Diese Arbeit wurde von den National Institutes of Health (NIH) AI091759 und dem Morbus Crohn und Colitis Foundation of America von William und Shelby Modell Family Foundation-Forschungspreis (bis Mn) unterstützt. Schistosome-infizierte Mäuse wurden von der NIAID Schistosome Resource Center (NIAID Contract N01 A130026) geliefert.

1. Reinigende Schistosoma mansoni Eier <ol><li> Infect Swiss-Webster-Mäusen mit schistosome Cercarien, die das infektiöse Stadium des Lebenszyklus Schistosoma 8 ist. Alternativ erhalten schistosome-infizierten Mäusen aus dem NIAID Schistosomiasis Resource Center ( <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). Lassen Mäuse für 6-7 Wochen nach der Infektion, in welcher Zeit-Punkt, Eier sind in der Leber vorhanden und können wiederhergestellt werden, wie unten beschrieben (siehe Abb. 1). </li><li> Einschläfern Mäuse mit CO 2, und benetzen mit 70% Ethanol. Bewegen Sie die Maus auf dem Rücken. Verwenden Sie stumpfen Pinzette und Schere weggeschnitten Haut und des darunterliegenden Bauchfell. Excise die Leber, die unter dem Brustkorb und Zwerchfell entfernt. </li><li> Fein hacken Lebern und 20 ml pro Leber der Verdauung Mischung von Collagenase / Dispase (0,5 mg / ml), Penicillin (100 U / ml) und Streptomycin (100 ug / ml) in PBS zusammengesetzt. Setzen Sie mixture in einem 50 ml Falcon-Röhrchen und über Nacht in einem 37 ° C geschüttelt. </li><li> Zentrifugieren Mischung bei 400 xg / 3 min / 20 ° C. Vorsichtig abgießen und die überschüssige Flüssigkeit füllen Rohr mit PBS. Zentrifugieren und wiederholen PBS waschen. Nach letzten Zentrifugation, Pellet in 25 ml PBS. </li><li> Strain die resuspendierten Pellets über eine Standard-Küche metallischen Sieb (mit einem 50-ml-Kolben der Spritze, um die Maische durch Leber Stücke in ein Becherglas), indem man das Gemisch durch ein feines Sieb (10 ml Spritze verwenden, um durch die Mischung feines Sieb drücken, gefolgt in ein zweites Becherglas). </li><li> Setzen gespannten Mischung in einem 50 ml-Falcon-Röhrchen. Zentrifuge bei 400 xg / 5 min / 20 ° C. Füllen Sie vorsichtig heraus, ohne Überstand Pellet. Pellet in 3 ml PBS. </li><li> Overlay vorsichtig mischen, auf einen 20% Percoll, 40 ml Gradienten von 8 ml Percoll und 32 ml 0,25 M Saccharose (FW = 342,3 so 8,56 g Saccharose/100 ml H 2 0). </li><li> Zentrifuge 10 min / 800 × g / 20 ° C Abpipettieren eind verwerfen gallertartige oberste Schicht der Leberzellen. Entfernen Ei Pellet am Boden mit einer Transferpipette und in ein 15 ml Falcon Röhrchen. </li><li> Wash Pellet 3X in 10 ml PBS / 1 mM EDTA / 1 mM EGTA mittels Zentrifuge Einstellungen: 3 min / 30 ug / 20 ° C Resuspendieren in 500 ul PBS. </li><li> Overlay Mischung auf eine neue 25% Percoll, 10 ml-Gradienten (2,5 ml Percoll und 7,5 ml 0,25 M Saccharose) unter Verwendung Zentrifuge Einstellungen: 10 min / 800 × g / 20 ° C </li><li> Waschen in 10 ml PBS mit Zentrifuge Einstellungen 3X: 3 min / 30 xg / 20 ° C. </li><li> In den letzten 10 ml Wasch-, entfernen Sie 100 ul, um Eier zu zählen. Eier schnell absetzen, so dass die Falcon-Röhrchen und muss vor dem Entfernen Probe gemischt werden. Verdünnen Sie die Probe mit 100 ul 900 ul PBS. 100 ul der Mischung sollte als Tröpfchen auf einem Objektträger mit einem Binokular (1:10 Verdünnung) gezählt werden. Wide-Bohrung Pipettenspitzen verwendet werden sollte. </li><li> Resuspendieren Eier bei 50.000 Eier / ml in PBS. Eier können für mehrere Monate bei -80 ° C gelagert werden und aufgetaut einmal oder TWICe für die Verwendung. Vor dem Einsatz unter einem Mikroskop, um sicherzustellen, dass Eier noch intakt sind (siehe Abb. 1) zu inspizieren. </li></ol> 2. Intraperitoneale Sensibilisierung mit S. mansoni Eier: Tag 0 <ol><li> Planen Eier bei 5000 eggs/100 ul PBS in