We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

Tüm deneyler Baylor Araştırma Enstitüsü Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) yönergelerine uygun olarak yapıldı. 1. Kültürleme tipi belirsiz Haemophilus influenzae (NTHi) ve aşı hazırlama <ol><li> Bir çikolata agar plakası üzerinde Plaka NTHi ve 37 ° C&#39;de ve% 5 CO2 seviyesinde bir gece boyunca nemlendirilmiş bir CO2 kuluçka makinesi içinde başaşağı plaka tutun. Ertesi gün, kültür beyin kalp infüzyon suyu bakteri. Daha sonra, steril 1.5 ml&#39;lik tüp içine suyu 1 ml. </li><li> Oda sıcaklığında 5 dakika boyunca 4000 x g&#39;de santrifüjleyin. Sonra, süpernatantı atmak ve steril 1x PBS 1 ml pelet yeniden askıya. kez bu adımı yineleyin. </li><li> Bir spektrofotometrede 600 nm dalga boyunda seyreltilmiş kültür absorbansı 1x PBS 900 ul içinde yeniden süspansiyon haline çözeltisi 100 ul aktarın ve ölçün. </li><li> canlılık ve koloni oluşturan birim belirleyinçukulata agar plakaları üzerinde 1:10 seri dilüsyonları kültürlenmesi ve CO2 37 ° C&#39;de gece boyunca inkübe ve% 5 inokulum S (CFU). </li><li> Önceki aşamada belirlenen canlılık ve CFU göre, 20 x 10 6 CFU / ml&#39;lik bir son konsantrasyona inokulum ayarlayın. </li></ol> 2. İçi trakea (o) damlatma <ol><li> 150 mg / ml ketamin + 20 mg / ml ksilizin çözeltisi ile fare, 0.1 ml / 10 g vücut ağırlığına sahip intraperitonal fare (ip) enjekte edilir. prosedür hayvan sıcak tutmak için bir ışık kaynağı ile kaputu yapılmalıdır. </li><li> Fare yeterli anestezi olduğunu doğrulamak için fare birlisme alan sıkıştırın. </li><li> Sırtında fare yerleştirin. boyun ventral alan tıraş ve klorheksidin ve% 70 etil alkol ile dezenfekte edin. </li><li> Sıçan diş forseps kullanarak boyun üst karın kısmının cilt kaldırın. Bir neşter kullanılarak timus üzerinde küçük bir kesi (0.5-1.0 cm) yapmakve dikkatle trakea maruz kas teşrih. <ol><li> 27 G iğne ile 1 ml hava ile yavaş yavaş başka bir 0.05 ml hava ve ardından, bir kontrol olarak, Bölüm 1 veya tuz elde edilen 0.05 mi NTHi, ardından 0.05 mi hava, enjekte edilir. yaklaşık 1 dakika boyunca dikey hayvan tutun. </li></ol></li><li> cerrahi yapıştırıcı ile kesi kapatın. yaklaşık 10 dakika yanal yaslanmış ve sıcak fare tutmak. </li></ol> 3. BALS Koleksiyonu <ol><li> enfeksiyondan sonra servikal dislokasyon 24 saat ile fare Kurban. </li><li> anatomik makas ile fare karın boşluğuna açın ve kanamaya ventral aort kesti. Akciğerin ventral kısmını Açığa. </li><li> trakea Açığa ve 20 gauge kateter ile entübe. BALF tamponu 0.8 ml (1 x PBS,% 0.1 dekstroz, 10 U / ml heparin) aşılamak ve yumuşak aspirasyon ile lavaj toplanır. Bu işlemi üç kez tekrarlayın. </li><li> Bir hemocyto kullanılarak BALF süspansiyon 100 ul toplam hücre sayısınıTripan Mavisi boyama ile 10X büyütme altında ölçer. </li><li> modifiye Giemsa boyama için slaytlar hazırlayın. 5 dakika boyunca 500 xg&#39;de Sitospin ve santrifüj uygun oyuklarına BAL&#39;da alikosu 100 ul. 10 dakika için slaytlar kurutun ve üretici protokollerine göre modifiye Giemsa kullanarak hücreleri leke. </li><li> Bu floresans (FACS) aktive hücre, konfokal mikroskopi, gen ifadesi ve western blot 11-13 kadar bir sonraki analiz için geri kalan hücreler, kullanın. </li></ol> 4. Histopatolojisi Hazırlık <ol><li> Adım 2.4 açıklandığı gibi histopatoloji analizi için, NTHi ile fareleri enfekte. enfeksiyonun 24 saat sonra, servikal dislokasyon ile hayvanlar kurban. akciğerler ve daha önce de belirtildiği gibi trakea maruz anatomik makas ile göğüs boşluğu açmak kesin. Sonra, trakea içine 20 gauge kateter yerleştirin. </li><li> % 4 formalin içinde hazırlanan% 2 sıcak agaroz çözeltisi ile akciğer doldurun. Bir kereAkciğerler tam agaroz çözüm katılaşmaya akciğerlere üstüne yeri buz, şişirilmiş. </li><li> Sonra, dikkatle göğüs fişi çıkarın ve hematoksilen ve eozin (H &amp; E) bölümleri için işlenir kadar 1x PBS içinde% 4 formalin 50 ml çözelti koydu. </li></ol> BAL&#39;da Hücreleri Boyama 5. FACS <ol><li> 96 oyuklu V-tabanlı mikro titre plaka BALS sıvısından 1 x 10 5 hücre koyun. </li><li> 4 ° C&#39;de 3 dakika boyunca 4000 x g&#39;de 300 soğuk 1 x PBS ul ve spin hücreleri yıkayın. </li><li> (1: 1000 seyreltme) 100 ul 1x PBS hücre leke çözeltisi ile hücreleri leke ve 15 dakika boyunca oda sıcaklığında tutulması. </li><li> 4 ° C&#39;de 3 dakika boyunca 4000 x g&#39;de soğuk 1 x PBS, 300 ul hücre yıkayın. </li><li> 1 içeren FACS yıkama tamponu [Yıkama tamponu (1 x PBS,% 2 FBS, 1 mM EDTA,% 0.1 sodyum azid] 100 ul hücre edin: 100 seyreltilmiş Fc Block, 4 ° C&#39;de 15 dakika karıştırıldı. </li><li> 4.000 xg&#39;de Santrifüj hücreleri 3 dakika ve sup atınernatant. </li><li> 4 ° C&#39;de 30 dakika boyunca bir FACS yıkama tamponu (100 seyreltme 1) 100 ul yüzey boyama kokteyli ile Leke hücreleri. </li><li> 4 ° C&#39;de 3 dakika boyunca 4000 x g&#39;de FACS yıkama tamponu 300 ul ile üç kez hücreleri yıkayın. </li><li> Oda sıcaklığında 15 dakika süreyle% 4 paraformaldehid, 100 ul hücre düzeltildi. </li><li> 4 ° C&#39;de 3 dakika boyunca 400 x g&#39;de PBS 1X 300 ul hücreler iki kez yıkanır ve FACS yıkama tamponu 300 ul içinde yeniden askıya. </li><li> boncuk flow sitometri kullanılarak tek renkli boyama kontrolleri yapın. <ol><li> 96 oyuklu V-tabanlı mikrotiter plakası içine tanelerin bir damla ekleme ve PBS 1X 200 ul ile yıkayın. </li><li> PBS ve boncuk 1x 100 ul lekeleme antikorun 1 ul ilave edin ve 5 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> PBS 1X 300 ul iki kez boncuk yıkayın. </li></ol></li><li> 14 flow sitometri tek lekeli kontrolleri ve hücreleri analiz edin. </li></ol> RNA izole etmek Akciğer Doku 6. Homojenizasyon <ol><li> Enfeksiyondan sonra, akciğer (20-40 mg), küçük bir parça toplamak ve gece boyunca 4 ° C &#39;de reaktif stabilize edici RNA tutun. Daha sonra, bir sonraki aşamada kadar -80 ° C&#39;de saklayın. </li><li> RNA stabilizasyonu ayıracı kurtulmak için soğuk 1x PBS akciğer dokuları yıkayın. </li><li> 1.5 ml santrifüj tüpü ihtiva eden liziz tamponu (1 mi liziz tamponu, 10 ul β-merkaptoetanol) halinde doku yerleştirin. </li><li> sivri makas kullanarak küçük parçalar halinde doku doğrayın ve 20-40 saniye boyunca bir kablosuz motorlu pelet havaneli kullanarak homojenize. </li><li> 5 dakika boyunca buz üzerinde lizat tutun ve adım 7.1 ile devam edin. </li></ol> Akciğer 7. RNA İzolasyon <ol><li> 3 dakika boyunca 18.000 x g&#39;de lizat santrifüjleyin. pipetle ile süpernatantı ve dikkatle yeni bir 1.5 ml tüp aktarın. </li><li> lizat% 70 etanol içinde 1 hacim. Pipetleme tarafından hemen karıştırın ve 2-4 dakika bekleyin. </li><li> yerleştirilmiş bir döndürme kolonuna lizat 700 ul kadar transfer2 ml&#39;lik bir toplama tüpü içinde. </li><li> 15 saniye boyunca 9.600 xg&#39;de Santrifüj ve flow-through atın. </li><li> 15 saniye boyunca 9.600 xg&#39;de 350 mcL sıkı yıkama tamponu ve santrifüj ekleyin. flow-through atın. </li><li> Doğrudan spin kolon zarına 80 ul DNaz eklenir ve 15 dakika için oda sıcaklığında saklayın. </li><li> 15 saniye boyunca 9.600 xg&#39;de spin kolon ve santrifüj 350 ul sıkı yıkama tamponu ekleyin. flow-through atın. </li><li> 15 saniye boyunca 9.600 xg&#39;de 500 ul RNA yıkama tamponu ve santrifüj ekleyin. flow-through atın. </li><li> 2 dakika süreyle 9600 xg&#39;de 500 ul RNA yıkama tamponu ve spin ekleyin. flow-through atın. </li><li> 1 dakika boyunca 9.600 xg&#39;de yeni 2 ml toplama tüpü ve santrifüj spin kolon yerleştirin. flow-through atın. </li><li> 1 dakika boyunca 9.600 x g&#39;de spin kolon ve santrifüj doğrudan 30-50 mL RNaz içermeyen su ilave edilir. -80 ° C&#39;de saklayın RNA. </li><li> RNAseq analizi 14 gerçekleştirin. </li></ol>

