Gli autori desiderano ringraziare il Dr. Jay Pepose per aiutare insegnare il modello da noi. Questo lavoro è stato sostenuto dal National Institutes of Health sovvenzioni EY11885 (PMS), EY21247 (PMS) e una sovvenzione illimitata di ricerca per prevenire la cecità al Dipartimento di Oftalmologia.

Cura degli animali Nota: Gli analgesici non sono utilizzati in questo modello in quanto sono anti-infiammatori e quindi si invaliderebbe il modello. 1. Preparazione di virus <ol><li> Grow HSV-1 su cellule Vero (80% confluenti) utilizzando una diluizione iniziale di 0,01 molteplicità di infezione (in T-150 beuta, circa 5 x 10 5 unità formanti placca) per 3-4 giorni finché tutte le celle sono arrotondate-up e sono facilmente sloggiato colpendo il pallone. </li><li> Rimuovere i supporti per provette da centrifuga e centrifugare a sterili Sorvall Leggenda RT a 4 ° C e 3.500 rpm per 15 min. Dopo aver rimosso il surnatante (vedi 1.3), risospendere grande pellet da ciascuna provetta 50 ml con 5 ml di totale media. Ultrasuoni per 1 min in due burst 30 sec. </li><li> Rimuovere il surnatante e mettere in 50 provette sterili Oak Ridge ml e centrifugare a 9000 rpm per 1 ora a 4 ° C in una Beckman J2-21 centrifuga. Questo è di produrre un piccolo pellet virale. Eliminare il surnatante. </li><li> Combina ultrasuoni da 1,2 spiritoh pellet da 1,3 e sonicare ancora per 45 sec. Centrifuga in Sorvall Legenda RT a 1.500 giri al minuto per 5 minuti a 4 ° C. Salvare il surnatante. Questo costituisce il magazzino virus che viene suddiviso in aliquote e conservato a -70 ° C. </li><li> Titer virus su cellule Vero. Io di solito tavola 10 a 0 su 10 -8 diluizione (ciascuno dei quali è a 10 volte più diluita) 2x da 2 serie di diluizione separati. Titolazione dei virus viene eseguita su piastre da 12 pozzetti sterili. </li></ol> 2. Infezione mouse <ol><li> Iniettare ciascuna mouse con 0,1 ml di anestetico peso cocktail/20 corpo del mouse g (ketamina, 60 mg / kg + TMA 5 mg / kg che è diluito in HBSS). Questo cocktail è usato perché è uno dei mezzi più sicuri di anestesia quando dosaggi adeguati sono utilizzati e gli effetti di questo farmaco sono molto breve. </li><li> Quando i topi vengono anestetizzati graffiare la cornea dell&#39;occhio destro con un ago calibro 30 utilizzando un microscopio per dissezione per assicurare che la cornea non è perforato. </li><li> Ogni topo received una iniezione intraperitoneale di 1 ml di pool di siero umano (Sigma, Temecula, California; anti-HSV reattività con una dose efficace per il 50% di neutralizzazione virale 1:800) per proteggere cornee da danni durante l&#39;infezione primaria. </li><li> Numero i topi sia con clip orecchio o ad orecchio pugno. </li><li> Dopo che tutti i topi sono stati numerati, utilizzando una pipetta 20 microlitri, si infettano ogni occhio con 10 6 PFU (5 pl). Abbiamo infettare un solo occhio e in genere l&#39;occhio destro. Abbiamo anche posto 5 ml di HBSS per l&#39;altro occhio per mantenerlo umido mentre sono anestetizzati. 5 ul è sufficiente a mantenere l&#39;idratazione degli occhi poiché le procedure sono eseguite in meno di 10 minuti, e gli animali vengono dosati con anestetico conseguenza. Infezione è confermata tamponando topi 3 giorni dopo l&#39;infezione con un applicatore di cotone sterile che è stata idratata in 1 ml di mezzo Vero. Quindi 100 pl viene aggiunto alle cellule Vero coltivate in 48 pozzetti. Queste piastre sono il monitoraggio giorno per effetto citopatico<html> (CPE). </li></ol> 3. La riattivazione di topi da Latenza <ol><li> I topi vengono anestetizzati almeno 5 settimane dopo l&#39;infezione primaria. Va notato che abbiamo riattivato topi fino ad un anno dopo l&#39;infezione primaria e non sono osservate differenze significative nella risposta. </li><li> Una volta anestetizzato sono collocati TM20 cromatografia Vu transilluminatore, che emette UV-B alla lunghezza d&#39;onda di picco di 302 nm tali che solo un unico occhio è esposto alla luce UV-B. Ogni mouse è esposto a 250 mJ di UV-B luce cm 2. </li><li> Riattivazione è determinato dal tampone occhi con applicatore cotone sterile che viene dapprima collocato in 1 ml di mezzo Vero giorno 0 (Prima UV-B per controllare l&#39;esposizione per shedders spontanee) e poi il 1-7 post-UV-B irradiazione giorni. Il materiale tampone