einem 5 ml SnapCAP Rohr. Für die Injektion schätzen 50% mehr Eier als nötig. </li><li> Legen Sie eine 23 Gauge 3/4inch Nadel und einer 1 ml Spritze mit 100 ul Ei Suspension pro Maus. Eier absetzen, so Last Nadel mit Eiern unmittelbar vor der Verwendung. </li><li> Felsen Spritze hin und her, um Eier vor jeder Injektion zu mischen, und injizieren intraperitoneal 100 L Suspension pro Maus. </li></ol> 3. Retroorbitalen intravenöse Challenge mit S. mansoni Eier: Tag 14 <ol><li> Bereiten Eier wie oben bei 5.000 eggs/100 ul. </li><li> Anesthetize Mäuse in speziellen Kammer mit Isofluran (4%) oder intraperitoneal mit Xylazin (10 mg / kg) und Ketamin (120 mg / kg). Tiefe von Anesthesia wird durch Quetschen der Fußsohle und die Gewährleistung keine körperliche Reaktion bestimmt. </li><li> Nach der Beladung mit 1 ml Spritze und 23 Gauge Nadel mit 3/4inch Eier, mit einer Pinzette, um die Nadel in einem Winkel von 90 ° mit der Schräge nach unten in den Winkel zu biegen. Sicherstellen, dass keine Luftblasen entstehen. </li><li> Bewegen Sie die Maus auf einer Seite. Einfahren Haut so Auge ragen, und injizieren 100 ul in die Gefäße hinter den Augapfel in einem 45 ° Winkel zur Nase. </li><li> Ziehen Sie Nadel und leichten Druck auf Blutungen zu kontrollieren. </li></ol> Alle diese Verfahren sind mit besonderer Sorgfalt durchgeführt werden, und Mäuse müssen überwacht, bis sie aus der Narkose erholen werden. Diese Protokolle wurden von der University of Pennsylvania Institutional Animal Care und Use Committee (IACUC) zugelassen. 4. Experimentelle Ernte: Tag 22 <ol><li> Einschläfern Mäuse mit CO 2. </li><li> Nasse Maus mit 70% Ethanol, und am Rücken. </li><li> Mit stumpfer FORCeps, fassen Sie die Maus Bauch und machen einen kleinen Schnitt in der Haut. Entfernen Sie vorsichtig die Haut mit einer Schere und entlarven den Brustkorb und Bauchfell. </li><li> Schneiden Sie das Bauchfell. Schneiden Sie die Bauchaorta, um das Blut mit einer Pasteurpipette zu erholen. Auf Eis lagern. </li><li> Bewegen Sie die Leber nach unten, um die Membran freizulegen. Verwenden einer feinen Schere, um die Membran geschnitten. Vorsichtig aufgeschnitten den Brustkorb auf das Lungengewebe zu entlarven. </li><li> Recover parathymic die Lymphknoten, die Lunge mit feinen Pinzetten abtropfen lassen. Diese werden unter dem Brustkorb gesteckt, auf beiden Seiten des Thymus 9. Store auf dem Eis in 1ml steriles Medium (DMEM mit 10% Hitze-inaktiviertem fötalem Kälberserum, 100 U / ml Penicillin, 100 ug / ml Streptomycin, 2 mM L-Glutamin, 50 uM 2-Mercaptoethanol, alle erhältlich von Invitrogen ergänzt). </li><li> Um die BAL zu erholen, und pumpen die Lungen für die histologische Analyse, müssen intratracheale Intubation durchgeführt werden. Setzen Sie die Luftröhre, indem Sie die Speicheldrüsen zur Seite, und pLacing feinen Pinzette unter der Luftröhre. Mit feinen Schere, schnitt ein Loch in der Mitte der Luftröhre, und legen Sie Schlauch in die Lunge. Befestigen Sie Schlauch durch das Binden mit chirurgischen Faden. </li><li> Bringen Sie PBS-gefüllten 1 ml-Spritze auf den Schlauch und pumpen die Lungen mit 1 ml PBS. Sorgfältig abrufen BAL Wäsche mit Spritze. Auf Eis lagern. </li><li> Bringen Sie 4% Paraformaldehyd (PFA) in PBS-gefüllten 1 ml-Spritze mit dem Schlauch aufpumpen und in die Lunge. </li><li> Entfernen Sie die Schläuche, und ziehen Sie die chirurgischen Faden zu verhindern Auslaufen des PFA. </li><li> Sorgfältig sezieren aus Lungengewebe und in einem 50 ml Falcon-Röhrchen mit 5 ml 4% PFA. </li></ol> 5. Vorbereitung der Recovered Gewebe <ol><li> Serum: Nach der Lagerung auf Eis für 30 min bis 2 h für Blutgerinnung, Serum aus dem Blut durch Zentrifugieren (13.000 × g / 10 min / 4 ° C). Serum wird bei -20 ° C gelagert und kann für