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The authors have nothing to disclose.

Bu yazıda, bakteriyel akciğer patojen ile farelerin akciğerleri aşılamak için eşsiz ve minimal invaziv prosedürü açıklar. Bu prosedür, inflamatuar sinyal yollarının genleri eksik olan fareler kullanılarak, farklı genlerin fonksiyonunu incelemek için kullanılabileceğini göstermektedir. Bu prosedür, aynı zamanda, viral ve mantar akciğer enfeksiyonu enflamatuvar yanıtları incelemek için kullanılabilir. Böyle intranazal veya aerosol inhalasyon gibi diğer yöntemlere göre bu prosedürün avantaj...

Enflamasyon enfeksiyöz ajanlara karşı savunma temel bir bağışıklık mekanizmadır. Bu patojen ortadan kaldırılması ve hasarlı doku onarımını teşvik eder. Aynı zamanda normal sağlıklı durumuna 1&#39;e kurtarma kolaylaştırır. Ancak, düzensiz inflamasyon sıklıkla kronik enflamatuar hastalıklar 2 yol açar. Hava yolu inflamasyonu, kronik obstrüktif akciğer hastalığı (COPD), astım ve pulmonar fibrosis 3 farklı akciğer hastalıkları için bir başlangıç ​​tetikleyicidir. Tiplendirilemeyen (kapsüllenmemiş), Haemophilus influenzae (NTHi) kronik üst ve alt akciğer enflamatuar hastalıklar 4,5 ile ilişkilidir. Kronik obstrüktif akciğer hastalığı 4,6,7 çocuk ve yetişkinlerin alt solunum yolları izole baskın türdür. NTHi enfeksiyon sonrası inflamatuar yanıtı (örneğin, TNF ve IL-1 gibi) proenflamatuar sitokinlerin regülasyonu ile karakterize edilir ve b aracılıky mitojen aktive protein kinaz (MAPK) ve toll benzeri reseptörler aracılığıyla nükleer faktör-KB (NF-KB) (TLR) 8. Fare modelleri nedeniyle farklı gen eksikliği hatlarının durumu akciğer inflamatuar hastalığın altında yatan patolojiyi analiz etmek için çok yararlı araçlardır. Çeşitli metotlar, intranazal uygulaması ve aerosol haline getirilmiş inhalasyon 9,10 dahil olmak üzere, canlı / zayıflatılmış bakterileri ve bakteriyel ürünler, aşılamak için kullanılmıştır. Burada, içi trakeal instilasyon göstermektedir. daha az sıklıkta kullanılmasına rağmen, bu yaklaşım daha verimli ve çünkü daha düşük solunum sistemine inokulum doğrudan teslimat yüksek tekrarlanabilir.

