The authors thank Therwa Hamza, John E. Tidwell, Nina Clovis, and Suzanne Smith for experimental assistance and Suzanne Smith for proofreading. The authors also thank John Thomas, PhD for providing the bacterial clinical isolates and John B. Barnett, PhD for his support and the use of the biological safety lab at the Department of Microbiology, Immunology and Cell Biology at West Virginia University. The authors acknowledge financial support from the Osteosynthesis and Trauma Care Foundation and National Science Foundation (#1003907). Microscope experiments and image analysis were also performed in the West Virginia University Imaging Facility, which is supported in part by the Mary Babb Randolph Cancer Center and NIH grant P20 RR016440.
Animal use for blood draws were approved by the West Virginia University Institutional Animal Care and Use Committee. All experiments were executed in compliance with all relevant guidelines, regulations, and regulatory agencies.

1。 PRPの作製とその活性化 1.1採血<ol><li>イソフルランの吸入（誘導のためのO 2中2％、保守のための1％）でウサギを麻酔。 </li><li> 20mlのシリンジに2ミリリットル0.129 Mのクエン酸三ナトリウムを（anticoagulentソリューション）を描画します。クエン酸三ナトリウム溶液を50 mlの蒸留H 2 O中の1.897グラムクエン酸三ナトリウムを溶解し、0.22μmの滅菌フィルターで濾過することによって調製される。 </li><li> 70％エタノールを用いてウサギの耳を殺菌。 </li><li>翼状針（25g）をシリンジに接続を介して、ウサギの耳静脈から（ 例えば 5ミリリットル）の血を引く。 </li><li>穏やかな攪拌によってクエン酸三ナトリウム溶液を用いて血液を混ぜる。血液とクエン酸三ナトリウム水溶液の体積比は9:1である。 </li></ol> 1.2 PRPの調製（ 図1） <ol><li> 50mlプラスチック遠心管に抗凝固処理血液を転送します。 10μlのアリコートを取る血球計算法を用いてベースライン血小板数を決定するために、血液。 </li><li>スイングアウトローター付き遠心機で室温（RT）で10分間、300×gで血液を遠心分離します。低い（ 図1）への加速とブレーキの速度を設定します。 </li><li>遠心後、血液は3層に分かれています。底層は、中間層（一般的に &quot;バフィーコート&quot;と呼ばれる）は、主に赤血球で濃縮血小板や白血球から構成され、最上位層は、主に血液の液体成分である血漿、血小板である（ 図1）。細胞培養用フードに遠心管を慎重に輸送;層を乱すことがありません。 1ミリリットルプラスチックピペットを用いて、15​​ mlプラスチックチューブに、血漿、バフィーコート、および2-3 mm厚赤血球層のすべてを転送します。 </li><li>室温で15分間3000 xgで転送サンプルをもう一度遠心します。上層（上清）を乏血小板血漿（PPP）と見なされますdは、新しいチューブに移す。 </li><li> 2.0×10 6血小板/μlを（血球計算法によって決定される）を得るために、PPPを使用して、残りの血液サンプル中の血小板濃度を調整することによって、PRPを得る。 </li></ol> 1.3 PRPの活性化<ol><li> 1,000 IU / mlの濃度になるように取り組んで5ミリリットル10％塩化カルシウムで5,000 IUウシトロンビンを溶解させることによって、PRPの活性化溶液を調製します。 </li><li> PRPとPPPにライセンス認証ソリューションを追加して、繰り返して、PRP-およびPPP-ゲルを形成するために、ピペッティングにより溶液を混ぜて使用する。 PRPまたはPPPへの活性化溶液の体積比は1:4です。 </li></ol> 2キルカーブアッセイを使用して、PRP の in vitro抗菌試験（ 図2） では、 <ol><li>無菌接種ループを使用して、Sのいくつかのコロニーを追加プラスチックチューブでミューラーヒントンブロス（MHB）5mlにその晩平板培養から黄色ブドウ球菌 。 Vortexは簡潔にしてから、37℃で2時間サンプルをインキュベート次の、細菌のメディアの光学濃度を分光光度計を用いて決定し、予め決められた標準曲線に基づいて〜1×10 8 cfu / mlに等しい光学密度に調整した。 </li><li> 1×10 6 CFU / mlを取得し、氷の上で接種を配置するPBSを用いて100倍希釈を作る。 </li><li>セットアップおよび滅菌、使い捨て5ミリリットル丸底ポリスチレンチューブにラベルなど、各チューブに2 mlの最終容積については、 表1に示されている以下のサンプル·グループを準備。 </li><li>活性化のためのトロンビン溶液（ゲル形成）が続いポリスチレンチューブにPRP、PPP、またはPBSを最初に追加します。次に、MHBを追加してから、S.球菌接種（1×10 6 CFU / ml）を1×10 5 CFU / mlの最終濃度を得ることができる。 </li><li> ℃で150rpmで攪拌​​しながら軌道37でチューブをインキュベートする。 </li><li>予め決められた時点（ 例えば 0,1、および2時間）で、繰り返しピペッティング（これを経由して各チューブ内の溶液を混合細菌は、PRPゲルの内部に閉じ込められるかもしれないので、ステップ）が重要である。 、サンプルの10μlを取り滅菌0.9％生理食塩水で順次希釈し、トリプシン大豆寒天（TSA、5％羊血液付き）CFUカウントのためのプレート上に各希釈液の100μlアリコートをピペット。 </li><li> 37℃で一晩培養寒天プレート°Cは、その後プレートのコロニーをカウントし、記録します。 y軸上のx軸およびCFU / mlの上の時間（hr）とlogarithimicスケールでデータをプロットします。 </li></ol>

