私たちは、動物のケアとモニタリングのために、S.、本研究と米国農務省で使用されるカラスを提供するためのヴェルナー、APHIS、WS、NWRC動物管理スタッフに感謝したいと思います。言及または製品を使用するには、米国農務省の承認を意味するものではありません。この研究のための資金は、米国農務省、APHIS、獣医サービスによって提供された。

我々のプロトコルは、我々が以前に7を発表した1から採用されている。動物に関わるすべての手続きは、米国農務省（USDA）、動植物衛生検査局（APHIS）、野生動物サービス（WS）、国立野生生物研究センター（NWRC）の施設内動物管理使用委員会によって承認された。 1。カラスGavaging <ol><li>アメリカのカラスの消化管を通して「疑似脳物質 &#39;の通過時間を見積もります。 <ol><li>調理の5ミリリットルを混合し、強制経口投与針2（ 図1）を用いた青色色素および強制経口投与1カラスと全卵をスクランブル。 </li><li>青/緑に染色された糞はもはや排泄されるまで、30分ごとにカラスないことを確認してください。 </li></ol></li><li>感染していないと末期RML（チャンドラー株）に感染したC57BL / 6マウスの脳を入手します。 </li><li> 1Xと弾丸ブレンダーホモジナイザーの通常20％重量/容量、感染したマウス脳乳剤を生成リン酸緩衝生理食塩水（PBS）とガラスビーズを、次いで、より大きな粒子状物質を除去するために1分間3,000×gで遠心する。上清を除去し、無菌のPBS中の10％重量/体積を生成するために、1×PBS等容量の希釈した。必要になるまで-80℃で凍結する。 </li><li>前胃管栄養への17時間は、カラスのペンから、水食料を削除ではなく。 </li><li>無作為に処置群にカラスを割り当て、強制経口強制栄養針2を使用して正常または感染したいずれかのマウス脳ホモジネート5mlでそれぞれカラス。 </li><li>転送は、個々のケージにカラスや4時間後、胃管栄養法（ 図2）のために、各ケージ内のすべての糞を集め、プール。 </li><li>均一な組織が得られるまでガラスビーズと弾丸ブレンダーホモジナイザーの各カラスのためにプールされた糞を均質化する。 *カラスの糞は主に液体であり、容易に混合することができる。 </li><li>各カラスのために、10ミリリットルの全容量のために9.5ミリリットルの1X PBSに糞便ホモジネート500μlの希釈する。 </li><li>糞便homogenaを遠心分離1400×gで15分間のTE、上清を抽出します。 </li><li>二微生物感染の脅威を最小限ホモジネート、100μlの1〜100単位の液/ mlペニシリンおよび100μg/ mlのストレプトマイシン（Invitrogen社、NY）を用いて上清を治療するためにその後減少させるために20分間室温でUV光下に置くウイルスおよび細菌汚染のリスク。 UV露光した後、残りの微生物の膜を破壊するために30秒間70を設定することで、3000 Mpは超音波処理における超音波処理のサンプル。 </li></ol> 2。マウス接種<ol><li>無作為に以下の処置群（ 表1）にマウスを割り当てる。 <ol><li>グループ1 - 陽性の処置マウスは経口5ミリリットル感染マウスの脳を強制飼養カラスからの糞便ホモジネート1mlで腹腔内（IP）接種した。 </li><li>グループ2 - 負の処置マウスは、経口5ミリリットル、通常のマウスの脳を強制飼養カラスからの糞便ホモジネート1mlのIPアドレスを接種した。 </li><li> infecteを希釈1×PBSで1:100重量/容量のグループ3および4のdおよび正常マウス脳ホモジネート。 </li><li>第3群 - 陽性対照マウスは、感染マウス脳ホモジネート1mlのIPを接種した。 </li><li>グループ4 - 陰性対照マウスは正常マウス脳ホモジネート1mlのIPを接種した。 </li></ol></li></ol><table border="1"><tbody><tr><td></td><td>治療グループ</td><td>動物の数</td></tr><tr><td>グループ1 </td><td>スクレイピー+クロウ糞</td><td> 100 </td></tr><tr><td>グループ2 </td><td>スクレイピー - クロウ糞</td><td> 25 </td></tr><tr><td>グループ3 </td><td>スクレイピー+マウス脳</td><td> 10 </td></tr><tr><td>グループ4 </td><td>スクレイピー - マウス脳</td><td> 5 </td></tr></tbody></table> 表1スクレイピー接種の状況（負正+、 - ）と動物の数が7を使用していました。 <ol start="2"><li>腹腔内にマウスに接種： <ol><li>優しく首筋の親指と人差し指で背面の首の毛皮によるマウスとは、腹側を露出させ回転させます。 </li><li>ヘッドがやや低いので、マウスの後端部を上昇させる。 </li><li>針ロック注射器を用いて仙腸関節にCM前部皮膚を通して25 G針1センチ、正中線の左右1cmの1-2を挿入します。 </li><li> 1mlを、マウスの体腔内に徐々に接種注入する。 </li></ol></li></ol> 3。マウスの監視<ol><li>彼らはマウススクレイピーの臨床徴候を表現まで毎日マウスを監視します。臨床症状は含まれる：脊柱後、運動失調、硬い尾、グルーミング、衰弱、および嗜眠の欠如を。 </li><li> 0目に見える=なし、1 =中等度、および2 =が激しいところ目に見える兆候は、明らかであるとき6臨床徴候のそれぞれにマウスを獲得。 </li><li> 3日間、または365日後に接種（DPI）で連続6それぞれの記号の1日の総得点は1日の≥8に到達したときに、マウスを安楽死させる≥。 </li><li>収穫脳直後のeuthan-70℃でのアジアと店舗</li><li> 、スクレイピー診断を確認、次のように希釈されたプロテイナーゼK溶液（PK）の50μg/ mlのの3μlの脳サンプルを消化するために：3.1。 μlのPK、500mMのEDTA12.5μlの、pH8で、45℃で30分間、1×PBSの109.39μlを、次いで、8μlのローディング緩衝液を添加し、95℃で5分間、サンプルをインキュベートすることによってPKを不活性化する。 12％SDS-PAGEゲル電気泳動させると、室温で1時間、PBS中の0.2％のTween 20でイモビロンPVDF膜を5％脱脂乳でブロックへの転送上に負荷サンプル。次いで、室温で1時間のSuperblock中で希釈西洋ワサビペルオキシダーゼ、Bar224にコンジュゲート抗プリオンモノクローナル抗体でプローブする。 PBS-0.2％Tween 20で1時間、膜を洗浄します。可視化するために、G-ボックスゲルドキュメンテーションシステムに化学発光基質およびイメージを使用してウエスタンブロット5分間インキュベートする。 </li></ol>

