著者らは、欧州委員会第7次フレームワーク計画（PIOF-GA-2009から255466）、およびアイルランドの健康研究会（HRA_POR/2010/138）からこの原稿に関連する支援を承諾したいと思います。 ルクス -タグE.大腸菌は博士コーマックガーン、コーク大学から親切な贈り物だった。

1。腫瘍誘導<ol><li>ルーチン腫瘍誘発については、無血清培地を200μlに懸濁された細胞の腫瘍形成性最小用量は、感染フリー6〜8週齢の雌BALB / cまたは無胸腺MF1-nu/nuマウスの脇腹に皮下（sc）注射したN = 21ゲージの注射針を用いて6（ハーラン、オックスフォード、英国）（1×10 6 4T1細胞）。血球計およびトリパンブルー色素排除（Gibco）を用いて、視覚カウントによって決定された接種に用いた細胞の生存率は95％以上であった。 </li><li>腫瘍確立後、腫瘍は成長と発展させ、そして週に2回モニターした。腫瘍体積は、腫瘍の最長径、b は直径と長径と直交する方向である式V =（AB 2）Π/ 6によると、計算された。 </li></ol> 2。細菌の準備<ol><li>このprotocoで使用されて細菌の菌株lは、E.だっ細菌は、BLIによって検出することができるようになり、そのluxABCDEエンコードプラスミドを保持する大腸菌 K-12 MG1655、非タンパク質毒素発現株、。E.は統合luxABCDEを含むコリ MG1655は37で好気的に成長させた°LB培地のC（Sigma-Aldrich社、アイルランド）は300μg/ mlのエリスロマイシン（EM）を補充した。 MG1655の生物発光誘導体はプラスミドp16S ルクス構成のP HELP luxABCDEオペロン1が含まれているを使用して作成しました。 </li><li>マウスへの投与のための準備のために、培養物を中間対相（600nmでの光学密度）に200rpmで振とう機で37℃でLB培地中でインキュベートした。細菌は胃管栄養のための静脈内投与、または1×10 10のために、（6000×gで5分間）遠心分離により回収し、PBS（Sigma）で洗浄し、PBSで希釈した1×10 7個のコロニー形成単位（CFU）/ mLであった。 </li></ol> 3。細菌の管理者権限る<ol><li>腫瘍が体積で約100mm 3に達したときにマウスを無作為に実験群に分けた。静脈内投与のためには、拘束されたマウスはそれぞれ28G注射針を使用して外側尾静脈に直接注入し、100μlの10 6個の細胞を受けた。各接種の生菌数は、回顧メッキによって決定された。 </li><li> GITに植民地化研究のために、10 9細菌細胞は、経口的に3日連続で、強制経口投与により、マウスあたり100μlで投与した。既存の共生細菌のレベルが前経口 ​​胃管栄養法の開始1〜7日間マウスの飲料水の5 mg / mlのストレプトマイシンを添加することによって供給する前に減少した。 </li></ol> 4。生物発光画像法<ol><li> 生体 BLIイメージングの 2Dは​​IVIS100（キャリパー）を用いて行った。定義された時点ポスト細菌投与では、マウスはキャリパーのXGI-8ガス麻酔を使用して麻酔をかけた3％イソフルラン、および全身画像解析を使用したシステムは、高感度で2から5分間IVIS 100システムで行われた。 </li><li> 3Dイメージングのために、麻酔したマウスは、背イメージング用光学イメージングシステム（IVISスペクトラム、キャリパー）の内側にマウスイメージングシャトルの中に置かれた。 