私は中の助けを実験計画を概念と素材を支援、治療レジメンと剖検を確立する助けA. Pessier分析し、MWHチャットフィールド、MJ Robak、K.タスカーとDレンゲルヘルプCLリチャーズ-Zawackiに感謝したいと思います研究室や撮影中にそのスペースを使用するためのサポートとタロンガ動物園でのM·マクファデンとP.ハーロウ。プロジェクトは、部分的に、LA Brannellyに授与ルイジアナ州環境教育委員会の許可を経てライフ·テクノロジーズによって野生生物水産ルイジアナ州省によって資金を供給された。

倫理承認：本研究は、そのメソッドは、チューレーン大学の動物への使用、およびケア委員会（。プロトコルなし0407）によって承認された。 1。 Bdの感染症の検査<ol><li> BD用拭き取り<ol><li>無菌綿棒で綿棒動物ventrumに5回、各太もも、それぞれの側、および45ストロークの合計各肢に5回。拭き取りは両生類の皮膚にBD用のサンプリングの非侵襲的な方法である。 </li><li>静かに、DNAの最大量を綿棒で収集されていることを確認するために、ストロークの際との間で綿棒を回転させます。 </li><li> -20℃で1.5ミリリットルマイクロチューブに保管して綿棒</li></ol></li><li> DNA抽出および定量PCRアッセイ<ol><li>動物の組織のための取扱説明書に従って、市販のDNA単離キットを使用してスワブからのゲノムDNAを抽出する。 </li><li>ボイル以下の定量的リアルタイムPCR（定量PCR）を用いて抽出したDNAを分析する<eM&gt;ら17、以下の変更を加えて。： </li><li> DNA抽出サンプルを二重脱イオン水で1:10に希釈する。 </li><li> PCR阻害を防止するために、ウシ血清アルブミン（BSA）0.7μlを添加する。 </li><li> PCR阻害が結果に影響を与えていなかったことを確認するために、各サンプル中の内部陽性対照を含める。 </li><li>正と負の制御と遊走子（感染症）の負荷を推定するための希釈一連の標準を使用して実行サンプル。 </li></ol></li></ol> 2。接種<ol><li>文化のBd <ol><li> （40分間121°C）オートクレーブ処理し、冷却させ、1 Lの逆浸透水または脱イオン水に、10gのトリプトンを添加することによってトリプトンブロスを調製する。一度23℃に冷却し、1ミリリットルのペニシリン - ストレプトマイシンを追加します。 </li><li> Bdの株 （J. LongcoreからJEL411、PhyllomedusaキツネザルからGuabal、パナマから単離された）、23℃で7日間、リプトンブロスで成長</li></ol></li><li> Bを転送リプトンと寒天平板にロス文化。 <ol><li>プレートを作るために、1Lの逆浸透または脱イオン水、オートクレーブ（40分間121℃）に10グラムのトリプトンと10グラム寒天を加える。 </li><li>オートクレーブ処理した混合物を、層流フードでは、タッチするのに十分なクールですが、それは凝固する前に、ペトリ皿に入れ、フル1/4-1/3たら。 </li><li>寒天が完全に凝固し、冷却したときに、各プレートに0.5ミリリットルのBdブロスを追加し、プレートの周りに均等に分散。乾燥することができます。 </li><li>プレートは 、BD の遊走子、パラフィルムでシールプレートを接種したら。 </li><li>プレートは5〜7日、23℃でインキュベートすることができます。接種前に実行可能性を確保するために運動を遊走子確認してください。 </li></ol></li><li>遊走子接種ソリューションの洪水プレート<ol><li> Bdのインキュベーションおよび成長の5〜7日後に、エージング水道水5mlで各プレートをあふれさせると遊走子を10分間メディアを入力することができます。 </li><li>遊走子suspeを注ぐポータブル容器にnsion。 </li><li>血球計数器で遊走子濃度を推定。 </li><li>高齢者水道水で3ミリリットル当たり1×10 6の濃度に混合物を希釈。 </li></ol></li><li>動物に接種<ol><li>個々の50ミリリットルの容器では、そのventrum上で3ミリリットルの接種液を注ぐことにより、各動物に接種。余分な接種物接種用容器の底に滴下することができます。 </li><li>各動物には感染を確実にするために24時間接種容器内に残ります。 </li><li>接種の24時間後に、個々の消毒をterrariaに動物を戻す。 </li><li>感染は2週間用にビルドすることができます。綿棒と定量PCRの結果の感染を確認し、治療を開始。 </li><li> BD-ネガティブコントロールの場合、5ミリリットルと洪水BD-フリーの寒天プレートは、上記と同じ方法を使用して、水道水、かつモック接種動物歳。 </li></ol></li></ol> 3。治療計画<ol><li> PREPA処理槽の再<ol><li> 1Lの両生類リンゲル液（ - 最小有効治療濃度0.0025％のイトラコナゾール）に2.50ミリリットルの水イトラコナゾール（1％イトラコナゾール内用液）を追加します。 <ol><li>両生類リンゲル溶液レシピ：1 Lの逆浸透水または脱イオン水、6.6グラムの塩化ナトリウム、0.15グラムの塩化カリウム、0.15グラムの塩化カルシウム、および0.2グラムの重炭酸ナトリウム。オートクレーブ溶液（40分間121°C）し、室温まで冷却する。 </li><li> 0.01％イトラコナゾール処理濃度については、1Lの両生類リンゲル液に10.00ミリリットル水イトラコナゾール（1％イトラコナゾール内用液）を追加</li><li> 0.005％イトラコナゾール処理濃度については、1Lの両生類リンゲル液に5.00ミリリットルの水イトラコナゾール（1％イトラコナゾール内用液）を追加します。 </li></ol></li><li> BD-陽性およびBD-ネガティブコントロールの場合、無イトラコナゾールと両生類のリンゲル液中の動物を入浴。 </li></ol></li><li>治療&lt;OL&gt; <li>処理液の35ミリリットルで消毒50ミリリットルの容器内の個々の両生類を配置します。 </li><li>動物が完全に静かにコンテナを回転させることによって5分間カバーされていることを確認してください。 </li><li>消毒しterrariaに動物を返します。 6日間繰り返します。 </li></ol></li><li>治療の成功を確実にする<ol><li>日間の治療上のBd感染の試験動物が始まる。 </li><li>テスト1週間の治療が終了した後のBd感染の動物、および感染がクリアされたことを確認するために4週間、週に一度。 </li></ol></li></ol>バイオハザードとして4、BD <ol><li>個々のterrariaを毎週消毒。 <ol><li> 10％の漂白剤溶液浴中terrariaと治療コンテナを浴びる。 </li><li>流水で二回、各漂白コンテナを洗浄します。 </li><li>容器は再使用する前に24時間乾燥させます。 </li></ol></li><li>クリーンニトリル手袋を着用し、交差汚染を防ぐために、動物を取り扱う際Bdは病原体の。各動物の取り扱いの間に手袋を変更します。 </li><li> 10％の漂白剤の解決策をもたらすことにより、廃棄前に、すべての液体廃棄物を消毒する。 </li><li>可能性のある廃棄前にBdのにさらされているすべての固形廃棄物をオートクレーブ。 </li></ol>

