Name: an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses
Uploaded: 2017-07-07T12:30:00
Description: Watch this Scientific Journal Video about An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses at JoVE.com

Dette arbejde blev støttet af det italienske Cystic Fibrosis Foundation Project FFC # 18/2013, FFC # 29/2015 og af den italienske Cystic Fibrosis League gennem Veneto Branch-Associazione Veneta Lotta Contro Fibrosi Cistica Onlus.

Alle dyreforsøg, der blev beskrevet, blev godkendt af det intramurale dyrevelfærdskomité for dyreforsøg af Interdepartmental Center for Eksperimentel Forskningstjeneste ved University of Verona og i overensstemmelse med det europæiske direktiv 2010/63 UE, italienske D.Lgs 26/2014 og den reviderede &quot;Guide til pleje og brug af laboratoriedyr&quot;, Washington, DC: National Academy Press, 1996. Denne protokol og eksperimenter blev godkendt af National Institutes of Health (n 273/15). Dyr havde fri adgang til stan...

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	<li>Barnes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+approaches+to+asthma-chronic+obstructive+pulmonary+disease+overlap+syndromes.">Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes.</a> J Allergy Clin Immunol. 136 (3), 531-545 (2015).</li><li>Cohen-Cymberknoh, M., Kerem, E., Ferkol, T., Elizur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+inflammation+in+cystic+fibrosis:+molecular+mechanisms+and+clinical+implications.">Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications.</a> Thorax. 68 (12), 1157-1162 (2013).</li><li>Dhooghe, B., Noel, S., Huaux, F., Leal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+inflammation+in+cystic+fibrosis:+pathogenesis+and+novel+therapies.">Lung inflammation in cystic fibrosis: pathogenesis and novel therapies.</a> Clin Biochem. 47 (7-8), 539-546 (2014).</li><li>Durham, A. L., Caramori, G., Chung, K. F., Adcock, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+anti-inflammatory+therapeutics+in+asthma+and+chronic+obstructive+lung+disease.">Targeted anti-inflammatory therapeutics in asthma and chronic obstructive lung disease.</a> Transl Res. 167 (1), 192-203 (2015).</li><li>Sagel, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biomarkers+of+airway+inflammation+in+cystic+fibrosis.">Noninvasive biomarkers of airway inflammation in cystic fibrosis.</a> Curr Opin Pulm Med. 9 (6), 516-521 (2003).</li><li>Starkey, 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=Murine+models+of+infectious+exacerbations+of+airway+inflammation.">Murine models of infectious exacerbations of airway inflammation.</a> Curr Opin Pharmacol. 13 (3), 337-344 (2013).</li><li>Cacalano, G., 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=Neutrophil+and+B+cell+expansion+in+mice+that+lack+the+murine+IL-8+receptor+homolog.">Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.</a> Science. 265 (5172), 682-684 (1994).</li><li>Simonet, W. 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=Long-term+impaired+neutrophil+migration+in+mice+overexpressing+human+interleukin-8.">Long-term impaired neutrophil migration in mice overexpressing human interleukin-8.</a> J Clin Invest. 94 (3), 1310-1319 (1994).</li><li>Stellari, F. F., 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=In+vivo+imaging+of+transiently+transgenized+mice+with+a+bovine+interleukin+8+(CXCL8)+promoter/luciferase+reporter+construct.">In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct.</a> PLoS One. 7 (6), e39716 (2012).</li><li>Stellari, F., 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=In+vivo+imaging+of+the+lung+inflammatory+response+to+Pseudomonas+aeruginosa+and+its+modulation+by+azithromycin.">In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.</a> J Transl Med. 13, 251 (2015).</li><li>Stellari, F., 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=In+vivo+monitoring+of+lung+inflammation+in+CFTR-deficient+mice.">In vivo monitoring of lung inflammation in CFTR-deficient mice.</a> J Transl Med. 14 (1), 226 (2016).</li><li>De Simone, 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=Host+genetic+background+influences+the+response+to+the+opportunistic+Pseudomonas+aeruginosa+infection+altering+cell-mediated+immunity+and+bacterial+replication.">Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.</a> PLoS One. 9 (9), e106873 (2014).</li></ol>

I et tidligere arbejde 11 blev der vist en kontrast mellem bIL-8-Luc-afhængige BLI og BAL markører. Den var afhængig af den differentielle grad af følsomhed inden for musestammer 12 . Af denne årsag kræver den første anvendelse af bIL-8-Luc-modellen til en anden musestamme en indledende undersøgelse af inflammatorisk respons, både hvad angår BLI og mere standardiserede inflammatoriske markører. Mustransfektion medfører mild lungebet...