Zytokin-ELISAs oder S verwendet werden mansoni Ei Antigen-spezifischen IgG-Isotyp ELISAs <sup&gt; 6, 10. </li><li> Lymphknoten: Einzelne Zellsuspensionen hergestellt und Zellzahl verwendet, um die Größe der Immunantwort zu beurteilen. Um Th2-Zell-Polarisation zu untersuchen, werden die Zellen mit S. restimuliert mansoni Ei-Antigen für 72 Stunden. Intracellar Zytokinfärbung kann mittels Durchflusszytometrie untersucht werden, und die Überstände kann zurückgewonnen und bei -20 ° C für ELISA von Th2-Zytokinen (IL-4, IL-5, IL-13) 6. </li><li> BAL: Zellen werden pelletiert (400 × g / 5 min / 4 ° C). BAL-Flüssigkeit gewonnen und bei -20 ° C zur Analyse von Th2-Zytokinen durch ELISA. BAL-Zellen Zahlen werden als eine Anzeige für Entzündung der Atemwege verwendet. Zubereitungen von 10.000-100.000 Zytozentrifuge Zellen (500 U / 5 min / 4 ° C), wird gefolgt von Färbung Diffquik ermöglichen Aufzählung entzündliches Infiltrat 4, 6. Alternativ kann BAL-Zellen mittels Durchflusszytometrie 6 untersucht werden. </li><li> Lungengewebe: Lungengewebe ist stored über Nacht bei 4 ° C. Feste Gewebe in Paraffin eingebettet, geschnitten und auf Objektträger für histologische und Immunfluoreszenz-Analyse. </li></ol> 6. Repräsentative Ergebnisse Dieses Protokoll Details alle notwendigen Schritte, um (i) Vorbereitung gereinigt Schistosoma mansoni Eier zur Verwendung in vivo (Abb. 1), (ii) zu sensibilisieren und fordern Mäuse mit diesen Eiern und (iii) erholen Organe zur Untersuchung der Lunge Entzündungsreaktion. Diese In-vivo-Modell reproduzierbar treibt eine starke Th2-Lungen-Entzündung. Dies wird durch Entzündung der Atemwege und Eosinophilie gezeigt als visualisiert von BAL-Zellzahlen und Zyto Zubereitungen (Abb. 2). H &amp; E-gefärbten Lungen-Abschnitte können für Ei-induzierte Granulome (Abb. 3a) untersucht werden, und Immunfluoreszenzfärbung von Lungen-Abschnitten ermöglicht die Visualisierung der alternativ aktivierten Makrophagen und Th2-Zytokin-induzierte Gene wie ResisZinn-ähnliches Molekül (RELM) α (Abb. 3b). Die Antigen-spezifische CD4 Th2-Cytokin-Antwort wird durch ELISA für IL-5 untersucht, IL-4 und IL-13 der Antigen-stimulierten parathymic Lymphknotenzellen (4). <img alt="1" src="/files/ftp_upload/3905/3905fig1.jpg" /> Abbildung 1 Schistosoma mansoni Ei Injektion Modell repräsentative Bilder von granulomatösen Lebern, die charakteristisch für hohe Belastungen S.mansoni-Ei, und der S.mansoni Eier werden gezeigt -.. Durchschnittliche Größe (L) 120 mu m durch (W) 50 um. <img alt="2" src="/files/ftp_upload/3905/3905fig2.jpg" /> Abbildung 2. S. mansoni Ei Injektion treibt Entzündung der Atemwege. A. BAL Zellzahlen (x10 5) aus naiven (N) oder S.mansoni (Sm) Ei-injizierten Mäusen. * P &lt;0,05. B. Zytozentrifuge Vorbereitung der BAL-Zellen. Mac, Makrophagen; Eos, Eosinophilen, Lymphe, Lymphozyten. Bar, 10 um. <img alt="Abbildung 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> Abbildung 3. Visualisierung der pulmonalen Granulome und alternativ aktivierten Makrophagen rund um das Ei S.mansoni. A. Pulmonale Granulom rund um das Ei Sm (schwarzer Pfeil) visualisiert wurde in H &amp; E-gefärbten Lungen-Abschnitten. B. Immunfluoreszenzfärbung für RELMα (grün), Mannose-Rezeptor (rot) und DAPI (blau) zeigt alternativ aktivierten Makrophagen (weißer Pfeil) in der Umgebung des Granuloms autofluoreszierenden Sm Ei (schwarzer Pfeil). Bar, 50 um. <img alt="Abbildung 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> 4. S.mansoni Ei Antigen-spezifischen Th2-Cytokin-Antwort. Entleeren parathymic Lymphknotenzellen aus naiven (N) oder Sm Ei-injizierten Mäusen wurden mit Sm stimuliert &lt;/ Em&gt; Ei-Antigen für 72 Stunden, durch ELISA der Überstände für IL-4, IL-5 und IL-13 an. Scale, ng / ml. * P &lt;0,05.

