Wir danken Hye-Sook Oh, Joanne Morello und Alan Collmer, Department of Plant Pathology und Pflanzen-Mikroben-Biologie, Cornell University, für wertvolle Diskussionen. Wir danken Jesse Munkvold für Kommentare zu diesem Artikel. Die Finanzierung wurde von der National Science Foundation Pflanzengenom-Programm bereitgestellt, Vergabe-Nummer DBI-0605059 (GBM).

Teil 1: Pflanzenwachstum und Wartung Nicotiana benthamiana Pflanzen im Assay verwendet werden sollte ca. 7 Wochen alt. Sie sollten getrimmt mindestens 4-5 Tage vor dem Test werden alle axillären Zweige und Blüten entfernen. Es ist eine gute Idee, axilläre Zweige bald entfernen, nachdem sie entstehen, damit die Pflanzen mehr überschaubar. Teil 2: Bakterienkultur TAG 1: <ol><li> Platten oder Kulturen für alle Stämme im Assay verwendet werden zur gleichen Zeit gestartet. Für die Pseudomonas spp. die sind 2-Induktoren und 3 Herausforderung Stämme, Streifen Bakterien auf KBM-Platten mit den entsprechenden Antibiotika aus gefrorenen Glycerin Aktien (Tabelle 2). Eine kleine Menge von Bakterien sollte auf der Mitte der Platte verteilt werden. Die Platte bei 30 ° C für ca. 18-24 Stunden. Für Agrobacterium, starten Sie eine flüssige Kultur in 2 ml LB aus einer frischen Platte und wachsen über Nacht bei 250 rpm, 30 ° C. </li></ol> TAG 2: <ol start="2"><li> Für Pseudomonas, fügen 150-200 ul Flüssigkeit KBM, um die Bakterien und breitete sie über die gesamte Platte mit einem sterilen Glas-Spreader. Legen Sie die Platte wieder in den Inkubator und lassen Sie es für ein anderes 20-24 Stunden wachsen. Es sollte ein Bakterienrasen auf der Platte am nächsten Tag unter Angabe gutes Wachstum. Für Agrobacterium, starten Sie eine zweite Kultur in ca. 5-10 ml LB mit 20 uM Acetosyringon und wachsen lassen über Nacht auf 20 Stunden bei 250 rpm, 30 ° C. </li></ol> Teil 3: Herstellung von Pflanzen für den Test TAG 2: <ol><li> Einen Tag vor dem Test sollten die Pflanzen in einen Raum bei 22-24 ° C bewegt, ~ 35-40% relativer Luftfeuchtigkeit und konstantem Licht. </li><li> Mark Kreise auf den Blättern, die mindestens 1,5 cm im Durchmesser mit einem dicken, schwarzen Marker. Zwei bis vier Kreise können pro Blatt gekennzeichnet werden. Die Kreise sollten gut verteilt und bevorzugt nicht über eine große Vene. Wählen Sie gut entwickelte Blätter. Vermeiden älteren Blättern oder Blätter, die harte oder dicke im Griff und vermeiden Kennzeichnung Kreise auf den unteren Abschnitten des Blattes. </li></ol> Kennzeichnung Kreisen einen Tag vor dem Experiment ist nicht unbedingt erforderlich, um das Protokoll. Allerdings spart Zeit, wenn es eine große Anzahl von Pflanzen, die getestet wird, und macht es einfacher, präzises Timing zwischen der Induktion von PTI und Herausforderung Impfungen zu erreichen. Teil 4: Induktion von PTI TAG 3: <ol><li> Ernten Sie die Zellen von der Platte in 10 mM MgCl 2 für P. fluorescens und P. putida. Für Agrobacterium, Zentrifuge der Kultur, um Zellen zu sammeln und resuspendieren in 10 mM MgCl 2 + 10 mM MES pH 5,6. Wiederholen Sie die Zentrifugation und Resuspendieren in der MgCl 2-MES-Lösung. Die optische Dichte (OD) der Zellen bei 600 nM. Passen Sie die ODS die endgültige geforderten Werte wie in Tabelle 2 beschrieben. In der MgCl Pseudomonas schließlich sollte in 10 mM MgCl 2 und Agrobacterium resuspendiert 2-MES-Lösung. Ein Gesamtvolumen von 25 ml ist ausreichend, um etwa 40-50 Punkte zu impfen. </li><li> Infiltrate der PTI-Induktoren in die vorgezeichneten Kreise auf den Blättern mit einer 1 ml Spritze ohne Nadel. Notieren Sie sich die Reihenfolge, in der die Pflanzen eingeschleust und die genaue Zeit, als die Infiltration begonnen wurde. </li></ol> Teil 5: Challenge Impfung <ol><li> Bereiten P. syringae pv.. Tomaten DC3000, DC3000 Pst Δ hopQ1-1 und Ps pv. tabaci für die Herausforderung, wie in Teil 4.1 und Tabelle 2 beschrieben. </li><li> Sieben Stunden nach dem Induktor Infiltration, führen Sie die Herausforderung Infiltration