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E., Barnett, J. B., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+effects+of+exogenous+IL-12+supplementation+and+antibiotic+treatment+in+infection+prophylaxis.">Additive effects of exogenous IL-12 supplementation and antibiotic treatment in infection prophylaxis.</a> J. Orthop. Res. 30 (2), 196-202 (2012).</li></ol>

著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

多血小板血漿は、ますますその治癒促進特性15から17に起因する臨床応用に使用されている。本研究では、PRPは、感染予防のための新しいアプローチとして提示された。 PRPは、MRSA、MSSA、A群連鎖球菌 、および淋菌に対しても強い抗菌性を有することが見出された。感染予防のための従来の抗生物質治療と比較して、PRPの主な利点は、以下を含む：（1）現在の抗生物質療?...

インプラント関連感染は重要な臨床合併症である。 黄色ブドウ球菌 （ 黄色ブドウ球菌 ） は、インプラント関連感染症から分離された最も一般的な微生物の一つである。これは、インプラントの表面をカバーし、抗生物質耐性感染1,2につながる可能性がバイオフィルムを製造することができる。インプラント関連感染症の治療は、頻繁に繰り返さdebridementsかつ長期の非経口抗生物質治療のために長期入院を必要とします。抗生物質耐性例では、インプラントの除去が必要になることがあります。抗生物質に対する細菌の耐性が上昇するとしても、疾病管理予防センター（CDC）によって参照されている &quot;世界で最も緊急の健康問題の一つ。&quot;時間では、新しい効果的な抗菌治療法の開発がなければ、多剤耐性病原体は、従来の抗生物質による治療不可能となる可能性があります。インプラント関連の防止感染症は、ことが重要であると新規予防剤またはアプローチはこのような感染症を予防するために必要とされる。 多血小板血漿（PRP）は、骨と骨移植の治癒3から5を助けることができる30以上の成長因子が含まれている自己血の濃度である。骨再生や軟組織の成熟を高めるために、PRPの応用はますますため血小板がリリースし、様々な成長因子のその高濃度の診療所で報告されています。 PRPのいくつかの特徴では、PRPも抗菌特性6-9を持っているかもしれないことを示します。 PRPは、血小板の数が多い、白血球の高濃度（細菌および真菌に対する宿主防御アクションを持っているかもしれない）、複数の抗菌ペプチド7,8,10が含まれています。心臓外科患者の大規模コホートの最近の研究では、創傷閉鎖signif時PRPゲルの術中使用することが明らかになったicantly浅と深い胸骨感染11の発生率を減少させた。これらの理由及び観察のために、私たちは、PRPは、そのよく研究された治癒促進特性以外にも、抗菌特性を持っているという仮説を立てた。感染を防ぐためにPRPを使用しての潜在的な利点があり得る：（i）PRPが従来の抗生物質治療に比べて抵抗性を誘発する可能性が低くなります。 （ⅱ）PRPがまた感染予防に相乗効果があるかもしれ治癒を促進する性質をもっています。PRPの治癒促進特性は、病原体とヒトの細胞がインプラント表面12にはレースをしていると感染の確率を減らすことにより、細菌の付着を防止するためにシールを提供することができます、13。 （ⅲ）PRPは本質的に、生体適合性、安全かつ伝染性疾患のリスクがない。 私たちの長期的な目標は、インプラント関連infectiを防ぐための新たなアプローチとして、PRPを使用することですアドオン。本研究の目的は、PRP は 、in vitro抗菌特性の PRPのを調べるために、二回遠心分離のアプローチを使用して、そのような抗菌性を評価するためのプロトコルを記述するために準備することでした。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

