Teilweise Unterstützung durch Zuschüsse zu MRS ist von Natural Sciences and Engineering Research Council of Canada (NSERC), die Harry Botterell Foundation for Neuroscience Research, ARC, und die Garfield Kelly Cardiovascular Research und Development Fund. SRA ist ein Empfänger der Queens University Research Award und dem Robert J. Wilson Fellowship. PJ ist ein Empfänger der Königin Graduate Award und dem Robert J. Wilson Fellowship. AG und SA werden die Empfänger der Königin Graduate Award. SF war der Empfänger der Ontario Graduate Scholarship in Science and Technology (OGSST). AG ist der Empfänger der Königin Franklin Bracken Graduate Scholarship. SA ist der Empfänger der Königin RS McLaughlin Graduate Scholarship.

1. Resurrecting Gefrorene Makrophagen <ol><li> Vor Wiederbelebung eingefrorenen Zellen aus dem -80 ° C Gefrierschrank, muss man Kulturmedium Verwendung sterilfiltriert Dulbecco modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum (FBS) und 5 g / ml plasmocin ergänzt vorzubereiten. Plasmocin ist eine Antibiotika-Lösung in unsere Forschung zur Beseitigung und Vermeidung von Mykoplasmen-Kontaminationen von Zellkulturen. </li><li> Für ein Fläschchen von gefrorenen Zellen gibt man 4 mL, 1 mL der kultivierten Medium bis zu einer 25 cm 2 Zellkulturflasche in ein steriles biohazard Containment Kapuze. </li><li> Wenn die Flasche mit der eingefrorenen Zellen aus dem -80 ° C Gefrierschrank genommen werden, werden sie schnell von Hand Erwärmung in den sterilen Haube, die etwa 2-3 min dauert aufgetaut. </li><li> Sobald die Zellen aufgetaut sind, sind sie schnell in den Kolben mit dem konditionierten Medium mit einer 1 ml sterilen Pipette übertragen. </li><li> Die Flasche mit den Zellen werden in einer Gewebekultur-Inkubator bei 37 ° C und 5% CO 2 platziert. </li><li> Die Zellen für das Wachstum und die Einhaltung täglich überprüft, bis sie 75% bis 90% konfluent mit einem inversen Mikroskop werden. </li><li> Während der Zellkultur Inkubation, 12 mm runde Glasplatten durch Abdecken in 70% igem Ethanol in einem großen Glas-Petrischale für 1 Stunde in den Biohazard Containment Gehäuse sind sterilisiert, wird der Alkohol nachträglich entfernt und gespeichert reusage, und das nasse Glas Seiten bleiben ausgesetzt, um die sterilfiltriert Luftstrom im Gehäuse unter einer UV für 24 Stunden oder bis sie trocken sind. </li></ol> 2. Plating Cells für das Sialidase Assay <ol><li> Vor der Beschichtung der Zellen ist eine einzige sterile runde Glasplatte sorgfältig in eine Vertiefung einer BD Falcon 24-well Flachboden mit Deckel Gewebekultur-behandelte Polystyrol Platte gelegt mit einem sterilisierten flammenden voll gebogen, 4 &quot;, gezahnt, Edelstahl ForceP . Zum einen Kolben von Zellen bei 75% Konfluenz verwenden wir im Allgemeinen 12 Wells der Kulturplatte. </li><li> Für adhärente Makrophagen, die DMEM konditioniertem Medium aus der Flasche mit einer 5 mL sterilen Pipette in die sterile Containment Gehäuse entfernt werden. Waschen Sie die adhärenten Zellen einmal mit 1x Tris sterile Kochsalzlösung pH 7,4 auf jedem Serum enthaltenden Medium zu entfernen, und fügen Sie 1 ml Calcium-freien Medium (CMF) Phosphat-gepufferter Kochsalzlösung bei pH 7,4 genug, um die Zellen zu decken. Die Zellen sind in der 37 ° C Inkubator für etwa 10 min oder in Verkehr gebracht, bis die Zellen zum Abheben in die Flasche zu beginnen. Der Kolben kann sanft geschaukelt werden oder geschüttelt, um alle verbleibenden adhärenten Zellen zu lösen. </li><li> Um die 1 ml suspendierten Zellen in der Flasche, 3 ml DMEM konditioniertem Medium. Nehmen Sie 0,5 ml der suspendierten Zellen und steril übertragen sie auf das Gewebe gezüchtet Platte mit den 12 mm runden Glasplatten. Rund, 0,5 x 10 6 Zellen, die runde Glasplatten übertragen. </li><li> Die