עבודה זו ממומנת על ידי RJ Reynolds חברת הטבק (RJRT) במסגרת הסכם לשיתוף פעולה מחקרית עם בית הספר לאוניברסיטת Wake Forest לרפואה. GL פראסאד הוא עובד במשרה מלאה של RJRT.

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease, A Report of the Surgeon General. , (2010).</li><li>Zeller, M., Hatsukami, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Strategic+Dialogue+on+Tobacco+Harm+Reduction:+a+vision+and+blueprint+for+action+in+the+US.">The Strategic Dialogue on Tobacco Harm Reduction: a vision and blueprint for action in the US.</a> Tob. Control. 18, 324-332 (2009).</li><li>Sopori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cigarette+smoke+on+the+immune+system.">Effects of cigarette smoke on the immune system.</a> Nature Reviews Immunology. 2, 372-377 (2002).</li><li>Birrell, M. A., Wong, S., Catley, M. C., Belvisi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+tobacco-smoke+on+key+signaling+pathways+in+the+innate+immune+response+in+lung+macrophages.">Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages.</a> J. Cell Physiol. 214, 27-37 (2008).</li><li>Laan, M., Bozinovski, S., Anderson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+inhibits+lipopolysaccharide-induced+production+of+inflammatory+cytokines+by+suppressing+the+activation+of+activator+protein-1+in+bronchial+epithelial+cells.">Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells.</a> J. Immunol. 173, 4164-4170 (2004).</li><li>Moodie, F. 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=Oxidative+stress+and+cigarette+smoke+alter+chromatin+remodeling+but+differentially+regulate+NF-kappaB+activation+and+proinflammatory+cytokine+release+in+alveolar+epithelial+cells.">Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells.</a> Faseb J. 18, 1897-1899 (2004).</li><li>Oltmanns, U., Chung, K. F., Walters, M., John, M., Mitchell, 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=Cigarette+smoke+induces+IL-8,+but+inhibits+eotaxin+and+RANTES+release+from+airway+smooth+muscle.">Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle.</a> Respir. Res. 6, 74 (2005).</li><li>Vayssier, M., Favatier, F., Pinot, F., Bachelet, M., Polla, 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=Tobacco+smoke+induces+coordinate+activation+of+HSF+and+inhibition+of+NFkappaB+in+human+monocytes:+effects+on+TNFalpha+release.">Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release.</a> Biochem. Biophys. Res. Commun. 252, 249-256 (1998).</li><li>Witherden, I. R., Vanden Bon, E. J., Goldstraw, P., Ratcliffe, C., Pastorino, U., Tetley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+human+alveolar+type+II+epithelial+cell+chemokine+release:+effects+of+cigarette+smoke+and+neutrophil+elastase.">Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase.</a> Am. J. Respir. Cell Mol. Biol. 30, 500-509 (2004).</li><li>Hatsukami, D. K., Biener, L., Leischow, S. J., Zeller, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+and+nicotine+product+testing.">Tobacco and nicotine product testing.</a> Nicotine Tob. Res. 14, 7-17 (2012).</li><li>Ashley, D. L., Backinger, C. L., van Bemmel, D. M., Neveleff, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Regulatory+Science:+Research+to+Inform+Regulatory+Action+at+the+Food+and+Drug+Administration's+Center+for+Tobacco+Products.">Tobacco Regulatory Science: Research to Inform Regulatory Action at the Food and Drug Administration's Center for Tobacco Products.</a> Nicotine Tob. Res. , (2014).</li><li>Arimilli, S., Damratoski, B. E., Bombick, B., Borgerding, M. F., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytotoxicity+of+different+tobacco+product+preparations.">Evaluation of cytotoxicity of different tobacco product preparations.</a> Regul. Toxicol. Pharmacol. 64, 350-360 (2012).</li><li>Gao, H., Prasad, G. L., Zacharias, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cell-specific+cytotoxic+responses+of+oral+cavity+cells+to+tobacco+preparations.">Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.</a> Toxicol In Vitro. , (2012).</li><li>Arimilli, S., Damratoski, B. E., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combustible+and+non-combustible+tobacco+product+preparations+differentially+regulate+human+peripheral+blood+mononuclear+cell+functions.">Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.</a> Toxicol. In Vitro. 27, 1992-2004 (2013).</li><li>Arimilli, S., Damratoski, B. E., Chen, P., Jones, B. A., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+leukocyte+subsets+from+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells+in+clinical+research.">Rapid isolation of leukocyte subsets from fresh and cryopreserved peripheral blood mononuclear cells in clinical research.</a> Cryo Letters. 33, 376-384 (2012).</li><li>Schmid, I., Krall, W. J., Uittenbogaart, C. H., Braun, J., Giorgi, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dead+cell+discrimination+with+7-amino-actinomycin+D+in+combination+with+dual+color+immunofluorescence+in+single+laser+flow+cytometry.">Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.</a> Cytometry. 13, 204-208 (1992).</li><li>Kim, G. G., Donnenberg, V. S., Donnenberg, A. D., Gooding, W., Whiteside, 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=A+novel+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells:+comparisons+to+a+4+h+51Cr-release+assay.">A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.</a> J. Immunol. Methods. 325, 51-66 (2007).</li><li>Taga, K., Yamauchi, A., Kabashima, K., Bloom, E. T., Muller, J., Tosato, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-induced+death+by+apoptosis+in+human+lymphokine-activated+natural+killer+cells.">Target-induced death by apoptosis in human lymphokine-activated natural killer cells.</a> Blood. 87, 2411-2418 (1996).</li></ol>