<table><tbody><tr><td>Chocolate agar plate</td><td>Fisher Scientific</td><td>CAS50-99-7</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Themo Scientific</td><td>R01300</td><td></td></tr><tr><td>Heparin</td><td>Hospira,Inc</td><td>RL-3010</td><td></td></tr><tr><td>Deft quick solution</td><td>Sigma</td><td>GS500-500ML</td><td></td></tr><tr><td>Syringe needle 20/26G&amp;nbsp;</td><td>BD</td><td>(REF305115/175)</td><td></td></tr><tr><td>Iml syringe</td><td>BD</td><td>REF 309602</td><td></td></tr><tr><td>Catheter 20GA</td><td>BD</td><td>REF 381433</td><td></td></tr><tr><td>Dissecting Scissors, straight, 10 cm long</td><td>kentscientific</td><td>INS600393</td><td></td></tr><tr><td>Iris Forceps, serrated, 10cm long</td><td>kentscientific</td><td>INS650915</td><td></td></tr><tr><td>Tweezer #5 Stainless steel, 11cm long</td><td>kentscientific</td><td>INS600095</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>CAS 67-56-1</td><td></td></tr><tr><td>Agarose</td><td>peqlab</td><td>35-1020</td><td></td></tr><tr><td>5ml polystyrene round-bottom tubes</td><td>BD</td><td>REF 352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Light Labs</td><td>A-7001-R</td><td></td></tr><tr><td>Reasy Mini&amp;nbsp; kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Pellet pestile motor (Tissue homoginizer)</td><td>Sigma</td><td>Z359971-1EA</td><td></td></tr><tr><td>96 well microtiter plates V bottom</td><td>Thermo</td><td>2605</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>CD45.2-APC</td><td>eBioscience</td><td>17-0454-81</td><td>Working dilution 1:100</td></tr><tr><td>Ly-6G-eFlor 450</td><td>eBioscience</td><td>48-5931-82</td><td>Working dilution 1:100</td></tr><tr><td>BSA</td><td>HyClone</td><td>SH30574.03</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Live/Dead fixable aqua dead cell stain kit</td><td>Invitrogen</td><td>L-34957</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>lifetechnologies</td><td>15575-020</td><td></td></tr><tr><td>CD16/CD32 FcBlock</td><td>BD</td><td>553142</td><td></td></tr><tr><td>Facs tubes polystyrene round bottom tube</td><td>BD</td><td>352052</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience</td><td>4018</td><td></td></tr></tbody></table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

Burada, verimli ayrıntılı bir prosedür tanımlamak ve doğrudan Haemophilus influenzae teslim farelerin alt solunum yollarına. Biz H. hazırlanması için prosedür göstermek influenza aşı, H. içi trakeal damlatma akciğere influenzae, farklı gen ekspresyon analizi için bronko-alveolar lavaj sıvısı (BALF), BAL sıvısında bağışıklık hücrelerinin analizi ve RNA izolasyonu toplanması. Bu prosedür, bir bakteri, virüs ya da mantar akciğer enflamatuar yanıtı çalışma için kullanılabilir. daha az belirsizlik ile daha verimli alt solunum yollarına bakteriyel inokulum sunar, çünkü doğrudan trakea damlatma çoğunlukla burun veya aerosol inhalasyon prosedürleri tercih edilir.

Intra-trakeal instilasyon tuzlu su ile yükleme daha BALS (Şekil 1A, sol panel) lökositlerin belirgin artan sayıda sonuçlandı. Lökositlerin diferansiyel sayısı analizi açık bir şekilde artmış nötrofil infiltrasyon (Şekil 1, sağ panel) göstermiştir. BAL sıvısında hücrelerinin FACS analizi daha da nötrofillerin sayısını (Şekil 1B) doğruladı. Akciğer dokusunda H &amp; E-boyanan kesitler histolojik analiz havayo...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

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.

intra-trakeal İdaresi<em&gt; Haemophilus influenza</em&gt; Fare Modelleri Havayolu inflamasyonu Eğitim için

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

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Research

JoVE Journal

Immunology and Infection

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

10.1K Görüntüleme.  Baylor Research Institute. 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. 

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

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.

Akış Sitometri Akciğerler ve Akciğer Boşaltma lenf düğümleri Analizi fareler cerrahi olmayan intratrakeal damlatma

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

İmmünoloji ve Enfeksiyon

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Noninvaziv Entübasyon Fare Akciğer Patolojisi ve Fizyolojisi Eğitim için

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Tıp

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

Farelerde Host immün yanıtı değerlendirmek için Orofarengeal intratrakeal PAMP İdaresi ve Bronchoalveolar Lavage Kullanımı

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Kimerik fare modellerinde Rekombinant Grip aşıların burun içinde yoluyla mukozal bağışıklık Okumak

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Mikrobiyal Toplulukları incelenmesi<em&gt; İn Vivo</em&gt;: Arası Ana aracılı Etkileşim Modeli<em&gt; Candida albicans</em&gt; Ve<em&gt; Pseudomonas aeruginosa</em&gt; Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis 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.

Entübasyon-aracılı intratrakeal (IMIT) damlatma: A Noninvaziv, Lung-özel Dağıtım Sistemi

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Farelerde Trakea hızlı intübasyon için geliştirilmiş bir yöntem

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

İki fotonlu Intravital mikroskobu kullanılarak grip virüsü enfeksiyonu sırasında trakeal mukozasında görüntüleme hücre etkileşimi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

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.

Ana bilgisayar-patojen yanıt ve aşı etkinliği farelerde değerlendirilmesi

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

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