Kroniske lungesygdomme, såsom astma, kronisk obstruktiv lungesygdom (COPD), cystisk fibrose (CF) og bronchiectasis, er karakteriseret ved luftvejsinflammation. Luftvejsinflammation er kendetegnet ved ødem, cellulær infiltration, T-lymfocyt- og mastcelleaktivering, øgede luftvejsafskærmninger og overdreven kollagenaflejring. CF er en multisystemforstyrrelse, og dens største årsag til dødelighed og morbiditet er lungebakterieinfektion med øget lungekræftdannelse. Nedgang i lungefunktion forudsiger et signifikant fattigere resultat 1 , 2 , 3 , 4 . Betændelsestilstande i luftvejene observeres sædvanligvis ved evaluering af immunologiske markører, der rekrutteres under den inflammatoriske proces i materiale afledt fra de nedre og øvre luftveje, såsom sputum, som tilvejebringer variabel resÜLTS. Bronkoskopier udføres også 5 . Murine-modeller er værdifulde værktøjer til undersøgelse af patogenese og udvikling af sygdomme præget af luftvejsinflammation, og for hvilke effektive behandlinger eller helbredelser endnu ikke er blevet identificeret. Dyremodeller af lungeinfektion og betændelse er blevet brugt til at studere astma og værtspatogeninteraktioner, herunder kemikaliernes rolle som simulerer menneskelige forhold ( f.eks. Eksponering af cigaretrøg, LPS, elastase, ovalbumin, poly I: C osv . Som Såvel som kombinationer af ovenstående) 6 . Måling af inflammationsrelaterede parametre kræver dyrets ofre, da invasive tilgange er nødvendige for at måle faktorer som bakteriel belastning, cytokiner i lungerne og indsamlet bronkoalveolær lavage (BAL) væske. Histologiske undersøgelser er ofte også nødvendige. Muligheden for at indhente oplysninger om inflammatorisk responskinetik kræver anvendelse af numHævede mus. Derfor er en teknik, der giver mulighed for at indhente sådanne oplysninger uden behov for at ofre dyrene værdifulde på tekniske, etiske, økonomiske og operationelle grundlag. IL-8 er en vigtig spiller i inflammationsprocessen, der rekrutterer leukocytter til det betændte væv. Det repræsenterer en molekylær udlæsning til undersøgelsen af ​​inflammatorisk pathwayaktivering. MIP-2 og KC kan være funktionelle homologer af human IL-8 hos mus. Mus udtrykker kun en potentiel IL-8 receptor, en homolog af human CXCR2 7 , 8 , men de er i stand til at modulere en heterolog IL-8-genpromotor, der driver et reportergen. En lungeinflammationsmine-model er for nylig udviklet efter observationen, at en bovin IL-8-promotor / luciferase-reporterkonstruktion kan transaktiveres i mus. Denne funktion gør det muligt at anvende bioluminescensbilleddannelse (BLI) til at overvåge det inflammatoriske respons i levende aNimals 9 . Denne model er blevet tilpasset til at studere inflammation udløst af bakterielle exoprodukter ( fx LPS eller produkter frigivet af bakteriestammer) eller TNFalpha 10 , 11 . Lægemiddelopdagelsesprocessen er fokuseret på udvikling og optimering af gamle og nye antiinflammatoriske molekyler, der kan behandle lungesygdomme, såsom CF, astma og COPD. Disse nye kemiske enheder skal hurtigt og bekvemt testes i dyremodeller, der kan knyttes til specifikke kliniske fænotyper for at lette udformningen af ​​klare kliniske forsøg.