<ol>
	<li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216 (4542), 136-144 (1982).</li><li>Miller, M. W., Williams, E. S., 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=Epizootiology+of+chronic+wasting+disease+in+free-ranging+cervids+in+Colorado+and+Wyoming.">Epizootiology of chronic wasting disease in free-ranging cervids in Colorado and Wyoming.</a> Journal of Wildlife Diseases. 36 (4), 676-690 (2000).</li><li>Miller, M. W., Williams, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+prion+transmission+in+mule+deer.">Horizontal prion transmission in mule deer.</a> Nature. 425 (6953), 35-36 (2003).</li><li>Sigurdson, C. J., Adriano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Wasting+Disease.">Chronic Wasting Disease.</a> Biochimica et Biophysica Acta. 1772 (6), 610-618 (2007).</li><li>Miller, M. W., Williams, E. S., Hobbs, N. T., Wolfe, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+sources+of+prion+transmission+in+mule+deer.">Environmental sources of prion transmission in mule deer.</a> Emerging Infectious Diseases. 10 (6), 1003-1006 (2004).</li><li>Mathiason, C. K., Hays, S. A., 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=Infectious+prions+in+pre-clinical+deer+and+transmission+of+chronic+wasting+disease+solely+by+environmental+exposure.">Infectious prions in pre-clinical deer and transmission of chronic wasting disease solely by environmental exposure.</a> PLoS ONE. 4 (6), e5916 (2009).</li><li>VerCauteren, K. C., Pilon, J. L., Nash, P. B., Phillips, G. E., Fischer, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+remains+infectious+after+passage+through+digestive+system+of+American+crows+(Corvus+crachyrhunchos).">Prion remains infectious after passage through digestive system of American crows (Corvus crachyrhunchos).</a> PLoS ONE. 7 (10), e45774 (2012).</li><li>Imran, M., Mahmood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+animal+prion+diseases.">An overview of animal prion diseases.</a> Virology Journal. 8 (493), (2011).</li><li>Watts, J. C., Balachandran, A., Westaway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+universe+of+prion+disease.">The expanding universe of prion disease.</a> PLoS PATHOGENS. 2 (3), e26 (2006).</li><li>Bruce, M. E., 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=Transmissions+to+mice+indicate+that+'new+variant'+CJD+is+caused+by+the+BSE+agent.">Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent.</a> Nature. 389 (6650), 498-501 (1997).</li><li>Ryder, S., Dexter, G., Bellworty, S., Tongue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+lateral+transmission+of+scrapie+between+sheep+kept+under+natural+conditions+using+lymphoid+tissue+biopsy.">Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy.</a> Research in Veterinary Science. 76 (2004), 211-217 (2004).</li><li>Collinge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+prion+zoonoses.">The risk of prion zoonoses.</a> Science. 335 (6067), 411-413 (2012).</li><li>Beringue, V., Vilotte, J. L., Laude, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+agent+diversity+and+species+barrier.">Prion agent diversity and species barrier.</a> Veterinary Research. 39 (47), (2008).</li><li>Harrington, R. D., Baszler, T., 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=A+species+barrier+limits+transmission+of+chronic+wasting+disease+to+mink+(Mustela+vison).">A species barrier limits transmission of chronic wasting disease to mink (Mustela vison).</a> The Journal of General Virology. 89 (4), 1086-1096 (2008).</li><li>Wisniewski, H. M., Sigurdarson, S., Rubenstein, R., Kascsak, R. J., Carp, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mites+as+vectors+for+scrapie.">Mites as vectors for scrapie.</a> Lancet. 347 (9008), 1114 (1996).</li><li>Post, K., Riesner, D., Walldorf, V., Mehlhorn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fly+larvae+and+pupae+as+vectors+for+scrapie.">Fly larvae and pupae as vectors for scrapie.</a> Lancet. 354 (9194), 1969-1970 (1999).</li><li>Matthews, D., Cooke, B. 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+potential+for+transmissible+spongiform+encephalopathies+in+non-ruminant+livestock+and+fish.">The potential for transmissible spongiform encephalopathies in non-ruminant livestock and fish.</a> Revue Scientifique Et Technique-Office International Des Epizooties. 22 (1), 283-296 (2003).