viene valutata come descritto in 2.5). Per quei tamponi che hanno virus infettivo la quantità di virus è ridacchiarono da un saggio di placca standard utilizzando 12 pozzetti. </li></ol>ove_title &quot;&gt; 4. valutazione clinica <ol><li> I topi vengono valutati per la malattia di occhio clinico da un osservatore mascherato utilizzando un microscopio binoculare da dissezione. </li><li> Opacità corneale è valutato su una scala da 0 a 4, dove 0 indica stroma chiaro, 1 indica lieve opacizzazione stromale, 2 indica l&#39;opacità moderata con distinguibili le caratteristiche dell&#39;iride, 3 indica l&#39;opacità densa con perdita di dettaglio definito dell&#39;iride ad eccezione dei margini, degli alunni e 4 indica opacità totale senza vista posteriore. </li><li> Neovascolarizzazione è valutato su una scala da 1 a 8 dividendo la cornea in quattro quadranti uguali e determinare il grado di vascolarizzazione in ciascuno di questi quadranti. </li><li> Blefarite è misurata come segue: 0, nessuna lesione, 1, minimo gonfiore delle palpebre, 2, scarico moderato gonfiore oculare e croccante, 3, gonfiore grave, moderata perdita di capelli perioculare, e lesioni cutanee, e 4, forte gonfiore con intercettazione occhi crosta, grave perdita di capelli perioculare, e lesioni cutanee. </li><li> Prove di encefalite inclusioneDED curvo indietro, capelli arruffati, e evidenti alterazioni neurologiche. I topi sono eutanasia quando mostrano segni significativi di encefalite. </li></ol> 5. Risultati rappresentativi Il modello periodico che è stato sopra descritto è stato pubblicato da Shimeld, et al. 1-3. Hanno testato e il nostro laboratorio ha utilizzato una versione modificata di questo modello a pubblicare i manoscritti nel corso degli anni 4-15. Il modello è stato utilizzato per definire il ruolo che la citochina interferone-γ gioca a ricorrenti HSV-1 malattia 10. Come mostrato in figura 3, topi privi IFNy visualizzato malattia peggiore corneale di fatto topi wild-type 14. È stato anche usato con successo per definire il valore terapeutico di diversi vaccino diverso costruisce 6,10,11. Si noti che in un caso il vaccino testato funzionava bene profilatticamente ma non rivelarsi terapeuticamente efficace 6. Tuttavia,quando una replica-incompetente vaccino virale è stato testato, non era solo profilatticamente efficace, ma è anche molto efficace terapeuticamente 10,11, vedi Figura 5 nel riferimento 10. Più recentemente abbiamo usato questo modello per determinare se le differenze di genere erano rilevabili in topi sottoposti a HSK ricorrente. Come mostra la Figura 1 mostra, maschio topi C57BL / 6 visualizzare opacità corneale molto meno di quanto non facciano C57BL femmina / 6 topi. Analogamente, quando la neovascolarizzazione corneale è stata valutata, topi maschi dimostrato crescita dei vasi significativamente meno nuovo nella cornea di fatto topi femmina (Figura 2). Al momento stiamo testando se questo fenotipo è ceppo specifico eseguendo analisi simili in altri ceppi di topi. Abbiamo anche determinare se la differenza è dovuta alla mancanza di testosterone o la presenza di estrogeni. <img alt="Figura 1" src="/files/ftp_upload/4276/4276fig1.jpg" /> Figura 1. Malattia corneale in C57BL maschio e femmina / 6 ceppi di topi a seguito UV-B riattivazione indotta. Topi latentemente infette sono stati indotti a riattivare con irradiazione UV-B e topi sono stati monitorati per opacità corneale per 35 giorni. I numeri di topi utilizzati per questi studi sono stati i seguenti: maschi, n = 15; femmine, n = 15. I risultati indicano media ± SEM. * Si è significativamente maggiore indotta da virus della malattia in topi femmina C57BL / 6 per i giorni 14-35 (P = 0,001-0,01). <img alt="Figura 2" src="/files/ftp_upload/4276/4276fig2.jpg" /> Figura 2. Malattia corneale in C57BL maschio e femmina / 6 ceppi di topi a seguito UV-B riattivazione indotta. Topi latentemente infette sono stati indotti a riattivare con irradiazione UV-B e topi sono stati monitorati per la neovascolarizzazione corneale per 35 giorni. I numeri di topi utilizzati per questi studi sono stati i seguenti: maschi, n= 15; femmine, n = 15. I risultati indicano media ± SEM. * Si è significativamente maggiore indotta da virus della malattia in topi femmina C57BL / 6 per i giorni 14-28 (P = 0,001-0,01).

