We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

著者らは、開示することは何もありません。

利用可能な農地が減少し、人口が増加すると、世界中の研究者が作物改良のためのニーズを押すでチャレンジされています。収率が大幅に様々な生物的および非生物的ストレスによって影響を受ける可能性があります。その中でも、病原体感染だけでは45米国では約12％の損失を担当する作物収量の減少の主な原因の一つです。この問題を解決するには、大規模な研究は、モデルのシ?...

それらの固着性のために、植物は、常に様々なライフスタイルや栄養戦略1を示す病原体の過多によって脅かされています。壊死栄養性病原体積極的に秘密の毒素や酵素が宿主組織を殺すために、死んだ細胞1に供給するようにしながら、最初の近似として、生物栄養性病原体は、栄養素を取得する生きている彼らのホストを維持します。病原体と呼ばれるhemibiotrophsの別のグループは、病原体の蓄積2の特定のしきい値に達した時に壊死栄養段階への生物栄養段階とシフトとの感染のコースを開始します。効果的にこれらの微生物に対して自身を守るために、植物は、病原体の攻撃を検出してトリガするために、複数の監視機構を搭載した複雑な先天性免疫系を進化させてきた細胞死3だけでなく、全身獲得抵抗性（SAR）を4プログラム局在しました。現在の研究は不可欠SIGを特徴付けるに焦点を当てていますnalingコンポーネントと植物免疫系5内のクロストーク。 「ジグザグ」モデル5で提案されているように、植物の先天性免疫応答の最初の層は、微生物の侵入を検出するために、原形質膜局在パターン認識受容体（のPRR）の存在を必要とします。 PRRは、微生物関連分子パターン（ミリアンペア）を認識し、MAMP・トリガ免疫（MTI）6を確立することができます。抗菌PRタンパク7をコードする遺伝子の転写のアップレギュレーションを誘導するだけでなく、MTIは、細胞壁の活性酸素種の産生（ROS）および活性窒素種（RNS）、カロースの堆積を含む病原体の増殖を、停止イベントの様々なつながり同様に、複数のキナーゼシグナル伝達の活性化は、8が経路として。 これまで、いくつかのミリアンペア、細菌flg22 9を含む 、シロイヌナズナでMTIをトリガするために同定されています</sup&gt;（フラジェリンに由来する22アミノ酸断片）、および構造細胞壁成分elf18 10（細菌の翻訳伸長因子Tuから18アミノ酸）11ペプチドグリカン。成功した感染を確立するには、いくつかの特殊な病原体は、細胞内または細胞間空間に秘密の病原性エフェクタータンパク質の能力を進化させ、その結果、MTIを抑制し、エフェクター・トリガ感受性（ETS）12,13をトリガしています。例えば、病原性エフェクターは感染組織14-16内の疾患の発症を誘導するためにMTIのマイトジェン活性化プロテインキナーゼ（MAPK）のリン酸化カスケードを不活性化することができます。ホストと病原体との間の動的共進化の間に、植物もエフェクタータンパク質を認識し、病原体の病原性分子17を減衰させるために反撃の戦略を開発しました。この直接的または間接的エフェクターの認識は、病害抵抗性（R）タンパク質18によって媒介されます。トンの大部分裾はNB-LRRのメンバー（ヌクレオチドが結合およびロイシンリッチリピート）ファミリー19です。 Rタンパク質によって非病原性エフェクターの認識は、エフェクタートリガ免疫（ETI）20として特徴付け強く、より広範な免疫応答を誘発します。防御遺伝子21の発現および防衛代謝物22の産生を誘導するほかに、ETIは、多くの場合、隣接する組織3に広がるの病原体を制限するために過敏反応（HR）として知られている急速なローカライズされたプログラムされた細胞死につながります。 プログラムされたローカライズされた細胞死23に加えて、植物は（SAR）4全身獲得抵抗性と呼ばれる長期的かつシステム全体の免疫応答を開始することができます。生物栄養性病原体でのチャレンジの際に、植物細胞は、局所および全身の組織24の双方において、内因性の植物ホルモンのサリチル酸（SA）とPRタンパク質の生合成および蓄積を誘発します。トンを通して彼のプロセスは、準備の高まり状態は、病原体24の広いスペクトルによってその後の感染の間に速い防御応答を取り付けるため可能に感染していない葉で達成されます。 