我々はTを提供するためのジョン・ケリーとマーティン・テイラー（衛生＆熱帯医学のロンドン・スクール）を感謝しますB。トリパノソーマ GVR35-VSL-2およびin vivoイメージングのアドバイス博士アンドレアZELMER（LSHTM）。この作品は、ビル・アンド・メリンダ・ゲイツ財団グローバル・ヘルス・プログラム（助成金番号OPPGH5337）によってサポートされていました。

倫理 すべての作業は、英国内務省動物（科学的手続）法1986と衛生＆熱帯医学動物福祉のロンドン・スクールと倫理審査委員会の承認の下で行いました。このレポートに続いているガイドラインに到着。 生物発光トリパノソーマブルセイのインビボ通路に1 <ol><li> Tの凍結保存された株式（と呼ばれるstabilate）を削除B。トリパノソーマ株 GVR35-VSL-2（MTAを通じて衛生＆熱帯医学のロンドン・スクール教授のジョン・ケリーから入手可能）9液体窒素から、室温に平衡化することを可能にします。グルコース生理食塩水（PBS-G）は、pH 8.0（1L PBS + 15グラムのブドウ糖）10を滅菌リン酸緩衝で2×10 4トリパノソーマ/ mlにstabilateを希釈します。 注：T. B。トリパノソーマ GVR35-VSL-2は、ヒトに対して非感染性であり、SAPOは、実験を行うための施設をライセンスが必要です。 </li><li>インジェクション電気ショック療法0.2ミリリットル20〜25グラムのメスのCD1マウスは、25 G針で腹腔内経路（IP）経由トリパノソーマを希釈しました。これは、「ドナーマウス」です。特定病原体フリー（SPF）-cageおよび飼料を自由摂取に感染したマウスを置きます。 注：CD1マウスは近交系株及びT.ですB。トリパノソーマ GVR35感染はこの株でよく特徴付けられています。しかし、このプロトコルはまた、近交系または遺伝的に改変された株にも適用可能であるが、毛皮の色がイメージング6,9の感度に影響を与える可能性があることに留意することが重要です。 </li><li>採血により末梢寄生虫を監視します。プラスチック製の制止にマウスを置き、21 G針や瀉血に静脈穿刺によって尾から血液の5μLを取り、赤血球を溶解し、より簡単にカウントを可能にするために、0.85％滅菌塩化アンモニウムで1/10を混ぜます。 </li><li>ノイバウアー血球計数器で希釈した血液をカウントします。 <ol><li>両室に希釈した血液をロードします。全体の中央グラムを数えます一方のチャンバのID は、nトリパノソーマを得ました。血液中のトリパノソーマの濃度= N×10 4トリパノソーマ/ mlで、任意の希釈倍率を調整し、2室の平均値を計算します。 注：後約5-7日後、感染、末梢寄生虫は、2×10 4トリパノソーマ/ mlの濃度で感染を行うために十分に上昇する必要があります。 </li></ol></li><li>トリパノソーマレベルが必要なマウスの数のために十分に高い場合には、1.6のステップに進みます。 1つのドナーマウスにおいて6×10 4トリパノソーマ/ mlの寄生虫血症レベルは、10匹のマウスの感染のために十分です。 注：統計的に有意なグループサイズは、N = 6匹のマウスの各処置群8を必要としています。感染症の他のモデルでは、電力の計算は、適切なグループサイズを決定するために適用される必要があるであろう。 </li><li> （約5〜10分間28-30℃で）熱ボックスに置きマウスは、尾静脈を拡張することを可能にします。終末麻酔を誘導するために、25 G針では20mg / kgのペントバルビタールの静脈内投与します。マウスは軽くない引っ込め反射がないことを確認するためにフットパッドを押すことによって麻酔されていることを確認。 </li><li>ビジューチューブに5000 USP単位/ mlで5ミリリットルのヘパリンを等分し、1ミリリットル注射器に取り付けられた針の内外二回吸引することによりヘパリンで治療する21 G針。 </li><li>ヘパリン処理した21 G針および1mlシリンジを取り、胸郭の下中央に針を挿入することにより、麻酔をかけた通路マウスの心臓穿刺を行う（血液のビーズは、針の基部にプールされます）、静かにプランジャーに引き戻します心臓の室を崩壊させないように。血液の0.7ミリリットルの最小値を収集します。 </li><li>上記のステップ1.1のようにPBS-G pHが8.0で2×10 4トリパノソーマ/ mlの接種物を生成するために感染した血液を希釈します。 注：この時点で、残りの血液を8％グリセロールと混合することによってstabilateを生成するために凍結保存することができ、20％の熱不活性化胎児CALF血清（ハイFCS）および72％感染した血液。ゆっくりと液体窒素に移す前に、-1℃/分の冷却速度を達成するために、遅い凍結冷凍コンテナを使用して、-80℃で凍結します。 </li></ol>実験マウスの2.感染<ol><li>静脈を拡張するために優しく暖かいに加熱ボックスにCD1雌マウス（〜21〜25グラム）を配置します。プラスチック製の拘束に温めマウスを置き、マウス当たり4000トリパノソーマと同等の尾静脈に25 G針、静脈内に接種物を希釈した0.2ミリリットルを注入。出血を止めるために、その後、注射部位に圧力をかけます。 注：腹腔内感染症は、感染の有効なルートであり、10以前の研究で使用されてきたが、我々は、静脈内経路よりも、この少ない再現性を発見しました。 </li><li> SPF-通気ケージに3のグループでの感染マウスとケージをランダム。対照として、野生型の非生物発光寄生虫、TとCD1雌マウスを注入B。ブルセイ GVR35 た（n =3）。 </li><li>ブラッドフィルム顕微鏡を介して感染を監視します<ol><li>静脈穿刺または瀉血からスポット5μlの血液をスライドガラス上に血液塗抹標本行う（第2のガラススライドを使用して静かに薄膜を生成するために、スライド全体に一滴の血液を押します）。空気乾燥にスライドを許可します。 </li><li> dH 2 Oでリンスする前に10分間ギムザで100％のメタノールと染色で血液塗抹標本を修正し、空気乾燥させ。 100Xの目的の下で10視野（FOV）内のトリパノソーマの数を数えます。 注：血液撮影が早くも1日、感染後のような、動物全体の非侵襲的イメージングと一緒に行われます。動物の数を処理するときにサンプルを室温で保存し、後にカウントすることができるように、トリパノソーマ定量化のこの方法は、血球計計数よりも好ましいです。 </li></ol></li></ol> 3.生物発光イメージングは​​、感染を追跡します注意：感染を監視するには、全体のアニメーションアル非侵襲的イメージングを使用することができます。 <ol><li> 30 mg / mlとでダルベッコのリン酸緩衝生理食塩水（DPBS）中のD-ルシフェリン、ホタルルシフェラーゼ基質のストック溶液を準備します。フィルタは、滅菌し、-20℃でアリコートで凍結します。 注：D-ルシフェリンは光に敏感である、したがって箔にアリコートを包むことにより、光から保護します。それは活動11に影響を与えることができるように繰り返しルシフェリン凍結融解しないでください。 </li><li>室温にD-ルシフェリンのアリコートを温めます。 （nonbioluminescent野生型寄生虫株に感染対照マウスを含む）ケージから各マウスを取り外し、25 G針を腹腔内（DPBSで希釈）温めD-ルシフェリン150mgの/キロを注入。 /分2.5 Lで2％のイソフルラン/ O 2混合物で麻酔を誘導するために約5分間イソフルランチャンバー内にマウスを置きます。 注：彼らは麻酔下にあるときにマウスを不動になります。ここで使用される撮像装置では、3匹のマウスを一度に処理することができます。マウスはanesthetある場合30分より長く化された、眼科用人工涙軟膏乾燥からマウスの眼を防止するために適用することができます。 </li><li>ここで説明したイメージャの場合は、ソフトウェアを開き、製造元の指示に従ってソフトウェアを使用してマシンを初期化します。カメラや加熱されたプレートは温度に達した後の画像を取得します。 注：この機器は、通常、一晩バックグラウンドキャリブレーションを実行するためにマシンを可能にする、オフにされていません。それが再起動する必要がある場合には、それは最初の校正チェックを実行する必要があります。 </li><li> （3.5を参照）イメージャに麻酔したマウスを置きます。任意の明るい生物発光信号の滲み出し最小限にするためにマウスとの間に大きな黒のセパレータを使用してください。露光時間のセットを使用して画像マウス。 