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.

Tutti gli esperimenti sono stati eseguiti in conformità con le linee guida del Comitato Istituzionale Animal Care e Usa (IACUC) del Baylor Research Institute. 1. Coltura non tipizzabile Haemophilus influenzae (NTHi) e preparazione del Inoculo <ol><li> Piatto NTHi su una piastra di agar cioccolato e mantenere la piastra capovolta in un umidificata CO 2 incubatore notte a 37 ° C e 5% CO 2. Il giorno seguente, la cultura i batteri in brodo Brain Heart Infusion. Quindi, aggiungere 1 ml di brodo in una provetta sterile da 1,5 ml. </li><li> Centrifugare a 4000 xg per 5 min a temperatura ambiente. Poi, scartare il surnatante e risospendere il pellet in 1 ml di PBS sterile 1x. Ripetere questa operazione una sola volta. </li><li> Trasferire 100 ml di soluzione di ri-sospeso in 900 ml di PBS 1x e misurare l&#39;assorbanza della cultura diluito a 600 lunghezze d&#39;onda nm in uno spettrofotometro. </li><li> Determinare l&#39;unità di vitalità e formazione di colonies (CFU) del inoculo di coltura 1:10 diluizioni seriali su piastre di agar cioccolato e incubando overnight a 37 ° C e 5% CO 2. </li><li> Sulla base della vitalità e CFU determinata dal passaggio precedente, regolare l&#39;inoculo ad una concentrazione finale di 20 x 10 6 CFU / ml. </li></ol> 2. intra-tracheale (esso) instillazione <ol><li> Iniettare il mouse per via intraperitoneale (ip) con 0,1 ml / 10 g di peso corporeo di mouse con un / ketamina + 20 mg / ml soluzione xylazina ml 150 mg. La procedura deve essere eseguita nel cappuccio con una sorgente luminosa per mantenere l&#39;animale caldo. </li><li> Pizzicare lo spazio interdigitale del mouse per verificare che il mouse sia adeguatamente anestetizzato. </li><li> Posizionare il mouse sul dorso. Radere la zona ventrale del collo e disinfettare con clorexidina e il 70% di alcol etilico. </li><li> Sollevare la pelle della parte ventrale superiore del collo usando ratto pinze dente. Fai una piccola incisione (0,5-1,0 cm) sopra il timo con un bisturie sezionare accuratamente il muscolo per esporre la trachea. <ol><li> Usando una siringa da 1 ml con un ago 27 G, iniettare lentamente 0,05 ml di aria, seguito da 0,05 ml NTHi ottenuti dalla sezione 1 o soluzione salina come controllo, seguita da un&#39;altra 0,05 ml di aria. Tenere animali verticale per circa 1 min. </li></ol></li><li> Chiudere l&#39;incisione con la colla chirurgica. Mantenere il mouse lateralmente reclinata e caldo per circa 10 min. </li></ol> 3. Raccolta BALF <ol><li> Sacrificare il mouse cervicale dislocazione 24 ore dopo l&#39;infezione. </li><li> Aprire la cavità addominale del mouse con forbici anatomiche e tagliare l&#39;aorta ventrale a sanguinare. Esporre la parte ventrale del polmone. </li><li> Esporre la trachea e intubare con un catetere 20 gauge. Instillare 0,8 ml di tampone BAL (1x PBS, 0.1% di destrosio, 10 U / ml di eparina) e raccogliere il lavaggio aspirando delicatamente. Ripetere questa procedura per tre volte. </li><li> Contare il numero totale di cellule in 100 microlitri di sospensione BALF utilizzando un hemocytometro a 10 ingrandimenti con Trypan Blue colorazione. </li><li> Preparare vetrini per modificare la colorazione Giemsa. Un&#39;aliquota di 100 ml di BAL nei rispettivi pozzetti della cytospin e centrifugare a 500 xg per 5 min. Asciugare le diapositive per 10 minuti e macchiare le cellule utilizzando modificato macchia Giemsa secondo protocolli del produttore. </li><li> Utilizzare le cellule rimanenti per la successiva analisi, come la fluorescenza delle cellule attivate (FACS), microscopia confocale, l&#39;espressione genica e Western Blot 11-13. </li></ol> 4. istopatologia Preparazione <ol><li> Per l&#39;analisi istopatologia, infettare i topi con NTHi come descritto al punto 2.4. Dopo 24 ore di infezione, sacrificare gli animali da dislocazione cervicale. Tagliare aprire la cavità toracica con le forbici anatomiche per esporre i polmoni e la trachea come accennato prima. Quindi, inserire il catetere 20 gauge nella trachea. </li><li> Riempire il polmone con il 2% soluzione di agarosio calda preparata in formalina 4%. Una volta che ilpolmoni sono completamente gonfiati, posto ghiaccio sulla parte superiore dei polmoni a solidificare la soluzione di