The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

Tutti gli esperimenti sugli animali sono stati eseguiti secondo le linee guida per la cura e l&#39;uso di animali approvati dal Comitato Etico locale Karolinska Institutet, Stoccolma. Il Comitato Etico locale segue le direttive dell&#39;Unione europea in materia di cura degli animali. NOTA: I principali cambiamenti rispetto al protocollo originale sono il tempo di recupero dopo l&#39;intervento chirurgico (6 ore contro 1 ora) e il livello di LPS iniettati (2 mg / kg contro 15 mg / kg). In caso contrario, le diverse impostazioni relative alla chirurgia stessa non sono stati modificati. 1. preparazione del materiale per la stimolazione <ol><li> Accendere il computer e il sistema di acquisizione dati collegato alla stimolazione elettrodo (Figura 1A). </li><li> Inserire il Riconoscimento programma. </li><li> Preparare una soluzione stock di LPS ad una concentrazione di 5 mg / ml in PBS 1x, aliquota, e conservarlo a -20 ° C. Il giorno dell&#39;esperimento, scongelare un&#39;aliquota e preparare un adequate campione di LPS (0,5 mg / ml) per iniettare circa 100 microlitri nell&#39;animale, in base al peso. </li></ol> 2. Preparare l&#39;animale per anestesia <ol><li> Utilizzare C57BL 6 topi /. Mantenerle in condizioni climatiche controllate con un ciclo luce-12 hr / scuro, alimentarli chow roditori standard e dare loro acqua ad libitum. </li><li> Eseguire un intervento chirurgico su topi che pesano circa 25 g al giorno dell&#39;esperimento. NOTA: Quando reazioni infiammatorie sono indotti, è particolarmente importante effettuare regolari controlli e osservazioni delle reazioni cliniche negli animali. L&#39;eutanasia precoce da CO 2 inalazione è necessaria se la condizione animale non soddisfa i criteri etici. </li><li> Impostare la macchina anestetico. <ol><li> Assicurarsi che il tubo sia collegato correttamente e non sia danneggiato in alcun modo. Assicurarsi che la zona ventilata funziona correttamente. Collegare il filtro chiave per la bottiglia isoflurano e riempire il vaporizer con una quantità sufficiente di isoflurano. </li><li> Aprire l&#39;alimentazione del gas (aria e ossigeno) e fare in modo che le bottiglie contengono abbastanza gas per l&#39;esperimento. Un connettore a tre vie è quindi in grado di inviare il flusso isoflurano alla camera di induzione o la maschera. </li></ol></li><li> Ruotare il connettore a tre vie per la camera di induzione. Scegli un mouse dalla gabbia casa e inserire l&#39;animale nella camera. Regolare il regolatore di flusso di 1,0 L / min di ossigeno e 1,0 L / min di aria. Regolare la concentrazione isoflurano al 4-5%. </li><li> Quando si raggiunge il livello desiderato di anestesia, quando si raggiunge il livello desiderato di anestesia, radere l&#39;area chirurgica e spostare l&#39;animale dalla camera alla maschera. Girare il connettore a tre vie per il flusso maschera. Regolare il regolatore di flusso di 0,25 L / min ossigeno e 0,25 L / min di aria. <ol><li> Regolare la concentrazione isoflurano al 1,5-2,5%. Controllare il livello di anestesia per il controllo del riflesso e la frequenza respiratoria prima di iniziare SEGUIRE IL PROCEDIMENTO chirurgicae. </li></ol></li><li> Fissare le gambe del mouse al banco di lavoro con nastro adesivo. Assicurarsi che il