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著者は、競合する経済的利益を宣言していません。

このプロトコルを用いて、我々は、安全かつ効果的に両生類でBdのためのイトラコナゾール処置の治療投与量と時間を削減することができた。最低治療用量および時間（6日間0.0025％）は無症状感染Iについて治療の成功だったnebulifer。低用量と治療時間の両方の有効性は、両生類のすべての種と年齢のクラスに使用すべき安全かつ効果的な治療法はまだありませんよう?...

両生類は現在、すべての脊椎動物の分類群1の生物多様性の最大の下落を経験している。ツボカビ、ツボカビ真菌病原体Batrachochytriumのdendrobatidis（BD）によって引き起こされる皮膚病、これらの劇的な下落2の主要な原因の1つであるBdは生物多様性の損失を生じさせるための記録最悪病原体である：それは両生類の600以上の種に感染し、持ってできるだけ多く200種が絶滅危惧または2,3絶滅したそのうちの300種以上の減少を引き起こした。両生類ツボカビ菌は、特定のホストされていない、それは 、BD 感染が深刻な病気を引き起こす進行するためにホストの資質が許すかは不明である。ツボカビは、電解質の損失および心不全4で得られた、皮膚におけるイオンチャネルを破壊することによって死を引き起こす。また、（何のBdの遊走子を含まない） のBd上清オタマジャクシ5最先端レベルの免疫応答を引き起こすGの研究者は 、BD は 、副生成物6毒性を生産信じています。 多くの種が急速に減少しているため、キャプティブ保証コロニー繁殖プログラムは、重要な保全対策であり、いくつかの種のための唯一の生命線である。確立し、無病コロニーを維持することは両生類管理施設のための挑戦である。 Bdの感染症を治療するための現在の化学療法の方法は、有効であるが、治療される動物に有害であることができ、単一の治療法は、種および両生類7歳クラスの両端に成功しなかった。 Bdの感染の治療のいくつかの臨床試験では、07行われている。治療の三最も一般的に使用される方法は、温熱療法、クロラムフェニコールおよびイトラコナゾールである。温熱療法は、ほとんど副作用を有すると思われるnonchemotherapeutic治療選択肢である複数のライフステージで使用することができ、GENERである同盟国効果的。治療試験では、チャットフィールドとリチャーズ- Zawacki 8は、処置した動物の96％が10日間30°Cに収容された後、感染をクリアしていたことが分かった。この試験は100パーセント成功しませんでしたが、それは他の人が自分の種のための温熱療法を評価するためのベースラインを提供しています。温熱療法は、多くの種に有効であるが、長時間にわたって高温に留まることができない多くの高山種について非実用的である。クロラムフェニコール、抗菌性は、多くの種でのBdを治療するために成功裏に使用され、 インビトロでの真菌の成長を阻害されている。抗菌ように、クロラムフェニコールは、具体的には真菌を攻撃しないが、真核細胞9においてアポトーシスを引き起こすことが知られている。これは、作用の正確なメカニズムは不明であるが、クロラムフェニコール療法は、細胞内の胞子を死滅させる、皮膚におけるアポトーシスを引き起こすことが推測されている。臨床試験は行われないので、したが用量および治療 ​​時間が評価されておらず、現在の治療プロトコルは、典型的には、地上両生類のためのこの治療は実用的でない、2〜4週間10,11のための浸水を必要とする。ヒトへの暴露は、再生不良性貧血12のまれな形態と関連していたので、クロラムフェニコールはまた、獣医学的使用のためのいくつかの国で制限されています。 イトラコナゾール、アゾール系抗真菌は、最も広く飼育されている両生類のBd感染の治療のために使用される。最も広く使用されるプロトコルは、5分11日間連続日の0.01％イトラコナゾール溶液中に入浴動物である。このプロトコルは、両生類のために有効であるが、それは両生類11,13での臨床試験に基づくものではない、哺乳動物への投与に使用される基準に由来した。このプロトコルは、特に属ラナにし、オタマジャクシ、最近metamorphs 11で、特定の種の負の副作用を引き起こす可能性があります。オタマジャクシ処理は、イトラコナゾールのはるかに低い濃度（0.0005％）を用いて成功しているが、脱色14と関連していた。これと同じ濃度は、最近のmetamorphsでトライアルが行われ、感染15の治療に失敗したことが判明した。イトラコナゾール治療によるマイナスの副作用の危険性がありますので、いくつかの専門家は、11日間連続して1の半分の通常の濃度（0.005％）を使用し、これが BD 感染の16を硬化させることに成功したことが判明しました。 本研究では 、BD 感染 15を治療するためのイトラコナゾールを使用して臨床治療試験の要約である。プロトコルは、イトラコナゾールの BD感染動物の治療のための安全かつ効果的である最低用量や治療時間を見つけるための試みを説明しています。本研究で使用した動物は、湾岸のヒキガエルの最近のmetamorphs、Incilius nebuliferた。

