この研究は、NIH / NIDDKからの助成金1K01DK079019A2（PT）によってサポートされていました。

1。マウス接種のための細菌の準備以下は無菌条件下で行わなければなりません。 <ol><li>キャップで1 17×100mmのポリプロピレンチューブを取り、新鮮なルリア培地（LB）培地3 mlを加える。オートクレーブしたチップを使用して、ひずみCP1 2の一部凍結細菌のグリセロールストックを取り、LB培地を含むチューブに培養液の少量を転送します。酸素がまだ管を入力することができるように、チューブのキャップを交換します - 文化は好気的条件下で成長する必要がある。で37箇所管℃で一晩220rpmで振盪インキュベーターのC。 </li><li>翌日、3 mlのLB培地で新しいチューブに一晩培養した培養液の5μlを転送し、37℃で一晩インキュベーター内の静的条件下で増殖</li><li> 40mlのLB培地それぞれを含むAA 50mlチューブに培養液40μlの3日目の転送で。一晩37℃のインキュベーター内で静的な場所。 </li><li>電源をオンにして、4℃に遠心分離機を設定一度遠心機が最終温度に達しており、40ミリリットルナルゲン遠心管にそれぞれ40 mlの培養液を移す。 </li><li> LB培地で音量を調整して、必要に応じて - 遠心分離機のバランスをとるためにチューブの重量を量る。 20分間6,000 rpmでチューブを軽くスピン。 </li><li>上清を慎重に除去し、穏やかに40ミリリットル滅菌PBSに細菌ペレットを中断します。 </li><li> PBSでステップ1.5を繰り返します。 </li><li>上清を吸引除去し、500μlの滅菌PBS中の細菌を一時停止。 1.5 mlのマイクロチューブに溶液を移します。懸濁液10μlを取り、氷冷したPBS 990μlに希釈します。接種に必要な量を決定するために、OD 420 nmを読み取るために分光光度計を使用しています。の適切な音量を決定するために、氷冷PBSでペレットを一時停止するには：外径420 nmの番号 （mlで）に乗り、細菌ペレット[元の推定量を減算します。 ODを420nmで = 0.545、ペレット約0.075ミリリットル。 0.545から0.075 = 0.470]。 </li><li>細菌懸濁液10μlを取り出して、ディ990μlの氷冷PBSでリュート。 OD 420 nmをお読みください。目標は1.000±0.010希釈懸濁液のOD 420 nmの値を達成することです。あなたの希釈の数がこの目標を上回っている場合、懸濁液に複数のボリュームを追加し、別の値を読み取ります。数が0.990を下回っている場合は、懸濁液をスピンダウンし、所望の読み取り値を取得するまで、外径420 nmの読みを繰り返す。 </li></ol> 2。動物の感染症マウス（5から7週齢、グループ当たり10）はジャクソン研究所（バーハーバー、メイン州）から購入した。 <ol><li>マウスはプレキシグラス室におけるイソフルラン1〜4％の吸入により麻酔し、リカンベントまで監視されています。 </li><li>一度マウスはガラスハミルトンシリンジ（1.5〜2.0センチメートルの長さ）の変更された30gの針に取り付けポリエチレン（PE10）カテーテルとカテーテル法によって細菌の点滴のために取られています。カテーテルは針のハブに挿入されます。マウスはplacですedは麻酔下で背側表面にノーズコーンを使用して維持。 </li><li>マウスの陰​​茎は陰茎尿道の入り口の潤滑に使用されている穏やかな圧力と綿棒で、潤滑剤のリベラルな量によって押し出される。 </li><li> 1×10 8個の細菌を含有するリン酸緩衝生理食塩水、10μlのカテーテル挿入に続いて麻酔したマウスの尿道内に導入される。 </li><li>細菌の付着を可能にするために、直ちに排尿を防ぐために、細菌の導入後マウスはプレキシグラス室中で15分間、麻酔状態に維持されている。 </li><li>マウスをケージに戻し、次の24時間監視されています。 </li></ol> 3。動作テストマウスは、感染前（ベースライン）へと3日、7日、14日、21日、感染後28日に試験した。呼ば痛覚過敏と触覚アロディニアは、腹部と足11,12 Rに適用von Freyフィラメントを用いて試験した後足13 egion。テストは固定時刻、​​標準的な方法論と、すべての動物の単一実験試験採用された時に施行した。グループの盲検試験は、動物モデルにおいて行動ベースの痛み試験の限界と戦うために利用された。グラム（Stoelting、米国）が撤退応答の腹部と周波数に適用されていた0.04、0.16、0.4、1.0、4.0の力を持つ5つの個々のvon Freyフィラメントを算出した。 <ol><li>順応の30分の期間の後、マウスはステンレススチールワイヤーグリッドの床が社内で行われた個々のプレキシグラス室（6×10×12 cm）で配置されます。 </li><li>呼ば痛覚過敏と触覚アロディニア5 von Freyフィラメントを用いて試験した。各フィラメントは合計10回に5秒の間刺激間隔で1〜2秒のために適用され、毛は力の昇順で試験した。 </li><li>各フィラメントは前立腺の一般付近で下腹部領域に適用されると注意が脱感作を回避したり、エフェクトを &quot;巻き上げる&quot;ために、この領域内の異なる領域を刺激するために取られた。フィラメントは、10回の合計のための刺激の間に少なくとも5秒の間隔をおいて1〜2秒のための力の低い順に適用された。 </li><li>行動は、3種類のフィラメント刺激に対する肯定的な反応であると考えています：腹部の1）鋭い引き込み、2）即時舐めるまたはフィラメント刺激の面積の傷、3）ジャンプ。 </li><li>応答周波数は、肯定応答の割合（10点、10 = 50％の例えば 5回答）として算出したとのデータが応答周波数の平均パーセンテージ±SEとして報告されました</li><li> 25以上の正の合計ベースライン応答を持つ動物は研究から除外されます。 </li><li>接触性アロディニアは0.04、0.16、0.4、1.0と4.0グラムの力とvon Freyフィラメントを用いて後肢の足の領域でテストされました。中央値50％引っ込め閾値（5）は、アップダウンを用いて評価したフィラメントが少し曲げるまでテストが0.04グラムのフィラメントで開始されたメソッドは、後肢の足底表面に垂直に印加。フィラメントは、肯定的な反応が観察されるまで、昇順で試験した。フィラメントへの積極的な応答は試験足の足や舐めるのシャープ撤退のいずれかとして定義されていました。肯定応答が記録されたときに次の弱いフィラメントが適用され、否定応答が観察された場合は、次の強いフィラメントを適用した。 </li><li>記載された実験と方法論は、ノースウェスタン大学の動物のケアと使用委員会によって審査され、承認されています。 </li></ol>