The authors declare that there are no conflicts of interest.

אנו ואחרים הוכיחו בעבר כי הטיפול בPBMCs עם עקר מדכא כמה תגובות, כוללים ביטוי והפרשה של ציטוקינים ופעולות פונקציונליות כגון תא המטרה להרוג 14. השיטות הניסיוניות שתוארו בעבודה הקודמת דורשות תקופות דגירה ארוכות יותר והיו צנועים בגודל 14. בהתחשב ביישומים הפוטנצ?...

גוף משמעותי של נקודות ידע לתופעות לוואי הבריאותיות של עישון סיגריה כרוני, כולל מחלות לב וכלי דם (CVD), מחלת ריאות חסימתית כרונית (COPD) וסרטן 1,2. עישון סיגריות כרוני כבר ידוע לגרום לדלקת ודיכוי מערכת חיסונית, ושינויים אלה דיווחו לתרום לעלייה בסיכון לזיהום חיידקים וסרטן במעשנים 3. במבחנה וטכניקות vivo לשעבר שימושיות בהבהרת הבסיס המולקולרי של תופעות pathophysiological של עשן סיגריות 4-9 (טבלת 1) ומוכרות ככלים חשובים להדרכת תקנה החדשה של מוצרי טבק שונים 10,11. לדוגמא, יש לנו הפגנו כי הכנות דליקים מוצר טבק (בעקר) כגון בינוני כל-מותנה עשן (WS-CM) וחומר חלקיקים כולל (TPM) הן הרבה יותר רעילות לתאים ומזיק לDNA מאשר בעקר או ניקוטין 12,13 אינו דליקים. עולה בקנה אחד עם העבודה שפורסמה, היא דיווחה לאחרונה כי בעקר או ניקוטין 12,13 דליקים. עולה בקנה אחד עם העבודה שפורסמה, שדיווחנו לאחרונה כי בעקר דליק גרם דיכוי חיסוני משמעותי. זה מעיד דיכוי Toll- כמו קולט -ligands (TLR), מגורה הפרשת ציטוקינים, וממוקד תא (K562) ההמתה בPBMCs במודל vivo לשעבר 14. בהתחשב במשמעות של דלקת בתהליכי מחלה נגרמים על-עשן סיגריות, אופטימיזציה נוספת של תנאי assay כדי להעריך את השפעות modulatory חיסון של עשן סיגריות מוצגת בדוח זה. Vivo לשעבר מבחני בדרך כלל נמדדים תוך-תאיים ומופרשים ציטוקינים כמו גם הפונקציה של cytolytic ציטוטוקסיות תאי T וNK בתא K562 הורגת מבחני 14. מבחני מעורבים מראש דגירה עם WS-CM וניקוטין והגירוי הבא של PBMCים עם TLR אגוניסטים על פני תקופה של 3 ימים; הקריאות הסופיות מבוצעות באמצעות מבחני צמודי אנזים immunosorbent (ELISAs) ו / או cytometry זרימה. אנו מנוצלים lipopolysaccharide חיידקים (LPS), אשר נקשר לקולטנים TLR-4 ומעורר PBMCs כתוצאה מכך הייצור של ציטוקינים והפרשה של ציטוקינים תאיים. בנוסף לאופטימיזציה של צעדי assay השונים להערכת השפעות המערכת החיסונית של עקר, אנחנו גם שיטות הנוכחיות לPBMCs בידוד, מבחני מוות של תאים, ו- IL-8 כימות. שיטות אלה עשויות להיות מיושמות מטרה לענות על שאלות מחקר אחרות ומעודנות יותר כדי להעריך מוצרי טבק בהקשר הרגולטורי. טבלת 1. פורסם דיווחים על במבחנה ושיטות לשעבר vivo משמשות ללמוד אפקטי pathophysiological varilus של הכנות מוצר טבק CS, בינוני עשן סיגריות.; CSC, עיבוי עשן סיגריות; מנוע חיפוש מותאם אישית, תמצית עשן סיגריות; ELISA, אנזים צמוד immunosorbent assay; GADPH, glyceraldehyde dehydrogenase 3-פוספט; qPCR, תגובת שרשרת של פולימראז כמותיים; RT, תגובת השרשרת של פולימראז כמותיים זמן אמת; TS, עשן טבק. <table style=";text-align:right;direction:rtl"><tbody><tr><td> מחבר (שנת הלימוד) </td><td> אל Laan et. (2004) </td><td> אל מודי et. (2004) </td><td> אל Oltmanns et. (2005) </td><td> Vayssier (1998) </td><td> אל Witherden et. (2004) </td><td> אל Birrell et. (2008) </td></tr><tr><td> תאים המשמשים </td><td> תאים אנושיים הסימפונות האנדותל (BEAS-2B), נויטרופילים אדם </td><td> תאים אנושיים alveolar אפיתל (A549) </td><td> תאי שריר חלקים בדרכי נשימה אנושיות (HASMC) </td><td> תאים אנושיים premonocytic U937, מונוציטים אדם </td><td> סוג מכתשי II תאי אפיתל (ATII) </td><td> monocytic אדםשורת תאים (THP-1), מקרופאגים ריאות אנושיים </td></tr><tr><td> TPP משמש </td><td> מנוע חיפוש מותאם אישית </td><td> CSC </td><td> מנוע חיפוש מותאם אישית </td><td> TS </td><td> מנוע חיפוש מותאם אישית </td><td> CS </td></tr><tr><td> שיטה </td><td> ELISA, qPCR, הגירה, שינוי electromobility </td><td> Immunohisto-כימיה, אלקטרופורזה, ערכת Arrayscan, RT-PCR, ELISA </td><td> ELISA, RT-PCR, qPCR, אלקטרופורזה </td><td> משמרת ג&#39;ל ניידות </td><td> מיקרוסקופ אור, מיקרוסקופ אלקטרונים, אלקטרופורזה, ELISA </td><td> qPCR, ELISA, ערכת E-toxate (Sigma), assay צלחת p65 (TransAM), אלקטרופורזה, ערכות immunoassay שונות </td></tr><tr><td> מדד </td><td> IL-8, GM-CF, AP-1, NF-κB, הגירה </td><td> acetyltransferases היסטון, deacetylases היסטון, NF-κB, IL-8, PI κB-α, GADPH </td><td> HO-1, GADPH, RANTES, IL-8, eotaxin </td><td> חלבוני הלם / עומס חום (HSP / Hsp70), גורם שעתוק HF, NF-κB,TNF-α </td><td> חלבון פעילי שטח (SP-A, SP-C), IL-8, MCP-1, GRO-α, TNF-α, IL-1β, IFN-γ </td><td> IL-8, IL-1β, IL-6, TNF-α, MIP1-α, GRO-α, MAPK / JNK / זירחון ERK, cJUN: DNA מחייב, גלוטתיון, p65: DNA מחייב </td></tr><tr><td> תוצאה סופית </td><td> מנוע חיפוש מותאם אישית מטה-מווסת את ייצור ציטוקינים באמצעות דיכוי ההפעלה AP-1. </td><td> H 2 O 2 וCSC לשפר acetylation של חלבוני היסטון, להקטין פעילות deacetylase היסטון, באופן דיפרנציאלי להסדיר שחרור ציטוקינים מעודדי דלקת. </td><td> עשן סיגריות עלול לגרום לשחרורו של IL-8 מHASMC, משופר על ידי TNF-α, CSE 20% פחות שחרור IL-8, עיכוב של eotaxin וטווחים על ידי עשן סיגריות. </td><td> TS הופעל גורם שעתוק HF, שהיה קשור עם ביטוי יתר Hsp70 ועיכוב של NFkB מחייב פעילות ושחרור TNF-α. </td><td> alveo רמות chemokine תאים שמקורם ATII מופחת פשרהתיקון lar, תורם לנזק סיגריה מושרה עשן alveolar ואמפיזמה. </td><td> נתונים לספק הסבר מכניסטי לזיהומים בדרכי הנשימה מדוע מעשנים גדלו. דיכוי התגובה המולדת מלווה בעלייה בIL-8. </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">12 X 75 tubes</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">2 mL Microtubes</td>
