Name: sublingual immunotherapy as an alternative to induce protection against acute respiratory infections
Uploaded: 2014-08-30T14:00:00.000+00:00
Duration: 16 min 56 s
Description: Watch this Scientific Journal Video about Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections at JoVE.com

We acknowledge Dr. Jean-Claude Sirard from the Center for Infection and Immunity of Lille, Institute Pasteur de Lille-France, for kindly providing the purified flagellin and Dr. Teresa Camou, Director of the National Reference Laboratory, Ministry of Health of Uruguay for kindly providing the pneumococcal strain. 
The authors would like to express their acknowledgement to Mr. Diego Acosta and Mr. Ignacio Turel form BichoFeo Producciones-Uruguay for their commitment and hard work during the entire video production and edition. 
This work was supported by the grants PR_FCE_2009_1_2783 and BE_POS_2010_1_2544 from the National Agency of Research and Innovation, ANII from Uruguay, the Program for Development of Basic Sciences, PEDECIBA of Uruguay and Sectoral Commission of Scientific research, CSIC-Universidad de la Rep&#250;blica, Uruguay.

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The authors have nothing to disclose.

治療剤の舌下投与は気道における免疫応答を調節するための有用な手段として実証されている。呼吸器疾患の治療のためのSLITの主な利点は、鼻腔内投与31に基づく治療法よりも安全である、肺や鼻孔への化合物の直接の送達を必要としないことである。 舌下免疫療法は、いずれかのアレルギー性炎症および喘息32の症状を改善するか、またはここに示されているように、急性肺感染症を治療するために先天性免疫機構の一時的活性化を誘導するために、調節応答の誘導のために、さまざまな方法で免疫応答を調節するために使用することができる。 このビデオで提示マウスモデルは、SLITための治療剤としての異なる化合物のスクリーニングのための便利な方法である。 この動物モデルは影響を決定するために有用な手段を提供肺の免疫応答、ならびに他の器官（ 例えば 、リンパ節または遠位粘膜部位を排出する）in vitroモデルを使用することによって模倣することができないでSLITの。舌下免疫療法を用いて得られた結果を説明するいくつかの論文がありますが、舌下投与の手順のための詳細な方法は、まだ利用可能とされていない。さらに、モデルは、気道における全身ならびに局所防御を付与することを目的舌下ワクチンの評価のために用いることができる。 添付のビデオに示されるように、化合物の舌下投与を容易に広範囲なトレーニングを必要とせずに行うことができる簡単な手順である。一般的には、動物取扱に習熟人が、このプロトコールに記載されているように注射用麻酔薬を使用した10匹のマウスのグループにスリットを実行するために1時間が必要になります。肺炎球菌チャレンジが同様に実行される場合、追加の90分を準備する必要があります細菌懸濁液と動物の鼻腔内チャレンジを行う。 ここで紹介するのFACSプロトコルは、リンパ節だけでなく、肺の「細胞動態への影響を排液、投与ローカルサイトのSLITの影響の便利な特性評価を可能にする。 気管支コンテンツおよび肺実質の個別の分析では、組織内にとどまるものから、気道の免疫常駐し、浸潤細胞型を区別することが重要です。 BALの内容の分析は、肺胞マクロファージの代謝回転の研究だけでなく、異なる処理によって誘導された肺胞腔、 例えば 、PMNの、好酸球、単球への細胞動員のダイナミクスを可能にする。 BALはまた、酵素結合免疫吸着アッセイ（ELISA）または舌下ワクチン接種後に誘発された分泌されたIgA抗体の検出によって分泌されるサイトカインおよびケモカインの存在を評価するために使用することができる。肺の「組織に関する研究他の細胞型、古典的樹状細胞、T細胞およびB細胞の特徴付けを可能にする。 FACS分析のためのBAL試料及びリンパ節の調製は簡単である。サンプル収集の後、通常は60分〜20のサンプルについて染色プロトコルを完了するために必要とされる。対照的に、肺または舌下組織からの細胞の単離は、細胞外マトリックスの消化が必要とされるので、より多くの時間を必要とする。舌下経路により送達される治療剤の吸収は、 生体内撮像系を用いて 、蛍光または放射性標識された分子を追跡することによって対処することができる。 舌下免疫療法は効果的に呼吸器疾患を治療または予防するために使用することができ、気道ならびに全身的に免疫応答を誘導するための魅力的な方法である。スリットiの後に気道における免疫応答の寛容対活性化を決定メカニズムの解明単独でまたは別の呼吸器状態に対する利用可能な治療と組み合わせて使用​​することができる新たな治療戦略の合理的設計を可能にするために重要だ。