<ol>
	<li>Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+uropathogenic+Escherichia+coli.">Molecular epidemiology of uropathogenic Escherichia coli.</a> Journal of infection and chemotherapy official journal of the Japan Society of Chemotherapy. 13, 68-73 (2007).</li><li>Rudick, C. N., 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=Uropathogenic+Escherichia+coli+induces+chronic+pelvic+pain.">Uropathogenic Escherichia coli induces chronic pelvic pain.</a> Infect. Immun. 79, 628-635 (2011).</li><li>Pontari, M. A., Ruggieri, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+in+prostatitis/chronic+pelvic+pain+syndrome.">Mechanisms in prostatitis/chronic pelvic pain syndrome.</a> The Journal of urology. 172, 839-845 (2004).</li><li>Schaeffer, 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=Clinical+practice.+Chronic+prostatitis+and+the+chronic+pelvic+pain+syndrome.">Clinical practice. Chronic prostatitis and the chronic pelvic pain syndrome.</a> The New England journal of medicine. 355, 1690-1698 (2006).</li><li>Rudick, C. N., Schaeffer, A. J., Thumbikat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+induces+chronic+pelvic+pain.+American+journal+of+physiology.">Experimental autoimmune prostatitis induces chronic pelvic pain. American journal of physiology.</a> Regulatory, integrative and comparative physiology. 294, R1268-R1275 (2008).</li><li>Elkahwaji, J. E., Ott, C. J., Janda, L. M., Hopkins, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+acute+bacterial+prostatitis+in+genetically+distinct+inbred+strains.">Mouse model for acute bacterial prostatitis in genetically distinct inbred strains.</a> Urology. 66, 883-887 (2005).</li><li>Hultgren, S. J., Porter, T. N., Schaeffer, A. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+type+1+pili+and+effects+of+phase+variation+on+lower+urinary+tract+infections+produced+by+Escherichia+coli.">Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.</a> Infect. Immun. 50, 370-377 (1985).</li><li>Jarrell, J., Giamberardino, M. A., Robert, M., Nasr-Esfahani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bedside+testing+for+chronic+pelvic+pain:+discriminating+visceral+from+somatic+pain.">Bedside testing for chronic pelvic pain: discriminating visceral from somatic pain.</a> Pain research and treatment. 2011, 692102 (2011).</li><li>Jarrell, J. <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+Cutaneous+Allodynia+in+Association+with+Chronic+Pelvic+Pain.">Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain.</a> J. Vis. Exp. (28), e1232 (2009).</li><li>Giamberardino, M. 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=Viscero-visceral+hyperalgesia:+characterization+in+different+clinical+models.">Viscero-visceral hyperalgesia: characterization in different clinical models.</a> Pain. 151, 307-322 (2010).</li><li>Laird, J. M., Martinez-Caro, L., Garcia-Nicas, E., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+visceral+pain+and+referred+hyperalgesia+in+the+mouse.">A new model of visceral pain and referred hyperalgesia in the mouse.</a> Pain. 92, 335-342 (2001).</li><li>Laird, J. M., Souslova, V., Wood, J. N., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+visceral+pain+and+referred+hyperalgesia+in+Nav1.8+(SNS/PN3)-null+mice.">Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice.</a> J. Neurosci. 22, 8352-8356 (2002).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of neuroscience. 53, 55-63 (1994).</li><li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect. Immun. 55, 373-380 (1987).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> The Prostate. 72, 307-317 (2012).</li><li>Rivero, V. E., Cailleau, C., Depiante-Depaoli, M., Riera, C. M., Carnaud, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-obese+diabetic+(NOD)+mice+are+genetically+susceptible+to+experimental+autoimmune+prostatitis+(EAP).">Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP).</a> Journal of autoimmunity. 11, 603-610 (1998).</li><li>Pakarainen, T., Zhang, F. P., Makela, S., Poutanen, M., Huhtaniemi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+replacement+therapy+induces+spermatogenesis+and+partially+restores+fertility+in+luteinizing+hormone+receptor+knockout+mice.">Testosterone replacement therapy induces spermatogenesis and partially restores fertility in luteinizing hormone receptor knockout mice.</a> Endocrinology. 146, 596-606 (2005).</li><li>Taguchi, O., Kojima, A., Nishizuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+after+neonatal+thymectomy+in+the+mouse.">Experimental autoimmune prostatitis after neonatal thymectomy in the mouse.</a> Clinical and experimental immunology. 60, 123-129 (1985).</li></ol>

