Name: a murine model of dengue virus-induced acute viral encephalitis-like disease
Uploaded: 2019-04-28T14:00:00
Description: Watch this Scientific Journal Video about A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease at JoVE.com

Questo studio è stato sostenuto da sovvenzioni del Ministero della Scienza e della Tecnologia (MOST107-2321-B-038-001) e dal finanziamento intramurale 106TMU-CIT-01-2, Taipei, Taiwan.

I protocolli sperimentali di studio sugli animali sono stati approvati dal Comitato Istituzionale per la cura degli animali e l'utente del National Defense Medical Center (numero IACUC: 16-261), secondo le linee guida stabilite dal Ministero della Scienza e della Tecnologia di Taiwan.
1. Procedura di infezione
<ol><li>Preparare le scorte non adattate dENV2 (strain PL046)9 (originariamente ottenute dai Centers for Disease Control di Taiwan, che vanno da 2,5 x 107<...

<ol>
	<li>Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E., Halstead, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+infection.">Dengue infection.</a> Nature Reviews Disease Primers. 2, 16055 (2016).</li><li>Katzelnick, L. C., Coloma, J., Harris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue:+knowledge+gaps,+unmet+needs,+and+research+priorities.">Dengue: knowledge gaps, unmet needs, and research priorities.</a> Lancet Infectious Diseases. 17 (3), e88-e100 (2017).</li><li>Carod-Artal, F. J., Wichmann, O., Farrar, J., Gascon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+complications+of+dengue+virus+infection.">Neurological complications of dengue virus infection.</a> Lancet Neurology. 12 (9), 906-919 (2013).</li><li>Geneva: World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. , (2009).</li><li>Amaral, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+infection+with+dengue-3+virus+induces+meningoencephalitis+and+behavioral+changes+that+precede+lethality+in+mice.">Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice.</a> Journal of Neuroinflammation. 8, 23 (2011).</li><li>de Miranda, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue-3+encephalitis+promotes+anxiety-like+behavior+in+mice.">Dengue-3 encephalitis promotes anxiety-like behavior in mice.</a> Behavioural Brain Research. 230 (1), 237-242 (2012).</li><li>Velandia-Romero, M. L., Acosta-Losada, O., Castellanos, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+infection+by+a+neuroinvasive+neurovirulent+dengue+virus.">In vivo infection by a neuroinvasive neurovirulent dengue virus.</a> Journal of Neurovirology. 18 (5), 374-387 (2012).</li><li>Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte Dos Santos, C., Deubel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+mouse+central+nervous+system+in+response+to+infection+with+mouse-neurovirulent+dengue+viruses.">Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses.</a> Journal of Virology. 72 (1), 823-829 (1998).</li><li>Tsai, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+retard+dengue+virus-induced+acute+viral+encephalitis.">Microglia retard dengue virus-induced acute viral encephalitis.</a> Scientific Reports. 6, 27670 (2016).</li><li>Cheng, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Nrf2+by+the+dengue+virus+causes+an+increase+in+CLEC5A,+which+enhances+TNF-alpha+production+by+mononuclear+phagocytes.">Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-alpha production by mononuclear phagocytes.</a> Scientific Reports. 6, 32000 (2016).</li><li>Ho, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+dengue+virus+infection+and+viral+cytotoxicity+in+neuronal+cells+in+vitro+and+in+vivo+by+targeting+endocytic+pathways.">Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways.</a> Scientific Reports. 7 (1), 6910 (2017).</li><li>Jhan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-TNF-alpha+restricts+dengue+virus-induced+neuropathy.">Anti-TNF-alpha restricts dengue virus-induced neuropathy.