This work was supported by "Spanish Ministry of Economy and Competitiveness" [grant number BIO2014-5258P], "European Commission" by the H2020 programs [grant numbers TBVAC2020 643381].

<ol>
	<li>Zumla, A., 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+WHO+2014+global+tuberculosis+report--further+to+go.">The WHO 2014 global tuberculosis report--further to go.</a> Lancet Glob Health. 3 (1), e10-e12 (2015).</li><li>Mangtani, 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=Protection+by+BCG+vaccine+against+tuberculosis:+a+systematic+review+of+randomized+controlled+trials.">Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials.</a> Clin Infect Dis. 58 (4), 470-480 (2014).</li><li>Aguilo, N., 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=Pulmonary+Mycobacterium+bovis+BCG+vaccination+confers+dose-dependent+superior+protection+compared+to+that+of+subcutaneous+vaccination.">Pulmonary Mycobacterium bovis BCG vaccination confers dose-dependent superior protection compared to that of subcutaneous vaccination.</a> Clin Vaccine Immunol. 21 (4), 594-597 (2014).</li><li>Chen, L., Wang, J., Zganiacz, A., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+intranasal+mucosal+Mycobacterium+bovis+BCG+vaccination+confers+improved+protection+compared+to+subcutaneous+vaccination+against+pulmonary+tuberculosis.">Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis.</a> Infect Immun. 72 (1), 238-246 (2004).</li><li>Giri, P. K., Verma, I., Khuller, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+intranasal+vaccination+with+Mycobacterium+bovis+BCG+against+airway+Mycobacterium+tuberculosis+challenge+in+mice.">Protective efficacy of intranasal vaccination with Mycobacterium bovis BCG against airway Mycobacterium tuberculosis challenge in mice.</a> J Infect. 53 (5), 350-356 (2006).</li><li>Lagranderie, 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=BCG-induced+protection+in+guinea+pigs+vaccinated+and+challenged+via+the+respiratory+route.">BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route.</a> Tuber Lung Dis. 74 (1), 38-46 (1993).</li><li>Gopal, 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=Interleukin-17-dependent+CXCL13+mediates+mucosal+vaccine-induced+immunity+against+tuberculosis.">Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis.</a> Mucosal Immunol. 6 (5), 972-984 (2013).</li><li>Khader, S. A., 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=IL-23+and+IL-17+in+the+establishment+of+protective+pulmonary+CD4++T+cell+responses+after+vaccination+and+during+Mycobacterium+tuberculosis+challenge.">IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge.</a> Nat Immunol. 8 (4), 369-377 (2007).</li><li>Aguilo, N., 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=Pulmonary+but+Not+Subcutaneous+Delivery+of+BCG+Vaccine+Confers+Protection+to+Tuberculosis-Susceptible+Mice+by+an+Interleukin+17-Dependent+Mechanism.">Pulmonary but Not Subcutaneous Delivery of BCG Vaccine Confers Protection to Tuberculosis-Susceptible Mice by an Interleukin 17-Dependent Mechanism.</a> J Infect Dis. , (2015).</li><li>Middlebrook, G., Cohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriology+of+tuberculosis:+laboratory+methods.">Bacteriology of tuberculosis: laboratory methods.</a> Am J Public Health Nations Health. 48 (7), 844-853 (1958).</li><li>Brosch, 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=A+new+evolutionary+scenario+for+the+Mycobacterium+tuberculosis+complex.">A new evolutionary scenario for the Mycobacterium tuberculosis complex.</a> Proc Natl Acad Sci U S A. 99 (6), 3684-3689 (2002).</li><li>Kaushal, D., 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=Mucosal+vaccination+with+attenuated+Mycobacterium+tuberculosis+induces+strong+central+memory+responses+and+protects+against+tuberculosis.">Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.</a> Nat Commun. 6, 8533 (2015).</li><li>Lochhead, J. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Adv Drug Deliv Rev. 64 (7), 614-628 (2012).</li><li>Lochhead, J. J., Wolak, D. J., Pizzo, M. E., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transport+within+cerebral+perivascular+spaces+underlies+widespread+tracer+distribution+in+the+brain+after+intranasal+administration.">Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration.</a> J Cereb Blood Flow Metab. 35 (3), 371-381 (2015).</li><li>Griffiths, K. 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=Cholera+toxin+enhances+vaccine-induced+protection+against+Mycobacterium+tuberculosis+challenge+in+mice.">Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.</a> PLoS One. 8 (10), e78312 (2013).</li><li>Hirota, 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=Plasticity+of+Th17+cells+in+Peyer's+patches+is+responsible+for+the+induction+of+T+cell-dependent+IgA+responses.">Plasticity of Th17 cells in Peyer's patches is responsible for the induction of T cell-dependent IgA responses.</a> Nat Immunol. 14 (4), 372-379 (2013).</li><li>Jaffar, Z., Ferrini, M. E., Herritt, L. A., Roberts, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+lung+mucosal+Th17-mediated+responses+induce+polymeric+Ig+receptor+expression+by+the+airway+epithelium+and+elevate+secretory+IgA+levels.">Cutting edge: lung mucosal Th17-mediated responses induce polymeric Ig receptor expression by the airway epithelium and elevate secretory IgA levels.</a> J Immunol. 182 (8), 4507-4511 (2009).</li></ol>