SAおよびそのようなベンゾなどの合成類似体（1,2,3） -チアジアゾール-7-カルボチオ酸 S -メチルエステル（BTH）および2,6-ジクロロ酢酸（INA）、化学的にサリチル酸を誘導することができる（SA）外部アプリケーション24時-Triggeredイミュニティ（STI）。病原性関連遺伝子1（NPR1）のNonexpressorは、局所および全身の組織21,25,26の両方でSA媒介防御応答時の主要な転写調節因子としてのSA受容体および機能の一つであることが提案されています。それは決定的にNPR1は、SARの確立のために必要とされ、NPR1の損失はシュードモナスシリン 25の方に劇的な感受性につながることが実証されています。 広く植物の分子の寄与を特徴づけるために、植物病原体相互作用のコンポーネントは、複数のバイオアッセイは、ROSは27バーストを含め、具体的な防衛のイベントを測定するために、それらのタンパク質生成物21のカロース沈着28、防御遺伝子の発現および蓄積を開発されてきました。これらの個々のアッセイは、植物免疫応答の特定のフォームへの洞察を提供することができるが、それらのどれも、しかし、植物全体レベルでの完全な防御応答を表現することができません。逆に、感染後の病原体の成長の定量化は、生物レベルでの免疫応答の全体的な推定値を提供します。したがって、正確性の高い標準化された病原体接種アッセイの開発と最適化は、シロイヌナズナの免疫応答の研究と発見を燃料することが重要です。 シュードモナスシリン 、グラム陰性細菌は、Arabidops含む植物宿主の範囲の疾患を引き起こすことができる植物病原体として同定されました29です。 P. -モデル植物病原体システム、シロイヌナズナとして、 syringaeの相互作用は、広く植物の防御応答に29の基礎となる分子メカニズムを理解するために適用されています。今までは、P。50を超えますシリンゲの pathovarsは、異なる植物種30に感染する能力に基づいて同定されました 。P. syringaeの PV。 トマトDC3000（PST DC3000）31 と P. syringaeのPV。maculicola ES4326（PSM ES4326）32は、二つの最も広く使用され、広範囲に特徴付け病原性株です。別に植物によって認識され、MTI応答を誘発されることから、pstファイル DC3000とPSM ES4326は、MTIを抑制し、病原体の成長31,33を支持するETSをトリガするために病原性エフェクタータンパク質を分泌することができます。 機能的にシロイヌナズナとPとの間の相互作用を分析するために、複数のシリン、0;病原体感染の方法は、病原体の送達アプローチに基づいて開発されてきました。土壌生育した植物は、病原体は、シリンジ浸潤、真空浸透、浸漬、噴霧接種接種29,34によって送達することができます。最近、苗洪水接種アッセイは、組織培養での大規模なスクリーニングを行うために開発された若いアラビドプシス植物35の成長プレート。シリンジ浸潤は、最も一般的に使用される手法の一つとして、手動で気孔29と呼ばれる自然の葉の開口部を通してアポプラストに病原体を提供しています。 Pのこのアプローチにより、等量syringaeのは、感染した葉に浸潤することができ、植物の免疫応答の強度が反比例病原菌の成長レベルと相関しています。したがって、病原体の成長の定量化は、植物全体レベルでの免疫機能を評価するための最適なアプローチとして機能します。また、シリンジ浸潤がbのできる局所および全身の組織を、区別することができますSAR 36の根底にある分子機構の特性を明らかにするのに適切な電子。 以下のプロトコルでは、強化された疾患感受性（EDS）のためのシロイヌナズナ変異体をスクリーニングするために、PSM ES4326と最適化された注射器浸潤アッセイを説明します。このプロトコルは、2つのシロイヌナズナの遺伝子型を採用する：野生型生態型コロンビア-0（COL-0）植物（コントロール）とnpr1-1の機能喪失型変異体を （hypersusceptible） それは、病原性細菌株PSM ES4326 37で感染します。 npr1-1変異体は、チロシンに高度に保存されたヒスチジンを変更し、25の非機能性タンパク質をレンダリングNPR1分子のアンキリンリピートのコンセンサス配列内の点変異を保有します。また、シリンジ浸潤アッセイの改変の数は、MTIおよびSTIを含む免疫応答の特定の層の欠陥の定量化を可能にすることが記載されています。