</a> Journal of Leukocyte Biology. 104 (5), 961-968 (2018).</li><li>Kao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antiparasitic+drug+niclosamide+inhibits+dengue+virus+infection+by+interfering+with+endosomal+acidification+independent+of+mTOR.">The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR.</a> PLoS Neglected Tropical Diseases. 12 (8), e0006715 (2018).</li><li>Tsai, T. T., Chen, C. L., Tsai, C. C., Lin, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+heat+shock+factor+1+as+an+antiviral+strategy+against+dengue+virus+replication+in+vitro+and+in+vivo.">Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo.</a> Antiviral Research. 145, 44-53 (2017).</li><li>Jhan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+virus+infection+increases+microglial+cell+migration.">Dengue virus infection increases microglial cell migration.</a> Scientific Reports. 7 (1), 91 (2017).</li><li>Benskey, M. J., Manfredsson, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraparenchymal+Stereotaxic+Delivery+of+rAAV+and+Special+Considerations+in+Vector+Handling.">Intraparenchymal Stereotaxic Delivery of rAAV and Special Considerations in Vector Handling.</a> Methods in Molecular Biology. , 199-215 (2016).</li><li>Fong, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+encephalitis/encephalopathy+with+reversible+splenial+lesion+(MERS)+due+to+dengue+virus.">Mild encephalitis/encephalopathy with reversible splenial lesion (MERS) due to dengue virus.</a> Journal of Clinical Neuroscience. 36, 73-75 (2017).</li><li>Sarathy, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lethal+murine+infection+model+for+dengue+virus+3+in+AG129+mice+deficient+in+type+I+and+II+interferon+receptors+leads+to+systemic+disease.">A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease.</a> Journal of Virology. 89 (2), 1254-1266 (2015).</li><li>Schul, W., Liu, W., Xu, H. Y., Flamand, M., Vasudevan, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dengue+fever+viremia+model+in+mice+shows+reduction+in+viral+replication+and+suppression+of+the+inflammatory+response+after+treatment+with+antiviral+drugs.">A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs.</a> The Journal of Infectious Diseases. 195 (5), 665-674 (2007).</li><li>Tyler, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Viral+Encephalitis.">Acute Viral Encephalitis.</a> New England Journal of Medicine. 379 (6), 557-566 (2018).</li><li>Yauch, L. E., Shresta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+dengue+virus+infection+and+disease.">Mouse models of dengue virus infection and disease.</a> Antiviral Research. 80 (2), 87-93 (2008).</li><li>Assir, M. Z., Jawa, A., Ahmed, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dengue+syndrome:+subacute+thyroiditis+and+intracerebral+hemorrhage.">Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage.</a> BMC Infectious Diseases. 12, 240 (2012).</li><li>Kumar, R., Prakash, O., Sharma, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+hemorrhage+in+dengue+fever:+management+and+outcome:+a+series+of+5+cases+and+review+of+literature.">Intracranial hemorrhage in dengue fever: management and outcome: a series of 5 cases and review of literature.</a> Surgical Neurology. 72 (4), 429-433 (2009).</li><li>Simanjuntak, Y., Liang, J. J., Lee, Y. L., Lin, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+of+prochlorperazine+for+use+against+dengue+virus+infection.">Repurposing of prochlorperazine for use against dengue virus infection.</a> Journal of Infectious Diseases. 211 (3), 394-404 (2015).</li><li>Rocha, B. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue-specific+serotype+related+to+clinical+severity+during+the+2012/2013+epidemic+in+centre+of+Brazil.">Dengue-specific serotype related to clinical severity during the 2012/2013 epidemic in centre of Brazil.</a> Infectious Disease Poverty. 6 (1), 116 (2017).</li></ol>