<ol>
	<li>Shimeld, C., Hill, T. J., Blyth, B., Easty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+model+of+recurrent+herpetic+eye+disease+in+mice.">An improved model of recurrent herpetic eye disease in mice.</a> Curr. Eye Res. 8, 1193-1205 (1989).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+infection+and+induction+of+recurrent+herpetic+eye+disease+in+mice.">Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.</a> J. Gen. Virol. 71, 397-404 (1990).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+protects+the+mouse+eye+from+damage+after+herpes+simplex+virus+infection+by+limiting+spread+of+virus+in+the+nervous+system.">Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.</a> J. Gen. Virol. 71, 681-687 (1990).</li><li>Laycock, K. A., Lee, S. F., Brady, R. H., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+model+of+recurrent+herpes+simplex+viral+keratitis+induced+by+ultraviolet+B+radiation.">Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.</a> Invest. Ophthalmol. Vis. Sci. 32, 2741-2746 (1991).</li><li>Keadle, T. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+ameliorates+corneal+disease+in+a+mouse+model+of+recurrent+herpetic+keratitis.">IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis.</a> Microbial Path. 38, 13-21 (2005).</li><li>Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+recombinant+glycoprotein+D+subunit+vaccine+on+the+development+of+primary+and+recurrent+ocular+infection+with+herpes+simplex+virus+type+1+in+mice.">Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.</a> J. Inf. Dis. 176, 331-338 (1997).</li><li>Keadle, T. L., Laycock, K. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+cytokines+IL-1+and+TNF-a+are+required+for+recurrent+herpetic+keratitis+in+NIH+mice.">Proinflammatory cytokines IL-1 and TNF-a are required for recurrent herpetic keratitis in NIH mice.</a> Invest. Ophth. Vis. Sci. 41, 96-102 (2000).</li><li>Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+cytokine+expression+in+a+murine+model+recurrent+herpetic+stromal+keratitis.">Corneal cytokine expression in a murine model recurrent herpetic stromal keratitis.</a> Ocular Immunol. Inflam. 9, 193-205 (2001).</li><li>Keadle, T. L., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++and+CD8++cells+are+key+participants+in+the+development+of+recurrent+herpetic+stromal+keratitis+in+mice.">CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice.</a> Microbial Path. 32, 255-262 (2002).</li><li>Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+immunization+with+a+virion+host+shutoff-defective,+replication-incompetent+herpes+simplex+virus+type+1+strain+limits+recurrent+herpetic+ocular+infection.">Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection.</a> J. Virol. 76, 3615-3625 (2002).</li><li>Keadle, T. L., Laycock, K. A., Morris, J. L., Leib, D. A., Morrison, L. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+vaccination+with+vhs(-)+herpes+simplex+virus+reduces+the+severity+of+recurrent+herpetic+stromal+keratitis+in+mice.">Therapeutic vaccination with vhs(-) herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice.</a> J. Gen. Virol. 83, 2361-2365 (2002).</li><li>Keadle, T. L., Morris, J. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aminoguanidine+on+primary+and+recurrent+ocular+herpes+simplex+virus+infection.">The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection.</a> Nitric Oxide. 13, 247-253 (2005).</li><li>Stuart, P. M., Morris, J. E., Sidhu, M., Keadle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL3+protects+mice+from+corneal+pathology+during+recurrent+HSV-1+infection.">CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.</a> Front. Biosci. 13, 4407-4415 (2008).</li><li>Keadle, T. L., Alexander, D. E., Leib, D. A., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+is+not+required+for+recurrent+herpetic+stromal+keratitis.">Interferon gamma is not required for recurrent herpetic stromal keratitis.</a> Virology. 380, 46-51 (2008).</li><li>Carr, D. J., Austin, B. A., Halford, W. P., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Interferon-gamma+by+an+adenovirus+vector+blocks+herpes+simplex+virus+Type+1+reactivation+in+vitro+and+in+vivo+independent+of+RNase+L+and+double-stranded+RNA-dependent+protein+kinase+pathways.">Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.</a> J. Neuroimmunol. 206, 39-43 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Il modello ricorrente di erpetica cheratite stromale (HSK) qui descritto consente al ricercatore di studiare HSK umano in un modello che è più coerente con quella che si osserva durante la malattia umana. Così, i punti di forza del modello sono che la malattia si verifica nel contesto di un sistema immunitario che ha già da stimolati durante la malattia primaria. Poiché questi topi già hanno una risposta immunitaria a HSV-1, i fattori che riattivano risposta immunitaria che sono probabilmente diverse da quelle che...