in der gleichen Reihenfolge von Pflanzen wie die Induktion wurde. Im Allgemeinen kann ein Punkt auf der Peripherie der ersten Impfung Kreis als Mitte der zweiten Impfung Kreis eingesetzt werden. Wenn es mehrere Punkte auf ein Blatt werden dann sicherstellen, dass die Infiltrationen nicht überschneiden. </li><li> Platte Verdünnungsreihen aller Kulturen in den Assay auf KBM oder LB-Platten mit den entsprechenden Antibiotika verwendet werden, um die genaue CFU Zahl zu bestimmen. </li></ol> Teil 6: Scoring für den Abbau von PTI Tag 5: <ol><li> Achten Sie auf das Auftreten von Zelltod durch ETI in den Spots, die mit Pst DC3000 herausgefordert wurden. Der Zelltod im überlappenden Bereich der Infiltration zeigt eine Aufschlüsselung der PTI und sollte als positive Phänotyp erzielt werden. </li></ol> Tag 7: <ol start="2"><li> Achten Sie auf das Auftreten von Zelltod durch Krankheit in den Flecken, die mit Pst DC3000 Δ hopQ1-1 oder Ps pv in Frage gestellt wurden. Tabaci </em&gt;. Ergebnis für eine positive Phänotyp in der gleichen Weise wie in Teil 6.1 beschrieben. </li></ol> Wenn die Kontroll-Pflanzen (das sollte nicht für PTI beeinträchtigt werden), um den Zelltod in den überlappenden Bereich der Infiltration zeigen beginnen, aufhören, weitere Beobachtungen. Teil 7: Repräsentative Ergebnisse Abbildung 1 zeigt das Ergebnis eines Tests, wenn P. fluorescens wurde als Induktor und Pst DC3000 als Herausforderung genutzt. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. P. fluorescens wurde auf N. infiltriert benthamiana Blätter (schwarzer Kreis) zu induzieren PTI und 7 Stunden später, vor Ort wurde mit Pst DC3000 (weißer Kreis) in Frage gestellt. Pflanzen für FLS2 Schweigen zeigte eine Aufschlüsselung der PTI in der Region, wo P. fluorescens wurde infiltriert (A), geändert durch Zelltod zu sehen. Control-Anlagen, die nicht zum Schweigen gebracht wurden, zeigten keine Zelltod in dem überlappenden Bereich aufgrund der Induktion von PTI (B). Rote Pfeile zeigen Mangel an oder das Vorhandensein von Zelltod in dem überlappenden Bereich der Infiltration. Fotos wurden 2 Tage nach der Infiltration genommen. Bitte <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen. <table border="1"><tbody><tr><td> PTI-Induktor </td><td> Zelltod-auslösenden Herausforderung </td><td> Natur des Zelltods </td><td> Code </td></tr><tr><td> Pseudomonas fluorescens 55 </td><td> Pseudomonas syringae pv tomato (Pst.) DC3000 6 </td><td> ETI </td><td> Pf / DC </td></tr><tr><td> P. putida KT2440 </td><td> Pst DC3000 </td><td> ETI </td><td> Pp / DC </td></tr><tr><td> P. fluorescens 55 </td><td> Pst DC3000 Δ hopQ1-1 6 </td><td> Krankheit </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae pv.. tabaci 11528R </td><td> Krankheit </td><td> Agro / PTAB </td></tr></tbody></table> Tabelle 1: Kombinationen von PTI induzieren und Zelltod-auslösenden Mikroben in den Zelltod Assay für PTI verwendet. <table border="1"><tbody><tr><td> Bakterienstamm </td><td> Selektionsmedium </td><td> Finale OD verwendet im Experiment </td><td> Entsprechende CFU / ml </td></tr><tr><td> P. fluorescens 55 </td><td> KBM Ampicillin (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM Ampicillin (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampicin (100 ug / ml) </td><td> 0,5 </td><td> 5 x 10 8 </td></tr><tr><td> Pst DC3000 </td><td> KBM Rifampicin (100 ug / ml) </td><td> 0,02 </td><td> 2 x 10 7 </td></tr><tr><td> Pst DC3000 Δ hopQ1-1 </td><td> KBM Rifampicin (100 ug / ml) </td><td> 100-fache Verdünnung von 0,1 </td><td> 1 x 10 6 </td></tr><tr><td> P. syringae pv.. tabaci 11528R </td><td> KBM Rifampicin (100 ug / ml) </td><td> 100-fache Verdünnung von 0,1 </td><td> 1 x 10 6 </td></tr></tbody></table> Tabelle 2: Culture Bedingungen und Inokulum Ebenen in den Test eingesetzt. Die OD und die entsprechenden CFU Werte können zwischen verschiedenen Marken von Spektralphotometern variieren.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

