Questo lavoro è stato sostenuto da un finanziamento di Crohn e Colite Foundation of America, (CCFA; 3711) la prevenzione del cancro e Research Institute del Texas (CPRIT; RP160169), e UT Southwestern Medical Center dati a MHZ

Tutti gli esperimenti descritti qui sono stati effettuati utilizzando 6-8 settimane di età topi maschi wild-type (C57BL6 / J) mantenuto in libera una struttura specifica patogeno (SPF) presso il Centro risorse animali (ARC), UT Southwestern Medical Center. Tutti gli studi sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) e sono state condotte in conformità con le linee guida IACUC e il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio. 1. Raccolta e preparazione del colon <ol><li> Euthanize topi con CO 2 asfissia seguita da dislocazione cervicale. </li><li> Spray topi con il 70% di etanolo e il pin arti per una scheda di pinning mantenendo il lato del mouse dorsale verso il basso sul tabellone. </li><li> Utilizzando forbici e pinze pre-sterilizzati dissezione, fare una incisione linea mediana nel peritoneo dell&#39;addome. Aprire l&#39;addome piegando il peritoneo con una pinza. </li><li> Rimuovere l&#39;intestino dalla cavità addominale wiesimo dissezione pinze e forbici. Separare il colon dal piccolo intestino tagliando al fondo cieco e l&#39;altra estremità al retto. </li><li> Posizionare i due punti in una capsula di Petri sterile contenente ghiacciata PBS. Lavare il contenuto del lume del colon con PBS freddo utilizzando una siringa da 20 ml in possesso di un ago G 20 o un ago sonda gastrica fino feci nel lume del colon è completamente rimosso. </li><li> Tagliare i due punti con le forbici in senso longitudinale. Lavare il colon agitando vigorosamente in PBS ghiacciato in una piastra di Petri sterile. </li><li> Tagliare il tessuto del colon in pezzi lunghi circa 1 cm con un bisturi sterile o forbici. </li><li> Registrare il peso dei pezzi colon. NOTA: tutti i passaggi della sezione 1 possono essere eseguite sia all&#39;interno che all&#39;esterno di una cappa di sicurezza biologica. Una volta che i due punti sono pronti per la cultura, tutti i passaggi devono essere eseguiti all&#39;interno di una cappa di sicurezza biologica per mantenere la sterilità. </li></ol> 2. Colon Orguna cultura <ol><li> Inserire un filtro cella (100 micron) su una piastra di coltura cellulare 6 pozzetti. Trasferire tutti i pezzi colon raccolti da un topo nel filtro cella. </li><li> Aggiungere 5 ml di terreno DMEM / F12 contenente il 5% di FBS, penicillina-streptomicina (1x), e Gentamicina (20 mg / ml). coprire completamente i pezzi del colon con i media. </li><li> Incubare per 2 ore a 37 ° C in un incubatore con 5% di CO 2 e dell&#39;aria 95%. </li><li> Sollevare il filtro delle cellule e aspirare i media. </li><li> Posizionare il filtro delle cellule di nuovo sul bene e aggiungere 5 ml di mezzi freschi senza antibiotici. Sollevare il filtro delle cellule e aspirare i media. </li><li> Ripetere la stessa operazione di lavaggio (passo 2,5) altre due volte (3x totale) per rimuovere tutti i residui antibiotici. </li><li> Trasferire i pezzi colon da una singola colino cellula in un singolo pozzetto di una piastra di coltura cellulare sterile 12 pozzetti. </li><li> Aggiungere DMEM medio / F12 contenente 5% FBS (senza antibiotici). Regolare il volume del mezzo con il weight dei pezzi colon, ad esempio, 1 medio ml per 100 mg di tessuto. Incubare per 12 ore a 37 ° C in un incubatore iniezione 5% di CO 2 e dell&#39;aria 95%. </li><li> Raccogliere il supernatante della coltura in una provetta sterile da 1,5 ml. </li><li> Centrifugare a 12.000 xga 4 ° C per 5 min. Separare il surnatante in una nuova provetta da 1,5 ml per uso nei batteri uccidendo dosaggio e / o altri saggi immunitari. Il supernatante può essere conservato a -80 ° C fino alla dosaggio. </li><li> Raccogliere i pezzi del colon in un tubo per l&#39;isolamento di RNA. </li></ol> 3. E. coli Analisi Uccidere <ol><li> Seminare E. coli in 5 mL Luria-Bertani (LB) brodo in una provetta da 15 ml e incubare a 37 ° C con agitazione a 200 rpm durante la notte. Mantenere il tappo della provetta leggermente allentato. </li><li> Centrifugare la provetta di coltura batterica a 1.200 xg per 10 minuti a 4 ° C. Rimuovere il surnatante e risospendere il pellet batterico in 5 ml di PBS ghiacciato. </li><li> Trasferire 1 ml della bactsospensione erial nella cuvetta e misurare OD a 600 nm. Utilizzare PBS come bianco. </li><li> Calcolare l&#39;unità formanti colonia (cfu) utilizzando una curva standard prestabilito. Qui, si supponga che 1 OD = 2 x 10 9 CFU / ml (circa). </li><li> Diluire la sospensione batterica per rendere la sospensione madre 1 