L'infezione da DENV è stata rilevata nel SNC di pazienti con dengue grave3,17, suggerendo la possibilità della manifestazione di encefalite virale acuta avvenuta durante la patogenesi dengue. Qui, riportiamo un modello murino in vivo dell'infezione da DENV per studiare il coinvolgimento della disfunzione del SNC nella dengue grave, in particolare con un focus sulle malattie acute simili all'encefalite acuta indotta da DENV. Rispetto ai modelli precedenti, in pa...

DENV, un virus artropode della famiglia Flaviviridae, contiene un genoma di RNA a senso positivo che codifica tre proteine strutturali virali (capside, premembrana e busta) e sette proteine non strutturali virali (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5). I quattro sierotipi di DENV (DENV1-4), che infettano circa 390 milioni di persone ogni anno, causano un onere globale anche se i governi hanno diretto notevoli sforzi verso il controllo dei vettori e delle malattie delle zanzare1. Attualmente, i vaccini protettivi e i farmaci antivirali terapeutici sono in fase di sviluppo e richiedono un'ulteriore convalida a lungo termine2. Nella pratica clinica, anche se un paziente dengue con infezione da SNC è raro, deve essere ulteriormente esplorato per comprendere la diversità dello sviluppo della malattia dengue3. Sono necessarie ulteriori indagini e validazioni; in particolare, l'Organizzazione Mondiale della Sanità (OMS) ha incluso il coinvolgimento del compromissione del SNC, come il danno cognitivo, le convulsioni, l'encefalopatia e l'encefalite, nella classificazione della dengue grave3,4. La costruzione di modelli animali di infezione da DENV è indispensabile per esplorare la neuropatogenesi dell'infezione da DENV.
Per generare l'infezione da CNS da parte di DENV, diversi studi hanno eseguito diverse vie di infezione da DENV, tra cui (1) l'inoculazione intracerebrale di topi C57BL/6 che hanno ricevuto 4 x 103 unità di formazione di placche (PFU) di DENV35non adattati, 6, (2) inoculazione intraperitoneale di topi BALB/c che hanno ricevuto 7 x 104 PFU di DENV47in vitro , (3) inoculazione intracerebrale di topi svizzeri che hanno ricevuto 1 x 105 PFU di DENV18in vivo e (4) co-inoculazione intracerebrale e intraperitale dei topi da allattamento ICR che hanno ricevuto 1 x 106 PFU di DENV29 nonadattati . Secondo i risultati di questi studi5,6,7,8,9, infezione DENV nei topi provocare la replicazione virale nel cervello, portando a virale acuta sindromi simili all'encefalite, cambiamenti comportamentali accompagnati da paralisi degli arti e instabilità posturale, neurotossicità e infiammazione del SNC, perdite plasmatiche generali e localizzate attraverso la barriera emato-encefalica (BBB) e letalità. Tutti i risultati di questi studi5,6,7,8,9hanno dimostrato la capacità di DENV di infettare il SNC e l'induzione di encefalite virale acuta malattia a seguito di infezione.
Sulla base dei nostri risultati attuali9,10,11,12,13,14,15, abbiamo creato un modello murino di infezione DENV come un in vivo per esaminare l'efficacia terapeutica di agenti/fattori mirati contro la replicazione virale, nonché la neurotossicità. Qui, riportiamo il protocollo utilizzato per creare un topo ICR immunocompetente per studiare l'infezione da CnS e per monitorare lo sviluppo di neuropatie con diverse gravità causate da DENV. I risultati mostrano la progressione significativa della malattia simile all'encefalite nei topi infetti da DENV in modo dipendente dal tempo.

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	<tbody>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Roswell Park Memorial Institute 1640 medium (RPMI)</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">11875-085</td>
			<td style="width:200px;">Diluting virus</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">0.3-mL Insulin Syringe&#160;</td>
			<td style="width:211px;">BD Ultra-Fine&#173;II</td>
			<td style="width:119px;">328838</td>
			<td style="width:200px;">Intracerebral and intraperitoneal injection</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Microbalance</td>
			<td style="width:211px;">METTLER TOLEDO&#39;s LabX</td>
			<td style="width:119px;">AL104</td>
			<td style="width:200px;">Weight mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-adapted DENV2 (strain PL046)</td>
			<td style="width:211px;">The Centers for Disease Control of Taiwan</td>
			<td style="width:119px;">-</td>
			<td style="width:200px;">Infect mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Institute of Cancer Research (ICR) suckling mouse</td>
			<td style="width:211px;">BioLASCO Taiwan Co., Ltd</td>
			<td style="width:119px;">-</td>
			<td style="width:200px;">Our murine model</td>
		</tr>
	</tbody>
</table>

a murine model of dengue virus-induced acute viral encephalitis-like disease

Il virus dengue (DENV), un virus trasmesso da artrodi trasmesso dalle zanzare, può causare la grave malattia nota come febbre emorragica dengue, caratterizzata da complicazioni letali dovute a perdite di plasma, asciti, effusioni pleuriche, grave emorragia e compromissione dell'organo. Alcuni casi di infezione Da DENV presentano manifestazioni neurologiche; tuttavia, gli studi non hanno esplorato ulteriormente la neuropatogene indotta da DENV. In questo studio, presentiamo un protocollo per utilizzare un topo ICR (Istituto di ricerca sul cancro) immunocompetente per studiare l'induzione dell'infezione del sistema nervoso centrale (SNC) con DENV, seguito dalla progressione di encefalite virale acuta malattia.

Gravi complicazioni neurologiche associate alla dengue sono state segnalate nei pazienti per la patogenesi dengue4. Anche se questi casi sono rari in clinica, la creazione di un modello murino immunocompetente di infezione Da DENV può essere utilizzato non solo per studiare l'immunopatogene, ma anche per esplorare l'infezione da SNC, la neuroinfiammazione, la neurotossicità e il virale acuto una malattia simile all'encefalite. In questo studio, secondo il nostro ...

Watch this Scientific Journal Video about A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease at JoVE.com

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Qui, presentiamo un protocollo per la creazione di un iCR immunocompetente (Istituto di ricerca sul cancro) modello murino di infezione del sistema nervoso centrale per visualizzare lo sviluppo della neuropatia. Il monitoraggio dei disturbi encefalitici virali con punteggi di malattia identici potrebbe essere eseguito per mostrare la neuropatia indotta dal virus dengue in vivo.

