著者らは、上述の研究への参加のために雲南省、中華人民共和国とAgboville、コートジボワール、地域社会に感謝しています。私たちは、プロトコルのテストや実行を支援している地元のフィールドチームの世話になっています。我々は、スイス熱帯公衆衛生研究所から以下の同僚には特に感謝しています：顕微鏡画像を提供するためのイヴェットEndriss、ハンスペーター·マルティと彼のチームは研究室のサポートのためとステファニーKrauth彼女の支援のためのビデオ撮影中。最後に、我々は有用なコメント、一連の二人の匿名レフェリーに感謝します。

1。加藤カッツテクニック<ol><li>顕微鏡スライド上にスツールの41.7ミリグラム（mg）を保持するための穴が標準で加藤カッツテンプレートを配置します。 </li><li>新聞やアルミ箔片の上に新鮮な糞便試料の2-3グラム（g）をスクープし、ふるい上にそれにワイヤーやプラスチック製のメッシュの部分を押してください。 </li><li>小さなプラスチック製のへらを使用して、メッシュオフふるいにかけた材料​​をこすり、完全にそれに加藤カッツテンプレートで穴を埋める。過剰糞便物質を除去するために、ヘラで穴の内容を平準化。 </li><li>垂直になりました顕微鏡スライドに付着糞便材料を乱すことなく、テンプレートを削除します。テンプレートとへらを洗剤と水で洗浄し、再利用することができます。 </li><li>セロファンの部分を置き、顕微鏡スライド上の糞便サンプルの上、少なくとも24時間（HR）のグリセリンマラカイトグリーン溶液に予め浸した。 </li><li>厚層塗抹標本に糞便材料を広めるために、優しくきれいなマイクロを押してくださいサンプルスライドに対するスコープのスライドは、均等に顕微鏡スライドの幅よりもわずかに小さい直径の円内に材料を分配する。 </li><li>スライドは、スライドを直射日光から遠ざけなければならない時に30から60分（min）、のためにクリアすることができます。その後、体系的に光学顕微鏡（40〜100倍の倍率）で厚層塗抹標本を調べます。 STHの卵の数を数えて、種（すなわち、A.lumbricoides、T.trichiura、および鉤虫）によってそれらを階層化する。注目すべきは、他の寄生虫卵（例えばマンソン住血吸虫）も、この方法により検出することができ、別々にカウントされ、記録されるべきである。 </li><li>感染の強さの標準化推定値を取得するには、従来スツール（EPG）のグラム当たりの卵の数として表され、24（24×41.7ミリグラム≈1グラム）によって卵の数を掛けます。 </li></ol> 2。 Baermannテクニック<ol><li>ガラス漏斗を保持し、ゴムチューブを取り付けることができ、スタンド、閉じたウィットを設定漏斗の底にヘクタールクリップ、。 </li><li>医療ガーゼの切れ端で漏斗および層それで、ふるいまたは円錐のような形金網のピースを置きます。 </li><li>水道水で漏斗をいっぱいにして、セットアップ全体が漏れないことを確認してください。 </li><li>医療ガーゼの真ん中の上に新鮮な便の20〜30 gをすくうと、それが完全に水中に置かれていることを確認してください。必要に応じてより多くの水を追加します。サンプル以上の医療ガーゼを折りたたみます。 </li><li>下から、2時間漏斗で人工光を照らす。 </li><li>静かにゴム管のクリップを放出することによって遠心管に漏斗で水の下部の50ミリリットル（ml）を排出します。 </li><li> 5分間600×gで（相対遠心力）で収集された水を遠心し、慎重に土砂を乱すことなく上澄みを注ぐ。チューブの最後の数滴を保持します。 </li><li>顕微鏡スライド上に溶液の沈殿物及びピペット2滴を再び中断し、スライドを調べる（検出と種の確認のための100-400X倍率40倍の倍率）光学顕微鏡下で。 Sの最初の段階の幼虫（L1） 線虫は生きていると積極的に顕微鏡下に移動します。 </li></ol> 3。 20メートルシャトルランテスト<ol><li> 20メートルフラットでストレートランニングコースを測定して、コーンでマーク。 </li><li>テスト参加者は胸周り18手首に心拍数モニターと対応するトランスミッターを装着しましょう。この機器は、参加者の心拍数は、試験前後で測定することができますので、参加者は最大の努力を発揮しているかどうかを監視します。 </li><li> 20メートルのラップを区切る事前に記録された音声信号のペースに従うことによってコース上を前後に実行するように参加者に指示する。時速8.5キロ（km /時）、0.5キロ/毎分HRと同等で20メートル間隔の増加を示す信号の周波数の走行速度で始まる。 </li><li>目を確認してくださいで参加者は、新しい20メートル間隔に信号を鳴らすたびに開始され、次の信号が到着する前に、それを終了します。 </li><li>彼/彼女は、2つの連続した​​20メートル間隔でペースに従わない場合、参加を停止します。 </li><li>参加者は参加者の年齢や性別に加えて、完全に完成した最後の間隔のために示さ最高走行速度を記録します。 </li><li>試験終了時に達成し、心拍数が19の子供の最大心拍数であることを確認します。この心拍数に達していない場合、子は、最大限の努力を発揮していない可能性があり、したがって、テストを繰り返すべきである。 </li><li> 3.248 *年齢（年）+ - VO 2 MAX = 31.025 + 3.238 *速度（km /時）：レジェと共同研究者によって述べられ式を使って参加者の最大有酸素能力（VO 2 ​​max）を、推定0.1536 *速度（km /時）*年齢（年数）。 </li></ol> 4。代表的な結果の面では加藤カッツの技術とSTH診断は、別の寄生虫卵のユニークな特徴は、20ほとんどの種の同定を可能にします。 A.lumbricoidesの卵（約45から75マイクロメートル（μm）長いと35から50μm幅、 図1）は T.trichiura（ 図2）、鉤虫（ 図3）の卵よりも厚いシェルの壁を持っています。非受精卵この外皮を欠いており、全体的にやや細長く、大きいながらA.lumbricoidesの受精卵（ 図1）は 、それが黄金色に見えるように外側のアルブミンコートを持っています。 T.trichiura（ 図2、約50μm長いと22μm幅）の卵は細長いあり、異なる極性のプラグがあります。鉤虫の卵（約64から76程度長いと36から40μm幅、 図3）は 、他の一方で、細胞の高密度クラスタの周りに透明なリングに続いて非常に薄い壁を持っている卵の中央に置きます。 S.stercoralis L 1の幼虫（ 図4）は、約180から380程度の長さで、非常に短い口腔を持っています。明確な生殖原基（ 図4の青矢印で強調表示）を観察し、幼虫の尾することができます（ 図4の赤い矢印で強調）鋭い端を持っています。 子どもの体力に関するSTH感染症の本当の影響がまだ13,14,15を議論されているように、この時点では、その影響のための代表的な結果ではありません。 20メートルシャトルランテストの結果の解釈は様々な方法で行うことができます。子どもの体力は、年齢や性別、その他の潜在的に関連する健康状態、社会経済的地位​​、文化的習慣の点で似ている感染および非感染者との間で比較することができます。これは、横断調査13,14,15で行うことができる場所STHの感染状況と感染および非感染した子供の体力は、特定の時点で評価される。学齢期の子どもたちの体力が駆虫薬/プラセボ治療の前後に評価されているランダム化比較試験の設計を行なった際、証拠の強さは、両方の短期および長期の評価を可能にする、高いであろう。一つは、さらにSTH感染症や体力の強さの間の可能な用量反応関係を識別し、体力との共寄生1の異なるレベルを比較してみることができます。 VO 2 maxによって示されるようにヤップや同僚15、体力、によって提示された仕事では、大幅T.trichiura（ 図5）に感染していない子どもに比べてT.trichiuraに感染した子どもたちに減少することが見出された。 <img alt="図1" src="/files/ftp_upload/3966/3966fig1.jpg" /> ふぃぎゅ<html> RE 1 A.lumbricoidesの卵の顕微鏡画像：受精卵（）と非受精卵（B）の500倍の倍率下。スケールバー=50μmである。 <img alt="図2" src="/files/ftp_upload/3966/3966fig2.jpg" /> 図2 500倍の倍率下T.trichiuraの卵の顕微鏡画像。スケールバー=50μmである。 <img alt="図3" src="/files/ftp_upload/3966/3966fig3.jpg" /> 図3 500倍の倍率下鉤虫の卵の顕微鏡画像。スケールバー=50μmである。 <img alt="図4" src="/files/ftp_upload/3966/3966fig4.jpg" /> 図4：250X倍率下S.stercoralis L 1は幼虫の顕微鏡画像。矢印はテール（赤）と生殖器原基（青）を示す。スケールバー=100μmである。 d/3966/3966fig5.jpg &quot;/&gt; 図5 Tに感染していない子どもたちの体力レベルと比較しT.trichiuraに感染して子供の体力レベルを示すグラフtrichiura。

