この作品は、NIHのRO1 AI079253と退役軍人局によって資金を供給された。

1。動物モデル<ol><li>マウス：P47-phoxの使用/ -マウスでは、年齢と性別をマッチC57BL6/DBAマウスを。インスティテューショナル·動物実験委員会の実験のための認可を受けなければならない。 </li><li>麻酔：麻酔を誘導するために、連続イソフルラン管理システムを使用してください。気化器システム（VetEquip）は（2-3％）イソフルランで満たされている。マウスが外部刺激に応答して、呼吸、動き、角膜反射を観察することによって、完全に麻酔されていることを確認。 </li><li>外科的処置：70％エタノールで手術領域（ベンチ）をスクラブ。滅菌手袋と清潔な手術衣とマスクを着用してください。髪をクリッピングし、切開が行われるエリアに、70％エタノールで交互ベタジンを適用することにより、マウスを準備します。無菌の表面に手術を行い、滅菌器を使用します。目を覚まして、自由に移動するまで、術後マウスを監視します。 </li><li>気管内ザイモサン管理<ol><li>麻酔を投与1.2で説明したように。 </li><li>ベタジンと70％エタノールで手術領域（首）を消毒し、外科切開により気管を露出させる。 </li><li> 27ゲージの針で突き刺す気管および滅菌PBSに溶解した0.5μg/μLの溶液（25gのマウスの総体積50μl）を用いて1μg/ gの用量でザイモサン（セントルイス、MO）を注入。 </li><li>クローズマウスの首は無菌条件下で5から0無菌絹縫合糸で巻いた。 </li><li> 4時間及び24時間後の画像。 </li></ol></li><li>盲腸結紮及び穿刺<ol><li> 1.2で説明したように麻酔を管理します。 </li><li>手術部位（腹部）で髪をクリップしてベタジンと70％エタノールで消毒してください。 </li><li>正中開腹術を行い、盲腸を識別します。 </li><li> 4から0絹縫合糸と、21ゲージの針を1パスで結紮に対して遠位穿刺盲腸で露光盲腸の先端50％をライゲーションする。 </li><li> 4から0無菌絹縫合糸で切開を閉じます。 </li><li> 4時間後の画像と（24時間）。 </li></ol></li><li>オーラル四塩化炭素（CCl 4）投与<ol><li> 1.2で説明したように麻酔を管理します。 </li><li>経口胃管栄養法によりトウモロコシ油に溶解CCl 4の （2μg/ gで、セントルイス、ミズーリ州）を管理する。四塩化炭素投与の手順はIBC / EHS方針に従って、指定された領域で実行する必要があります。 </li><li> 4時間及び24時間後の画像。 </li></ol></li></ol> 2。生物発光画像を取得する<ol><li>発光モードに設定IVIS 200（Xenogenコーポレーション、アラメダ、カリフォルニア州）イメージングシステムを、初期化して、電荷結合素子（CCD）をロックするために、温度を待つ。 </li><li>露光時間、ビニング（媒体）とF /ストップ（8）の設定を選択します。 </li><li>正常な体温を維持するために加熱されたステージ上でのイメージングチャンバー内に仰臥位にマウスを置きます。ノーズコーンを介して、1.2で説明したようにマウスがIVISイメージング室にいる間麻酔を管理します。 </li><li> L-012を投与する<html> （20μg/ g）をイメージングのために選択された各時点における眼窩注射により静脈内（10μg/μLの）滅菌PBSに溶解した。 </li><li> L-012注射後2分で始まる画像をキャプチャします。我々は通常、40秒露出を使用しています。 </li></ol> 3。関心領域を使用してデータ解析<ol><li>リビングImageソフトウェアV.4.2（Xenogenコーポレーション、アラメダ、CA）を使用してデータを分析します。 </li><li>画像を開いて、インタレスト·ツールの領域の測定を選択します。 </li><li>胸部および/または腹部の上にマウスの写真画像の上に擬似色のデジタル画像を（光子検出を表す）を重ね合わ後の利息の形状や大きさの領域を選択します。 </li><li>関心領域からの信号強度（光子束）を定量化する。 </li></ol> 4。統計<ol><li>個々の時点でBonferroniの事後テストで2-way ANOVAを実行するための統計解析ソフトウェア·パッケージを使用しています。 </li></ol>TLE &quot;&gt; 5。代表的な結果ザイモサンは、炎症誘発性酵母細胞壁製品とNADPHオキシダーゼ3の強力な活性化因子である。