			<td style="width:148px;">Axygen</td>
			<td style="width:119px;">MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">3R4F reference cigarettes&#160;&#160;</td>
			<td style="width:148px;">Univ. of Kentucky, College of Agriculture</td>
			<td style="width:119px;">3R4F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">50 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">500 ml bottle</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">7AAD</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">559925</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well flat bottom plate</td>
			<td>Termo Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">96 well round bottom plates</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture hood</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:119px;">1300 Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Centrifuge</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:119px;">58110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CFSE</td>
			<td style="width:148px;">Molecular Probes Life Technologies</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cluster tubes</td>
			<td style="width:148px;">Corning</td>
			<td>4401</td>
			<td>Harmful if swallowed, carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cytofix/Cytoperm (Permwash)</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555028</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMSO (Dimethyl sulfoxide )</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DPBS</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:119px;">17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FBS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">F2442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCAP Array</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">652099</td>
			<td>Software analyzes CBA data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Filter unit</td>
			<td style="width:148px;">Nalgene</td>
			<td style="width:119px;">156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Canto II</td>
			<td>&#160;8 colors, at Ex 405 and Em785.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Calibur</td>
			<td>&#160;4 colors at Ex 495 and Em 785.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Flow cytometry analysis software</td>
			<td style="width:148px;">Tree Star</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Freezing Container</td>
			<td style="width:148px;">Nalgene</td>
			<td>5100-0001</td>
			<td>Contains DMSO,&#160; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">GogliPlug</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td>Carcinogen, Irritant, Corrosive&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:223px;">H2SO4</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">339741</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human Inflammatory Cytokine Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IFN-&#947; V-500 Antibody</td>
			<td style="width:148px;">BD Horizon</td>
			<td style="width:119px;">561980</td>
			<td>&#160;skin sensitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IL-8 ELISA Kit</td>
			<td style="width:148px;">R and D Systems</td>
			<td style="width:119px;">DY208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isolation Buffer</td>
			<td style="width:148px;">Isolymph, CTL Scientific Corp.</td>
			<td style="width:119px;">1114868</td>
			<td>Flammable liquid, Irritant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Isopropyl alcohol</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">L-Glutamine</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LPS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIP1-&#945; PE Antibody</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">554730</td>
			<td>Acute toxicity, Oral</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Monensin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotine</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3876</td>
			<td>Acute toxicity, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Parafilm</td>
			<td style="width:148px;">Bemis</td>
			<td style="width:119px;">&#8220;M&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>Flammable, Skin irritation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Pen/strep</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Running buffer</td>
			<td style="width:148px;">MACS Running Buffer, Miltenyi Biotech</td>
			<td style="width:119px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Th1/Th2 CBA Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551809</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">TNF-&#945; Alexa Fluor 488 Antibody</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:119px;">502915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Transfer pipette</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tris Base</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations

בין שינויי pathophysiological אחרים, חשיפה כרונית לעשן סיגריות גורמת לדלקת ודיכוי מערכת חיסונית, אשר נקשר לרגישות מוגברת של מעשנים לזיהומי חיידקים ושכיחות גידולים. Ex vivo דיכוי של מערכת חיסונית בתיווך קולט בתאי הדם היקפיים mononuclear אנושיים (PBMCs) טופל במרכיבי עשן הוא גישה אטרקטיבית ללמוד מנגנונים ולהעריך את ההשפעות ארוכת הטווח הסבירה של חשיפה למוצרי טבק. כאן, אנו מותאמים שיטות לבצע מבחני vivo לשעבר באמצעות PBMCs מגורה על ידי lipopolysaccharide חיידקים, יגנד רצפטור-4-כמו אגרה. ההשפעות של מדיום כל ממוזג עשן (WS-CM), הכנת מוצר טבק דליק (TPP), וניקוטין נחקרו על הפרשת ציטוקינים והרג תאי מטרה על ידי PBMCs במבחני vivo לשעבר. אנו מראים כי ציטוקינים מופרשים IFN-γ, TNF, IL-10, IL-6, IL-8 וציטוקינים תאיים IFN-47 ;, TNF-α, וMIP-1α דוכאו בPBMCs החשופה-WS-CM. פונקצית cytolytic של PBMCs מפעיל, כפי שנקבעו על ידי assay תא מטרת K562 הורג גם מופחתת על ידי חשיפה לWS-CM; הניקוטין היה מינימאלי יעיל במבחנים אלה. לסיכום, אנו מציגים סדרה של מבחני השתפרו להעריך את ההשפעות של עקר במבחני vivo לשעבר, ושיטות אלה יכולים להתאים בקלות לבדיקת מוצרים אחרים בעלי עניין.