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本稿で説明した3つのプロトコルがBulang少数民族21のメンバーの中で、アフリカ6,13,14のさまざまな部分での雲南省、中国の人民共和国の南西部にテストされ、処刑されている。 加藤カッツ技術のための利用可能ないくつかのテンプレートがあります。穴のサイズはそれぞれの異なった糞便材料の異なる量をお届けします。 EPGはの計算のために、それはEPG?...

注：このリストでマテリアルが一般的であり、異なるソースから取得することができます。 <table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>加藤カッツキット 厚さ1.5mm、テンプレート内の6ミリメートル（mm）の（直径）の穴を持つ[400プラスチック製のテンプレート; 400プラスチック製のへら、80メッシュサイズのナイロン画面の20メートル役割;親水セロファンの20メートルのロール、34厚μmである。] </td><td>ベスタガードフランセン</td><td></td><td>グリセリン - マラカイトグリーン溶液はこのキットに含まれていませんが、どんな化学サプライヤーから購入することができますのでご注意ください。 </td></tr><tr><td>ガラス漏斗[約8センチメートル（cm）幅および6cm深く] </td><td></td><td></td><td> Baermann技術用ガラス漏斗は水の約60 mLを保持することができるはずです。木曜日ゴムホースとガーゼのEサイズは、使用されるガラス漏斗の種類に依存しています。 </td></tr><tr><td>生物顕微鏡</td><td>オリンポス</td><td> CX21LED/CX21 </td><td></td></tr><tr><td>遠心</td><td>上海医療機器グループ</td><td> 80 - 2T </td><td></td></tr><tr><td>事前に記録された信号</td><td> Bitworksデザイン</td><td>チーム一体全体テストバージョン1.3.1 </td><td> iPhoneアプリケーション</td></tr><tr><td>心拍数モニター</td><td> POLAR </td><td> FT1 </td><td>ウォッチとトランスミッタ</td></tr></tbody></table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