インプラント関連感染が増加して抗生物質耐性、動物と人間の間に抗生物質耐性菌の伝送、および感染症の治療コストの高さに起因する世界的なヘルスケア産業にますます困難になってきています。 そこで本研究では、多血小板血漿（PRP）の潜在的な抗菌特性に基づいてインプラント関連感染の予防に有効である可能性がある新たな戦略を開示している。治癒を促進するためのよく研究された特性のために、PRP（生物学的製剤）はますます整形外科手術、歯と口腔の手術、顎顔面手術、整形手術、スポーツ医学などを含む臨床応用に使用されている PRPはインプラント関連感染の防止に従来の抗生物質治療に高度な代替手段である可能性があります。 PRPを使用することにより、従来の抗生物質治療Siに比べて有利であるかもしれないPRPのNCE抗生物質耐性を誘導する可能性が低いとPRPの抗菌と癒しの促進特性は感染予防上の相乗効果があるかもしれません。それも病原体とヒトの細胞がインプラント表面のためにレースをしていることが知られており、治癒を促進するのPRPのプロパティには、それによって感染の確率を減らす人間の細胞接着を向上させることができます。また、PRPは本質的に、生体適合性、安全かつ伝染性疾患の危険性から自由である。 我々の研究のために、私たちは一般的に整形外科の感染症で発見され、PRPは、in vitroでこれらの細菌に対する抗菌性を持っているかどうかを検討されているいくつかの臨床菌株を選択しました。我々は、同じ血小板濃度がすべてのサンプルを得ることができるようになり二回遠心分離のアプローチを使用してPRPを用意しました。我々は、一貫性のある抗菌性の調査結果を達成し、PRPは、in vitroでメティのような細菌に対する抗菌性を強いていることを発見したウイルスバスター感受性およびメチシリン耐性黄色ブドウ球菌、A群連鎖球菌 、および淋菌 。したがって、PRPの使用は、感染を防ぐために、インプラント関連感染の高価な術後治療の必要性を低減する可能性を秘めているかもしれません。

PRPは、再現性回遠心分離法（ 図1）を用いて調製される。 PRPは、メチシリン耐性S.に対する in vitro抗菌特性で （CFUsで最大100倍の減少）は、強力な存在が判明した一般的に世界中の病院14に見られる黄色ブドウ球菌 （MRSA）（ 図3）。同様に、PRPはメチシリン感受性球菌に対して強い抗菌性を持って黄色ブドウ球菌 （MSSA）、A?...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

インプラント関連感染は重要な臨床合併症である。この研究は、インプラント関連感染を防ぐために、多血小板血漿（PRP）を用いたアプローチを説明し、一定の血小板濃度でPRPを調製するためのプロトコルを提示して、PRPの新たに同定された抗菌性や抗菌性を調べるための関連プロトコルを報告<emin vitroで&gt;。</em

感染を防ぐための新しいアプローチとして、PRP：準備と<em&gt;インビトロ</emPRPの&gt;抗菌特性

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

prp-as-new-approach-to-prevent-infection-preparation-vitro

Research

JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K ビュー.  West Virginia University. インプラント関連感染は重要な臨床合併症である。この研究は、インプラント関連感染を防ぐために、多血小板血漿（PRP）を用いたアプローチを説明し、一定の血小板濃度でPRPを調製するためのプロトコルを提示して、PRPの新たに同定された抗菌性や抗菌性を調べるための関連プロトコルを報告。

ビデオ: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

シンプルで堅牢な<emin vivoでの>と in vitroで</emウイルスの組み立てを学ぶための>アプローチ

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;ハチノスツヅリガ</em&gt;モデル生物として研究する<em&gt;レジオネラ·ニューモフィラ</em&gt;感染症

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

遺伝的分化を制御するメカニズムを研究するためのアプローチと機能などのヘルパーT細胞のレトロウイルス形質導入

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...