Die Autoren bedanken sich bei Michael Chou, Bingbing Dai, und Liang Xiao für einen Beitrag für diese Handschrift erkennen. Wir erkennen auch Dr. John Gray für die großzügigen Gaben der Reagenzien in dieser Studie verwendet. PB wird durch ein Postdoc-Stipendium von der National Cancer Center unterstützt. Diese Forschung wurde unterstützt durch Zuschüsse aus dem National Institute of Health (R01AI68978, P01CA132681 und RCA170820A), einen Zuschuss von der Bill and Melinda Gates Foundation, eine translatorische Beschleunigung Zuschuss aus dem Joint Center for Translational Medicine und einen Zuschuss von der California HIV / AIDS unterstützt Research Program.

DC-LV stabileres Zellen werden auf der Grundlage der GPR Verpackungs-Zelllinie 11, die die notwendigen Komponenten lentiviralen GagPol, rev und das Tet-Off-System enthält konstruiert. Zuerst wird retrovirale Transduktion verwendet, um eine GPRS Verpackungs-Zelllinie, die eine tet-abhängigen SVGmu Glykoprotein kodiert erzeugen. Dann wird Konkatemers Array Transfektion verwendet, um das GPRS-Zelllinie mit einem lentiviralen Vektor Transgen wie GFP transfiziert. Diese stabilen Erzeuger-Zellinie, wie LV-mGFP bezeichnet, können in vitro und in vivo auf ihre Fähigkeit, eine DC-LV Impfstoff gegen GFP erzeugen getestet werden. 1. Generieren eines Tet-abhängigen SVGmu Zelllinie Plasmid PRX-SVGmu ist ein Konstrukt, in dem DC-spezifische Glykoprotein SVGmu stromabwärts des tTA-advanced-Promotor von retroviralen Plasmid pRetroX-Tet-off 1 (Abbildung 1C) geklont. Kultur 293T-Zellen in D10 (Dulbecco modifiziertem Eagle-Medium mit10% fötalem Rinderserum, 2 mM L-Glutamin). Kultur GPR Verpackungs-Zelllinie in D10 mit Doxycyclin (1 ng / ml) und Puromycin (2 pg / ml). Alle Zellen werden in einem befeuchteten 37 ° C-Inkubator mit 5% CO 2 kultiviert. <ol><li> 16-18 Std. vor transienten Transfektion Platte 2 × 10 6 293T-Zellen in 4 ml D10 in einer 6-cm Zellkulturschalen so dass die Zellen 90% Konfluenz nähern zum Zeitpunkt der Transfektion. </li><li> Transfektion von retroviralen Plasmide: mix 100 ul 1,25 M CaCl 2-Lösung, steril Milli-Q-Wasser und die folgenden Plasmide: 5 ug PRX-SVGmu, 2,5 ug pgag-Pol, 2,5 ug pVSV-G. Das Endvolumen beträgt 500 ul. Zu einem 5 ml Polystyrol mit rundem Boden Rohr, 500 ul 2X HBS (50 mM HEPES, 10 mM KCl, 12 mM Dextrose, 280 mM NaCl, 1,5 mM Na 2 HPO 4 * 7H 2 O, pH 7,05). In das Plasmid Mischung tropfenweise in die 2X HBS-Puffer während sprudelnden den Puffer kräftig mit einem Glas Pasteur Pipette. Nach dem Hinzufügen der mixture weiterhin sprudeln für weitere 30 sec. Dann fügen Sie die gesamte Mischung auf die 293T-Zellen in 6-cm Kulturschale und Inkubation bei 37 ° C </li><li> 4 Stunden nach der Transfektion sorgfältig ersetzen das Medium in der Kulturschale mit 4 ml vorgewärmten D10. </li><li> Spin-Infektion: 48 Stunden nach der Transfektion Ernte SVGmu kodierende retrovirale Partikel im Überstand, indem der Überstand durch ein 0,45-um-Filter. Teller die GPR Verpackung Zellen in einer 24-Well-Platte mit 2 x 10 4 Zellen / well. In abfiltrierten Überstands