我々は以前にその気管ザイモサンを示しphoxのP47-/で、より堅牢な好中球の肺の炎症と炎症性サイトカインの産生を誘導する-マウス1、野生型と比較した。ザイモサンチャレンジ後のマウス-ここでは、私たちの目標は、野生型とP47-phoxの/の肺におけるROS産生を比較することであった。 4時間および24時間後のマウス-気管ザイモサン投与後4時間後に、我々は、ベースラインと同様にP47-phoxの/に比べて野生で光子放出の増加と比較して、野生型マウスでは胸の上に光子放出の大幅な増加が観察された。これとは対照的に、P47-phoxの/生物発光シグナル-マウスはザイモサン処理（ 図1-B）後4時間でベースラインレベルにとどまっている。したがって、NADPHオキシダーゼはbに表示されます電子ザイモサン投与後の肺内のROS生成の主要な源。 次に、我々は、CLP誘発性敗血症モデルにおけるROS産生を評価した。敗血症、急性肺損傷（ALI）、急性呼吸窮迫症候群（ARDS）4,5を含む末端器官の損傷に関連する生命を脅かす症候群です。 ROS媒介損傷は敗血症誘発多臓器不全を駆動する重要な因子であると考えられている。私たちは、ROSが有意に胸（4時間）の上とベースラインと比較して野生型マウスでCLPに続く腹部（24時間）以上に増加したことが分かった。しかし、P47-phoxの/のROSレベル-マウスの時点（ 図2 AC）の両方でベースラインレベルと同様であった。これらの結果は、CLP誘発性敗血症のマウスモデルにおけるROSの生成はNADPHオキシダーゼ依存であることを示している。 最後に、我々はCCl 4誘発肝傷害モードでROS産生を検出するために、生物発光イメージングを使用リットル。 四塩化炭素は、肝細胞の壊死を引き起こし、急性肝障害や肝線維症6の両方をモデル化するために使用されています。炎症やけがの前述のモデルとは対照的に、我々は、腹部におけるROS産生は控えめに（約35％）P47-phoxの/にベースラインと比較して増加していたことがわかった- 4時間以下のCCl 4投与のマウス。マウス（ 図3-B） -しかし、 四塩化炭素誘発性のROS生成の大きさは、P47-phoxの/よりも野生型マウスでは有意に大きかった。これらのデータは、両方P47 phoxの含有NADPHオキシダーゼ依存性および非依存性のROS産生が急性四塩化炭素誘発肝傷害で起こることを示唆している。 <img alt="図1" src="/files/ftp_upload/3925/3925fig1.jpg" /> ザイモサン処理後の肺の中の図1：ROS産生。 A）代表bioluminescenROS産生のCEイメージング。マウスや腹部の発光がザイモサン処理によって変更されていません-胸に加えて、発光が野生型とP47-phoxの/の両方で腹部上に検出可能であることに注意してください。野生型とP47-phoxの/の胸からB）の光子数-ザイモサンの（IT）の気管内単回注射後のマウス。生物発光イメージングは​​、ベースライン、4時間、および24時間で行った。胸上の関心領域からの発光は示さ擬似カラースケールを用いて同定した。結果は平均として提示されている±SE、nはグループあたり= 6-9、* = p &lt;0.05で。 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">拡大図はこちらをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/3925/3925fig2.jpg" /> 図2 ROSの検出は、以下のCLP。 ROSの産生、Bの）代表生物発光イメージング）胸から光子カウント、および野生型とP47-phoxの/の腹部からC）の光子カウント-ベースライン時にマウス、4時間、およびCLP後24時間。結果は平均として提示されている値±SE、群あたりn = 4-5、* = p &lt;0.05で。 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">拡大図はこちらをクリックしてください</a> 。 <img alt="図3" src="/files/ftp_upload/3925/3925fig3.jpg" /> 後腹部のCCl 4誘発肝傷害における図3 ROS産生。経口CCl 4の治療後、ベースライン時のマウス、4時間、および24時間- ROS産生と野生型とP47-phoxの/の腹部からB）の光子数の）代表生物発光イメージング。のn = 4-5グループごと、* = p &lt;0.05で、結果は平均±SEとして提示されている<a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">にはここをクリックして拡大図</a> </&gt;。