特別な利害関係は宣言されません。

UPECによるマウス前立腺の感染における生体内細菌性前立腺炎の病態に関与している可能性のある事象のモデリング、CP / CPPSまたは慢性炎症における素因イベントなどが可能になります。方法は女性の尿路感染症7,14でUPECモデルに文学の大きな体によって細菌準備や点滴の引き分けに記載。モデルは、潜在的なワクチン候補と免疫調節のメカニズムをテストする、病因を研究す?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>培養管</td><td> BDファルコン</td><td> 352059 </td></tr><tr><td> LBブロスミラー</td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td>ナルゲン遠心管</td><td>サーモ</td><td> 3118-0050 </td></tr><tr><td>イソフルレン</td><td>バトラーシャイン</td><td> NDC 11695-6776-1 </td></tr><tr><td>カテーテルニードル30G </td><td>ハミルトン</td><td> 91030 </td></tr><tr><td> PE10チューブ</td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> von Freyフィラメント</td><td> Stoelting </td><td> 58025から31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

尿路病原性大腸菌 （UPEC）は、尿路感染症や細菌性前立腺炎1に重要な役割を果たしている病原体である。我々は最近、UPECは、慢性骨盤痛2、慢性前立腺炎/慢性骨盤痛症候群（CP / CPPS）3,4の機能の開始に重要な役割を持っていることが示されている。臨床的に関連UPECによる前立腺の感染は、組織の損傷および自己免疫のメカニズム5の開始を含むことができるメカニズムを介して慢性痛を開始し、確立することができます。 前立腺内UPECの病因を理解するための課題は、操作に前立腺の相対的な到達不能です。我々は、それによって前立腺の上行性感染を確立雄マウスにUPECの臨床株を提供するために前述の尿道感染症法6を利用した 。ここでは、BAを標準化するための私達のプロトコルを記述cterial接種7と同様に細菌の点滴のために麻酔雄マウスをcatheterizingするための手順。 CP / CPPSは主に触覚アロディニア4の存在によって特徴付けられる。動作テストは呼ば内臓痛8-10起因する皮膚の痛覚過敏の概念に基づいていた。内臓組織における過敏焦点は通常非痛み刺激（アロディニア）に誇張された応答を可能にする皮膚の疼痛閾値を低減します。基盤となる内臓痛の強度に伴って増加する傾向がある異常反応で皮膚の結果に垂直力の応用。我々はUPEC細菌を持つ単一の感染に応答して時間をかけて接触性アロディニアを定量化するためのvon Frey繊維を利用したNOD / ShiLtJマウスでの方法論を記述する。

<html>我々は、細菌感染時に慢性的な痛みの有無をNOD / ShiLtjおよびC57BL/6J雄マウスを調べた。雄性マウス（5〜7週齢）尿道（ 図1）に生理食塩水や細菌を注入した。生理食塩水をたらしC57BL/6JマウスとUPEC感染C57BL/6Jマウスのvon Freyフィラメントと骨盤領域の機械的刺激は、実験の28日間コース（ 図1）の間に変更されなかった応答周波数が得られた。これとは対照的に、UPEC...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

前立腺の感染は、慢性前立腺炎における骨盤痛を媒介する要因かもしれません。我々は時間をかけて、マウスにおける接触性アロディニアを測定するための雄マウスと方法論の尿道への細菌の点滴、標準化された細菌の接種物の調製のための手順を記述している。

細菌性前立腺炎のマウスモデルにおける接触性アロディニアの測定

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

measurement-tactile-allodynia-murine-model-bacterial

Research

JoVE Journal

Immunology and Infection

15.7K ビュー.  Northwestern University Feinberg School of Medicine. 前立腺の感染は、慢性前立腺炎における骨盤痛を媒介する要因かもしれません。我々は時間をかけて、マウスにおける接触性アロディニアを測定するための雄マウスと方法論の尿道への細菌の点滴、標準化された細菌の接種物の調製のための手順を記述している。

ビデオ: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

生体内でマウスにおける活性酸素種のイメージング創モデル

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

下流の単一細胞分析のため尿路感染症のマウスモデルから生成された単一細胞内細菌群集の分離

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次細菌性肺炎のマウスモデルに対する精密病原体送達と回復システム

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部大動脈瘤マウスモデルにおける脈拍伝播速度、分解性およびひずみの測定

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

蛍光系抗生物質プローブを用いた細菌耐性の可視化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

グラム陰性細菌敗血症の新生児画像モデル

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...