3D光学復興のための細菌ルシフェラーゼ信号の画像を取得するには、500から580 nmの範囲で発光フィルターの波長は、信号対雑音比を最大にするためにフィルタごとに3〜4分のビン16アクイジション時間で使用されていました。この画像取得シーケンスの一部として、構造化された光像は高さマップを定義するために得られた。このマップは入力拡散光イメージング断層撮影（DLIT）非負最小二乗最適化2を使用して、3D光学像を形成するために使用された再構成アルゴリズムだった。 </li><li>画像解析：関心領域を特定し、リビングImageソフトウェア（キャリパー）を用いて定量した。 </li></ol> 5。代表者結果本研究では、非病原性の常在菌はluxABCDEオペロンを発現している大腸菌 K-12 MG1655はsc 4T1異種移植腫瘍を有するマウスに投与IVであった。細菌ルクス信号がマウスの腫瘍に特異的に検出された後のIV投与（ 図2）。サンプルマウス由来の細菌の培養の回復は生菌番号と検出された光の量（ 図3）との間に線形関係が存在することを検証します。GITに経口投与された共生細菌の in vivoイメージングでも3DのBLIを使用して達成される。 <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="図1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> 図1。プロトコルタイムライン 。皮下腫瘍をマウスで誘導し、細菌が、腫瘍の発達（100ミリメートル3）によって管理されています。生きたマウスでは、BLIのイムアール様々な時間ポイント·ポスト細菌投与（矢印は、典型的な時刻を表示）で熟成。 <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="図2" /> 図2。 Eの投与担癌マウスへのコリ MG1655 luxABCDE。4T1皮下腫瘍はMF1 nu / nuマウスとEに誘導されたコリ MG1655 luxABCDEは、腫瘍の開発時に投与した。各動物には外側尾静脈に直接注入10 6個の細胞を受けた。マウスはサンプル屠殺したマウスの腫瘍からの生菌のその後の回復（CFU）と一緒に勉強中の4時間点（黒丸z軸と画像）（棒グラフ）で撮像した。 （時点ごとに示す代表的なマウス）を経時的に観察された腫瘍に特異的に細菌数とプラスミド遺伝子発現の増加。 <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="図3" /> 図3。腫瘍内細菌数および生物発光との関係。生菌は腫瘍では、様々な時間点ポストIV投与でBLIへの後続の腫瘍からex vivoで細菌培養によって列挙された。 生体 bioluminesce単位での相対的な細菌数の値（CFU）のログがグラフ表示されます。細菌数および細菌の生物発光信号間の強固な相関が観察され、R 2 = 0.9717 1。 <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="図4" /> 図4。マウス消化管の3D IVISイメージはE.によって植民地化コリ MG1655。マウスのGITは、E. 10 9 cfuの経口投与によって植民地化された3日間連続して大腸菌 。植民地化されたマウスの3Dトモグラフィーから隔離されたサンプル画像が表示されます。3D画像は、解剖学的登録を提供するために、骨格のデジタルマウスアトラスを示しています。E.はコリ MG1655生物発光は、下位に ​​ある緑で表示され、より高いレベルで紫色です。