The authors have nothing to disclose.

Although current vaccine against tuberculosis, BCG, is the most widely administered vaccine in history, tuberculosis remains one of the leading causes of death and morbidity from infectious diseases worldwide. This paradox is explained by the lack of protection of this vaccine against pulmonary tuberculosis, the responsible form of transmission. New vaccination approaches effective against pulmonary forms of the disease are urgently needed, as they would have the greatest impact on disease transmission globally.
Our data clearly show that a change in the route of administration of BCG to mimic the natural route of infection could be a successful strategy to prevent pulmonary tuberculosis. Our results are in accordance with other authors showing equivalent data in different animal models, including guinea pigs and non-human primates (NHP) 6,9,12.
Remarkably, our unpublished data indicated that the volume of administration by the intranasal route is a critical step of the protocol. These results revealed that following intranasal delivery of 100 H37Rv bacteria resuspended in 10 &#956;l (instead of 40 &#956;l), we only recovered lung CFUs from around 20% of the animals (data not shown. 
Importantly, one of the possible limitations of intranasal administration as a vaccine delivery route in clinic could be its proximity with the central nervous system13,14. In this regard, aerosol immunization might be safer as delivery route for pulmonary tuberculosis vaccines in humans.
This protocol describes a standardized methodology that can be adapted to other studies, as comparison of pulmonary vaccines, or different routes of pulmonary immunization, including aerosol.
Our data suggest that the analysis in parallel of protective efficacy and immune response, using lung samples from the same animal, could be a useful tool to identify biomarkers of protection. In this regard, our results reveal that lung IL17, but not IFN&#947;, seems to correlate with a better vaccine protective efficacy. These data highlight a possible role of IL17 in the protective response induced by intranasal BCG, which is in accordance with data reported by other authors using mucosal subunit TB vaccines 7,15. Our data also indicate that presence of IgA (both total and MTB-specific) in BAL samples correlates with protection conferred by intranasal BCG. Importantly, we described previously, in agreement with other works, that IL17 contributes to traslocation of IgA to respiratory airways and gut lumen.9,16,17

שחפת (TB) היא אחת המחלות הזיהומיות המובילים בגרימת מותם מזוהה יותר HIV בעולם בשילוב עם העלייה הגואה של זנים עמידים multidrug גורם שחפת בעיה בריאותית עולמית מדאיגה 1. כלי אבחון חדשים, תרופות יעילות יותר ופחות רעילות, וחיסוני TB בטוחים ויעילים חדשים הם צורך דחוף, במיוחד במדינות המתפתחות. לחיות Bacille Calmette-גרן מוחלשים (BCG) היא כיום חיסון מורשה רק נגד שחפת, אשר כבר מנוהל intradermally בלידה מאז 1970 ברחבי העולם. BCG נחשב יעיל במניעת צורות חמורות של המחלה (דלקת קרום המוח שחפת miliary) בילדים, אך הראה יעילות עולה בקנה אחד נגד שחפת ריאתית אחראית של העברת מחלות 2. חיסון ריאתי, אשר מחק מסלול טבעי של זיהום שחפת, מייצג גישה אטרקטיבית עבור תחול תגובה מקומי חיסון של פונדקאיים. בהקשר זה, יצירות פרה-קליניים שונים במודלים של בעלי חיים TB רלוונטיים שונים הוכיחו יעילות החיסון יותר בעקבות חיסון ריאתי כפי בהשוואה לתוואי תת עורית או תוך-3-6. עם זאת, מנגנוני ההגנה מופעלים על ידי חיסון ריאתי אינם מובנים היטב. בשנים האחרונות, כמה יצירות הצביעו לעבר בתגובה בתיווך IL17 כגורם חשוב של תגובה חיסונית ברירית ספציפית TB, כמו במודלים של עכברים לוקים עבור יעילות מגן עקב חיסון רירי IL17 נפגע 7,8. לאחרונה הראינו לראשונה כי אפי ממשל BCG מוגן DBA / 2 עכברים, זן עכבר מאופיין בהיעדר הגנה לאחר תת עורית BCG חיסון 9. תוצאות אלו הראו כי חיסון TB נשימה יכול להיות יעיל יותר בהפחתת שיעור השחפת במדינות אנדמיות, שבו BCG תוך-עורית נחשב יעיל נגד pulmonTB האר&quot;י.