x 10 5 CFU / ml. </li><li> Trasferire il surnatante cultura colon organo (500 microlitri / pozzetto) raccolte nella sezione 2.10 nei pozzetti duplicati di una piastra di coltura cellulare di 24 pozzetti. </li><li> Aggiungere 10 ml cultura coli E. (1.000 ufc) in un pozzetto contenente 500 microlitri colon organo cultura surnatante. Lasciare l&#39;altra e senza alcun coli inoculazione E. per confermare che la cultura surnatante colon organo non contiene alcuna contaminazione. </li><li> Incubare lo stesso numero di batteri (1000 cfu) nei mezzi di coltura (DMEM / F12 più 5% FBS) senza antibiotici come controllo. </li><li> Incubare a 37 ° C per 1 h. </li><li> Mettere 50 ml di ogni campione drop-wiSE su piastre di agar MacConkey. Incubare le piastre MacConkey agar a 37 ° C durante la notte. </li><li> Contare il numero di colonie e calcolare la ufc / ml. </li></ol> 4. L&#39;effetto di fattori estrinseci ed intrinseci sul colon antimicrobici Host Difesa Risposte NOTA: Il saggio uccisione antimicrobico come descritto qui può essere adottato per esaminare l&#39;effetto dei modelli patogeni associati molecolari (PAMPs) e citochine sull&#39;attività battericida della cultura d&#39;organo surnatante. Un esempio di tale esperimento utilizzando IL-1β e IL-18 è descritta di seguito. <ol><li> Raccogliere i due punti di topi come descritto nella Sezione 1. </li><li> Posizionare i due punti su un tovagliolo di carta sterile. Tagliare il colon longitudinalmente in tre parti con le forbici (Figura 2). </li><li> Lavare ogni parte del colon in ghiaccio freddo PBS come descritto nella Sezione 1. </li><li> Tagliare ogni parte del colon in piccoli pezzi e pesare in modo asettico. Trasferire i pezzi di ogni partedel colon in tre filtri di cellule separate (100 micron) disposte su tre pozzetti di una piastra di coltura cellulare 6 pozzetti (Figura 2). </li><li> Aggiungere 2 ml di DMEM medio / F12 contenente il 5% di FBS, penicillina-streptomicina (1x), e Gentamicina (20 mg / ml). </li><li> Incubare per 2 ore a 37 ° C in un incubatore iniezione 5% di CO 2 e dell&#39;aria 95%. </li><li> Lavare i pezzi del colon, come descritto nella 2,4-2,6. Trasferire i pezzi colon di un singolo colino cellula in un singolo pozzetto di una piastra di coltura cellulare sterile 12 pozzetti. I tre pozzetti contenenti tre parti del colon da un singolo mouse dovrebbero essere designati come greggia, IL-1β e IL-18 (Figura 2). </li><li> Aggiungere DMEM medio / F12 contenente 5% FBS (senza antibiotici). Regolare il volume del mezzo con il peso dei pezzi colon, ad esempio, 1 ml di mezzo per 100 mg di tessuto. </li><li> Stimolare la cultura organo colon con IL-1β (20 ng / ml) o IL-18 (20 ng / ml) per 12 h. I due punti non trattati<html> cultura organo serve come controllo. </li><li> Dopo 12 h di incubazione a 37 ° C in un incubatore iniezione 5% di CO 2 e il 95% di aria, raccogliere il supernatante della coltura in una sterile 1,5 mL provetta. </li><li> Trasferire 500 microlitri cultura surnatante in pozzetti duplicati di un 24-pozzetti. </li><li> Seminare E. coli (1.000 CFU) nel colon organo cultura surnatante come descritto nella Sezione 3. Per ogni condizione, inoculare E. coli in un singolo pozzo. L&#39;altro pozzetto contenente stesso organo cultura surnatante senza E. coli servirà come controllo per la contaminazione batterica. </li><li> Incubare la stessa quantità di batteri in terreni di coltura senza antibiotici come controllo. </li><li> Incubare a 37 ° C per 1 h. </li><li> Mettere 50 ml di ogni campione goccia a goccia su piastre di agar MacConkey. Incubare le piastre MacConkey agar per una notte a 37 ° C. </li><li> Contare il numero di colonie e calcolare la ufc / ml (Figura 4). </li></ol>e_title &quot;&gt; 5. Misurazione della espressione dei geni antimicrobici <ol><li> Dopo incubazione per una notte (12 ore) di cultura d&#39;organo del colon, come descritto nelle sezioni 2 e 4, lavare i pezzi del colon con 2 ml ghiacciata PBS (2x). </li><li> Raccogliere i pezzi del colon in una provetta libera tappo a vite 2 ml RNasi / DNasi. Mettere i tubi in ghiaccio. </li><li> Aggiungere 1 ml di reagente Trizol commerciale e lisi perline matrice nel tubo. </li><li> Lyse il tessuto utilizzando un omogeneizzatore tessuto automatizzato. </li><li> Raccogliere il lisato di tessuto in una nuova provetta. </li><li> Isolare RNA utilizzando il protocollo standard. </li><li> Misurare la concentrazione di RNA. </li><li> Diluire RNA opportunamente con acqua deionizzata e utilizzando 500 ng di RNA per sintetizzare cDNA. </li><li> Utilizzare il cDNA per real-time RT-PCR dei geni mirati antimicrobici, citochine, chemochine, e altri geni di interesse. </li></ol>