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	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, 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=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

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Hier wird ein Modell beschäftigt Schistosoma mansoni Eier zu Typ-2-Lungen-Entzündung zu induzieren beschrieben. Charakteristische Merkmale dieses Modells sind die potenten Th2-Immunantwort, Entzündung der Atemwege und Lungen Granulombildung. Da diese Parameter in Abhängigkeit vom Th2 CD4-Zell-Antworten 11 sind, ist dies ein nützliches Modell zur Untersuchung der Auswirkungen von Protein-spezifischen oder Linie-spezifische Deletionen auf Th2-Zell-Reaktionen zu untersuchen. Hier die Anzeigen würd...

<table border="1"><tbody><tr><td> Reagens </td><td> Größe / Beschreibung </td><td> Firma </td><td> Katalog # </td></tr><tr><td> Collagenase / Dispase </td><td> 500 mg </td><td> Roche Applied Science </td><td> 11 097 113 001 </td></tr><tr><td> Diff Quik Hema 3 Stain </td><td> Packen </td><td> Fisher Scientific Co. LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> Flüssigkeit </td><td> Gibco </td><td> 11965 </td></tr><tr><td> Falcon 50 ml </td><td> Packen </td><td> Falke </td><td> 352070 </td></tr><tr><td> Intramedic PE-Schlauch </td><td> 0,58 mm </td><td> Becton Dickinson und Co. </td><td> 427410 </td></tr><tr><td> Isofluran </td><td> 250 ml </td><td> Abt Tiergesundheit </td><td> 52600405 </td></tr><tr><td> Ketamin </td><td> 100 mg / ml </td><td> Fort Dodge Animal Health </td&gt; <td> N / A </td></tr><tr><td> Mannose-Rezeptor-Antikörper </td><td> Biotin </td><td> AbD Serotec </td><td> MCA2235B </td></tr><tr><td> Metallic Schmutzfänger </td><td> Standard-Küchensieb </td><td> Ziel </td><td> Laurentiis </td></tr><tr><td> Paraformaldehyd </td><td> 16% </td><td> Electron Microsc. Wissenschaften </td><td> 15710 </td></tr><tr><td> Penicillin / Streptomycin </td><td> 100x </td><td> Gibco </td><td> 15070 </td></tr><tr><td> Percoll </td><td> 1 Liter </td><td> GE Healthcare Biosciences </td><td> 0891 17 01 </td></tr><tr><td> RELMα Antikörper </td><td> 50 ug </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> Chirurgischen Faden </td><td> 2,0 USP 100 yds </td><td> Chirurgische Spezialgebiete Corp </td><td> SP118 </td></tr><tr><td> Spritzennadel </td><td> 23g, 3/4 Zoll </td><td> BD Vacutainer Lab. Med. </td><td>305143 </td></tr><tr><td> US Standardtestsiebe Sieve </td><td> 150 um, 0,0059 &quot; </td><td> WS Tyler, Inc. </td><td> ASTME-11, Spec. Nr. 100 </td></tr><tr><td> Xylazin </td><td> 20 mg / ml </td><td> Butler </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Schistosoma Parasiten sind Blut-Egel, die schätzungsweise 200 Millionen Menschen weltweit ein infizieren. Bei der chronischen Infektion mit Schistosoma, die schwere Pathologie, einschließlich Leberfibrose und Splenomegalie, wird durch die Immunantwort gegen den Parasiten Eier anstatt den Parasiten selbst 2 verursacht. Parasiteneier Induktion einer Th2-Antwort durch die Produktion von IL-4, IL-5 und IL-13, die alternative Aktivierung von Makrophagen und die Ansammlung von Eosinophilen ist. Hier beschreiben wir Injektion von Schistosoma mansoni Eier als Modell, um Parasiten-spezifische Th2-Zytokin Reaktionen in der Lunge und Lymphknoten, die Bildung von Granulomen pulmonalen rund um das Ei, und Entzündung der Atemwege zu untersuchen. Nach intraperitonealer Sensibilisierung und intravenöse Herausforderung, S. mansoni Eier werden in die Lunge über den Lungenarterien, wenn sie im Lungenparenchym eingeschlossen transportiertdurch Granulome von Lymphozyten, Eosinophilen und alternativ aktivierten Makrophagen 3-6 besteht. Verbunden mit Granulombildung, Entzündung in den bronchoalveolären Raum, Ausdehnung der Lymphknoten-und CD4-T-Zell-Aktivierung beobachtet werden kann. Hier haben wir ausführlich das Protokoll zur Isolierung von Schistosoma mansoni Eier aus infizierten Lebern (modifiziert nach 7), Sensibilisierung und herausfordernd Mäuse, und die Rückgewinnung der Organe (bronchoalveoläre Lavage (BAL), Lunge und Lymphknoten) für die Analyse. Wir berücksichtigen auch Vertreter histologischen und immunologischen Daten und Vorschläge für weitere immunologische Analyse. Insgesamt liefert diese Methode ein in vivo Modell zu Helminthen ausgelösten immunologischen Reaktionen in der Lunge zu untersuchen, was ist breit anwendbar auf das Studium der Th2 entzündlichen Erkrankungen einschließlich Wurminfektionen, fibrotische Erkrankungen, allergische Entzündungen und Asthma. Vorteile dieses Modells für die Untersuchung von typ.e 2 Entzündung in der Lunge sind die Reproduzierbarkeit eines potenten Th2-Entzündungsreaktion in der Lunge und Lymphknoten, die Leichtigkeit der Beurteilung von Entzündungen, die durch die histologische Untersuchung der Granulome rund um das Ei, und das Potenzial für langfristige Lagerung des Parasiten Eier.