Gewebekulturplatte enthaltenden Zellen bei 37 ° C in 5% CO 2 für 24 Stunden, damit für die Zellen, die runde Glasplatten haften. </li></ol> 3. Sialidase Assay 1. Dass die Kontrolle <ol><li> Legen Sie einen Objektträger (gefrostet: 1 Ende, 1Schieben, 25 x 75 x 1 mm) in der Containment-Schrank oder Labortisch und fügen durch Mischen in der angegebenen Reihenfolge die folgenden Verbindungen: 1 ul DAKO + 1 ul der 4-Muñana und schließlich + 3 ul Tris-gepufferter Kochsalzlösung pH 7,4 (TBS). </li><li> Nach dem Entfernen der Medien aus einer gut mit den Zellen, heben die kreisförmige Glasplatte mit den 4 &quot;ForceP um die Ecke des Brunnens. Entfernen Sie vorsichtig das Glas Folie mit dem ForceP und legen Sie die Folie mit dem Zellen nach auf die Mischung Lösung auf dem Objektträger. </li><li> Sofort nehmen die microcope slide zu einem epi-Fluoreszenz-Mikroskopie, die für die UV-Filter gesetzt wurde und verfügt über eine digitale Kamera für die Aufnahme der fluoreszierenden Bildern. </li><li> Fokus des Mikroskops unter Phasenkontrast, bis Sie die Zellen sehen können und dann zu fluoreszierenden Modus. Machen Sie Fotos von den Zellen unter Fluoreszenzlicht bei 1 min, 2 min und 3 min. Nehmen Sie ein einzelnes Bild der Zellen unter Phasenkontrast. </li></ol> 2. Making the positiven Test <ol><li> Spot die folgenden Verbindungen in der Reihenfolge auf einem anderen Objektträger: 1 ul DAKO + 1 ul der 4-Muñana + 2 ul der LPS + 1 ul der TBS. </li><li> Wiederholen Sie die Schritte 3.1.2 bis 3.1.4. </li></ol> 3. Making the Positive Prüfung zusammen mit Neuraminidase-Hemmer Tamiflu <ol><li> Spot die folgenden Verbindungen in der folgenden Reihenfolge auf einem anderen Objektträger: 1μL DAKO + 1μL von 4-Muñana + 2 ul der LPS + 1 ul von Tamiflu. </li><li> Tamiflu-Konzentrationen werden in 1x Tris vorbereiteten Kochsalzlösung pH 7,4; die endgültige Konzentration der Verbindungen in Kontakt mit den lebenden Zellen auf der kreisförmigen Glasplatte wird mit einem Verdünnungsfaktor von 5 haben. </li><li> Wiederholen Sie die Schritte 3.1.2 bis 3.1.4. </li></ol> 4. Die Bestimmung der Konzentration des Inhibitors benötigt, um 50% der Sialidase-Aktivität (IC 50) Inhibit <ol><li> Zur Quantifizierung der Fluoreszenz um die Zellen, werden die Bilder analysiert mittels ImageJ 1.38x mit der Analyse-Plugin für die Messung RBG. Nehmen Sie 50 stichprobenartige Messungen der Fluoreszenz die Zellen umgebenden und berechnet den Mittelwert der gesamten Fluoreszenz. </li><li> Die IC 50 ist aus einem Grundstück von der Inhibitorkonzentration als log-Einheiten umgewandelt (zB log ng / mL) auf der x-Achse gegen die mittlere Fluoreszenz auf der y-Achse mittels nichtlinearer Regression erzeugt. </li></ol> 5. Secrets to Success <ol><li> Sicherstellen, dass die Zellen in Kultur sind frei von Verunreinigungen von Mykoplasmen. Wir verwenden routinemäßig Plasmocin in einem Kulturmedium, um für dieses Steuerelement. </li><li> Die künstliche Neuraminidase Substrat, 4-Muñana, sollte verwendet werden frisch zubereitet werden. Die vorbereiteten Untergrund kann innerhalb einer Woche für die Sialidase Assay verwendet werden. </li><li> Wenn die Zelllinie hat einen niedrigen Rezeptor-Expression auf der Zelloberfläche, kann man eine höhere Dosis des Liganden für die Stimulation zu verwenden. </li><li> Wir haben auch erlebt, dass die Zellen über 6 mal passagiert könnte ihren Rezeptor Phänotyp verlieren. Frisch, sollte wiederbelebt Zellen kultiviert werden. </li></ol> 6. Repräsentative Ergebnisse Siehe animierte Protokoll der Sialidase-Assay mit repräsentativen Ergebnissen in der beigefügten <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">Powerpoint-Datei</a> .

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRA und PJ trugen gleichermaßen als Erstautoren.