The overall goal of this work is to illustrate the benefits of sublingual immunotherapy for the treatment of acute respiratory infections (ARI) and present the advantages of this delivery route compared to other routes of administration, namely intranasal.
ARI cause millions of deaths every year especially in children under five. Streptococcus pneumoniae remains as one of the major etiological agents of bacterial pneumonia in infants and the elderly1,2. To present, the main available treatment relies on the use of antibiotics but resistant strains are continuously arising3,4.
SLIT induces broad responses at systemic and also mucosal level, particularly at the respiratory tract5. It has proven effectiveness against influenza infection, promoting long term protection with production of humoral and cellular responses6,7. Besides, it has been shown that prophylactic treatment with bacterial lysates delivered by sublingual route reduced exacerbations of chronic obstructive bronchitis in the elderly8 and prevented recurrent respiratory infections in children9. SLIT has been widely used for the treatment of allergies and asthma. Clinical studies had not only demonstrated its efficacy to modulate the immune response in the respiratory tract but also its safety10. Despite the growing interest of pharmaceutical companies and researchers in SLIT, the mechanisms involved in the induction of mucosal immune responses after sublingual delivery of compounds remain obscure. Recently, attention has been focused on the mechanisms promoting tolerance associated with allergen desensitization. It has been proposed that resident and recruited cells at the sublingual mucosa, like dendritic cells and macrophages, can promote tolerance after SLIT11-13. Dendritic cells of the oral mucosa can promote IFN-gamma and IL-10 producing T helper cells11 as well as recirculate to the distal genital mucosa and promote CD8+ T cells14. However, little is known about the impact of SLIT on innate cells or its capacity to improve pathogen clearance during acute respiratory infections.
The natural control of pneumococcal infection in the lungs greatly depends on the efficient and swift activation of local innate defences. We previously showed that enhancement of lungs&#8217; innate immunity by a single intranasal dose of flagellin (FliC), a TLR5 and NLRC4 agonist, protects 75-100% of mice challenged with a lethal dose of a clinical isolate of Streptococcus pneumoniae serotype 1. This protection was shown to be dependent on local recruitment of GR1+ cells (likely polymorphonuclear neutrophils, PMNs) and not dependent on antibodies, B or T cells15. 
Flagellin is the structural component of the bacterial flagellum. In its monomeric form it is recognized by two Pathogen Recognition Receptors (PRRs), TLR5 that senses extracellular FliC16 and NLRC4/NAIP5 inflammasome that detects intracellular flagellin17,18. When FliC is sensed by the PRRs an important inflammatory response is triggered. We and others have demonstrated that instillation of purified FliC from Salmonella enterica serovar Typhimurium into the lungs drives swift production of chemokines and cytokines specially when recognized by the lungs&#8217; epithelium that in turn orchestrate the recruitment of immune cells into the airways, mainly PMNs15,19-21. Although transient, the substantial neutrophil infiltration that takes place into the airways after nasal delivery of FliC could be a concern if moving towards clinical therapies for human use. Excessive inflammation could be detrimental for the lungs&#8217; function. Moreover, it has been shown that intranasal delivery of immunostimulatory molecules may cause facial paralysis and/or brain toxicity22-24. 
Sublingual immunotherapy offers a safer alternative to modulate the immune response in the respiratory tract compared to the intranasal route. It is non-invasive, painless, simple and has good patient compliance25. Furthermore, as mentioned before, it can induce protective responses in the respiratory mucosa without the risks associated to direct intranasal or intrapulmonary delivery of formulations. Sublingual route could be alternatively used to deliver molecules that have great effects onto the lung&#8217;s immune system but that have been proven to be toxic or to elicit great inflammation when administered intranasally. Besides these advantages, formulations for sublingual immunotherapy have lower cost of manufacture since non-sterile products can be delivered by this route and endotoxic shock is not a concern for SLIT. On the other hand, it is worth noticing that higher doses of the immunostimulatory compounds compared to those used by intranasal or parenteral routes are necessary to induce an immune response in the lungs; also highly concentrated solutions are needed when using the mouse model of SLIT since the anatomical site where the formulations are deposited is small. 
Based on our previous published data, we developed a model of protection using sublingual immunotherapy with flagellin as model immunostimulant. We demonstrated that a single dose of flagellin induced 60% survival against invasive pneumococcal pneumonia caused by the serotype 1 strain while all mice in the control group died of infection within 5 days. Flow cytometry analysis showed that higher numbers of PMN are recruited into the airways of protected animals after sublingual treatment with flagellin suggesting that these cells might be involved in the mechanism of protection induced by sublingual immunotherapy. 
This video shows in detail how to perform sublingual immunotherapy and also how to recover relevant tissue from the sublingual mucosa, draining lymph nodes as well as lungs and airways to perform further analysis. Additionally, it illustrates the general technique of cell preparation for FACS analysis and briefly shows how to prepare Streptococcus pneumoniae suspensions and how to perform intranasal infections in mouse to set up the acute infection model.