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H9 broth</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">271310</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook ADC Enrichment</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">211887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Tween 80</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">TW00800250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3-mm diameter Glass Beads</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">038-138003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H10 Agar</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">262710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">1-ml syringe 26GA 0.45x10 mm</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">301358</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GentleMACS dissociator</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">C tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">M tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Collagenase D</td>
			<td style="width:176px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">DNaseI</td>
			<td style="width:176px;">Applichem</td>
			<td style="width:119px;">A3778,0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:176px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">RPMI 1640</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R0883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Red Blood Cell Lysing Buffer</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GlutaMAX Supplement</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:119px;">35050-061</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Penicillin-Streptomycin Solution</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Fetal Calf Serum</td>
			<td style="width:176px;">Biological Industries</td>
			<td style="width:119px;">04-001-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">2-Mercaptoethanol</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">M3148-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter 2.0 Handheld Automated Cell Counter</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC20040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter Cell Counter Sensors, 40 &#181;m</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC40050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:409px;">Mycobacterium Tuberculosis - Tuberculin PPD</td>
			<td style="width:176px;">Statens Serum Institut (SSI)</td>
			<td style="width:119px;">2390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IFN-&#947; ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3321-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IL17A ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3521-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Brefeldin A</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">553047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">BD Cytofix/Cytoperm Kit</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">555028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC-Cy7 Rat Anti-mouse IL-17A</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">560821</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC Mouse Anti-mouse IFNg</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">554413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&#8221;</td>
			<td style="width:176px;">UNIMED</td>
			<td style="width:119px;">27.134</td>
			<td>Used as trachea cannula for BAL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td>
			<td style="width:176px;">NUNC</td>
			<td style="width:119px;">430341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Albumin, from bovine serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Goat Anti-Mouse IgA (&#945;-chain specific)&#8722;Peroxidase antibody</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB)&#160;</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">T0440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">MyTaq DNA Polymerase</td>
			<td style="width:176px;">Bioline</td>
			<td style="width:119px;">BIO-21107</td>
			<td>The kit Includes Buffer 5x</td>
		</tr>
	</tbody>
</table>

protective efficacy and pulmonary immune response following subcutaneous and intranasal bcg administration in mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