GPR Verpackungszellen in der Schale (2 ml / Vertiefung) und Zentrifuge Zellen für 90 min bei 1050 × g und 25 ° C. Ändern Medium in frischen D10 mit Doxycyclin (1 ng / ml) nach Spin-Infektion (2 ml / well). </li><li> 72 Stunden nach der Transfektion, erweitern Sie die Kultur der transduzierten Zellen in Verpackungen D10 mit Doxycyclin (1 ng / ml) und Puromycin (2 ng / ml). </li><li> Um die Expression von SVGmu, Kultur die Zellen ohne Doxycyclin für 48 Stunden bestätigen. Messen Oberfläche expression von SVGmu mit Durchflusszytometrie unter Verwendung anti-Sindbis Serum. Diese Zellen werden als GPRS Verpackungs-Zelllinie bezeichnet. </li></ol> 2. Construct DC-LV Producer-Zellen durch Transfektion Concatemer Array Lentivirale Transferplasmid TL20-GFP ist ein selbst-inaktivierenden lentiviralen Transfer Vektorplasmid basierend auf pCL20c-MSCV-GFP mit einer Dox-regulierbaren virale RNA-Genom Ausdruck und Austausch des Cytomegalovirus (CMV)-Enhancer mit 7 tet-Operatoren (1C) 11 , 12,15. Plasmid PGK-ble ist ein beständig Bleomycin (BLE) Kassette durch eine schwache PGK-Promotor 2 angetrieben. <ol><li> Digest 20 ug Plasmid TL20-GFP mit dem Restriktionsenzym SfiI bei 50 ° C Digest 20 ug Plasmid PGK-ble mit PflMI bei 37 ° C </li><li> Reinigen der DNA-Fragmente (die PGK-ble Kassette ist 1011 bp und der Vektor TL20-GFP ist 6861 bp) durch Agarosegelelektrophorese. </li><li> Ligieren die TL20-GFP-Vektor und PGK-BLE Kassette in einem Molverhältnis von 25:1 (NEB T4 DNA Ligase). Inkubieren über Nacht bei Raumtemperatur. </li><li> Nachdem über Nacht Ligation reinigen DNA aus dem Ligationsansatz (Qiagen DNeasy Blood and Tissue Kit). Sicherstellen, dass die Menge des gereinigten DNA ist etwa 5 ug. </li><li> 16-18 Stunden vor der Transfektion Platte GPRS Verpackungszellen in einer 6-cm Zellkulturschalen, dass die Konfluenz werden etwa 90% zum Zeitpunkt der Transfektion. </li><li> Verwendung der Calciumphosphat-Transfektion Verfahren in Schritt 1.2 bis 5 ug des gereinigten konkatemeren DNA in die Verpackungszellen GPRS in 6-cm Zellkulturschalen zu transfizieren. 4 Stunden nach der Transfektion, entfernen Sie vorsichtig das Medium und ersetzen mit 4 ml vorgewärmten D10 mit Doxycyclin (1 ng / ml). </li><li> 48 Stunden nach der Transfektion ändern das Medium in D10 mit Doxycyclin (1 ng / ml) und Puromycin (2 pg / ml). Wählen Sie für transfizierte Klone durch Zugabe von Zeocin (50 ug / ml) in dem Kulturmedium. Kultur die Zellen für ca. 2 Wochen bis Zelle colonies können an der Unterseite des Geschirrs zu sehen. </li><li> Beschriften Sie die Zelle Kolonien an der Unterseite des Geschirrs. Nehmen Sie 24-well Zellkulturschalen, die Anzahl der Brunnen, und fügen Sie in jede Vertiefung 2 ml D10 mit Zeocin (50 ug / ml), Doxycyclin (1 ng / ml), und Puromycin (2 pg / ml). Saugen Sie das Medium der transduzierten Zellen, dann einen Tropfen Trypsin auf jede der Kolonien für weniger als eine Minute. Dann fügen Sie eine oder mehrere Tropfen D10 auf den gleichen Kolonien. Pick up Kolonien eine nach der anderen mit einer Pipette und übertragen Sie sie in