<table><tbody><tr><td>Ketamine solution (50 mg/ml)</td><td>Pharma Service, Uruguay</td><td></td><td></td></tr><tr><td>Xilacine solution (2 %)</td><td>Portinco S.A., Uruguay</td><td></td><td></td></tr><tr><td>Sterile 1 ml syringe</td><td>Modern, Uruguay</td><td></td><td></td></tr><tr><td>Sterile 27 G needle</td><td>Modern, Uruguay</td><td></td><td></td></tr><tr><td>RPMI 1640</td><td>General Electric Health Care</td><td>E15885</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>302020</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution</td><td>SIGMA</td><td>P4333</td><td></td></tr><tr><td>Sterile PBS without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>PAA</td><td>H21002</td><td></td></tr><tr><td>Type-I Collagenase</td><td>Life Technologies/Gibco</td><td>17100017</td><td></td></tr><tr><td>Deoxyribonuclease I (DNAse-I)</td><td>SIGMA</td><td>D4513</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies/Gibco</td><td>17105041</td><td></td></tr><tr><td>PerCP-Cy5.5 conjugated rat anti mouse IgG2b anti CD11b</td><td>BD</td><td>550993</td><td>Clone M1/70</td></tr><tr><td>APC conjugated hamster anti mouse IgG1 anti CD11c</td><td>BD</td><td>550261</td><td>Clone HL3&amp;nbsp;</td></tr><tr><td>APC-Cy7 conjugated rat anti mouse IgG2a anti Ly6G</td><td>BD</td><td>560600</td><td>Clone 1A8</td></tr><tr><td>Sterile Saline Solution</td><td>Laboratorio Farmaco Uruguayo, Uruguay</td><td></td><td></td></tr><tr><td>Tryptic Soy Agar</td><td>BD Difco, France</td><td>236950</td><td></td></tr><tr><td>Defibrinated Sheep Blood</td><td>Biokey, Uruguay</td><td></td><td></td></tr><tr><td>Sterile Petri Dishes</td><td>Greiner</td><td>633180</td><td></td></tr><tr><td>p10 Pipette</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>p20 Pipette</td><td>Eppendorf</td><td>3120000097</td><td></td></tr><tr><td>p200 Pipette</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>p200 Pipette</td><td>Capp</td><td>C200</td><td></td></tr><tr><td>p200 Pipette</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>p1000 Pipette</td><td>Eppendorf</td><td>3120000062</td><td></td></tr><tr><td>Sterile Filter Tips p10</td><td>Greiner</td><td>771288</td><td></td></tr><tr><td>Sterile Filter Tips p200</td><td>Greiner</td><td>739288</td><td></td></tr><tr><td>Sterile Filter Tips p1000</td><td>Greiner</td><td>750288</td><td></td></tr><tr><td>Vortex</td><td>BIOSAN</td><td>V1-plus</td><td></td></tr><tr><td>Stainless steel fine tip forceps&amp;nbsp;</td><td>SIGMA</td><td>Z168785/Z168777</td><td>Curved and straight</td></tr><tr><td>Dressing tissue forceps</td><td>SIGMA</td><td>F4392</td><td>Length 8 inches</td></tr><tr><td>Micro-dissecting forceps</td><td>SIGMA</td><td>F4017</td><td>Straight&amp;nbsp;</td></tr><tr><td>Micro-dissecting forceps</td><td>SIGMA</td><td>F4142&amp;nbsp;</td><td>Curved</td></tr><tr><td>Mayo Scissors</td><td>SIGMA</td><td>Z265993&amp;nbsp;</td><td></td></tr><tr><td>Scalpel</td><td>SAKIRA MEDICAL</td><td></td><td></td></tr><tr><td>Sterile Biopsy Punch &amp;Oslash; 3mm</td><td>Stiefel Laboratories Ltd.