1、3、10、30、60および180秒、メディアビニング、1 F /ストップ、オープンフィルタ、およびビューE（12.5 X 12.5 cm 2）でのフィールドを持ちます。 注：野生型nonbioluminescent GVR35寄生虫に感染した対照マウスは、backgrを提供しますound生物発光値。このモデルとルシフェリン動態実験（ 図6）から、我々は10分で生物発光信号のピークは投与後とイメージングは、約3匹のマウスのための5分を取ることを見出しました。麻酔マウスを撮影するときに考慮し、このタイムスケールを取ります。 </li><li>マウスの完全な画像化を確実にするために、腹側、背側及び側面図を得るために回転させます。シーケンシャル動物間イメージングのためのオリエンテーションのための一貫性を維持します。 </li><li>撮影後、マウスはSPF-ケージに戻る前に観察下麻酔から回復することができます。 </li><li> 21日目、感染後まで7日毎にマウスを画像化を続行します。動物全体にわたる生物発光信号が着実に増加があるはずです。 </li><li> D21、画像および血液フィルムでのマウス前述したように、腹腔内に40mg / kgのジミナゼン・アセチュレートをマウスに投与量。この薬は、末梢寄生虫がクリアされますが、血液braiを横断しませんn個の障壁は、したがって、CNS感染のより良い可視化を可能にします。 注：ジミナゼンのaceturateは早期ナガナ病を治療するために使用される十分に確立された薬物です。 </li><li> D28とD35で撮影し、監視を続けます。 </li></ol> 4. CNS感染を確認<ol><li> D35では、上記で詳述したように、画像マウス（3.1〜3.4ステップ）。 </li><li>撮影後、さらなる分析のために血液を採取し、ステップ1.6から1.8で詳述したようにマウスが麻酔と放血させるマウスから回復することができます。 </li><li>臓器から血液をクリアすると、時間間隔にわたって脳の領域から除去されている可能性があるルシフェリンのラベルを再導入するために、心臓を経由して：心臓穿刺に続いて、20ミリリットルのPBS（灌流液にルシフェリンの3 mg / mlでの追加オプション）でマウスを灌流ステップ4.1から。 </li><li>静かに脳を可能にするために、湾曲したハサミを使用して、頭蓋骨の周囲ベースから切断し、頭蓋骨の上部を持ち上げて、脳を削除緩やかに湾曲鉗子を用いて持ち上げることができます。 </li><li>適切なルシフェリンを確保するために、撮像前に臓器を超える15 mg / mlとルシフェリン50μlの黒いプラスチックピペットのセクションに摘出した脳を置き切除した脳に追加されました。上記のように設定を使用してイメージ。これは、脳半球への感染の局在化を実証します。 注意：以前に8に記載のように下流摘出脳の寄生虫の定量化は、定量PCRにより行うことができます。他の代替ダウンストリームアプリケーションは、固定後の脳組織内の寄生虫を識別するために、組織学または免疫組織化学が含まれる場合があります。 </li></ol>生物発光イメージングの5定量注：生物発光は、イメージングソフトウェアを用いて関心領域（ROI）を用いて定量化し、バックグラウンド生物発光のために補正することができます。 <ol><li> ROIツールを使用して、動物全体の定量aの長方形のROIを作成しますNDマウスの尾の付け根に鼻の先端をカバーするために、マウスの画像の上にそれを配置します。すべての動物、すべての時点について、同じサイズのボックスを使用します。脳のための投資収益率を見ると、画像中のマウスの頭部領域の上に配置する、同じように円形のROIを使用。 </li><li>必要であれば、感染を視覚的に表現するために同じスペクトル上に表示されるように画像を調整します。 注：ここで使用されるソフトウェアは自動的に最小値と最大光子カウントのために調整画像ごとにカラーマップを生成します。一連の画像間の視覚的な比較を可能にするために、カラースケールは、すべての画像に同じ値に設定する必要があります。 <ol><li> （経験的に決定される）ツールパレット上で最高の生物発光と「画質調整」タブで画像を使用して、対数スケールを選択し、適切なカラースケールの最小値と最大値。実験を通してすべての画像にこのスケールを適用します。 </li></ol></li></ol>