separate Vertiefungen der 24-Well-Platte Gewebekulturen. </li><li> Kultur und erweitere alle Zellklone in D10 mit Zeocin (50 ug / ml), Doxycyclin (1 ng / ml) und Puromycin (2 pg / ml) für die Bewertung der viralen Fähigkeit zur Produktion. </li></ol> 3. Bewerten Viral Produktion jeder Zelle Clone <ol><li> Trypsinize die Produzenten-Zellen und Platte um 4x10 6 Zellen in 6-cm Gewebekulturschale in D10 ohne Doxycyclin, so dass die Konfluenz90% übersteigt. Ersetzen Sie das Medium durch frisches vorgewärmte D10 täglich zu sammeln und das Medium für den Titer-Test 72 Stunden nach dem Entfernen dox. </li><li> Ernte der viralen Überstand durch Filtration des Mediums mit einem 0,45-um-Filter. </li><li> Teller Zielzellen exprimieren den Rezeptor DC-SIGN (zB 293T.hDC-SIGN oder Knochenmark der Maus abgeleitet dendritischen Zellen 16) und 293T als negative Kontrolle in 96-Well-Kulturschalen, 1 x 10 4 Zellen / well. In 2-fach serielle Verdünnungen des viralen Überstand in die Vertiefungen (100 ul / well). Üblicherweise werden 6-8 Verdünnungen ausreicht, um den Bereich, in dem GFP-positiven Zellen in einer linearen Beziehung zu der Menge an Virus hinzugefügt transduziert erreichen. </li><li> Zentrifuge Zellen und ersetzen Sie das Medium wie in Schritt 1.4 beschrieben. BMDCs sollte in RPMI-Medium mit 10% FBS und GM-CSF (1:20 J558L konditioniertes Medium) kultiviert werden. </li><li> Messen Sie die GFP-Expression in Zellen mittels Durchflusszytometrie 4-6 Tage nach der Transduktion transduzierten. Lentivirales vector-Titer in den Vektor Verdünnungsbereich berechnet, wenn der Prozentsatz von GFP-positiven Zellen und Vektor Betrag in einer linearen Beziehung stehen. Wählen Sie die Zell-Klon mit der höchsten Produktion viraler für spätere Anwendungen. </li></ol> 4. Produzieren und Konzentrat Lentivirusvektoren <ol><li> Kultur der Produzent Zelllinie (LV-mGFP) in 15-cm-Zellkulturschalen. </li><li> Trypsinize die Produzenten-Zellen und Zellen Platte in 15-cm-Zellkulturschalen bei mehr als 90% Konfluenz in frischen D10 ohne Doxycyclin. Ändern Medium mit frischem, vorgewärmten D10 täglich. </li><li> Zum Zeitpunkt des Peak virale Produktion (vom Anwender bestimmt), ernten die virale Überstand und filtern es mit einem 0,45-um-Filter. Laden Sie die gefilterten Überstand in dickwandigen 32,5 ml Ultrazentrifugenröhrchen, mit Parafilm versiegeln und Zentrifuge bei 50.000 xg, 4 ° C für 90 min. Gründlich das Pellet in 50 ul oder einem geeigneten Volumen PBS oder HBSS abhängig von der Anwendung. &lt;/ Li&gt; </ol> 5. Impfen Mäuse in vivo und Analysieren Antigen-spezifische Immunantwort <ol><li> Produzieren mit hohem Titer-Virus, wie in Kapitel 4 beschrieben. </li><li> Spritzen BALB / c-Mäuse (6-8 Wochen alt, weiblich) über einen Wegelagerer Route mit dem Vektor Suspension (25 ul pro Fußballen). </li><li> 2 Wochen nach der Immunisierung isolieren Milz und Ernte Splenozyten. Kultur Splenozyten mit GFP-Epitop-Peptide und Analyse der Gegenwart von GFP-spezifische CD8 + T-Zellen mit intrazellulärem Zytokinfärbung, wie zuvor 17 (3B) beschrieben. </li></ol>

