Ringraziamo Hye-Sook Oh, Joanne Morello e Alan Collmer, Dipartimento di Patologia Vegetale e Plant-Microbe Biologia, Cornell University, per le discussioni importanti. Ringraziamo Jesse Munkvold per commenti su questo articolo. Il finanziamento è stato fornito dal Programma Nazionale Genoma Plant Science Foundation, il numero di riconoscimento DBI-0605059 (GBM).

Parte 1: La crescita delle piante e la manutenzione Nicotiana benthamiana impianti utilizzati nel test dovrebbe essere di circa 7 settimane di vita. Essi dovrebbero essere tagliati almeno 4-5 giorni prima del test di rimuovere tutti i rami ascellari e fiori. E &#39;una buona idea per rimuovere rami ascellari subito dopo emergono in modo da rendere le piante più gestibile. Parte 2: coltura batterica GIORNO 1: <ol><li> Piastre o culture per tutti i ceppi utilizzati nel test sono iniziati allo stesso tempo. Per la Pseudomonas spp. tra cui 2 induttori e 3 ceppi di sfida, i batteri striscia su piastre KBM contenenti antibiotici appropriati dalle scorte glicerolo congelati (tabella 2). Una piccola quantità di batteri dovrebbero essere diffuse al centro del piatto. Incubare la piastra a 30 ° C per circa 18-24 ore. Per Agrobacterium, avviare una coltura liquida in 2 ml di LB da un piatto fresco e crescere durante la notte a 250 rpm, 30 ° C. </li></ol> 2 ° GIORNO: <ol start="2"><li> Per Pseudomonas, aggiungere 150-200 ml di liquido KBM ai batteri e li stendevano sulla intera piastra utilizzando una spatola sterile di vetro. Mettere la piastra posteriore nell&#39;incubatrice e lasciarlo crescere per un altro 20-24 ore. Ci dovrebbe essere un prato batterica sul piatto il giorno seguente, che indica una buona crescita. Per Agrobacterium, avviare una cultura secondaria LB in circa 5-10 ml con 20 acetosyringone mM e lasciarlo crescere durante la notte per 20 ore a 250 rpm, 30 ° C. </li></ol> Parte 3: impianti di Preparazione per il test 2 ° GIORNO: <ol><li> Un giorno prima del test le piante dovrebbero essere spostati in una stanza a 22-24 ° C, ~ luce del 35-40% di umidità relativa e costante. </li><li> Segna cerchi sulle foglie che sono almeno 1,5 cm di diametro con un pennarello nero spesso. Da due a quattro cerchi può essere contrassegnato per anta. I cerchi dovrebbero essere ben distanziati e preferibilmente non attraversare un grande vena. Scegli bene espanso foglie. Evitare di foglie più vecchie o foglie che sono difficili o spesso al tatto e di evitare la marcatura cerchi sulle parti inferiore della foglia. </li></ol> Cerchi segnando un giorno prima l&#39;esperimento non è essenziale per il protocollo. Tuttavia, fa risparmiare tempo se ci sono un gran numero di piante di essere analizzati, e rende più facile per raggiungere tempistica precisa tra l&#39;induzione di PTI e vaccinazioni sfida. Parte 4: Induzione di PTI 3 ° GIORNO: <ol><li> Raccolta le cellule dalla piastra in mM MgCl 10 2 per P. fluorescens e P. putida. Per Agrobacterium, centrifuga della cultura per raccogliere le cellule e risospendere in 10mM MgCl 2 + 10 mM MES pH 5,6. Ripetere la centrifugazione e risospendere in MgCl 2-MES soluzione. Misurare la densità ottica (DO) delle celle a 600 nm. Regolare l&#39;OD per i valori finali richiesti come descritto nella tabella 2. Pseudomonas dovrebbe essere infine risospeso in 10 mM MgCl 2 e Agrobacterium nel MgCl 2-MES soluzione. Un volume totale di 25 ml è sufficiente per inoculare circa 40-50 punti. </li><li> Infiltrarsi nel induttori PTI nel pre-segnato cerchi sulle foglie utilizzando una siringa da 1 ml senza ago. Scrivere l&#39;ordine in cui sono stati infiltrati nelle piante e l&#39;ora esatta in cui l&#39;infiltrazione è stata iniziata. </li></ol> Parte 5: inoculazione sfida <ol><li> Preparare P. syringae pv. pomodoro DC3000, DC3000 Δ hopQ1 Pst-1 e Ps pv. tabaci per la sfida, come descritto nella parte 4.1 