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			<td>FB0875713</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
			<td>12-565-450&#160;</td>
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			<td>Bio Basic Inc</td>
			<td>MB0328</td>
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			<td>Bio Basic Inc</td>
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			<td>Fisher Scientific</td>
			<td>NC9992493&#160;</td>
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			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
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			<td>Denville Scientific</td>
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			<td>02-215-503</td>
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			<td>Fisher Scientific</td>
			<td>13-100-675</td>
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			<td>3120 000.038</td>
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bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

専門のモバイル免疫細胞の非存在下では、植物は、病原体の攻撃から身を守るために彼らのローカライズされたプログラム細胞死と全身獲得抵抗性を利用します。プラント全体の免疫応答に特有のシロイヌナズナ遺伝子の寄与は、特異的かつ定量的に感染した組織内の病原体の成長を測定することによって評価することができます。 30年以上にわたり、hemibiotrophic細菌シュードモナスシリンPV。maculicola ES4326（PSMの ES4326）が広くシロイヌナズナ免疫応答の根底にある分子メカニズムを研究するためのモデル病原体として適用されています。葉組織への病原体を提供するために、複数の接種方法が確立されている、 例えば、シリンジ浸潤、ディップ接種、スプレー、真空浸透、および洪水接種。以下のプロトコルは、大人の葉に毒性の強いPSM ES4326を提供するために最適化された注射器浸潤法を説明土壌栽培シロイヌナズナ植物と正確には、この病原体に向けて強化された疾患感受性（EDS）をスクリーニング。また、このプロトコルは、さらに、サリチル酸（SA）-Triggeredイミュニティ（STI）とMAMP・トリガ免疫（MTI）を含む植物防御の異なる層内に特異的な免疫欠陥を分析するために、複数の前処理で補充することができます。

私たちはここで説明するプロトコルは、最適化されたPを表し、 syringaeのシリンジ浸潤アッセイは、定量的に、シロイヌナズナ植物における免疫応答を評価しました。 図1に示すように 、PSM ES4326のシリンジ浸潤を連続希釈し、コロニー計数を介して病原体の抽出および定量化が続きます。 プロトコルテキスト内のステップ3で説明し...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

使用した植物防御応答のファインキャラクタリゼーションのための細菌の葉浸潤アッセイ<em&gt;シロイヌナズナシュードモナスシリン</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Plant Biology

Plant Responses to the Environment

SCIENTISTS IN ACTION

21.5K ビュー.  University of Alabama at Birmingham. Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

ビデオ: Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

病原体関連分子パターン（PAMP）トリガ型植物におけるイミュニティ（PTI）のアッセイ

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

免疫・感染学

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 ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身細菌感染や免疫防御表現型<em&gt;キイロショウジョウバエ</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

牛にエフェクターおよびメモリーT細胞応答を評価するための長期培養インターフェロンγ酵素結合免疫アッセイの応用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリン アイソタイプ反応の評価

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

プローブに細菌の病原体を用いた細胞・生体レベルのホスト応答

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

病原性細菌に対する免疫学的応答を評価するための Opsonophagocytic 殺害アッセイ

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...