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<p class="jove_content"&gt; Die Autoren erklären, dass sie keine finanziellen Interessen haben.</p

Hier haben wir ein Verfahren zur Herstellung großer Mengen von lentiviralen Vektoren mit 293T-Zellen, die stabil transduzierten mit allen lentiviralen Komponenten unter der tet off Regelungssystem beschrieben. Bisher stützen sich die meisten Protokolle lentiviralen Vektoren zu erzeugen auf Standard-Calciumphosphat transiente Transfektion (siehe 18, beispielsweise). Dieser Ansatz ist erfolgreich auf einem klinischen Maßstab, aber es kann von einigen Einschränkungen nicht vorhanden sein können bei der Ausw...

Viele Vektor-Systeme für den Gentransfer entwickelt worden. Vektoren auf Basis von Lentiviren haben unter den am häufigsten untersuchten viralen System gewesen. Diese Vektoren sind vorteilhaft, weil sie effizient Transduktion sowohl sich teilende und nicht teilende Zellen 1, eine langfristige Expression durch die Integration in das Wirtsgenom, weisen eine geringe natürliche Anti-Vektor-Immunität in den meisten menschlichen Populationen 2 und haben ein geringes Potenzial für Genotoxizität von Insertionsmutagenese 3,4. Produktion von Lentiviren wurde stets von Sicherheitsbedenken wurde gefärbt. Lentivirale Vektoren sind in der Regel von HIV-1, dem Erreger von AIDS abgeleitet. Die transiente Transfektion von einzelnen Komponenten des lentivector (Transfer, Umschlag und Verpackung Plasmide) ist eine häufige und flexible Mittel zur Bereitstellung von genetischem Material in Labor-Einstellungen. Allerdings ist Scaling-up transienten Transfektionen für klinische Anwendungen schwerfällig und may für die Entwicklung von Replikations-kompetente lentivirus 5,6 führen. Um diese Hürden zu überwinden, wurden mehrere stabile Verpackung und Produzent Zelllinien 6-11 entwickelt. Eine dieser Linien, die GPR Verpackungslinie 11, hat den Vorteil, dass sie attraktiv durch Tetracyclin reguliert. In diesem Beitrag zeigen wir, wie man dieses System anzupassen, um selbst-inaktivierenden lentiviralen Vektoren, die speziell auf dendritische Zellen (DC-LVs) 12 sind gezielt zu erzeugen. Dendritische Zellen (DCs) sind die robust Antigen-präsentierenden Zellen des Immunsystems. Sie haben das Thema von großem Interesse in der Krebs-Impfstoff-Entwicklung, weil sie direkt initiieren, Programm und regulieren Tumor-spezifische Immunantworten 13. Die Aufnahme einer Impfung Protokoll gehören DCs hat das Potenzial, eine stärkere Immunantwort als Antitumor-Peptid oder DNA-Impfstoffe zu entlocken. Vor kurzem haben wir einen lentiviralen Vektor entwickelt, specifically zielt dendritischen Zellen durch eine modifizierte Sindbisvirus Glykoprotein, SVGmu 14. Diese Vektoren sind einzigartig, weil sie eine hohe Spezifität für dendritische Zellen zeigen und erzeugen stärkere Immunantwort als unspezifische, VSVg-pseudotypisierten Vektoren. Hier berichten wir über ein Verfahren zur Herstellung von großen Mengen dieser DC-bezogene lentiviralen Vektoren. Wir zeigen, dass diese DC-LVs können DCs infizieren und erzeugen starke CD8 + T-Zell-Immunantwort. Alle Verfahren, die Tiere wurden artgerecht, unter Zustimmung des USC Intitutional Animal Care und Use Committee durchgeführt. Um in vivo und klinische Versuche durchzuführen, ist es wichtig, eine Zelllinie, die Viren mit einer hohen Titer produzieren zu erstellen. Die Durchführung der Transduktion und Transfektion Schritte genau wie beschrieben maximiert die Chancen der Erzeugung eines solchen Klons.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentiviralen Vektoren (LVs) sind ein mächtiges Mittel zur Bereitstellung von genetischem Material, viele Arten von Zellen. Wegen Sicherheitsbedenken mit diesen HIV-1 abgeleiteten Vektoren verbunden sind, große Mengen von LVs ist eine Herausforderung. In diesem Beitrag berichten wir über ein Verfahren zur Herstellung hohe Titer von Selbst-inaktivierende LVs. Wir retroviral transduzieren das tet-off stabilen Erzeuger-Zellinie GPR eine Zelllinie, GPRS, die alle viralen Komponenten, einschließlich einer dendritische Zellen spezifische Glykoprotein SVGmu Ausdruck erzeugen kann. Dann verwenden wir konkatemeren DNA Transfektion zur Transfektion der LV Transferplasmid Codierung ein Reportergen GFP in Kombination mit einem Selektionsmarker. Einige der resultierenden Klone können LV mit einem Titer 10-fach größer als das, was mit der transienten Transfektion erreichen erzeugen. Außerdem diese Viren effizient transduzieren dendritischen Zellen in vitro und erzeugen eine starke T-Zell-Immunantwort zu unserem Reporter-Antigen. Diese Methode kann eine gute Option sein, for Herstellung starke LV-basierte Impfstoffe für klinische Studien von Krebs oder Infektionskrankheiten.