agarosio. </li><li> Poi, rimuovere con attenzione la spina dorsale e metterlo in una soluzione 50 ml di formalina 4% in PBS 1x fino a quando non viene elaborato per (H &amp; E) sezioni ematossilina eosina. </li></ol> 5. FACS colorazione delle cellule nel BALF <ol><li> Mettere 1 x 10 5 cellule dal fluido BALF in un piatto titolo micro V-bottom 96 pozzetti. </li><li> Lavare le cellule con 300 ml di freddo 1x PBS e far girare a 4000 xg per 3 min a 4 ° C. </li><li> Macchiare le cellule con soluzione macchia cellule in 100 microlitri 1x PBS (1: 1.000 diluizione) e mantenere a temperatura ambiente per 15 min. </li><li> Lavare le cellule con 300 ml di freddo 1x PBS a 4.000 xg per 3 min a 4 ° C. </li><li> Mantenere cellule in 100 ml di FACS tampone di lavaggio [tampone di lavaggio (PBS 1x, 2% FBS, 1 mM EDTA, 0,1% di sodio azide] contenente 1: 100 diluito Fc-Block per 15 min a 4 ° C. </li><li> cellule centrifugare a 4000 xg per 3 min e scartare supernatant. </li><li> cellule macchia con 100 microlitri superficie colorazione cocktail (diluizione 1: 100) in tampone di lavaggio FACS per 30 minuti a 4 ° C. </li><li> Lavare le cellule tre volte con 300 ml di tampone di lavaggio FACS a 4.000 xg per 3 min a 4 ° C. </li><li> Fix cellule in 100 ml di paraformaldeide al 4% per 15 min a temperatura ambiente. </li><li> Lavare le cellule due volte con 300 microlitri di PBS 1x a 400 xg per 3 min a 4 ° C e risospendere in 300 ml di tampone di lavaggio FACS. </li><li> Effettuare controlli per la colorazione monocolore citometria a flusso perline. <ol><li> Aggiungere una goccia di perle nel piatto microlitro 96 pozzetti V-parte inferiore e lavare con 200 microlitri 1x PBS. </li><li> Aggiungere 1 ml di colorazione degli anticorpi a 100 l PBS 1x e perline e incubare a temperatura ambiente per 5 min. </li><li> Lavare perle due volte con 300 ml 1x PBS. </li></ol></li><li> Analizzare i controlli e le singole cellule macchiato mediante citometria di flusso 14. </li></ol> 6. L&#39;omogeneizzazione di tessuto polmonare per isolare l&#39;RNA <ol><li> Dopo l&#39;infezione, raccogliere un piccolo pezzo di polmone (20-40 mg) e conservarlo in RNA stabilizzante reattivo a 4 ° C durante la notte. Poi, conservare a -80 ° C fino alla fase successiva. </li><li> Lavare i tessuti del polmone a freddo 1x PBS per sbarazzarsi del reagente stabilizzazione RNA. </li><li> Posizionare il tessuto in un tampone di lisi contenente 1,5 ml tubo da centrifuga (1 ml di buffer di lisi + 10 ml β-mercaptoetanolo). </li><li> Tritare il tessuto in piccoli pezzi con le forbici a punta ed omogeneizzare con un cordless pestello pellet motore per 20-40 secondi. </li><li> Mantenere il lisato in ghiaccio per 5 minuti e procedere con passo 7.1. </li></ol> 7. Isolamento RNA dal polmone <ol><li> Centrifugare il lisato a 18.000 xg per 3 min. Rimuovere il surnatante pipettando e trasferire con cura in una nuova provetta da 1,5 ml. </li><li> Aggiungere 1 volume di etanolo 70% al lisato. Mescolare immediatamente pipettando e aspettare 2-4 minuti. </li><li> Trasferire fino a 700 ml di lisato ad una colonna di spin localein un tubo di raccolta 2 ml. </li><li> Centrifugare a 9.600 xg per 15 sec e scartare il flow-through. </li><li> Aggiungere 350 microlitri tampone di lavaggio stringente e centrifugare a 9.600 xg per 15 sec. Gettare il flow-through. </li><li> Aggiungere 80 microlitri DNasi direttamente alla membrana Spin Column e mantenere a temperatura ambiente per 15 min. </li><li> Aggiungere 350 microlitri tampone di lavaggio stringente alla colonna di spin e centrifugare a 9.600 xg per 15 sec. Gettare il flow-through. </li><li> Aggiungere 500 microlitri tampone di lavaggio RNA e centrifugare a 9.600 xg per 15 sec. Gettare il flow-through. </li><li> Aggiungere 500 microlitri tampone di lavaggio RNA e far girare a 9.600 xg per 2 minuti. Gettare il flow-through. </li><li> Porre la colonna di spin in un nuovo tubo di raccolta da 2 ml e centrifugare a 9.600 xg per 1 min. Gettare il flow-through. </li><li> Aggiungere 30-50 acqua priva di RNasi microlitri direttamente alla colonna di spin e centrifugare a 9.600 xg per 1 min. RNA Conservare a -80 ° C. </li><li> Eseguire l&#39;analisi RNA-Seq 14. </li></ol>

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The authors have nothing to disclose.