e Tabella 2. </li><li> Sette ore dopo l&#39;infiltrazione induttore, eseguire l&#39;infiltrazione sfida nello stesso ordine delle piante come l&#39;induzione è stato fatto. In generale, un punto sulla periferia del cerchio prima inoculazione può essere utilizzato come il centro del cerchio seconda inoculazione. Se ci sono macchie più su una foglia quindi assicurarsi che le infiltrazioni non si sovrappongano. </li><li> Diluizioni seriali di tutte le culture usato nel test su piastre KBM o LB contenente l&#39;antibiotico appropriato per determinare il numero esatto CFU. </li></ol> Parte 6: punteggio per la ripartizione dei PTI Giorno 5: <ol><li> Cercare la comparsa di morte cellulare a causa di ETI nei luoghi che si sono sfidati con Pst DC3000. La morte cellulare dentro l&#39;area di sovrapposizione di infiltrazione indica la ripartizione dei PTI e risposta deve essere un fenotipo positivo. </li></ol> Giorno 7: <ol start="2"><li> Cercare la comparsa di morte cellulare a causa di malattia nei luoghi che si sono sfidati con Pst DC3000 Δ hopQ1-1 o Ps pv. Tabaci </em&gt;. Punteggio per un fenotipo positivo nello stesso modo come descritto nella parte 6.1. </li></ol> Quando le piante di controllo (che non deve essere compromessa per PTI) iniziano a mostrare la morte cellulare nella zona di sovrapposizione di infiltrazione, smettere di fare ulteriori osservazioni. Parte 7: I risultati rappresentativi La figura 1 mostra il risultato di un test in cui P. fluorescens è stato utilizzato come induttore e DC3000 Pst come la sfida. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. P. fluorescens era infiltrata sul N. benthamiana foglie (cerchio nero) per indurre PTI e 7 ore dopo, il luogo è stato contestato con Pst DC3000 (cerchio bianco). Impianti a tacere per FLS2 ha mostrato un crollo del PTI nella regione in cui P. fluorescens era infiltrata (A), come si vede dalla morte cellulare. Impianti di controllo che non sono stati ridotti al silenzio non ha mostrato la morte delle cellule nella zona di sovrapposizione a causa dell&#39;induzione di PTI (B). Frecce rosse indicano la mancanza o la presenza di morte cellulare nella zona di sovrapposizione di infiltrazione. Fotografie sono state scattate 2 giorni dopo le infiltrazioni. Si prega di <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <table border="1"><tbody><tr><td> PTI induttore </td><td> Cellule morte suscitando sfida </td><td> Natura della morte cellulare </td><td> Codice </td></tr><tr><td> Pseudomonas fluorescens 55 </td><td> Pseudomonas syringae pv pomodoro (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> Malattia </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae pv. tabaci 11528R </td><td> Malattia </td><td> Agro / ptab </td></tr></tbody></table> Tabella 1: Combinazioni di indurre la morte delle cellule e PTI-suscitando microbi utilizzati nel test delle cellule morte per PTI. <table border="1"><tbody><tr><td> Ceppo batterico </td><td> Selezione di media </td><td> Finale OD utilizzati nell&#39;esperimento </td><td> Corrispondenti CFU / ml </td></tr><tr><td> P. fluorescens 55 </td><td> KBM ampicillina (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM ampicillina (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampicina (100 ug / ml) </td><td> 0,5 </td><td> 5 x 10 8 </td></tr><tr><td> Pst DC3000 </td><td> KBM Rifampicina (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 Rifampicina (100 ug / ml) </td><td> 100 volte diluizione di 0,1 </td><td> 1 x 10 6 </td></tr><tr><td> P. syringae pv. tabaci 11528R </td><td> KBM Rifampicina (100 ug / ml) </td><td> 100 volte diluizione di 0,1 </td><td> 1 x 10 6 </td></tr></tbody></table> Tabella 2: condizioni di coltura e livelli di inoculo utilizzato nel saggio. L&#39;OD e corrispondenti livelli di CFU può variare tra di marca diversa spettrofotometri.