naso dell&#39;animale è ancora accuratamente posizionato nella maschera. </li></ol> 3. Chirurgia e stimolazione del nervo vago <ol><li> Disinfettare l&#39;area chirurgica con il 70% di etanolo. </li><li> Usando un bisturi, incidere con cura la pelle a livello del collo (un&#39;incisione di circa 1 a 1,5 cm). Nota: Nel protocollo, la procedura di chirurgia finisce qui per gli animali sham-operati. Infatti, è stato dimostrato che sfiora il nervo con un attrezzo metallico stimola già in una certa misura. Pertanto, per ottenere un più accurato controllo della chirurgia degli animali quando si utilizza una piccola quantità di LPS, fermare l&#39;operazione in questa fase. </li><li> Con l&#39;aiuto di un microscopio (obiettivo 12,5X), isolare il nervo vago sinistro dalla carotide utilizzando dissettore. In primo luogo individuare il muscolo sternocleidomastoideo rimuovendo pelle e grasso strati, e poi rientrare inordinare di mettere la pinza dietro sia il nervo e l&#39;arteria. Nota: Il passo successivo è molto difficile, come il nervo e arteria sono strettamente aderire l&#39;uno all&#39;altro. Pertanto, è molto facile da tagliare la nave e uccidere l&#39;animale. Tuttavia, ponendo molta attenzione la pinza tra il nervo e l&#39;arteria, che alla fine si separano, ed è possibile isolare il nervo. </li><li> Posizionare l&#39;elettrodo (Figura 1B) sotto il nervo vago. L&#39;elettrodo ad ago è piuttosto lungo, anche se il nervo muove leggermente durante la stimolazione, sarà sempre in contatto con l&#39;elettrodo. </li><li> Eseguire un intraperitoneale (ip) iniezione di LPS (2 mg / kg) con l&#39;aiuto di una siringa (per la lettura su livelli di citochine, cioè per misurare l&#39;effetto down-regulation). </li><li> Attendere 5 minuti prima di iniziare la stimolazione. </li><li> Stimolare il nervo vago per 5 minuti a 5 V e 1 Hz premendo il pulsante di avvio nel programma riconoscere. </li><li> Rispostare l&#39;elettrodo e suturare la ferita dell&#39;animale con filo di sutura chirurgica. </li><li> Spruzzare un No Sting Barriera Film (NSBF) sulla ferita al fine di migliorare la guarigione e per proteggere dalle infezioni. </li></ol> 4. Recupero del Animal <ol><li> Dopo l&#39;intervento chirurgico, spostare l&#39;animale torna alla sua gabbia casa per il risveglio e il recupero. Sotto la luce infrarossa al fine di mantenere la temperatura corporea, assicurarsi di monitorare l&#39;animale fino piena coscienza è stato ripreso. </li><li> Lasciate che l&#39;animale recuperare nella sua gabbia per 6 ore prima del sacrificio per l&#39;analisi. Nota: L&#39;effetto della stimolazione del nervo vago è molto veloce e ha anche dimostrato di essere di lunga durata (fino a 48 ore), in modo che il tempo di recupero può essere impostato dallo sperimentatore secondo le esigenze dello studio. </li></ol> 5. Il sacrificio per ulteriori analisi <ol><li> Posto l&#39;animale in una gabbia collegato a un dispositivo di somministrazione di CO 2. </li><li> Impostare il dispositivo su un 5Ciclo -min di CO 2 inalazione. </li><li> Quando l&#39;eutanasia è fatto, raccogliere gli organi di interesse e direttamente congelarli in ghiaccio secco per ulteriori analisi (ad esempio, la misurazione dei livelli di citochine negli estratti di milza usando un topo TH1 / TH2 9-Plex assay) 3. </li></ol>