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The authors have no competing financial interests.

Le cellule epiteliali intestinali sono molto sensibili in termini di requisiti di crescita e quindi difficile da coltura. Le cellule epiteliali isolate mediante trattamento EDTA non sopravvivono in mezzi di coltura cellulare convenzionali come DMEM 8. Pertanto, studi di interazione ospite-patogeno utilizzando cripta isolata o cellule epiteliali primarie sono molto impegnativo. Recentemente, Sato et al. descritto un sistema di coltura organoide cripta che è molto promettente e utile per gli studi...

L&#39;intestino rappresenta un sistema dinamico che agisce come una barriera per i microrganismi commensali, combatte contro patogeni, e regola la composizione microbica 1. Le cellule epiteliali intestinali, composto da enterociti, cellule caliciformi, le cellule e le cellule Paneth enteroendocrine, sono le principali popolazioni di cellule che forniscono accoglienza risposte di difesa contro microbiota intestinale. Le cellule caliciformi producono mucine che creano una zona smilitarizzata sulla parte superiore dello strato epiteliale 2. Le cellule Paneth e enterociti producono peptidi antimicrobici, citochine, e specie di ossigeno e di azoto reattivo che costituiscono antimicrobici risposte di difesa dell&#39;ospite e contribuiscano a dare forma alla composizione microbica intestinale 3, 4. In aggiunta alle cellule epiteliali, le cellule immunitarie compresi macrofagi, cellule dendritiche, neutrofili, cellule natural killer, linfociti e inna<html> cellule linfoidi te nella lamina propria e sottomucosa giocano un ruolo critico nella intestinali risposte di difesa ospite antimicrobici da citochine, chemochine, che producono e altri mediatori 5 - 7. Per comprendere come il sistema immunitario mucosale regola microflora e fornisce protezione contro l&#39;infezione microbica, è importante considerare la complessa interazione delle popolazioni cellulari eterogenee dell&#39;intestino. Tuttavia, un modello in vitro che comprende tutte le caratteristiche dell&#39;intestino non è disponibile. Pertanto, gli studi molecolari sulla interazione ospite-patogeno a livello intestinale sono altamente impegnativo. Nel corso degli ultimi anni, diversi sistemi modello che imitano gli aspetti della mucosa intestinale sono stati sviluppati per indagare i processi fisiopatologici coinvolti nelle malattie infiammatorie croniche intestinali (IBD) e altri disturbi gastrointestinali 8 -= &quot;xref&quot;&gt; 14. linee di cellule epiteliali intestinali immortalati sono spesso usati per studiare le risposte specifiche delle cellule epiteliali. Tuttavia, a causa di espressione genica differenziale e funzione in cellule immortalizzate, i dati ottenuti dal usando tali cellule non coincidono spesso con quelli osservati negli studi in vivo. Cripta intestinale organoide cultura è emerso recentemente come un potenziale strumento per valutare la risposta del epitelio intestinale a stimoli diversi 13. In questo sistema, le cellule staminali cripta sono autorizzati a crescere e sviluppare una struttura organoide 3D. Mentre il sistema di coltura organoide è molto utile per studiare molti aspetti dell&#39;epitelio intestinale, che non imita la complessa interazione di cellule immuni, cellule epiteliali e prodotti microbici. La cultura ex vivo del tessuto intestinale offre una migliore rappresentazione in vivo ospitanti risposte di difesa. In questo metodo, una parte dell&#39;intestino è coltivato in un piatto bianco coltura cellulareh mezzi appropriati che offrano i diversi tipi di popolazioni cellulari nell&#39;intestino di essere metabolicamente attivi per almeno 48 ore. Così, un ex vivo coltura dell&#39;organo può essere usato per misurare l&#39;espressione di geni antimicrobici e l&#39;host risposte di difesa dell&#39;intestino