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

両生類は、任意の脊椎動物クラスの最大の下落を経験しているし、これらの減少の主な原因は、疾患ツボカビを引き起こす真菌の病原体、Batrachochytriumのdendrobatidis（BD）、である。キャプティブ保証コロニーが絶滅両生類で世界的に重要であり、絶滅の重大な脅威のそれらのための唯一の生命線である。無病コロニーを維持することは、キャプティブ経営者の優先事項である、まだすべての種のため、ライフステージ全体で安全かつ効果的な治療が同定されていない。一般的に使用される投与量は、いくつかの個人や種に害を及ぼす可能性があるが、最も広く使用されている化学療法は、イトラコナゾールである。我々は、イトラコナゾールの低い、より安全で効果的な投与量は、Bdの感染症を治すために見つけることができるかどうかを評価するための臨床治療の治験を行った。我々は発見した0.01から0.0025パーセント処理濃度を低下させ、11から処理時間を減少させることによって5分浴の-6日間、カエルは副作用が少ないと少ない治療に関連した死亡の BD感染の硬化させることができた。

すべての動物は、治療が終わった4週間後にBD-陰性であった場合は、イトラコナゾール治療が成功したと考えられた。治療が開始される前に、2週間接種後、全てのBD-接種した動物は、治療後4週が終了している間、BD-陽性対照動物は（16,765.12の平均遊走子負荷を持っていた、（17.34±）1,121.7の平均遊走子負荷で、感染していた）14643.08±。 Incilius nebuli...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

両生類におけるBatrachochytriumのdendrobatidis感染が低減濃度（0.0025％）、および一般的に使用されるよりもイトラコナゾールの短い持続時間（6日間5分浴）を用いて除去することができる。以下で死亡率およびより少ない負の副作用は、この修飾治療プロトコルを用いて観察した。

減少イトラコナゾール濃度および期間は、治療するのに成功している<em&gt; Batrachochytriumのdendrobatidis</em&gt;両生類の感染

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K ビュー.  James Cook University. 両生類におけるBatrachochytriumのdendrobatidis感染が低減濃度（0.0025％）、および一般的に使用されるよりもイトラコナゾールの短い持続時間（6日間5分浴）を用いて除去することができる。以下で死亡率およびより少ない負の副作用は、この修飾治療プロトコルを用いて観察した。 

ビデオ: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

のAlexa Fluor ®標識を通じてデング熱ウイルスの可視化

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

免疫・感染学

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

全タンパク質の抽出および2次元ゲル電気泳動法のための<em&gt;バークホルデリア</em&gt;種

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

小さなインドのマングースにおけるイオフェノキ酸類似体の分析 (ヘルペステス・オーロプンクタトゥス)経口狂犬病ワクチン接種生物学的マーカーとしての使用のためのセラ

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

感染性単核球症の小児からの末梢血サンプル中の免疫細胞亜集団の分離

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

日本脳炎ワクチン接種小児におけるリンパ球検出のためのフローサイトメーター間ラボ間移入の標準化

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...