Un modello Murine di encefalite virale acuta indotta da virus dengue

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Questo protocollo di un modello di malattia si vede l'infezione da virus dengue nel cervello diventare una piattaforma in vivo per lo screening di vettori virali e ospiti associati alla neuropatogenesi. A differenza dei modelli precedenti, i nostri modelli utilizzano strumenti dell'infezione con un basso titer virale, per infettare i topi che allattano immunocompetente Institute of Cancer Research per l'induzione della procreazione della malattia encefalitica. A dimostrare la procedura sarà Ting-Jing Shen, dottorando del mio laboratorio.Prima di iniziare la procedura di infezione, diluire il virus dengue non adattato due stock, a una per 10 alla quinta unità di formatura della placca per 40 microlitri di concentrazione media RPMI 1640. Successivamente, caricare una siringa da 0,3 millilitri dotata di un ago calibro 30 con 10 microlitri di virus diluito. E una siringa da 0,3 millilitri dotata di un ago calibro 30, con 30 microlitri di virus per animale.Per l'erogazione intercerebrale del virus, premere l'oracolo tra il pollice e l'indice per trattenere delicatamente un topo dell'Istituto di ricerca sul cancro di sette giorni in una posizione incline e iniettare il volume di 10 microliter del virus diluito nell'area lambda, all'intersezione della sutura sagittale e lambdoide. Dopo l'iniezione intercerebrale, utilizzare il pollice e l'indice per tenere il mouse in posizione supina e, delicatamente intraperitonealmente, iniettare il volume di 30 microliter del virus diluito, nell'addome murino. Per il punteggio della progressione della malattia, assegnare un punteggio da zero a cinque a ogni topo inoculato ogni giorno, in base al grado di malattie virali acute simili all'encefalite, e tracciare i punteggi per ogni giorno come una figura basata sulla curva, utilizzando il più e meno medio la deviazione standard dei punteggi giornalieri dei test in ogni gruppo.Rispetto ai cambiamenti di peso corporeo misurati in topi finti e solo inoculati, gli animali inoculati del virus dengue mostrano una diminuzione del peso corporeo in ogni momento durante lo studio. Inoltre, gli animali infetti da dengue virus dimostrano un aumento significativo delle posture della schiena dell'intuizione, convulsioni limbiche, debolezza limbica, paralisi e morte rispetto ai sintomi mostrati da finti animali inoculati. Infatti, i topi infetti da dengue mostrano una riduzione dipendente dal tempo dei tassi di sopravvivenza che non si osserva nei finti animali inoculati, confermando la creazione di un modello infettivo di virus dengue all'interno del sistema nervoso centrale che dimostra la progressione di malattie virali acute simili all'encefalite nei topi.La puntura del sito sbagliato può causare il blocco dell'ago da parte del punteggio e l'overflow della soluzione virale al di fuori della testa, con conseguente iniezione fallita e un'infezione fallita. Seguendo la procedura, i campioni cerebrali possono essere raccolti per danni patologici, infiltrazioni di cellule immunitarie e analisi di replicazione virale rispettivamente mediante citometria del flusso di colorazione ematossilina ed eosina e test di formazione della placca. Il nostro modello murino immunocompetente, fornisce una piattaforma per esplorare i meccanismi patogeni dell'infezione da virus dengue, per lo sviluppo di vaccini contro il virus dengue o farmaci anti-dengue virus.Il virus Dengue è un agente patogeno di livello due di biosicurezza e, in quanto tale, tutti gli esperimenti con questo virus dovrebbero essere completati in un laboratorio qualificato di biosicurezza animale di secondo livello.

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Research

JoVE Journal

Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induzione di Graft-versus-host disease e<em&gt; In Vivo</em&gt; T monitoraggio cellulare Utilizzando un MHC-abbinato modello murino

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Ricerca

Immunologia e infezioni

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Tampone Metodo di campionamento per la rilevazione di Norovirus umana sulle superfici

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Valutazione delle risposte a cellula T Zika Virus-specifici in organi di nuova di Ifnar1 infetti- / - Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Induzione e punteggio della malattia di Graft-Versus-Host in un modello di Murine Xenogeneico e quantificazione delle cellule T umane nei tessuti del topo utilizzando pcR digitale

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Un modello di lesione polmonare acuta autolimitata dall'instillazione unilaterale dell'acido intra-bronchiale

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

Modello murino di sindrome da distress respiratorio acuto indotto da acido oleico

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Ipersensibilità da contatto come modello murino di dermatite allergica da contatto

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...