</td><td>2079D</td><td>5 mm diameter can also be used</td></tr><tr><td>Sterile 1.5 ml Tubes</td><td>Deltalab</td><td>200400P</td><td></td></tr><tr><td>Sterile 15 ml Tubes</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>Sterile 50 ml Tubes</td><td>Greiner</td><td>227261</td><td></td></tr><tr><td>Sterile serological pipettes 5 ml</td><td>Greiner</td><td>606160</td><td></td></tr><tr><td>Sterile serological pipettes 10 ml</td><td>Greiner</td><td>607160</td><td></td></tr><tr><td>Sterile serological pipettes 25 ml</td><td>Greiner</td><td>760180</td><td></td></tr><tr><td>Biological safety cabinet, class II</td><td>Thermo Scientific</td><td>1300 series, type A2</td><td></td></tr><tr><td>Micro-Isolator&amp;nbsp;Rack</td><td>RAIR&amp;nbsp;IsoSystem&amp;nbsp;</td><td>76144W</td><td>Super&amp;nbsp;Mouse&amp;nbsp;1800 AllerZone&amp;nbsp;</td></tr><tr><td>Refrigerated Microcentrifuge</td><td>Eppendorf</td><td>Legend Micro 21R</td><td></td></tr><tr><td>Microcentrifuge</td><td>Heraeus</td><td>Biofuge-pico</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>Sorval ST40R</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>Model&amp;nbsp; 3111</td><td></td></tr><tr><td>Sterile Thin-tip pasteur pipettes</td><td>Deltalab</td><td>D210022</td><td></td></tr><tr><td>Sterile pasteur pipettes</td><td>Deltalab</td><td>200007</td><td></td></tr><tr><td>Sterile 24-well plate</td><td>Greiner</td><td>662160</td><td></td></tr><tr><td>Trypan Blue Solution&amp;nbsp;</td><td>Life Technologies</td><td>T10282</td><td></td></tr><tr><td>Automatic Cell Counter - Countess</td><td>Life Technologies</td><td>C10227&amp;nbsp;</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Life Technologies</td><td>C10312</td><td></td></tr><tr><td>Flow Cytometry Tubes</td><td>BD</td><td>343675</td><td></td></tr><tr><td>Flow Cytometer - FACS Canto-II</td><td>BD</td><td></td><td></td></tr><tr><td>Real Time PCR Instrument - Rotor Gene Q or ABI 7900</td><td>Qiagen / Applied Biosystems</td><td></td><td></td></tr><tr><td>Trizol Reagent</td><td>Life Technologies</td><td>15596-026</td><td>Molecular Biology Grade</td></tr><tr><td>DNAse-I</td><td>Life Technologies</td><td>18068-015</td><td>Molecular Biology Grade</td></tr><tr><td>DNAse-I Buffer 10X</td><td>Life Technologies</td><td>18068015</td><td>Molecular Biology Grade</td></tr><tr><td>EDTA 25 mM</td><td>Life Technologies</td><td>18068015</td><td>Molecular Biology Grade</td></tr><tr><td>Ultra-Pure Water</td><td>Life Technologies</td><td>10977</td><td>Molecular Biology Grade</td></tr><tr><td>RNAse Out</td><td>Life Technologies</td><td>100000840</td><td>Molecular Biology Grade</td></tr><tr><td>Random Hexamer Primers</td><td>Life Technologies</td><td>N8080127</td><td>Molecular Biology Grade</td></tr><tr><td>M-MLV-RT buffer</td><td>Life Technologies</td><td>18057-018</td><td>Molecular Biology Grade</td></tr><tr><td>M-MLV-RT enzime</td><td>Life Technologies</td><td>28025-021</td><td>Molecular Biology Grade</td></tr><tr><td>QuantiTect Syber Green PCR Kit</td><td>Qiagen</td><td>204143</td><td>Molecular Biology Grade</td></tr><tr><td>Specific primers&amp;nbsp;</td><td>Life Technologies</td><td></td><td>Molecular Biology Grade</td></tr></tbody></table>