<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>

Morte a base della cellula test può essere usato per determinare il coinvolgimento di un gene nel PTI. Per esempio, la perdita di funzione mutanti per un gene di interesse può essere provata da questo test. Dei 4 combinazioni di induttore e inoculazioni sfida riportati nella tabella 1, è possibile che una sola combinazione può causare un fenotipo in fondo dell&#39;impianto. Abbiamo osservato che le piante a tacere per FLS2 mostrano una ripartizione del PTI nel Pf / DC e Pf/Q1-1 combinazioni di induttore e vaccinazioni sfida (tabella 1). E &#39;essenziale che le condizioni ambientali per il saggio sono il più vicino possibile a quelli descritti nella Parte 3.1. Bassa umidità provoca la morte cellulare per procedere troppo velocemente, rendendo difficile il test di segnare. Se le condizioni descritte nel nostro protocollo non lavorano nel vostro laboratorio, poi provare a modificare il livello di induttore o inoculazione sfida. Un altro parametro che può essere modificato è il divario di tempo tra induzione e di sfida, anche se abbiamo trovato che le lacune più breve tempo ha causato il saggio di abbattere troppo in fretta negli impianti di controllo. Si consiglia di almeno 4-5 impianti essere testato in un esperimento e un fenotipo positivo deve essere ripetuto in almeno 3-4 esperimenti indipendenti.

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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

Percepire potenziali patogeni nel loro ambiente, le piante usano recettori pattern recognition (PRR) presenti sulla loro membrana plasmatica. PRR riconoscere caratteristiche microbiche conservato chiamato pattern molecolare patogeni associati (PAMPs) e questo porta alla rilevazione PAMP-triggered immunità (PTI), che previene efficacemente la colonizzazione dei tessuti vegetali da parte di non-patogeni 1,2. Il sistema più ben studiata nel PTI è il FLS2-dipendente 3 percorso. FLS2 riconosce il flg22 PAMP che è un componente di flagellina batterica. Agenti patogeni di successo possiedono fattori di virulenza o effettori che possono sopprimere PTI e permettere al patogeno di causare malattie 1. Alcune piante, a loro volta in possesso di geni di resistenza che rilevano effettori o la loro attività, che porta a effettrici-triggered immunità (ETI) 2. Descriviamo una morte saggio basato su cellule di PTI modificato da Oh e Collmer 4. Il test è stato standardizzato in N. benthamiana, che viene utilizzato sempre più come sistema modello per lo studio delle interazioni pianta-patogeno 5. PTI è indotta da infiltrazione di un non-patogeno ceppo batterico in foglie. Sette ore dopo, un ceppo batterico che causa la malattia sia o che attiva ETI è infiltrato in una zona di sovrapposizione la zona di infiltrazione originale. PTI indotta dalla infiltrazione prima è in grado di ritardare o impedire la comparsa di morte cellulare a causa delle infiltrazioni seconda sfida. Al contrario, l&#39;aspetto della morte cellulare nella zona di sovrapposizione di inoculazione indica la ripartizione dei PTI. Quattro diverse combinazioni di induttori del PTI e vaccinazioni sfida sono stati standardizzati (Tabella 1). Il test è stato testato in materia di non tacere N. piante benthamiana che serviva come il controllo e le piante a tacere per FLS2 che sembravano destinate a essere compromessi nella loro capacità di sviluppare PTI.

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

Una morte saggio basato su cellule di PTI in piante di Nicotiana benthamiana è descritto.

Test per patogeni associati modello molecolare (PAMP)-trigger Immunità (PTI) nelle piante

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

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Research

JoVE Journal

Immunology and Infection

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

27.0K Views.  Boyce Thompson Institute for Plant Research. Una morte saggio basato su cellule di PTI in piante di Nicotiana benthamiana è descritto.

Video: 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.

Selezione di Plasmodium falciparum Parassiti per Cytoadhesion alle cellule cerebrali umane endoteliali

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

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Immunologia e infezioni

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.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza 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.

Un flusso di Adesione saggio per studiare Leucocyte reclutamento a epatici umani sinusoidali Endotelio in condizioni di stress Shear

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 Purificazione di<em&gt; Plasmodium falciparum</em&gt; Merozoiti Per l&#39;uso in opsonizzante anticorpo saggi

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.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

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.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

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.

Modelli murini di patologie gastriche e infezione da Helicobacter

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.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus 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.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

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;