Das Zelltod-basierte Test kann verwendet werden, um die Beteiligung eines Gens in PTI bestimmen. Zum Beispiel kann loss-of-function-Mutanten für ein Gen von Interesse unter Verwendung dieses Tests sein. Von den 4 Kombinationen von Induktor und Herausforderung Impfungen in Tabelle 1 angegeben, ist es möglich, dass nur eine Kombination kann in einem Phänotyp in Ihrer Anlage Hintergrund führen. Wir haben beobachtet, dass Pflanzen für FLS2 Schweigen eine Aufschlüsselung der PTI in der Pf / DC und Pf/Q1-1 Kombinationen von Induktor und Herausforderung Impfungen (Tabelle 1) zeigen. Es ist wichtig, dass die ökologischen Bedingungen für die Tests sind so nah wie möglich an die in Teil 3.1 beschrieben. Zu trockene Luft führt zum Zelltod, zu schnell zu fahren, so dass der Test nur schwer zu punkten. Wenn die Bedingungen in unserem Protokoll beschrieben werden, nicht in Ihrem Labor funktionieren, dann versuchen Sie die Änderung der Höhe der Induktor oder Herausforderung Impfung. Ein weiterer Parameter, die geändert werden können, ist die zeitliche Lücke zwischen Induktion und der Provokation, obwohl wir festgestellt, dass kürzere Zeitabstände der Test zu brechen zu schnell unter Kontrolle Pflanzen verursacht haben. Wir empfehlen, dass mindestens 4-5 Pflanzen in einem Experiment getestet werden und eine positive Phänotyp sollte mindestens 3-4 unabhängigen Experimenten wiederholt werden.