sublingual immunotherapy as an alternative to induce protection against acute respiratory infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

舌下免疫療法は、正常肺の免疫応答を調節するために使用することができる。私たちは、フラジェリン、TLR5及びNLRC4アゴニストの単回投与は、ケモカインCXCL1、CCL20および生理食塩水処置対照と比較してサイトカインIL-6をコードするmRNAの有意なアップレギュレーションを誘導することができることを示した。 mRNAレベルの誘導倍率は、スリット後の8時間でピークに達し、20時間（ 図1）の後に基礎レベルに戻ります。 SLITはS。肺炎連鎖球菌で2時間前に鼻腔内感染を行った場合しかし、CXCL1およびIL6 mRNAのレベルは、有意に非処置動物（ 図2）と比較して、SLIT後においても24時間後にアップレギュレートされたままであった。 FACSによってBAL中の細胞集団および肺組織の分析は、舌下経路によりFliCで処置した動物は、気道内ではなく肺&#39;組織（ 図3）で好中球の数が増加したことが明らかになった。 図4に示すように、フラジェリンとSLIT、保護を促進し、急性の肺炎球菌性肺炎に対する生存を増加させた。 <img alt="図1" fo:content-width="6.7in" src="/files/ftp_upload/52036/52036fig1highresV2.jpg" width="680" /> 図1フラジェリンと舌下免疫療法後の肺の「転写プロファイルの動態。八〜10週齢のBALB / cマウス（n = 4）は麻酔下で舌下経路によりフラジェリンまたは生理食塩水を10μgで処理した。肺は、異なる時点で採取し、核酸の保存剤中に置いた。全RNA抽出を実施し、cDNAを合成した。 mRNAレベルはTABLに記載されている特異的なプライマーを用いてリアルタイムPCRによって評価した電子1相対的定量化は、正規化のためACTB mRNAレベルを使用したΔCt法に従って行った。結果は、中央値±SEMとして生理食塩水処置群と比較して増加倍数として示されている。アスタリスクは、マンホイットニー検定に従って計算し、統計的に有意な差（p &lt;0.05）を示す。結果は、2つの独立した実験の代表である。 <img alt="図2" fo:content-width="6.2in" src="/files/ftp_upload/52036/52036fig2highresV2.jpg" width="660" /> とフラジェリン。エイト10週齢のBALB / cマウス（n = 4対照群であり、n = 7処理されたグループのために） 舌下免疫療法後に肺炎球菌性肺炎の間に、図2の肺&#39;転写プロファイルは、によってフラジェリンまたは生理食塩水を10μgで処理した麻酔下で舌下経路。 2時間後、マウス10を起こす最小致死量（MLD）を鼻腔内経路により攻撃したS.の臨床分離株の0％の死亡率4×10 5 CFU /50μlに対応する肺炎連鎖球菌血清型1 E1585、。肺は、チャレンジ後24時間後に回収し、RNA抽出およびcDNA合成を実施するまでの核酸保存剤で保存した。リアルタイムPCRを行った（表1のプライマーのリストを参照）および相対的定量化は、正規化のためACTB mRNAレベルを用い応じたΔCt法を実施した。結果は、中央値±SEMとして生理食塩水処置群と比較して増加倍数として示されている。アスタリスクは、マンホイットニー検定に従って計算し、統計的に有意な差（p &lt;0.05）を示す。 <img alt="図3" fo:content-width="6.5in" src="/files/ftp_upload/52036/52036fig3highresV2.jpg" width="680" /> 多形核好中球の図3。分析（PMN）SLIT後に肺の&#39;組織や気道における募集。エイトへ10週齢のBALB / cマウス（N = 4）を麻酔下で舌下経路により、フラジェリンまたは生理食塩水を10μgで処理した。 