</li><li>Jennelle, C. S., Samuel, M. D., Nolden, C. A., Berkley EA, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deer+carcass+decomposition+and+potential+scavenger+exposure+to+chronic+wasting+disease.">Deer carcass decomposition and potential scavenger exposure to chronic wasting disease.</a> Journal of Wildlife Management. 73 (5), 655-662 (2009).</li><li>Scherbel, C., Pichner, R., 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=Degradation+of+scrapie+associated+prion+protein+(PrPSc)+by+the+gastrointestinal+microbiota+of+cattle.">Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle.</a> Veterinary Research. 37 (5), 695-703 (2006).</li><li>Jeffrey, M., Gonza&#225;lez, L., 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=Transportation+of+prion+protein+across+the+intestinal+mucosa+of+scrapie+susceptible+and+scrapie-resistant+sheep.">Transportation of prion protein across the intestinal mucosa of scrapie susceptible and scrapie-resistant sheep.</a> Journal of Pathology. 209 (1), 4-14 (2006).</li><li>Nicholson, E. M., Richt, J. A., Rasmussen, M. A., Hamir, A. N., Lebepe-Mazur, S., Horst, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+sheep+scrapie+brain+homogenate+to+rumen-simulating+conditions+does+not+result+in+a+reduction+of+PrP(Sc)+levels.">Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels.</a> Letters in Applied Microbiology. 44 (6), 631-636 (2007).</li><li>Motes C, M. a. l. u. q. u. e. r. d. e., Grassi, J., 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=Excretion+of+BSE+and+scrapie+prions+in+stools+from+murine+models.">Excretion of BSE and scrapie prions in stools from murine models.</a> Veterinary Microbiology. 131 (1-2), 205-211 (2008).</li><li>Kruger, D., Thomzig, A., Lenz, G., Kampf, K., McBride, P., Beekes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faecal+shedding,+alimentary+clearance+and+intestinal+spread+of+prions+in+hamsters+fed+with+scrapie.">Faecal shedding, alimentary clearance and intestinal spread of prions in hamsters fed with scrapie.</a> Veterinary Research. 40 (1), 4 (2009).</li><li>Mathiason, C. K., Nalls, A. V., 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=Susceptibility+of+domestic+cats+to+chronic+wasting+disease.">Susceptibility of domestic cats to chronic wasting disease.</a> Journal of Virology. 87 (4), 1947-1956 (2013).</li><li>Bjorndal, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexibility+of+digestive+responses+in+two+generalist+herbivores,+the+tortoises+Geochelone+carbonaria+and+Geochelone+denticulate.">Flexibility of digestive responses in two generalist herbivores, the tortoises Geochelone carbonaria and Geochelone denticulate.</a> Oecologia. 78 (3), 317-321 (1989).</li><li>Clark, R. G., Gentle, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+grain+passage+time+in+captive+mallards.">Estimates of grain passage time in captive mallards.</a> Canadian Journal of Zoology. 68 (11), 2275-2279 (1990).</li><li>Dierenfeld, E. S., Koontz, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feed+intake,+digestion+and+passage+of+proboscis+monkey+(Nasalis+larvatus)+in+captivity.">Feed intake, digestion and passage of proboscis monkey (Nasalis larvatus) in captivity.</a> Primates. 33 (3), 399-405 (1992).</li><li>Thompson, A. K., Samuel, M. D., Van Deelen, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+feeding+strategies+and+potential+disease+transmission+in+Wisconsin+white-tailed+deer.">Alternative feeding strategies and potential disease transmission in Wisconsin white-tailed deer.</a> Journal of Wildlife Management. 72 (2), 416-421 (2008).</li><li>Pulford, B., Spraker, T. A., 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=Detection+of+PrPCWD+in+feces+from+naturally+exposed+Rocky+Mountain+elk+(Cervus+elaphus+nelsoni)+using+protein+misfolding+cyclic+amplification.">Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification.</a> Journal of Wildlife Diseases. 48 (2), 425-433 (2012).</li><li>Hicks, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guano+deposition+in+an+Oklahoma+crow+roost.">Guano deposition in an Oklahoma crow roost.</a> Condor. 81 (3), 247-250 (1979).</li><li>Aldous, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Winter+habits+of+crows+in+Oklahoma.">Winter habits of crows in Oklahoma.</a> Journal of Wildlife Management. 73 (4), 290-295 (1944).</li></ol>