ad uno stimolo particolare. Gli investigatori hanno utilizzato il sistema d&#39;organo ex vivo della cultura per studiare ospitanti risposte di difesa contro l&#39;infezione microbica nell&#39;intestino 15-21. Recentemente abbiamo adottato il sistema di coltura di organi per studiare il ruolo del inflammasome nelle risposte di difesa antimicrobici ospitanti in due punti di topo 22. Il inflammasome è una piattaforma molecolare per l&#39;attivazione della caspasi-1, che è richiesto per la produzione di IL-1β maturato e IL-18. Abbiamo dimostrato che IL-1β e IL-18 inducono peptidi antimicrobici che uccidono efficacemente pathobionts commensali come E. coli </em&gt;. Questa osservazione è coerente con un aumento coli onere E. in due punti del mouse inflammasome-difettoso 22. Questo sistema può quindi essere utilizzato per studiare il ruolo dei recettori pattern recognition (PRR) e di altre molecole immunitarie innate in intestinali risposte di difesa ospite antimicrobici nonché patogenesi dei disturbi intestinali come la malattia infiammatoria intestinale (IBD) e il cancro del colon-retto (CRC). Ci sono più di 200 geni di suscettibilità IBD, e mutazioni in molti di questi geni sono associati con alterata composizione microbica nell&#39;intestino. E &#39;di grande importanza clinica per determinare il meccanismo preciso attraverso il quale i geni IBD-suscettibilità regolano flora intestinale. L&#39;obiettivo generale di questo metodo è quello di introdurre un protocollo di base di ex vivo della cultura d&#39;organo del colon e dimostrare come questo metodo di coltura può essere usato per studiare antimicrobici risposte di difesa ospite dell &#39;intestino.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/ F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, modified, w/o Calcium chloride &#38; Magnesium chloride</td>
			<td>Sigma</td>
			<td>5634</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, heat inactivated</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma</td>
			<td>G1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-1b recombinan</td>
			<td>Reprokine</td>
			<td>RKP10749</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-18 recombinant</td>
			<td>Reprokine</td>
			<td>RKP70380</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Luria-Bertani Broth&#160;</td>
			<td>BD Bioscience</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Difco Dehydrated Culture Media: MacConkey Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0075-17-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Uded to measure RNA concentration</td>
		</tr>
		<tr>
			<td>UV/Vis Spectrophotometer</td>
			<td>BECKMAN</td>
			<td>DU 530</td>
			<td>Used to determine E. coli count</td>
		</tr>
		<tr>
			<td>iScript RT Supermix, 100 rxns</td>
			<td>Bio-Rad</td>
			<td>1708841</td>
			<td></td>
		</tr>
		<tr>
			<td>iTaq Univer SYBR Green Supermix&#160;</td>
			<td>Bio-Rad</td>
			<td>1725125&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix S (1/8&#34;), 2 mL Tube</td>
			<td>MP Biomedicals</td>
			<td>116925500</td>
			<td>Used to homgenize colon organ for RNA isolation</td>
		</tr>
		<tr>
			<td>FastPrep-24 5G System</td>
			<td>Bio-Rad</td>
			<td>116005500</td>
			<td></td>
		</tr>
		<tr>
			<td>100x15 Petri Dish</td>
			<td>Falcon</td>
			<td>5687</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate 6well ps TC CS100, Cellstar, 6w, tc, F-bottom (Flat), w/lid, sterile</td>
			<td>Cellstar</td>
			<td>5085</td>
			<td></td>
		</tr>
		<tr>
			<td>100 micron cell strainer</td>
			<td>Falcon</td>
			<td>5698</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST40R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Scientific orbital shaker</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo colon organ culture and its use in antimicrobial host defense studies