土壌伝播蠕虫（STH）感染が一般的である。確かに、10億以上の人々は、主に貧困が優先され、衛生行動、水供給、衛生、しばしば欠損1,2です。 回虫 、 鞭虫 、および2鉤虫種、 ズビニ鉤虫と鉤虫属発展途上国で、影響を受けているamericanus、最も普及しているSTHs 3アール 。鉤虫病、回虫、鞭虫症に起因すると推定され世界的な負担は、10.5 22.1で、それぞれ640万障害調整生存年数（デーリー）、4。さらに、推定30から100000000の人は間違いなくもかなりの公衆衛生への影響5,6を引き起こす糞線虫 、グローバルな重要性の最も無視STH種に感染している。複数種感染症（すなわち、単一の個人に抱い異なるSTHs）が共通しており、感染症があった林<html>下げ、生産性と途上国の経済見通しこうして1,3ヒツジシラミバエ。 共通STHsの診断のために、世界保健機関（WHO）はこのような感染症の有病率と強度を決定するための比較的簡単な方法である加藤カッツテクニック7,8を推奨しています 。それは、感染した被験者が糞便中に渡すこと寄生虫卵の検出を容易にする。 S.stercoralisの診断に関しては、使用可能な、シンプルで正確なツールは今のところありません。 Baermann技術は、その診断のために最も広く使用される方法です。 Baermann技術の背後にある原理は、その幼虫は走光9アールとして活躍S.stercoralisの幼虫が照らされ、新鮮な糞便サンプルからの移行です。それはあまり洗練された実験用の材料を必要とし、文化や免疫学的方法が5未満の時間がかかります。 t &quot;のような腹痛、下痢、掻痒症、貧血などの慢性症状に、アンダーや栄養失調、および認知障害10などの急性しかし一般的な症状、。からSTH感染範囲に関連付けられた&gt;病的症状は、一般的に非特異的であるためと微妙には、彼らはしばしば見過ごさ、影響を受ける個人が正常な状態であると見なされたり、与えられた設定の方が一般的かもしれない他の病気の症状として扱われます。したがって、それはSTH感染症の真の負担は評価によって過小評価されていることが考えられる通常、人口ベースの調査の場合のようにツールが自己宣言徴候や症状に頼る。 1980年代後半から1990年代初頭には、スティーブンソンらは6-12年の11,12歳の少年たちの体力を下げるSTH感染の可能性を強調した。科学的探究のこの行は、最近13,14,15新たな勢いを得た。 20メートル（m）シャトルランテストが開発されましたdは16レジェらによって検証され、子供17の有酸素運動を測定するために世界中で使われている。テストでは、標準化が容易であり、20メートル長いとフラットランニングコースとオーディオソースが使用可能な限り行うことができ、リソースに制約が設定13での使用は魅力的。 STH感染は学齢児童の体力に影響を及ぼすかどうかを評価する試みを容易にし、標準化するために、我々は実行すると、まだ、正確かつ再現性の成果を提供するために簡単ですSTH感染またはメジャー体力の診断方法論を提示する。これは、STH感染症の健康への影響に関する新たな証拠を生成するのに役立ちます。

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

土壌送信蠕虫（STHs）による慢性感染症が発育し、成長期の子供に無駄に吸収不良を引き起こします。それゆえ、これらの感染症はまた、子どもたちの体力を減らすことが妥当である。ここでは、STHsと子供の体力を評価するため、20メートルシャトルランテストの診断のための二つの技術を可視化する。

土壌伝播蠕虫の感染状況及び学齢児童の体力を決定

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

Research

JoVE Journal

Immunology and Infection

23.1K ビュー.  Swiss Tropical and Public Health Institute, Basel, Switzerland. 土壌送信蠕虫（STHs）による慢性感染症が発育し、成長期の子供に無駄に吸収不良を引き起こします。それゆえ、これらの感染症はまた、子どもたちの体力を減らすことが妥当である。ここでは、STHsと子供の体力を評価するため、20メートルシャトルランテストの診断のための二つの技術を可視化する。

ビデオ: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

臨床抗体サンプルの貪食活性を決定する

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

免疫・感染学

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

から卵を使用して、<em&gt;マンソン住血吸虫</em&gt;として<emインビボ&gt;</em蠕虫によって誘発される肺の炎症の&gt;モデル

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

アレルギー性​​気道炎症時の肺樹状細胞の成熟および移行の解析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

哺乳動物細胞に関連して細菌の生存を決定するための蛍光顕微鏡法

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

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

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

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

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

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

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

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

ヒト免疫不全ウイルスの複製フィットネスを決定するためのペアごとの成長競合アッセイ

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

自己免疫性脱髄に薬理学的介入のためのCNS保護に対する免疫系の抑制を決定します

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

コットンボールワームでデンソウイルスのホストフィットネス上のホスト組織分布、伝送モード、および効果を調査するためのプロトコル

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

レプトスピラトランスポゾン挿入変異体中に黄金シリアのハムスター感染の測定フィットネスにハイスループット並列配列

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...