、コレラ毒素の治療あたり&lt;30 ngのように。このデータは、予防またはコレラ感染の緩和中の脂肪酸の保護粘膜の役割を見つけることを目指して今後の研究を支援することがあります。

脂肪酸の最適濃度の決定脂肪酸の最適な濃度は、細胞増殖に匹敵するか、または結果のばらつきが比較的低いと、コントロール細胞とを超えた最大濃度として定義される。オレイン酸の最適濃度を決定するために、リノール酸およびリノレン酸の細胞は、最初は少しずつで大きな増分以降では、各脂肪酸の濃度を変化させることで処理した。 図1は、使用する?...

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Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

我々は大幅な悪影響脂肪酸と毒素を可溶化し、細胞生存率アッセイでそれらを使用することによって、細胞の生存に影響を及ぼさなかった3脂肪酸（オレインリノール酸およびリノレン酸）とコレラ毒素の許容濃度を決定するために設定してください。

ヒト腸管上皮細胞およびBALB / cマウスのマクロファージを用いた耐容脂肪酸とコレラ毒素の濃度の測定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

determination-tolerable-fatty-acids-cholera-toxin-concentrations

Research

JoVE Journal

Immunology and Infection

9.9K ビュー.  Kingsborough Community College. 我々は大幅な悪影響脂肪酸と毒素を可溶化し、細胞生存率アッセイでそれらを使用することによって、細胞の生存に影響を及ぼさなかった3脂肪酸（オレインリノール酸およびリノレン酸）とコレラ毒素の許容濃度を決定するために設定してください。

ビデオ: Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

M細胞による細菌の取り込みを評価するパイエル板腸管ループアッセイを連結したマウスのアプリケーション

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

免疫・感染学

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

腸管上皮細胞の腸内細菌の侵入<em&gt;インビトロ</em&gt;劇的垂直拡散室モデルを用いた強化され

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳動物細胞の細菌攻撃後のストレス顆粒形成の免疫蛍光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

飽和脂肪酸は、セラミド関連マクロファージの細胞死を誘導します。

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

再生の研究のための主な胃上皮単層培養のマウスおよびひと由来

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

BALB/cマウスにおける薬物誘発性自己免疫性肝炎の誘導による発症機序の研究

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

サルモネラおよび他の病原体に対する上皮細胞保護を研究・修飾するためのヒト誘導多能性幹細胞由来腸管オルガノイドの使用

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...