Qui, si descrive una procedura unica e minimamente invasiva per inoculare i polmoni di topi con un agente patogeno batterico ai polmoni. Abbiamo dimostrato che questa procedura può essere utilizzata per studiare la funzione di geni differenti utilizzando topi che sono carenti di geni di vie di segnalazione infiammatorie. Questa procedura può anche essere usato per studiare le risposte infiammatorie alle infezioni polmonari virali e fungine. I vantaggi di questo procedimento rispetto ad altri metodi come intranasale o ...

L&#39;infiammazione è un meccanismo immunitario fondamentale di difesa contro gli agenti infettivi. Promuove eradicazione e la riparazione dei tessuti danneggiati patogeno. Inoltre facilita il recupero ad un normale stato di salute 1. Tuttavia, l&#39;infiammazione disregolato spesso porta a malattie infiammatorie croniche 2. Infiammazione delle vie aeree è un trigger iniziale per diverse malattie polmonari come la broncopneumopatia cronica ostruttiva (BPCO), asma e fibrosi polmonare 3. L&#39;Haemophilus influenzae non tipizzabile (non incapsulata) (NTHi) è associata a malattie infiammatorie superiori ed inferiori polmonari croniche 4,5. E &#39;la specie dominante isolati dalle vie aeree inferiori di bambini e adulti con cronica ostruttiva 4,6,7 malattia polmonare. La risposta infiammatoria dopo l&#39;infezione NTHi è caratterizzata dalla sovraregolazione di citochine proinfiammatorie (come TNF e IL-1β), ed è mediata by mitogeno proteina chinasi attivata (MAPK) e fattore nucleare-kB (NF-kB) attraverso i recettori Toll-like (TLR) 8. Modelli murini sono strumenti molto utili per analizzare la patologia sottostante del polmone malattia infiammatoria a causa della disponibilità di diverse linee gene-carenti. Diversi metodi sono stati utilizzati per inoculare batteri vivi attenuati / e prodotti batterici, tra intranasale instillazione e aerosolized 9,10 inalazione. Qui, dimostriamo intra-tracheale instillazione. Sebbene utilizzato meno frequentemente, questo approccio è più efficace e altamente riproducibili a causa della consegna diretta dell&#39;inoculo al tratto respiratorio inferiore.

<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

Qui, descriviamo una procedura dettagliata in modo efficiente e fornire direttamente Haemophilus influenzae attraverso le vie respiratorie inferiori di topi. Dimostriamo la procedura per la preparazione H. influenzae inoculo, instillazione intra-tracheale di H. influenzae nel polmone, raccolta di liquido di lavaggio bronco-alveolare (BAL), l&#39;analisi delle cellule immunitarie nel BAL, e l&#39;isolamento di RNA per differenziale analisi di espressione genica. Questa procedura può essere utilizzata per studiare la risposta infiammatoria polmonare a qualsiasi batteri, virus o funghi. instillazione tracheale diretta è per lo più preferito su procedure intranasale o inalazione di aerosol in quanto fornisce in modo più efficiente l&#39;inoculo batterico nel tratto respiratorio inferiore, con meno ambiguità.

Instillazione intra-tracheale provocato un marcato aumento del numero di leucociti nel BALF (Figura 1A, pannello di sinistra) di installazione con soluzione salina. L&#39;analisi conteggio differenziale dei leucociti mostrato chiaramente migliore infiltrazione dei neutrofili (Figura 1, pannello di destra). L&#39;analisi FACS delle cellule nel BALF confermato ulteriormente l&#39;aumento del numero di neutrofili (Figura 1B). L&#39;analisi ...

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-tracheale Amministrazione<em&gt; Haemophilus influenzae</em&gt; In modelli murini per studiare infiammazione delle vie aeree

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

10.1K Views.  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.

Non chirurgica instillazione di topi con analisi dei polmoni e dei linfonodi polmonari drenante mediante citometria di flusso

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

Ricerca

Immunologia e infezioni

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.

Non invasivo intratracheale intubazione per studiare la Patologia e Fisiologia del mouse polmone

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 ...

Medicina

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.

L&#39;utilizzo di orofaringea intratracheale PAMP Amministrazione e lavaggio broncoalveolare per valutare la risposta immunitaria nei topi Host

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.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

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.

Studiare comunità microbiche<em&gt; In Vivo</em&gt;: Un modello di interazione ospite-mediata tra<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nei 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.

Intubazione-mediata intratracheale (IMIT) instillazione: A non invasiva, Lung-specifico sistema di rilascio

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.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

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.

Un metodo migliore per Rapid intubazione della trachea nei topi

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.

La formazione immagine interazione cellula nella Mucosa trachea durante l'infezione da Virus influenzale usando la microscopia Intravital due-fotone

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.

Valutazione delle risposte ospite-patogeno e l'efficacia del vaccino nei topi

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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