Die stabile Zelllinie in diesem Verfahren beschriebenen können große Mengen von lentiviralen Vektoren, die spezifisch an dendritischen Zellen ausgerichtet sind. Wie in 1B gezeigt, ergab Isolierung einzelner Klone stabiler Zelllinien unterschiedlicher Qualität 12. Unter 26 getesteten Klonen erzeugt 8 lentivirale Partikel mit einem Titer von mehr als 10 6 Transduktion Einheiten pro ml (TU / ml), die ein typisches Benchmark für SVG-pseudotypisierten lentiviralen Vektoren durch tra...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Wir verwenden hier retrovirale Transduktion und Transfektion konkatemeren eine Zelllinie, die die Komponenten eines lentiviralen Vektors (LV) in der Abwesenheit von Tetracyclin Ausdruck zu erstellen. Das LV kodiert GFP und mit einem Glykoprotein, SVGmu, die spezifisch für einen Rezeptor auf dendritischen Zellen pseudotypisiert.

Eine Tetracyclin-regulierten Zelllinie produziert qualitativ Titer lentiviralen Vektoren, die gezielt Dendritische Zellen

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K Aufrufe.  University of Southern California, Los Angeles. Wir verwenden hier retrovirale Transduktion und Transfektion konkatemeren eine Zelllinie, die die Komponenten eines lentiviralen Vektors (LV) in der Abwesenheit von Tetracyclin Ausdruck zu erstellen. Das LV kodiert GFP und mit einem Glykoprotein, SVGmu, die spezifisch für einen Rezeptor auf dendritischen Zellen pseudotypisiert.

Serie: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Optimiertes Protokoll für die effiziente Transfektion von dendritischen Zellen ohne Zell-Reifung

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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 ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolierung von Maus Lung Dendritischen Zellen

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Ausbau zytotoxische T-Lymphozyten aus Nabelschnurblut, dass Target Cytomegalovirus, Epstein-Barr-Virus und Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Überwachung dendritische Zellen mit Migration<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Generierung eines neuartigen Dendritisch-Zell-Impfstoffs mit Melanom- und Plattenkrebs-Stammzellen

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Die Erzeugung von Unreife, Ältere und Tolerogene Dendritischen Zellen mit unterschiedlichen Metabolic Phänotypen

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Eine einfache Protokoll ortspezifisch generieren acetyliert Proteine in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Entwicklung und Validierung eines empfindsamen Einzelmolekül Array digitale Enzym-linked Immunosorbentprobe Assay für menschliche Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...