עבודה זו מתארת ​​את ההשוואה של שני מסלולים של ממשל של BCG: תת עורי אף. מסלול תת עורי ניתן להשוות את intradermal, המהווה את הציר הקליני הנוכחי עבור BCG ברחבי העולם. מסלול אפי חיסון שואף לחקות את הקו הטבעי של זיהום של M. שחפת, במטרה להשרות תגובה חיסונית ישירות לריאות, איבר היעד העיקרי של הפתוגן. איור 1 מתאר את זרימת העבודה אחריו. שמונה עד עשר בן שבוע הנקבה DBA / 2 עכברים מחוסנים עם 10 6 CFU של BCG דנית ידי תוואי תת עורית או אפי של הממשל. שמונה שבועות לאחר מכן, קבוצה של עכברים הוא הקריב לנתח בתגובת ריאות חיסונית הנגרמת על ידי חיסון. דגימות BAL מתקבלות ראשונות ולאחר מכן אנחנו קוצרים ריאות. על מנת ללמוד יעילות מגן-נתונות חיסון, אנו לחסן differeקבוצה של עכברים NT עם אתגר אפי במינון נמוך של M. זן שחפת H37Rv. חודש לאחר מכן, נקריב חיות וריאות קציר. במקרה זה, עבור כל חיה שאנו משתמשים הריאה השמאלית כדי לקבוע עומס חיידקים ואת הריאה הימנית להעריך-אתגר פוסט תגובה חיסונית עקב חיסון. המטרה היא ליצור עומס חיידקים ונתוני תגובה חיסוניים בריאות עבור כל חיה כדי ללמוד וקושרת פוטנציאל של הגנה. כפי שניתן לראות בתרשים 2, הריאות היו מנותקים להשיג גם של homogenate איבר או השעיה הסלולר. ריאות הומוגני היו מצופות על מצע אגר מוצק לקבוע עומס חיידקים ארבעה שבועות לאחר אתגר. השעיה סלולרית ללמוד תגובה חיסונית עקב חיסון הושגה בעקבות עיכול אנזימטי ריאות עם D collagenase ו DNaseI. התוצאות שלנו מצביעות בבירור כי, כאשר t לעומתo התוואי תת עורית, תוואי אפי חיסון מקנה יעילות מגן הרבה יותר ריאות ארבעה שבועות לאחר האתגר (איור 3 א). בנוסף, אנו מאשרים כי עומס חיידקי בריאות מקבוצת BCG-חיסון intranasal מתאים H37Rv ולא חיסון BCG. לשם כך, ניתחנו מספר מייצג של מושבות מהקבוצה הזאת על ידי PCR ספציפיים עבור האזור הגנום RD9, אשר מגביר שברי אורך שונים BCG ומ שחפת (איור 3 ב). איור הראה בבירור כי כל המושבות מנותחות ספקו שבר של 0.4 KBP, אשר תואם H37Rv. הנתונים שלנו גילו קורלציה בין יעילות מגן שמעניקה חיסון BCG אפי ואת תגובה חיסונית עקב חיסון בריאות לפני לאתגר. אפי BCG בבירור מופעלות IL17 גבוה וייצור IFNγ הריאות, שהיא נמדדת על ידי ELISA (איור4A). נתונים אלה אושרו על ידי מכתים תאי (ICS) ו cytometry זרימה (מידע לא מוצג). בנוסף, מצאנו גם ריכוז גבוה של שניהם ריכוז IgA סך PPD ספציפי בדגימות BAL (איור 4 ב), המציין כי חיסון BCG ריאתי גורם הייצור של IgA ו טרנסלוקציה דרכי הנשימה. לבסוף, למדנו תגובה חיסונית הנגרמת BCG ריאות לאחר האתגר (איור 5). הנתונים שלנו נמצאו הבדלים בין IL17 ו IFNγ; IL17A לייצור CD4 + תאים התגלו רק בקבוצת BCG אפי, ואילו תאים מייצרים IFNγ נמצאו בכל הקבוצות נגועות H37Rv ללא קשר לחיסון. ייצוג הנתונים של כל חיה המתאימה תאי מייצרי IL17 ו עומס חיידקים הראה קשר מובהק בין הנוכחות של IL17 והפחתת עומס ריאות חיידקית, אשר לא נצפה במקרה של IFNγ. <img alt="איור 1" src="/files/ftp_upload/54440/54440fig1.jpg" /> באיור 1. Workflow להשוואת אפים ואת תת עורי כביש של BCG חיסון. שמונה שבועות לפרסם חיסון BCG, קבוצה של עכברים (6 לכל קבוצת ניסוי) משמש כדי לקצור ריאות ולבצע BAL, ולנתח תגובה חיסונית ריאתי מושרה על ידי חיסון. קבוצה נוספת של עכברים (6 / קבוצה), הוא מחוסן עם אתגר אפים במינון נמוך של M. זן שחפת H37Rv (100 CFU). חודש לאחר מכן, חיות מוקרבים ואת עומס חיידקי מנותחת הריאה השמאלית התגובה החיסונית PPD ספציפי בריאה הימנית. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/54440/54440fig2.jpg" /> <strאונג&gt; עיבוד איור 2. דוגמאות ריאות. Dissociator רקמות שמש לעבד ריאות. בניסויים כדי לקבוע עומס חיידקים, ריאות היו homogenised לפני הצלחת אותם במדיום אגר מוצק. בניסויים הדורשים השעיה הסלולר ריאות, זה נוצר בעקבות עיכול אנזימטי עם D collagenase ו DNaseI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/54440/54440fig3.jpg" /> איור 3. מגן יעיל שמעניק BCG חיסון. קבוצות של 6 DBA / 2 עכברים שחוסנו על ידי תת עורית (SC BCG), אפים (BCG ב) מסלול, או שלא חוסן (Unvacc) עם חיסון BCG דנית 10 6 CFU. בשתיים חודשים לאחר חיסון, עכברים היו מחוסנים intranasally עם מינון נמוך (100CFU) אתגר H37Rv, ושלושה חודשים לאחר