利害の対立が宣言されていません。

私たちは、カラスの消化器系を通じてプリオンのRES物質の通過を文書化するための手順を示しています。私たちは、カラスが無病の地域へのプリオン解像度素材を転位する能力を持っているかどうかを判断するために、従来の方法を使用していました。他はそれを排除することができなかった、どちらも19〜21と齧歯類22,23消化液を反芻するプリオン解像度...

伝達性海綿状脳症（TSE）は、野生生物に影響を与える致命的な感染性の神経変性疾患、家畜、そしてヒトである。 TSE疾患の感染性病原体はプリオンタンパク質1のミスフォールドまたは病原性アイソフォーム（PRP RES）であるように思われる。ヒツジおよびヤギにおけるスクレイピー;、牛海綿状脳症（動物TSE疾患はラバ鹿（ ミュールジカ ）、オジロジカ（ オジロジカ ）、エルク（ アカシカ ）、およびムース（ ヘラジカ ）の慢性消耗病（CWD）が含まれ国内の牛のBSE）;養殖ミンクにおける伝染性ミンク脳症、ネコのネコ海綿状脳症、ヒト以外の霊長類8にし、海綿状脳症、エキゾチックな動物園のエキゾチックな有蹄動物海綿状脳症は、家族ウシ科の反芻。単一のヒトTSE病、変異型クロイツフェルト·ヤコブ病は、稀で、 解像度のPrP-contaminを消費することによって取得されると考えられているated食品9。汚染された牛肉が10消費された場合も、BSEはヒトに感染することができます。すべてのTSE疾患のうち、スクレイピーおよびCWDは、動物から動物2,3,11にだけでなく、環境源4-6からのものであると推定される自律的な流行と感染のソースでのみ2である。研究では、ほとんどのTSE疾患は臨床徴候が発現する前にプリオン解像度素材の自然曝露イベントからの注目すべき拡張潜伏期間が必要2-4,6,8、見かけ種の壁を最小限に、しかし、種間伝播12月14日の可能性を排除しないことを示唆している。 感染性プリオン（PRP RES）材料の普及のためのメカニズムを識別することはTSE疾患が風景を越えて移動する方法についての質問に答えるために非常に重要です。実験的研究は、昆虫15,16、家禽および豚17、およびアメリカのカラス（ カラスのブラチことが示唆されているhyrhynchos）7,18は、PrP解像度材料の受動的なキャリアまたは分散機である。カラスの消化器系を通じてプリオンのRES物質の通過は最近、TSE疾患7の分散でプレイしてもよい役割を示す、文書化されています。これらの結果は、カラス、スカベンジャーは、遭遇する可能性があること、それがもっともらしい作る消費し、無病領域に、糞の堆積により感染性物質を輸送する。 我々はここに証明する手続きは、カラスの消化システムを介してプリオンのRES物質の通過を記録するために使用されたと大いに関連する今後の研究の他のスカベンジャーと肉食種特異的なモデルに、これらの方法の適用を容易にします。本研究では、従来の方法は、プリオン解像度素材の広がりと全体的な重荷に貢献できるプリオン解像度素材を、人身売買の型破りな手段を調査するために使用された。

