Name: selective harvesting of marginating-pulmonary leukocytes
Uploaded: 2016-03-11T15:00:00
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Forfatterne og dette arbejde blev støttet af NIH / NCI tilskud # R01CA172138 (til SBE).

Procedurer, der involverer dyr fag er blevet godkendt af Institutional Animal Care og brug Udvalg (IACUC) på Tel-Aviv University. 1. Rat-protokollen <ol><li> Forberedelser <ol><li> Arrangere 2 butterfly 21 g nåle. Eventuelt forberede 2 stumpe kanter sommerfugl 21 G kanyler ved indgivelse af den skarpe kant af nålen. </li><li> Sterilisere kirurgiske værktøjer: 2 par saks, stumpe-kantede pincet, hæmostat, tand-væv pincet steriliseret ved autoklave ved 121 ° C i mindst 30 min på (tør) indstilling tyngde...

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	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, 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=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, 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=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

Fremgangsmåden er præsenteret heri muliggør selektiv høst og læsning af den unikke population af MP-leukocytter. Sammenlignet med cirkulerende eller milt leukocytter, er MP befolkning kendetegnet ved en særskilt sammensætning leukocytsubpopulationer, højere aktivering niveauer højere frigivelse af forskellige cytokiner, og højere mRNA niveauer af pro- og anti-inflammatoriske cytokiner 2,3. Specifikt har vi vist, at MP-NK-celler er mere cytotoksiske end kredsløbssygdomme NK-celler mod forskellige m?...

Leukocytter klæber til kapillærerne i lungerne (dvs. marginating-pulmonal (MP) leukocytter) 1 blev vist at udvise forskellig leukocyt sammensætning og unik aktivitet sammenlignet med leukocytter fra andre immune rum (fx omsætning, milt, knoglemarv) 2- fire. For eksempel, MP-leukocytter udviser højere naturlige dræber (NK) celler cytotoksicitet mod forskellige tumorceller, sammenlignet med cirkulerende og spleniske NK celler, såvel som differentieret messenger-RNA (mRNA) -niveauer og forøget sekretion af pro- og anti-inflammatoriske cytokiner. Sammensætningen af ​​celler også differentieret fra cirkulerende leukocytter som MP-leukocytter har et højere forhold mellem medfødte / adaptiv immunitet i forhold til cirkulerende leukocytter (50% vs. 30%, henholdsvis). Målet med fremgangsmåden heri præsenterede er at muliggøre selektiv høst af MP-leukocytter, for at studere dette vigtige og unikke immun rum (cellepopulation), og at elucIDATE effekten af forskellige manipulationer (f.eks immun aktivering) på disse specifikke celler. For at forstå betydningen af denne unikke population, er det vigtigt at bemærke, at immunsystemet kan styre cirkulerende tumorceller, mikrometastaser og residual sygdom af in vivo funktioner af cellemedieret immunitet (CMI). Denne evne er indlysende trods af præcedens svigt af immunsystemet til at styre den primære tumor, og understøttet af rigelig in vivo evidens hos cancerpatienter og dyremodeller 5. Vigtigere er, disse resultater er ofte i modstrid med in vitro og ex vivo undersøgelser, der rapporterer, at de fleste autologe tumorceller er resistente over for cytotoksicitet ved cirkulerende leukocytter i blodprøver fra mennesker og dyr (målt ved cytotoksicitetsassays) 6,7. Denne uoverensstemmelse kan tilskrives in vivo findes særskilte leukocytpopulationer, såsom den ovenforførstnævnte MP leukocyt befolkning, og især dens delpopulation af aktiverede NK-celler 3. Faktisk syngene tumorceller (MADB106), som viste sig at være resistente over for cirkulerende og milt leukocytter, viste sig at være lyseres ved MP-NK-celler 3,8. Således er de angiveligt &quot;NK-resistente &#39;MADB106 celler, der metastaserer til lungerne af fischer344 (F344) rotter kontrolleret af MP-NK-celler, men ikke ved at cirkulere eller milt NK-celler, som er almindeligt studerede givet deres lette adgang. Rensede og aktive MP-leukocytter er utilgængelige gennem standard høstmetoder af leukocytter fra lungerne, som er baseret på lungevæv slibning eller biologisk nedbrydning 9. Vores tilgang har to store fordele sammenlignet med væv forarbejdning tilgange. For det første perfusion tilgang selektivt høster MP-leukocytter, der adskiller dem fra andre celler, der stammer fra lungerne parenkymatisk, interstitiel og broncho-alveolær Compartments. For det andet perfusion teknik bedre bevarer integriteten og den fysiologiske miljø i MP-leukocytter, i modsætning til slibning og biologisk behandling nærmer sig skader celler, ændre deres morfologi, og inducere produktionen og frigivelsen af ​​forskellige faktorer, der modulerer immun aktivitet og specifikt undertrykker NK cytotoksicitet 10. Lungerne er en vigtig målorgan for cancermetastase og til forskellige infektionssygdomme. Alle cirkulerende maligne celler og inficerede celler passerer gennem lungekapillærerne, hvor de skal deformere og interagere med kapillære endotelceller og residente leukocytter. Under disse betingelser, kan cirkulerende celler let målrettes af hjemmehørende MP-leukocytter. Det synes derfor biologisk fordel at have aktiverede leukocytter i denne immun rum, og det er vigtigt at studere denne unikke MP-befolkningen i forskellige biologiske, eksperimentelle og kliniske omgivelser. Det er værd at bemærke, at systemic immunaktivering ved forskellige biologiske respons-modifikatorer (f.eks polyinosin-polycytidylsyre acid (poly I: C) eller type-C CpG-oligodeoxynukleotider (CpG-C ODN)) er blevet vist at aktivere MP-leukocytter mere end cirkulerende leukocytter 3, 8,11.

<table border="0" cellpadding="0" cellspacing="0" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Autoclud Peristaltic pump</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>26G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>21G*3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>1 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>2.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>25 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blunted-edged forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Scissors</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">hemostat</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tissue forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Heparin sodium porcine mucosa (perservative free)</td>
			<td style="width:103px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

MP-rum udviser en anden leukocyt delmængde sammensætning i forhold til cirkulerende leukocytter. Ved anvendelse af flowcytometri analyse blev leukocytsubpopulationer identificeret og kvantificeret at karakterisere sammensætningen af ​​både cirkulerende og MP leukocytter. Granulocytter og lymfocytter blev identificeret baseret på forward og side scatters. Inden lymfocytterne blev NKRP-1 lyse celler identificeres som NK-celler, CD3 + T-celler, RM1 +</sup...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Selektiv Høst af Marginating-pulmonale Leukocytter

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

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