L&#39;intestino mostra un&#39;architettura di strutture cripta ripetitive costituite da diversi tipi di cellule epiteliali, propia lamina contenente cellule immunitarie, e stroma. Tutte queste cellule eterogenei contribuiscono a omeostasi intestinale e partecipare a difesa ospite antimicrobica. Pertanto, l&#39;identificazione di un modello di surrogato per studiare la risposta immunitaria e attività antimicrobica dell&#39;intestino in un ambiente in vitro è estremamente impegnativo. Gli studi in vitro utilizzando linee cellulari epiteliali intestinali immortalato o cripta addirittura primario organoide cultura non rappresentano l&#39;esatta fisiologia intestinale normale e del suo microambiente. Qui, si discute un metodo di tessuto del colon coltura del mouse in un piatto di cultura e di come questo organo ex vivo sistema di coltura può essere implementato in studi relativi a risposte di difesa antimicrobici ospitanti. In esperimenti di rappresentanza, abbiamo dimostrato che i due punti nella cultura d&#39;organo esprimono peptidi antimicrobici in response per esogena IL-1β e IL-18. Inoltre, le molecole effettrici antimicrobiche prodotte dai tessuti del colon nella cultura organo uccidono efficacemente Escherichia coli in vitro. Questo approccio, pertanto, può essere utilizzato per analizzare il ruolo dei modelli molecolari di patogeni e pericolo associati ed i loro recettori cellulari nella regolazione intestinale risposte immunitarie innate e risposte di difesa antimicrobiche dell&#39;ospite.

Un quadro rappresentativo di due punti nella cultura d&#39;organo è mostrato in Figura 1. I pezzi del colon nella cultura rimangono metabolicamente e fisiologicamente attiva. Essi rispondono in modo efficace agli stimoli esogeni aggiunti al mezzo di coltura. Un flusso di lavoro schematica della preparazione del tessuto colon per coltura ex vivo e stimolazione con stimoli esogeni, per esempio IL-1β e IL-18, è mostrata in figura 2. I d...

Watch this Scientific Journal Video about The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies at JoVE.com

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

Il<em&gt; Ex Vivo</em&gt; Colon Organ Culture e il suo uso in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

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Research

JoVE Journal

Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

13.9K Views.  UT Southwestern Medical Center. The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine. 

Video: The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imaging in vivo del transgenico parassiti Leishmania in un host live

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

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Immunologia e infezioni

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

L&#39;uso del Modello EpiAirway per Caratterizzare a lungo termine interazioni ospite-patogeno

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Il<em&gt; Ex Vivo</em&gt; Cultura e Pattern Recognition recettore stimolazione del mouse intestinale organoidi

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Culture placentare e deciduale organo umano allo Studio infezioni all&#39;interfaccia materno-fetale

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

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

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

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

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

Campionamento non invasivo del rivestimento mucoso fluido per la quantificazione dei livelli di Immune-mediatore In Vivo delle vie respiratorie superiore

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

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Ottimizzazione della tecnica del bracciale per il trapianto di cuore di Murine

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

Stabilire un modello di Cornea ex vivo suina per lo studio dei trattamenti farmacologici contro la cheratite batterica

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Un saggio ex vivo per studiare Candida albicans Morphogenesi ifale nel tratto gastrointestinale

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Test di efficacia antibiotica in un modello ex vivo di Pseudomonas aeruginosa e Staphylococcus aureus Biofilms nel polmone della fibrosi cistica

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...