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生きている動物での活性酸素種の &quot;リアルタイム&quot;測定（ROS）は、蛍光および化学発光プローブを使用することによって達成することができます。蛍光プローブは、弱い信号対雑音比12を有する苦しむ一方で、説明イメージング技術は、ルミノールベース基板のL-012とROSの化学反応後の発光の検出に敏感である。すべての生物発光イメージング技術と同様に、この方法論は、臓器...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> L-012 </td><td>和光ケミカルUSA社</td><td> 120から04891 </td><td></td></tr><tr><td>ザイモサン</td><td>シグマ、セントルイス、ミズーリ</td><td> Z4250 </td><td></td></tr><tr><td>四塩化炭素</td><td>シグマ、セントルイス、ミズーリ</td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPHオキシダーゼは、抗菌剤および抗真菌宿主防御を仲介する重要な酵素である。抗菌宿主防御における役割に加えて、NADPHオキシダーゼは、炎症反応を調節する重要な1シグナリング機能を持っています。したがって、NADPHオキシダーゼ由来のROS生成の &quot;リアルタイム&quot;の動態に測定する方法の開発が防衛、炎症やけがをホストするために関連するメカニズムを理解するための貴重な研究ツールであることが期待されています。 慢性肉芽腫症（CGD）は重篤な感染症や過度の炎症を特徴とNADPHオキシダーゼの遺伝性疾患です。食細胞NADPHオキシダーゼの活性化は、その細胞質サブユニット（P47 phoxの 、P67 phoxの 、およびp40 phoxの ）および膜結合flavocytochrome（gp91 phoxおよびP22 phoxのヘテロ二量体で構成される）へのRacの移行が必要。損失CGDでこれらのNADPHオキシダーゼコンポーネントの結果のいずれかの機能変異の。 CGDの患者と同様に、gp91 phoxの欠損マウスとP47 phoxの欠損マウスは、欠陥のある食細胞NADPHオキシダーゼ活性と障害ホスト防御13、14を持っています。上記のNADPHオキシダーゼ成分を含んでいて食細胞に加えて、他の種々の細胞型は、NADPHオキシダーゼの異なるアイソフォームを発現している。 ここで、我々は生きているマウスではROS産生を定量化すると炎症やけがのモデルでROS生成にNADPHオキシダーゼの貢献を線引きする方法を説明します。このメソッドは、電荷結合素子（CCD）によって記録される発光を発するように、L-012（ルミノールのアナログ）と反応し、ROSに基づいています。 L-012プローブの元の説明では、L-012-依存性化学発光は完全にこの反応で検出された主なROSはスーパーであったことを示す、スーパーオキシドジスムターゼにより廃止されましたオキシドアニオン15。その後の研究では、L-012は、反応性窒素種16、17を含む他のフリーラジカルを検出することができることが示されている。ヒェランら17 ホルボールミリスチン酸アセテート、NADPHオキシダーゼの強力な活性化因子、のその局所適用を示し、発光プローブのL-012を使用してマウスで検出することができオキシダーゼ依存ROS生成をNADPHに導いた。このモデルでは、彼らは、L-012-依存発光はP47 phoxの欠損マウスでは廃止されたことを示した。 我々は、野生型マウスとNADPHオキシダーゼ欠損P47 phoxの-/内ROS生成比較して-次の3つのモデルのマウス2：ザイモサン、NADPHオキシダーゼを活性化することができる壁由来製品プロ炎症性真菌細胞の1）気管内投与、2）盲腸結紮及び穿刺（CLP）、二次急性肺の炎症や損傷を伴う腹腔内敗血症のモデル、3）経口四塩化炭素（ 四塩化炭素）、ROS依存肝損傷のモデル。これらのモデルは、具体的にそれぞれ、非感染性の炎症、複数菌による敗血症、毒素誘発性臓器障害の文脈においてNADPHオキシダーゼ依存ROS生成を評価するために選択した。 P47に野生型マウスでは生物発光を比較phoxの-/ -マウスたちは、これらのモデルにおける生物発光信号にP47 phoxの含有NADPHオキシダーゼによって生成ROSの具体的な貢献を線引きすることができます。 P47に比べて野生型マウスでは増加したROSレベルを示した生物発光イメージング結果phoxの-/ -マウスでは、NADPHオキシダーゼは、炎症性刺激に応答して活性酸素の発生の主な原因であることが示された。この方法は、in vivoで炎症時ROS生成の&quot;リアルタイム&quot;モニタリングのための低侵襲なアプローチを提供します。

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPHオキシダーゼは、食細胞内の活性酸素種の主要な供給源（ROS）です。なぜならROSのはかない性質のために、生きている動物のROSレベルを測定し、監視することは困難である。生きたマウスにおけるROSのシリアル定量化のための低侵襲的方法が記載されている。

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 ...

bioluminescence-imaging-nadph-oxidase-activity-different-animal

Research

JoVE Journal

Immunology and Infection

16.1K ビュー.  Vanderbilt University School of Medicine. NADPHオキシダーゼは、食細胞内の活性酸素種の主要な供給源（ROS）です。なぜならROSのはかない性質のために、生きている動物のROSレベルを測定し、監視することは困難である。生きたマウスにおけるROSのシリアル定量化のための低侵襲的方法が記載されている。

ビデオ: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

実験用ラットとマウスにおけるエクト - とEndoparasitesの診断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

免疫・感染学

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

単離と異なる高齢マウス胸腺移植片の移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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.

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

生物発光イメージングを使用した多剤耐性アシネトバクターバウマニバーン感染症のための抗菌ブルーライトセラピーのインビボでの調査に

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

非侵襲的<em&gt;インビボ</em&gt;活性化可能な蛍光イメージング剤を用いた炎症性MMP活性の蛍光イメージング

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...