מכן נטל חיידקים הריאות נקבע. ניסוי נציג השני מוצג עצמאי. (א) נתונים בגרפים מיוצגים כממוצע + SD. כיוון אחד מבחן ANOVA עם ניתוח שלאחר Bonferroni בוצע לחשב מובהק סטטיסטי. (ב) מספר מייצג של מושבות אחת מקבוצת אפי BCG נותחו על ידי ספציפי PCR עבור אזור RD9 (שונה הגנומים BCG ו H37Rv) להבחין BCG ו מושבות H37Rv. (פורסם בעבר 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig3large.jpg" target="_blank">נא ללחוץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 4" src="/files/ftp_upload/54440/54440fig4.jpg" /> איור 4. חיסון ספציפי ריאה חיסון התגובה נתחה לפני אתגר עם H37Rv. קבוצות של 6 DBA / 2 עכברים היוחוסן על ידי תת עורית (SC BCG) או אף (BCG ב) מסלול, או שלא חוסן (Unvacc) עם חיסון BCG דנית 10 6 CFU. (א) בשעה חודשית-חיסון פוסט, השעיה הסלולר מריאות שנקטפו הושגה. תאים היו מגורה עם PPD כמתואר בסעיף שיטות IL17A (פאנל משמאל) IFNγ (פאנל מימין) ייצור נותחו על ידי ELISA. (ב) סה&quot;כ IgA, ומ שחפת (MTB) -specific IgA נותח ממדגם BAL ידי ELISA. נתונים ונקווה משני ניסויים בלתי תלויים מוצגים. נתונים בגרפים מיוצגים כממוצע + SD. (פורסם בעבר 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig4large.jpg" target="_blank">נא ללחוץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 5" src="/files/ftp_upload/54440/54440fig5.jpg" /> איור 5. חיסון ספציפיתגובת ריאתי חיסון נתחה לפני אתגר עם H37Rv. קבוצות של 6 DBA / 2 עכברים שחוסנו על ידי תת עורית (SC BCG) או אף (BCG ב) מסלול, או שלא חוסן (NV) עם חיסון BCG דנית 10 6 CFU. קבוצת ביקורת של עכברים שלא חוסנו, שאינם נגועים גם נכללה (NV / NI). בשניים חודשים לאחר חיסון, עכברים היו תיגר intranasally עם מנת H37Rv נמוכה (100 CFU), ומאוחר יותר של חודש אחד חיות היו מורדמות. הריאות על ימין ועל שמאל מאותה חיה שימשו כדי לקבוע עומס חיידקי ו IL17A- (א) או IFNγ- (B) בהפקת CD4 + תאים, בהתאמה. נתונים בפנלי שמאל מתאימים אחוז התאים מייצרי ציטוקינים נמדדו באמצעות flow cytometry, ומיוצגים כממוצע + SD. לוחות מימין לייצג נתוני עומס בקטריאלי cytokine- לייצר תאי CD4 + המתקבל עבור כל עכבר. רגרסיה לינארית חושבה ואת p-הערך המתקבל בכל מקרה מוצג המקרה של IL17A. אקווהנתונים משני ניסויים בלתי תלויים שמוצגים באיור. (פורסם בעבר 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig5large.jpg" target="_blank">נא ללחוץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

מגן היעילות ריאתי החיסונית בתגובה תת עורי ואת אפי BCG מינהל עכברים

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

protective-efficacy-pulmonary-immune-response-following-subcutaneous

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of Zaragoza. We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Video: Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Cytometry ססגוניות אנליזות זרימה של תגובה חיסונית תאית של קופי רזוס

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

שבעה צעדים Stellate תאים

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

מבוא של צרעות טפיליות<em&gt; תסיסנית</em&gt; ואת התגובה החיסונית Antiparasite

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

מדידת כמותית של התגובה החיסונית ובשינה<em&gt; דרוזופילה</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

מדידות נכונות חדרית סיסטולי לחץ בשילוב עם קציר של ריאה ודגימות רקמה חיסוניות בעכברים

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cecal קשירת ונקר-Induced אלח דם כמודל ללמוד Autophagy בעכברים

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

באמצעות RNA-התערבות לחקר התגובה החיסונית המולדת בהמקרופאגים עכבר

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

הניצול של לוע התחתון Intratracheal PAMP מנהל ושטיפה ברונכואלוואולרית כדי להעריך את תגובת מארח חיסון של עכברים

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasal המינהל של החיסונים שפעת רקומביננטי במודלי כימרי עכבר ללמוד חסינות רירית

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.