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Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni Eier sind potente Stimulatoren der T-Helferzellen vom Typ 2 (Th2) Immunantwort, die charakteristisch für Parasitenbefall, Asthma und allergische Entzündung. Dieses Protokoll nutzt S. mansoni Ei Injektion an einen CD4 Th2-Zytokin-induzierte Entzündungsreaktion in der Lunge, Lunge durch Granulombildung um das Ei gekennzeichnet, Eosinophilie und Makrophagen-Aktivierung Alternative zu generieren.

Mit den Eizellen<em&gt; Schistosoma mansoni</em&gt; Als ein<em&gt; In vivo</em&gt; Modell von Helminthen-induzierten Lungenentzündung

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

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Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K Aufrufe.  University of Pennsylvania. Schistosoma mansoni Eier sind potente Stimulatoren der T-Helferzellen vom Typ 2 (Th2) Immunantwort, die charakteristisch für Parasitenbefall, Asthma und allergische Entzündung. Dieses Protokoll nutzt S. mansoni Ei Injektion an einen CD4 Th2-Zytokin-induzierte Entzündungsreaktion in der Lunge, Lunge durch Granulombildung um das Ei gekennzeichnet, Eosinophilie und Makrophagen-Aktivierung Alternative zu generieren.

Serie: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Zerkarien Transformation und In-vitro- Anbau von Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Forschung

Immunologie und Infektion

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

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

Induktion der Graft-versus-Host-Krankheit und<em&gt; In Vivo</em&gt; T-Zell-Überwachung mit einem MHC-matched Mausmodell

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Ein<em&gt; Ex-vivo-</em&gt; Modell eines Oligodendrozyten-gerichtete T-Cell Attack in akuten Hirnschnitten

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza Virus Vermehrung in embryonierten Hühnereiern

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Ein optimiertes Verfahren zur Isolierung und Ausbau unveränderlichen natürlichen Killer T-Zellen aus der Milz der Maus

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Optische Überprüfung der neuartige Bakterien-spezifischen Sonden auf Ex Vivo menschlichen Lungengewebe durch konfokale Laser Endomikroskopie

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Bewertung der zytotoxischen und immunmodulatorische Wirkung von Substanzen im menschlichen Precision-Cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolierung und Charakterisierung von extrazellulären Vesikel von Erwachsenen Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...