2時間後、マウスにSのMLDで鼻腔内経路により攻撃した·ニューモニエ血清型1 E1585。 24時間チャレンジ後、BALを実施し、肺をFACS分析のために処理した。 PMNはLy6G 高 / CD11bの高 / CD11cの陰性細胞として同定され、FCS-SSCプロファイルに基づいていた。結果は、BALまたは肺中の全細胞数のに対して、PMNの割合として表されます。バーは、平均±SEM中央値を表す。アスタリスクは、一方向マンホイットニー検定に従って計算統計的に有意な差（p &lt;0.05）を示す。 <img alt="図4" fo:content-width="5.3in" src="/files/ftp_upload/52036/52036fig4highresV2.jpg" width="530" /> フラジェリンと図4 SLITは、急性肺炎球菌性肺炎に対してマウスを保護します。 </strong&gt;エイト10週齢のBALB / cマウス（n = 8）に麻酔下で舌下経路により、フラジェリンまたは生理食塩水を10μgで処理した。 2時間後、マウスにSのMLDで鼻腔内経路により攻撃した·ニューモニエ血清型1 E1585。生存率は、日常的に評価した。カプラン·マイヤー曲線をログランク（マンテル·コックス）検定に従って比較した。アスタリスクは統計的に有意な差（P &lt;0.05）.Resultsは2つの独立した実験の代表であることを示す。 <table align="center" border="1" cellpadding="0" cellspacing="0"><tbody align="center"><tr><td> 名前 </td><td> 配列5&#39;-3 &#39; </td><td> PCR産物lenght（bp）の </td></tr><tr><td> MB-actin_F </td><td> GCTTCTTTGCAGCTCCTTCGT </td><td rowspan="2"> 68 </td></tr><tr><td> MB-actin_R </td><td> CGTCATCCATGGCGAACTG </td></tr><tr><td>mCCL20_F </td><td> TTTTGGGATGGAATTGGACAC </td><td rowspan="2"> 69 </td></tr><tr><td> mCCL20_R </td><td> TGCAGGTGAAGCCTTCAACC </td></tr><tr><td> mCXCL1_F </td><td> CTTGGTTCAGAAAATTGTCCAAAA </td><td rowspan="2"> 84 </td></tr><tr><td> mCXCL1_R </td><td> ACGGTGCCATCAGAGCAGTCT </td></tr><tr><td> MIL-6_F </td><td> GTTCTCTGGGAAATCGTGGAAA </td><td rowspan="2"> 78 </td></tr><tr><td> MIL-6_R </td><td> AAGTGCATCATCGTTGTTCATACA </td></tr><tr><td> mTNFalpha_F </td><td> CATCTTCTCAAAATTCGAGTGACAA </td><td rowspan="2"> 63 </td></tr><tr><td> mTNFalpha_R </td><td> CCTCCACTTGGTGGTTTGCT </td></tr><tr><td> mCxcl2_F </td><td> CCCTCAACGGAAGAACCAAA </td><td rowspan="2"> 72 </td></tr><tr><td> mCxcl2_R </td><td> CACATCAGGTACGATCCAGGC </td></tr></tbody></table> リアルタイムPCR分析のために用いたプライマーを表1のリスト。qPCR分析のために使用される特定のプライマー配列。フォワードおよびマウスACTBのためのリバースプライマー、Cccl20、CXCL1、IL6、TNFAとCXCL1は 、5&#39;-3 &#39;配列として提示され、予想された生成物の長さは塩基対（bp）で示されている。