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Fin dalla sua scoperta nei primi anni 2000, i meccanismi della PAC sono stati accuratamente studiati. Ora abbiamo una buona immagine del percorso, e in particolare, l&#39;organo bersaglio, la milza, dove NE, le cellule T memoria, Ach, e macrofagi lavorare come una squadra molto efficiente per down-regolare mediatori infiammatori 2. Abbiamo inoltre recentemente pubblicato dati sulla importanza di un sistema prostaglandine funzionale nei topi, in particolare, PGE 2, che è ovviamente un ...

L&#39;immunità innata fornisce immediatamente prima linea di difesa contro le infezioni e malattie in una vasta gamma di organismi. Essa non solo avvia la risposta immunitaria primaria per eliminare la minaccia, ma svolge anche un ruolo fondamentale nell&#39;attivazione e educare l&#39;immunità adattativa che svolge risposte immunitarie secondarie in modo specifico agente patogeno. L&#39;infiammazione è orchestrato da una pletora di citochine e chemochine, che a loro volta hanno la capacità di attrarre altre cellule immunitarie al sito di infezione e di indurre i segni cardinali di infiammazione, come rossore, gonfiore, dolore, perdita di funzione, e la febbre . La durata e l&#39;intensità dell&#39;infiammazione dipendono da diversi fattori, ma risolvere l&#39;infiammazione e ripristinando l&#39;omeostasi è un passo fondamentale per evitare l&#39;insorgenza di malattie infiammatorie croniche. I recenti progressi nel campo delle neuroscienze e immunologia hanno svelato i meccanismi neurali specifiche con immenso potenziale terapeutico per il controllo inflinfiammazione sia nel sistema nervoso centrale e nella periferia. Uno di essi consiste nello colinergica via anti-infiammatori (PAC), noto anche come il riflesso infiammatoria, che è azionato dal sistema nervoso autonomo 4, 5. Attualmente è pensato che mediatori infiammatori attivano nervi sensoriali e inviare segnali relativi allo stato di infiammazione del sistema nervoso centrale. Una risposta riflessa viene quindi attivato attraverso il nervo vago efferente. Un ampio studio sui dettagli anatomici della PAC ha rivelato un modello parasimpatico-simpatico composto da due nervi, il nervo vago e nervo splenico, rispettivamente 6. Nella PAC, il nervo vago colinergica efferente attivato termina nel ganglio celiaco mesenterico, causando l&#39;attivazione del nervo splenico adrenergico da un meccanismo ancora da esplorare. Il nervo splenico, così attivato, si caratterizza per internovate in prossimità di cellule immunitarie nella polpa bianca, zona marginale, e polpa rossa della milza, la principale e organo obbligatorio della PAC 7, 8. Norepinefrina (NE) dalle terminazioni nervose splenici lega ai corrispondenti β 2 adrenergici espressi sui linfociti T splenici. Questo induce il rilascio colina acetil transferasi (ChAT) acetilcolina mediata (ACh), che a sua volta attiva i recettori nicotinici dell&#39;acetilcolina (a7) α7nACh sui macrofagi, limitando in tal modo la produzione di citochine e infiammazione 2. Di conseguenza, è chiaro che il sistema nervoso è in grado di regolare l&#39;infiammazione nei tessuti periferici e di ripristinare omeostasi immunitaria locale. Come il nome del percorso suggerisce, il sistema ACh è di importanza fondamentale per il funzionamento di questo percorso di regolazione neuro-immune. È interessante notare che i meccanismi coinvolti nell&#39;attivazione diPAC sembrano essere differente nella periferia e nel sistema nervoso centrale. Mentre l&#39;importanza dei recettori nicotinici (α7nAChR) nella milza è stata dimostrata in precedenza 9, recettori muscarinici (mAChR) sono obbligatorie per l&#39;attivazione centrale del percorso 10, 11. Più recentemente, la somministrazione periferica di una azione centrale M1 agonista muscarinico notevolmente soppressa fattore di necrosi tumorale siero e milza α (TNFa) durante letale endotossemia murina, un&#39;azione che richiedeva intatto nervo vago e nervo splenico segnalazione 12. Abbiamo inoltre recentemente dimostrato che topi privi prostaglandina E2 (PGE2) non erano in grado di rispondere alla stimolazione del nervo vago e non down-regolano il rilascio indotto da LPS di citochine nel siero e milza 3. Pertanto, la PAC potrebbe anche essere regolata da sistemi diversi dal principale pathw AChAy. Il nervo vago è stata indicata come tale per la sua naturalmente vagare nel corpo, innervano organi principali compreso il fegato, polmone, milza, reni, intestino e 13. Considerando questo grande innervazione e molto potente effetto immunosoppressivo del nervo vago, il potenziale terapeutico della PAC potrebbe coprire un&#39;ampia gamma di condizioni infiammatorie. Il nervo vago può essere elettricamente (o meccanicamente) attivato, con controllo della tensione e frequenza, e contrariamente al trattamento convenzionale, senza farmaci aggiunti al corpo. Le prove sono attualmente in corso in pazienti reumatici, per esempio, per testare il significato clinico di VNS nel trattamento di infiammazioni croniche 14. Complessivamente, la comunicazione e la regolazione di infiammazione neuro-immunitario sono attualmente allo studio, che fornirà un trattamento alternativo alla terapia convenzionale. Pertanto, l&#39;analisi del nervo vago stimulatioEffetto n nei diversi organi innervati, ma anche la caratterizzazione del potenziale azione terapeutica in modelli animali di infiammazione cronica, sarebbe sicuramente dare spunti e di speranza per nuovi potenziali bersagli terapeutici. La metodologia originale sviluppato da Tracey e colleghi 4 non poteva essere trasposto al nostro campo di ricerca a causa di eccesso di stimolazione della risposta infiammatoria (da una dose letale di LPS) e una troppo breve intervallo di tempo tra l&#39;attivazione della PAC e il read-out. Nel presente documento, presenteremo le modifiche apportate al protocollo originale, confrontare i due metodi diversi su livelli di citochine, ed evidenziare un nuovo fronte osservazione sull&#39;organo bersaglio (la milza).