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

感染性プリオン（PRP RES）材料は、おそらく致命的な神経変性伝達性 ​​海綿状脳症（TSE）の病気1の原因である。例えば、慢性消耗病（CWD）などのTSE疾患の送信は、動物から動物への2,3並びに環境源4-6からであると推定される。スカベンジャーと肉食動物は、CWDに汚染された死肉の摂取と排泄を通してプリオン解像度材料を移動させる可能性を持っている。最近の研究はアメリカのカラスの消化器系（ アメリカガラス ）、一般的な北アメリカのスカベンジャー7を通してプリオン解像度素材の通過を文書化しました。 我々はアメリカのカラスを通じてプリオン解像度素材の通過を記録するために使用される手順について説明します。カラスはRML - ひずみマウス適応スクレイピーで強制経口投与し、それらの糞は4時間後に強制経口投与を収集した。カラスの糞をし、その後プールし、腹腔内に注射したC57BL / 6マウス。彼らはマウススクレイピーの臨床徴候を表明し、その後、安楽死させたまではマウスを毎日モニターした。無症候性マウスは365日後に接種するまで監視した。ウェスタンブロット解析は、疾患状態を確認するために行った。結果は、プリオンがカラスの消化器系を通って移動した後に感染性のままであり、試験マウスにおいて疾患を引き起こす、糞便中に存在することが明らかになった。

使用手順は、カラスの消化器系は、スクレイピー脳ホモジネート7の強制経口投与した後にPrPの解像度感染性4時間を排除しないことを示している。プリオン解像度材料で強制経口投与されたすべての20カラスはその後、マウスに糞を経由したPrP 解像度素材を送信した。ウェスタンブロット分析によって完了した罹患したマウスは、臨床マウススクレイピー徴候...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

スカベンジャーは無病領域に糞中の感染伝達性海綿状脳症プリオンを移動させる可能性を持っている。私たちはマウス適応スクレイピープリオンはアメリカのカラスの消化管（ アメリカガラス ）、死んだ動物の共通の消費者も通過した後、感染のままかどうかを判断するために使用される詳細な方法。

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Research

JoVE Journal

Immunology and Infection

11.0K ビュー.  USDA. スカベンジャーは無病領域に糞中の感染伝達性海綿状脳症プリオンを移動させる可能性を持っている。私たちはマウス適応スクレイピープリオンはアメリカのカラスの消化管（ アメリカガラス ）、死んだ動物の共通の消費者も通過した後、感染のままかどうかを判断するために使用される詳細な方法。 

ビデオ: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

のための負の選択マーカーの開発赤痢アメーバ</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

免疫・感染学

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

伝播と緑色蛍光タンパク質を発現Maedi -ビスナウイルスの感染分子クローンの検出

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

プリオンのタンパク質のミスフォールディングサイクリック増幅

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

プラークアッセイを通じてウイルス濃度の測定：伝統と新しいオーバーレイシステムの使用

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

1.バイオセーフティレベル4戦士研究所スイートの入り口と出口の手順：（A）BSL-4実験室での安全上のご注意と操作手順

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

（A）BSL-4実験室での安全上のご注意と操作手順：2.一般プラクティス

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

（A）BSL-4実験室での安全上のご注意と操作手順：4.メディカルイメージング手順

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

（A）BSL-4実験室での安全上のご注意と操作手順：3.空中生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

ジカウイルス感染細胞培養系と<em&gt;インビトロ</emインターフェロンの&gt;予防効果

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...