Watch this Scientific Journal Video about Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections at JoVE.com

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

代替として舌下免疫療法は、急性呼吸器感染症に対する保護を誘導するために

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

sublingual-immunotherapy-as-an-alternative-to-induce-protection

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16.8K ビュー.  Universidad de la República. The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

ビデオ: Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

免疫・感染学

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

ワクチンには、マウスの応答における鼻咽頭関連リンパ網内系組織（NALT）の役割を調べる

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

マウスのリンパ管内免疫療法やワクチン

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

医学

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

の気管内の管理<em&gt;インフルエンザ菌</em&gt;マウスモデルにおいて気道炎症を研究します

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

防御効力および肺免疫応答は、マウスに皮下および鼻腔内BCG投与後の

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

疾患特異的抗体の生成のための抗原性リポソーム

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主-病原体応答とマウスでワクチンの有効性の評価

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

ニュージーランド白ウサギにおける母体免疫の研究における鼻腔内免疫とミルクコレクション (オリクトラグス・キュニクルス)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

代替として舌下免疫療法は、急性呼吸器感染症に対する保護を誘導するために

要約

さらに動画を探す

この動画の章

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

ワクチンには、マウスの応答における鼻咽頭関連リンパ網内系組織（NALT）の役割を調べる

マウスのリンパ管内免疫療法やワクチン

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

の気管内の管理

防御効力および肺免疫応答は、マウスに皮下および鼻腔内BCG投与後の

体内上部気道免疫メディエーターレベルの定量化のための粘膜内層流体の非侵襲的サンプリング

疾患特異的抗体の生成のための抗原性リポソーム

宿主-病原体応答とマウスでワクチンの有効性の評価

ニュージーランド白ウサギにおける母体免疫の研究における鼻腔内免疫とミルクコレクション (オリクトラグス・キュニクルス)

代替として舌下免疫療法は、急性呼吸器感染症に対する保護を誘導するために

要約

さらに動画を探す

この動画の章

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

ワクチンには、マウスの応答における鼻咽頭関連リンパ網内系組織（NALT）の役割を調べる

マウスのリンパ管内免疫療法やワクチン

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

の気管内の管理

防御効力および肺免疫応答は、マウスに皮下および鼻腔内BCG投与後の

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

疾患特異的抗体の生成のための抗原性リポソーム

宿主-病原体応答とマウスでワクチンの有効性の評価

ニュージーランド白ウサギにおける母体免疫の研究における鼻腔内免疫とミルクコレクション (オリクトラグス・キュニクルス)

体内上部気道免疫メディエーターレベルの定量化のための粘膜内層流体の非侵襲的サンプリング