התוצאות הוצגו כשגיאת ± סטנדרטית הממוצעת של הממוצע (ארבע דגימות תורם). מבחן t של הסטודנט בין דגימות בקרה שטופלו ולא טופלו בוצע באמצעות תוכנת Excel וכן השוואות מבחן t לכל הטיפולים עם הפקדים המתאימים שלהם. המובהקות הסטטיסטיות שצוינו על ידי: *, P &lt;0.05; **, P &lt;0.005; ***, P &lt;0.0005. <p cla...

Watch this Scientific Journal Video about Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations at JoVE.com

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

שיטות להערכת Cytotoxicity ודיכוי חיסוני של תכשירי מוצרים דליקים טבק

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

JoVE Journal

Immunology and Infection

11.3K Views.  Wake Forest University Health Sciences. Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. 

Video: Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

התלכדות חלקיקים שיטת זיהוי poliovirus

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

שתי שיטות של גיבוש Heterokaryon לגלות גורמים הגבלת HCV

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Assay כמותית תפוקה גבוהה מתא בודד Cytotoxicity לתאי T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

הקמה ואופטימיזציה של הגדרת תפוקה גבוהה ללמוד<em&gt; סטפילוקוקוס epidermidis</em&gt; ו<em&gt; Marinum Mycobacterium</em&gt; זיהום כמודל לגילוי תרופות

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

פיתוח של מערכת Reporter וירוס הפטיטיס B לפקח בשלבים המוקדמים של מחזור שכפול

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

שימוש Assay מונוציטים חד שכבתי להערכת Fcγ קולטן בתיווך Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

הרומן במבחנה פצע ריפוי הנועד להעריך את נדידת תאים

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

עיצוב של הIntranasal וניקוב, מודל כפול של דיכוי חיסוני מושרה באלח דם

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

תפירה Vivo ציטורעילות Assays ללמוד אימונומימינינס ב-CD8 התגובות הספציפיות לגידול של תאים

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

בדיקת מחוץ גופית מבחן להערכת כלבת חיסון עוצמת

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...