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			<td height="30" style="height:30px;width:347px;">FMT 2500 Fluorescence Tomography System</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td style="width:147px;">Experimental Builder</td>
			<td style="width:587px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">IVIS Lumina serie II Pre-clinical In Vivo Imaging System</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">MMPsense 750 FAST</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td style="width:147px;">NEV10001EX</td>
			<td>Protect from light, store the probe at 4 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Female inbred BalbC</td>
			<td style="width:457px;">Harlan Laboratories Italy</td>
			<td style="width:147px;"></td>
			<td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">bIL-8-Luc plasmid</td>
			<td style="width:457px;">Department of Medical Veterinary Science, University of Parma, Italy</td>
			<td style="width:147px;"></td>
			<td>Store the plasmid at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">pGL3basic vector</td>
			<td style="width:457px;">Promega</td>
			<td style="width:147px;">E1751</td>
			<td>Store the vector at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">JetPEI DNA transfection reagent</td>
			<td style="width:457px;">Polyplus transfection</td>
			<td style="width:147px;">201B-001G</td>
			<td>The DNA and JetPEI mix was formulated with a final N/P ratio of 7</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">D-luciferin potassium salt 1g</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td align="right" style="width:147px;">122796</td>
			<td>Protect from light, store at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Living Image software</td>
			<td style="width:457px;">Caliper Life Sciences,</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Isoflurane</td>
			<td style="width:457px;">ESTEVE spa</td>
			<td style="width:147px;">571329.8</td>
			<td>Do not inhale</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Bio-Plex Cytokine Assay Kit</td>
			<td style="width:457px;">Bio-Rad Laboratories</td>
			<td style="width:147px;">M60-009RDPD</td>
			<td>Store the unopened kit at 4 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Automated cell counter</td>
			<td style="width:457px;">Dasit XT 1800J</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Penn-century model DP-4M Dry power insufflator</td>
			<td style="width:457px;">Penn-century</td>
			<td>DPM-EXT</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Gas anesthesia system XGI-8</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">PE190 micro medical tubing</td>
			<td style="width:457px;">2biological instruments snc</td>
			<td style="width:147px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Syringe without needle 5ml</td>
			<td style="width:457px;">Terumo</td>
			<td style="width:147px;">SS*05SE1</td>
			<td>Cut the boards of the piston by a scissors</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Hamilton 0,10 ml (model 1710)</td>
			<td style="width:457px;">Gastight</td>
			<td align="right">81022</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Discofix 3-way Stopcock</td>
			<td style="width:457px;">Braun</td>
			<td align="right">4095111</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Syringe with needle 1ml</td>
			<td style="width:457px;">Pic solution</td>
			<td align="right">3,071,260,300,320</td>
			<td>Use without needle</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Plastic feeding tubes 18ga x 50mm</td>
			<td style="width:457px;">2biological instruments snc</td>
			<td>FTP-18-50</td>
			<td>Cut obliquely the tip&#160;</td>
		</tr>
	</tbody>
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an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses

Luftvejsinflammation er ofte forbundet med bakterielle infektioner og udgør en vigtig determinant for lungesygdomme. In vivo- bestemmelsen af ​​de proinflammatoriske evner hos forskellige faktorer er udfordrende og kræver terminale procedurer, såsom bronchoalveolær skylning og fjernelse af lunger til in situ- analyse, hvilket udelukker langsgående visualisering i den samme mus. Her induceres lungebetændelse ved intratracheal inddrivning af Pseudomonas aeruginosa- kultursupernatant (SN) i transientt transgeniserede mus, som udtrykker luciferase-reportergenet under kontrol af en heterolog IL-8 bovinpromotor. Luciferaseekspression i lungen overvåges ved in vivo bioluminescerende billede (BLI) analyse over en 2,5- til 48-timers tidsramme efter indstillingen. Proceduren kan gentages flere gange inden for 2 - 3 måneder, hvilket således muliggør evaluering af det inflammatoriske respons i de samme mus medUd behovet for at opsige dyrene. Denne fremgangsmåde tillader overvågning af pro- og antiinflammatoriske faktorer, der virker i lungen i realtid, og forekommer egnet til funktionelle og farmakologiske undersøgelser.

Den bIL-8-Luc transiente transgene musemodel blev anvendt til in vivo overvågning af lungebetændelse hos mus udfordret med koncentreret bakteriel supernatant (30x) indeholdende udskillede virulensfaktorer. Den inducerede inflammatoriske respons var detekterbar ved in vivo billeddannelse som en stigning i BLI signalet. Proinflammatorisk aktivitet var klart detekterbar 2,5 timer efter instillation, selvom BLI-signalet nåede højeste spids mellem 5 og 24 timer og var st...

Watch this Scientific Journal Video about An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses at JoVE.com

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

Fremgangsmåden beskrevet her tillader visualisering af IL-8-promotorafhængig inflammationsaktivering i lungerne af mus gennem ikke-invasiv bioluminescensbilleddannelse (BLI). Det samme dyr kan udsættes for BLI flere gange i op til to måneder fra leveringstidspunktet for luciferase-reporterkonstruktionen.

En IL-8 Transient Transgenized Mouse Model for<em&gt; In vivo</em&gt; Langsigtet overvågning af inflammatoriske reaktioner

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

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