Mit dem neu entwickelten Assay Sialidase-Aktivität in lebenden Makrophagen 2 zu erfassen, haben wir diese Technologie, um Sialidase-Aktivität in Liganden-induzierter Sialidase-Aktivität zu erkennen in Live-BMC-2 Makrophagen in einer dosisabhängigen Weise sowie in Live-DC-2.4 dendritischen Zellen , HEK-TLR4/MD2, HEK293, SP1 Mamma-Adenokarzinom-Zellen, menschliche WT und 1140F01 und WG0544 Typ I Sialidose Fibroblasten. Neuraminidase-Hemmer wie Tamiflu (Oseltamivir Phosphat) gehemmt thymoquinone-induzierte S...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
</tr>
<tr>
<td>Tamiflu (Oseltamivir Phosphate)</td>
<td>&nbsp;</td>
<td>Hoffman La Roche</td>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

Mammalian Toll-like Rezeptoren (TLRs) sind eine Familie von Rezeptoren, die pathogen-associated molecular patterns zu erkennen. Nicht nur, dass TLRs entscheidende Sensoren mikrobieller (zB Viren, Bakterien und Parasiten) Infektionen, sie spielen auch eine wichtige Rolle in der Pathophysiologie von Infektionskrankheiten, entzündliche Erkrankungen und möglicherweise bei Autoimmunkrankheiten. So muss die Intensität und Dauer der TLR-Reaktionen gegen Infektionskrankheiten streng kontrolliert werden. Daraus folgt, dass das Verständnis der strukturellen Integrität von Sensor-Rezeptoren, deren Liganden-Wechselwirkungen und Signalisierung Komponenten ist für die nachfolgenden immunologischen Schutz unerlässlich. Es wäre auch wichtig, Möglichkeiten für Krankheit Änderung durch den Sensor Manipulation. Obwohl die Signalwege der TLR-Sensoren sind gut charakterisiert, die Steuerung der Parameter Wechselwirkungen zwischen den Sensoren und deren Liganden immer noch schlecht definiert. Wir haben vor kurzem einen neuen Mechanismus der TLR-Aktivierung durch seine natürlichen Liganden, die nicht zuvor 1,2 beobachtet identifiziert. Er schlägt vor, Liganden-induzierte TLR-Aktivierung ist eng mit Neu1 Sialidase Aktivierung gesteuert. Wir haben auch berichtet, dass Neu1 dicht reguliert Neurotrophinrezeptoren wie TrkA und TrkB 3, die Neu1 und Matrix-Metalloproteinase-9 (MMP-9) cross-talk im Komplex mit dem Rezeptor 4 einzubeziehen. Die Sialidase Assay wurde zunächst verwenden, um einen neuen Liganden, thymoquinone zu finden, bei der Aktivierung von Neu4 Sialidase auf der Zelloberfläche von Makrophagen, dendritischen Zellen und Fibroblasten über GPCR Gαi Proteine ​​und MMP-9 5. Für TLR-Rezeptoren zeigen unsere Daten, dass Neu1 Sialidase bereits im Komplex mit TLR-2, -3 und -4 Rezeptoren, und ist bei der Ligandenbindung entweder Rezeptor induziert. Aktiviert Neu1 Sialidase hydrolysiert Sialyl α-2 ,3-verknüpften β-Galactosylresten entfernt von Ligandenbindung an sterische Hinderung an TLR-4 Dimerisierung, MyD88/TLR4 komplexen Rekrutierung, NFkB-Aktivierung und pro-inflammatorischen Zell-Antworten zu entfernen. In einer gemeinsamen Berichts hat Neu1 Sialidase wurde gezeigt, dass die Phagozytose in Makrophagen Zellen 6 zu regeln. Zusammengenommen hat die Sialidase Assay uns mit leistungsfähigen Einblicke in die molekularen Mechanismen von Liganden-induzierte Rezeptor-Aktivierung zur Verfügung gestellt. Obwohl die genaue Beziehung zwischen Neu1 Sialidase und die Aktivierung von TLR hat Trk-Rezeptoren noch nicht vollständig geklärt werden, wäre es eine neue oder bahnbrechende Ansatz Zellregulation Wege darstellen.

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Die Sialidase-Test ist eine einfache technische Ansatz, der neuartige molekulare Mechanismus (s) der TLR-Sensoren von mikrobiellen Infektionen und die Beteiligung an entzündlichen Erkrankungen an der Rezeptor-Ebene auf der Zelloberfläche von Live-Makrophagen aufzuklären wird.

Erkennung von Neu1 Sialidase-Aktivität bei der Regulierung der Toll-like-Rezeptor-Aktivierung

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K Aufrufe.  Queen's University - Kingston, Ontario. Die Sialidase-Test ist eine einfache technische Ansatz, der neuartige molekulare Mechanismus (s) der TLR-Sensoren von mikrobiellen Infektionen und die Beteiligung an entzündlichen Erkrankungen an der Rezeptor-Ebene auf der Zelloberfläche von Live-Makrophagen aufzuklären wird.

Serie: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Forschung

Immunologie und Infektion

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Basophilenaktivierungstest für die Untersuchung von IgE-vermittelten Mechanismen in Drug Überempfindlichkeit

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Quantitative Bildgebung von Lineage-spezifischen Toll-like Rezeptor-vermittelten Signaltransduktion in Monozyten und dendritischen Zellen aus kleinen Proben des menschlichen Blutes

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Methoden für die quantitative Bestimmung von Antikörper-induzierte Komplementaktivierung auf Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Aktivierung und Messung der NLRP3 Inflammasom Aktivität mit IL-1β in humanen Monozyten-abgeleiteten dendritischen Zellen

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.