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Computer</td>
			<td style="width:91px;">Toshiba</td>
			<td style="width:136px;">-</td>
			<td style="width:191px;">Any computer is actually compatible</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">MP-150 data acquisition system</td>
			<td style="width:91px;">Biopac Systems</td>
			<td rowspan="2" style="width:136px;">MP150WSW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Acknowledge software</td>
			<td style="width:91px;">Biopac Systems</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Mice C57Bl/6</td>
			<td style="width:91px;">Charles River</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anesthetic machine</td>
			<td style="width:91px;">Simtec Engineering</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical oxygen bottle</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">107563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical air bottle&#160;</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">108639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetflurane (1000mg/g)</td>
			<td style="width:91px;">Virbac</td>
			<td style="width:136px;">137317</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">LPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">L2630</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Saline</td>
			<td style="width:91px;">Merck Millipore</td>
			<td style="width:136px;">1024060080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS 10X</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5493</td>
			<td>Diluted 10 times for used concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe (1 ml)</td>
			<td style="width:91px;">BD Plastipak</td>
			<td style="width:136px;">303172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Needles 23G</td>
			<td style="width:91px;">KD-FINE</td>
			<td style="width:136px;">900284</td>
			<td>0.6 x 30 mm (blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Microdissecting forceps (curved)</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">F4142</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dissecting scissors</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z265969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical suture 4-0</td>
			<td style="width:91px;">Ethicon</td>
			<td style="width:136px;">G667G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Euthanasia unit</td>
			<td style="width:91px;">Euthanex Smartbox</td>
			<td style="width:136px;">EA-32000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Cavilon No Sting Barrier Film</td>
			<td style="width:91px;">3M Health Care</td>
			<td style="width:136px;">3346N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">TH1/TH2 9-Plex assay, ultrasensitive kit</td>
			<td style="width:91px;">MesoScale Discovery</td>
			<td style="width:136px;">K15013C-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Livello di TNFa e interleuchina-1β (IL-1β) dopo aver aumentato il Time Lapse dopo l&#39;intervento e ridurre la dose di LPS Come mostrato in precedenza, utilizzando il protocollo originale, VNS diminuzione dei livelli di TNFa (169,3 ± 24,9 pg / mg in SHAM contro 39,7 ± 10,8 pg / mg in VNS, p &lt;0.001) e IL-1β (360.0 ± 40.21 pg / mg SHAM contro 191,7 ± 27,2 pg / mg in VNS, p &lt;0,01) nella milza dopo l&#39;iniezione intra...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Aumento tempo di recupero e Diminuzione LPS Per studiare il nervo vago meccanismi di stimolazione in risposta infiammatoria limitati

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.8K Views.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Video: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Ricerca

Immunologia e infezioni

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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 ...

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.

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 ...

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

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

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

Trasduzione retrovirale di cellule T helper come un approccio genetico per studiare meccanismi che controllano la loro differenziazione e funzione

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

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Un modello di topo transgenizzato IL-8 per il<em&gt; In Vivo</em&gt; Monitoraggio a lungo termine delle risposte infiammatorie

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Saggi per caratterizzare nuovi endoteliale regolatori coinvolti nella risposta infiammatoria di screening

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.