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<td>MES</td>
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<td>Life Science UV/Vis Spectrophotometer</td>
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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

Um wahrnehmen potenzielle Krankheitserreger in ihrer Umwelt, verwenden Pflanzen pattern recognition receptors (PRR), die auf ihrer Plasmamembran. PRRs erkennen konservierte mikrobielle Eigenschaften genannt pathogen-associated molecular patterns (PAMPs) und diese Erkennung führt zu PAMP ausgelöste Immunität (PTI), das verhindert, Kolonisierung Pflanzengewebe durch Nicht-Erreger 1,2. Die gut untersuchte System in PTI ist die FLS2-abhängige Pfad 3. FLS2 erkennt die PAMP flg22, dass eine Komponente des bakteriellen Flagellin ist. Erfolgreiche Krankheitserreger besitzen Virulenzfaktoren oder Effektoren, die PTI unterdrücken und damit die Erreger Krankheit 1 führen kann. Einige Pflanzen wiederum besitzen Resistenzgene, die Effektoren oder ihre Tätigkeit, die Effektor-triggered Immunität (ETI) 2 führt zu erkennen. Wir beschreiben ein Zelltod-basierter Assay für PTI von Oh und Collmer 4 modifiziert. Der Assay wurde standardisiert in N. benthamiana, die verwendet wird zunehmend als ein Modellsystem für die Untersuchung von Pflanzen-Pathogen Interaktionen 5. PTI ist durch Infiltration eines nicht-pathogenen Bakterienstamm in den Blättern. Sieben Stunden später, ein Bakterienstamm, der entweder krank macht oder welche aktiviert ETI in einen Bereich ohne Zwischenraum über die ursprüngliche Infiltration Zone infiltriert. PTI vom ersten Infiltration induziert der Lage ist, verzögern oder verhindern das Auftreten von Zelltod durch die zweite Herausforderung Infiltration. Umgekehrt zeigt das Aussehen des Zelltods in den überlappenden Bereich der Impfung eine Aufschlüsselung der PTI. Vier verschiedene Kombinationen von Induktoren von PTI und Herausforderung Impfungen wurden standardisiert (Tabelle 1). Der Test wurde auf Nicht-Schweigen N. getestet benthamiana Pflanzen, wie die Kontrolle und Anlagen für FLS2 zum Schweigen, die voraussichtlich in ihrer Fähigkeit, PTI entwickeln beeinträchtigt werden serviert wurden.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Ein Zelltod-basierter Assay für PTI in Nicotiana benthamiana Pflanzen beschrieben.

Assay für Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Pflanzen

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

assay-for-pathogen-associated-molecular-pattern-pamp-triggered

Research

JoVE Journal

Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K Aufrufe.  Boyce Thompson Institute for Plant Research. Ein Zelltod-basierter Assay für PTI in Nicotiana benthamiana Pflanzen beschrieben.

Serie: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Auswahl der Plasmodium falciparum Parasiten für Cytoadhesion zu Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Forschung

Immunologie und Infektion

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Untersuchung der Effekte von Probiotika auf Pneumokokken-Kolonisation Mit ein<em&gt; In-vitro-</em&gt; Einhaltung Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Ein Flow-Assay zur Haftung Leukozyten-Rekrutierung Human Lebersinus Endothel unter den Bedingungen der Schubspannung Studieren

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield Reinigung von<em&gt; Plasmodium falciparum</em&gt; Merozoiten Zur Verwendung in opsonierende Antikörper-Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Fluoreszenz-basierte Neuraminidase-Inhibitionsassay die Empfänglichkeit der Influenza-Viren an die Neuraminidase-Inhibitor-Klasse von Anti-Virus zu beurteilen

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Maus-Modellen der Helicobacter -Infektion und Magen-Erkrankungen

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Gründung der Virusinfektion und Analyse von Host-Virus-Wechselwirkung in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Konfokale Abbildung von Double-Stranded RNA und Muster Anerkennung Rezeptoren im negativen Sinn-RNA-Virusinfektion

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;