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D., 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=Bioluminescence+imaging+of+chronic+Trypanosoma+cruzi+infections+reveals+tissue-specific+parasite+dynamics+and+heart+disease+in+the+absence+of+locally+persistent+infection.">Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.</a> Cell. Microbiol. 16 (9), 1285-1300 (2014).</li></ol>

The authors have nothing to disclose.

生物発光Tの開発B。トリパノソーマ GVR35株は、初期から後期までトリパノソーマ感染の可視化を可能にします。以前の感染モデルは、血液フィルム顕微鏡からリアルタイムに、寄生虫は、脳にある場合、後期を検出することができなかった、および寄生虫負荷12を決定するために、感染マウスからの脳の淘汰及び除去を必要としました。単一のマウスが感染の全?...

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4. 
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS). 
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.

<table><tbody><tr><td>PBS</td><td>Sigma, UK</td><td>P4417</td><td>tablets pH 7.4</td></tr><tr><td>Glucose</td><td>Sigma, UK</td><td>G8270</td><td>99.5% (molecular) grade</td></tr><tr><td>Ammonium chloride</td><td>Sigma, UK</td><td>A9434</td><td>99.5% (molecular) grade</td></tr><tr><td>Heparin (lithium salt)</td><td>Sigma, UK</td><td>H0878</td><td></td></tr><tr><td>Hi-FCS</td><td>Gibco, Life Technologies, UK</td><td>10500-064</td><td>500 ml</td></tr><tr><td>DPBS</td><td>Sigma, UK</td><td>D4031</td><td>Sterile filtered</td></tr><tr><td>Mr. Frosty</td><td>Nalgene, UK</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Sigma, UK</td><td>G5637</td><td></td></tr><tr><td>D-Luciferin</td><td>Perkin Elmer, UK</td><td></td><td></td></tr><tr><td>Sigma, UK</td><td>115144-35-9</td><td></td><td></td></tr><tr><td>Diminazene aceturate</td><td>Sigma, UK</td><td>D7770</td><td>Analytical grade</td></tr><tr><td>IVIS Lumina II</td><td>Perkin Elmer, UK</td><td></td><td>other bioimagers available &lt;em&gt;e.g.&lt;/em&gt; from Bruker, Kodak</td></tr><tr><td>Living Image v. 4.2</td><td>Perkin Elmer, UK</td><td></td><td>proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td></tr><tr><td>1 ml Syringe</td><td>Fisher Scientific, UK</td><td>10142104</td><td></td></tr><tr><td>20 ml Syringe</td><td>Fisher Scientific, UK</td><td>10743785</td><td></td></tr><tr><td>25 G Needles</td><td>Greiner Bio-one</td><td>N2516</td><td></td></tr><tr><td>21 G Needles</td><td>Greiner Bio-one</td><td>N2138</td><td></td></tr><tr><td>Twin-frosted microscope slide</td><td>VWR, UK</td><td>631-0117</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td>StarLab, UK</td><td>I1415-1000</td><td></td></tr><tr><td>7 ml Bijou tube</td><td>StarLab, UK</td><td>E1412-0710</td><td></td></tr><tr><td>Mouse restrainer</td><td>Sigma, UK</td><td>Z756903</td><td>our restrainer was made in-house, this is a similar model</td></tr></tbody></table>

bioluminescence imaging to detect late stage infection of african trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

このプロトコルは、Tを用いたマウスの感染後の疾患の進行を追跡するために方法を示しますB。トリパノソーマ 、人間のアフリカトリパノソーマ症のためのモデルが。1治療およびイメージングのステップのためのタイムテーブルを実証し、実験プロトコルのタイムラインを示す図 。 図2は 、末梢寄生虫を定量する?...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

アフリカトリパノソーマ症の後期感染を検出するための生物発光イメージング

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

bioluminescence-imaging-to-detect-late-stage-infection-african

Research

JoVE Journal

Immunology and Infection

7.7K ビュー.  London School of Hygiene & Tropical Diseases. This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

ビデオ: Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

免疫・感染学

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

HIV / AIDSの包括的＆コスト効率に優れた研究室の監視：アフリカのロールモデル

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

異なる動物モデルにおけるNADPHオキシダーゼ活性の生物発光イメージング

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物発光細菌のイメージング<emインビボで&gt;</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

神経侵襲性脳炎ウイルスのin vivoイメージングシステム（IVIS）検出で

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

の分離<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</em磁気手段によって&gt;後期感染赤血球

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

腫瘍進行と血管新生の二重生物発光イメージング

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

医学

シャーガス病の生体内発光モデルによる薬剤評価のためのプロトコール

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

著者スポットライト:寄生虫の負荷と宿主の応答のリアルタイムモニタリング

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

薬効研究のためのシャーガス病モデルマウスのIn vivo生物発光イメージングの謎解き

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

アフリカトリパノソーマ症の後期感染を検出するための生物発光イメージング

要約

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この動画の章

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

HIV / AIDSの包括的＆コスト効率に優れた研究室の監視：アフリカのロールモデル

異なる動物モデルにおけるNADPHオキシダーゼ活性の生物発光イメージング

生物発光細菌のイメージング

神経侵襲性脳炎ウイルスのin vivoイメージングシステム（IVIS）検出で

の分離

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

腫瘍進行と血管新生の二重生物発光イメージング

薬効研究のためのシャーガス病モデルマウスのIn vivo生物発光イメージングの謎解き

薬効研究のためのシャーガス病モデルマウスのIn vivo生物発光イメージングの謎解き