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-C1</td>
		</tr>
		<tr>
			<td>Pen/ Strep</td>
			<td>Sigma</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>52-21</td>
		</tr>
		<tr>
			<td>Transilluminatior</td>
			<td>UVP</td>
			<td>95-0452-02</td>
		</tr>
		<tr>
			<td>Sonicatior</td>
			<td>Branson</td>
			<td>Sonifier 450</td>
		</tr>
		<tr>
			<td>Oak Ridge Tubes</td>
			<td>Nalgene</td>
			<td>Z 720410</td>
		</tr>
		<tr>
			<td>Flasks- 7150</td>
			<td>Corning</td>
			<td>430823</td>
		</tr>
		<tr>
			<td>Plates- 12 well</td>
			<td>Corning</td>
			<td>CLS 3513</td>
		</tr>
		<tr>
			<td>Plates- 48 well</td>
			<td>Corning</td>
			<td>CLS 3548</td>
		</tr>
		<tr>
			<td>Sterile Conical Tubes- 30 ml</td>
			<td>Corning</td>
			<td>CLS 430828</td>
		</tr>
		<tr>
			<td>Sterile Cotton-tipped Applicators</td>
			<td>Fisher</td>
			<td>23-400-125</td>
		</tr>
		<tr>
			<td>30G Needles</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
		</tr>
		<tr>
			<td>25G Needles</td>
			<td>BD</td>
			<td>305122</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309604</td>
		</tr>
	</tbody>
</table>

recurrent herpetic stromal keratitis in mice, a model for studying human hsk

Malattie degli occhi erpetica, chiamato cheratite erpetica stromale (HSK), è un&#39;infezione potenzialmente accecante della cornea che si traduce in oltre 300.000 visite cliniche ogni anno per il trattamento. Tra 1 e 2 per cento di quei pazienti con malattia clinica sperimenterà perdita della vista della cornea infetto. La grande maggioranza di questi casi sono il risultato di riattivazione di una infezione latente da herpes simplex virus di tipo I e non causa di malattia acuta. È interessante notare che l&#39;infezione acuta è il modello più utilizzato per studiare questa malattia. Tuttavia, si è ritenuto che un modello ricorrente di HSK sarebbe più riflettente di ciò che avviene durante la malattia clinica. I modelli animali ricorrenti per HSK hanno utilizzato entrambi i conigli e topi. Il vantaggio di conigli è che essi sperimentano riattivazione dalla latenza in assenza di qualsiasi stimolo noto. Detto questo, è difficile esplorare il ruolo che molti fattori immunologici giocare in HSK ricorrenti in quanto il modello di coniglio non ha la immungici e le risorse genetiche che il mouse ha. Abbiamo scelto di utilizzare il modello di topo per HSK ricorrenti in quanto ha il vantaggio che vi siano molte risorse disponibili e anche sapere quando riattivazione avverrà perché riattivazione è indotta da esposizione a luce UV-B. Finora, questo modello ha permesso a tali laboratori di utilizzarlo per definire diversi fattori immunologici che sono importanti per questa malattia. Ha inoltre permesso di testare sia terapeutica e l&#39;efficacia del vaccino.

Watch this Scientific Journal Video about Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK at JoVE.com

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

La maggior parte degli studi sulla malattia erpetica corneale utilizzare un modello di infezione primaria. Tuttavia, l&#39;infezione primaria con HSV-1 non tipicamente portare a malattie umane. Qui si descrive un modello ricorrente della malattia erpetica corneale, che più da vicino imita malattia umana.

Ricorrente cheratite erpetica Stromal in Mouse, un modello per lo studio HSK umano

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

recurrent-herpetic-stromal-keratitis-mice-model-for-studying-human

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JoVE Journal

Immunology and Infection

11.8K Views.  Saint Louis University. La maggior parte degli studi sulla malattia erpetica corneale utilizzare un modello di infezione primaria. Tuttavia, l'infezione primaria con HSV-1 non tipicamente portare a malattie umane. Qui si descrive un modello ricorrente della malattia erpetica corneale, che più da vicino imita malattia umana.

Video: Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Il<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Studiare Contributi degli agenti patogeni e Host alla colite infettiva

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

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Immunologia e infezioni

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Un<em&gt; In vitro</em&gt; Modello per studiare eterogeneità di differenziazione dei macrofagi umani e polarizzazione

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D imaging multimodale di<em&gt; Citrobacter rodentium</em&gt; Infezioni in topi

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento di cellule murine Linfonodo stromali

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modello per lo studio delle interazioni tra l&#39;uomo di sangue intero e l&#39;endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infezione sperimentale con<em&gt; Listeria monocytogenes</em&gt; Come modello per lo studio Host Interferone-gamma Risposte

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologie per lo studio<em&gt; B. subtilis</em&gt; I biofilm come modello per Caratterizzare piccole molecole inibitori biofilm

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Impianto e monitoraggio da PET/CT di un modello ortotopico di mesotelioma pliologico umano in topi atimici

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...