<ol>
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M., 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=Tumor-targeting+prodrug-activating+bacteria+for+cancer+therapy.">Tumor-targeting prodrug-activating bacteria for cancer therapy.</a> Cancer Gene Ther. 15, 393-401 (2008).</li><li>Foucault, M. L., Thomas, L., Goussard, S., Branchini, B. R., Grillot-Courvalin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+the+study+of+intestinal+colonization+by+Escherichia+coli+in+mice.">In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.</a> Appl. Environ. Microbiol. 76, 264-274 (2010).</li><li>Collins, S. 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特別な利害関係は宣言されません。

遺伝子治療の文脈では、患者への治療遺伝子の送達のための生物学的製剤の使用は、非常に有望3-5で示されている。ウイルスと同様に、細菌の生得的な生物学的特性は、特に癌の文脈では、細胞または組織への効率的なDNA送達を可能にする。これは、いずれかの外部（非侵襲種）または腫瘍細胞（病原体）内の、全身局所的に複製から高い評価を得て投与した場合、細菌は腫瘍にホ?...

<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> 4T1細胞株</td><td> ATCC </td><td> CRL-2539 </td><td>自然発生的に生じたBALB / c乳腺腫瘍由来同系乳癌モデル</td></tr><tr><td> DMEM </td><td>シグマアルドリッチ</td><td> D6429 </td><td> ダルベッコ改変イーグル培地 </td></tr><tr><td> PBS </td><td>シグマアルドリッチ</td><td> D8537 </td><td>リン酸緩衝生理食塩水</td></tr><tr><td> Xenogen IVIS </td><td>キャリパーライフサイエンス</td><td></td><td> 2DイメージングのためのIVIS 100; 3DのIVISスペクトラム。 </td></tr><tr><td>ルリアブロスミラー（LB） </td><td>シグマアルドリッチ</td><td> L2542 </td><td> 大腸菌の増殖培地大腸菌の </td></TR&gt; <tr><td>エリスロマイシン</td><td>シグマアルドリッチ</td><td> E5389 </td><td>抗生物質</td></tr><tr><td>ストレプトマイシン</td><td>シグマアルドリッチ</td><td> S9137 </td><td>抗生物質</td></tr><tr><td> MF1nu/nuマウス</td><td>ハーラン（英国） </td><td> 069（NU）/ 070（NU / +） </td><td> HSD：ヌード-Foxn1nu </td></tr><tr><td> Balb / cマウス</td><td>ハーラン（英国） </td><td> 066 </td><td>ハプロタイプH-2 D </td></tr><tr><td>強制飼養針</td><td>獣医ハイテクソリューションズ（英国） </td><td> DE009 </td><td> 22Gさx 38mmストレート強制飼養針</td></tr><tr><td>静脈注射用シリンジ</td><td> BD Biosciences社</td><td> 309309〜1ミリリットル</td><td> 28 G X½インチマイクロファインIVの針でインスリン注射器。 </td></tr><tr><td>腫瘍接種用注射器</td><td>ブラウン</td><td> 9161376V </td><td> Omnifix 26 G X＆frac12;インチ針</td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

このビデオでは、がんの遺伝子と細胞療法における細菌の使用に重点を置いて、生きたマウスにおける細菌人身売買の研究のための全身bioluminesceイメージング（BLI）の使用について説明します。細菌が全身投与後に腫瘍内に優先的に成長する天然の能力を有する、癌治療のためのベクターの魅力的なクラスを紹介します。細菌はバクテリアと同時に腫瘍部位のlux遺伝子カセット許可BLIの検出を発現するように設計。時間をかけて腫瘍内の場所や細菌のレベルを容易に調べることができ、2または3次元で可視化した。この方法は、細菌種と腫瘍異種移植片タイプの広い範囲に適用されます。この記事では、皮下腫瘍を有するマウス内生物発光細菌の分析のためのプロトコルについて説明します。 BLIによる胃腸管（GIT）内共生細菌の可視化についても説明しています。この強力な、そして安価、リアルタイムイメージング戦略representsのがん研究、特に遺伝子治療において、感染症の文脈におけるin vivoでの細菌の研究のための理想的な方法。このビデオでは、 ルクスタグ付きEを研究するための手順の概要を説明IVISシステムと空間的および時間的な読み出し達成可能な利用のBLIを示す生きたマウスで、 大腸菌 、。

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

この記事はの管理について説明し<em&gt;ルクスタグ付き</em&gt;マウスに細菌およびその後<em生体内で&gt;</emIVISの生物発光イメージングを用い&gt;分析。

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 ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K ビュー.  University College Cork. この記事はの管理について説明しルクスタグ付きマウスに細菌およびその後分析。

ビデオ: Bioluminescent Bacterial Imaging In Vivo

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

カテゴリの検出細菌性病原体のための&quot;生物発光&quot;レポーターファージ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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 ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

急性及び慢性炎症を区別するために生体内イメージング法における

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

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

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

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

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

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

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

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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.