We thank Hye-Sook Oh, Joanne Morello and Alan Collmer, Department of Plant Pathology and Plant-Microbe Biology, Cornell University, for valuable discussions. We thank Jesse Munkvold for comments on the article. Funding was provided by the National Science Foundation Plant Genome Program, award number DBI-0605059 (GBM).

Part 1: Plant growth and maintenance
Nicotiana benthamiana plants used in the assay should be about 7 weeks old. They should be trimmed at least 4-5 days prior to the assay to remove all axillary branches and flowers. It is a good idea to remove axillary branches soon after they emerge in order to make the plants more manageable.
Part 2: Bacterial culture
DAY 1:
<ol>
<li>Plates or cultures for all the strains used in the assay are initiated at the same time. For the Pseudomonas spp. which include 2 inducers and 3 challenge strains, streak bacteria onto KBM plates containing the appropriate antibiotics from frozen glycerol stocks (Table 2). A small amount of bacteria should be spread on the center of the plate. Incubate the plate at 30&deg;C for about 18-24 hours. For Agrobacterium, start a liquid culture in 2 ml LB from a fresh plate and grow overnight at 250 r.p.m., 30&deg;C.</li>
</ol>
DAY 2:
<ol start="2">
<li>For Pseudomonas, add 150-200 &mu;l of liquid KBM to the bacteria and spread them over the entire plate using a sterile glass spreader. Put the plate back in the incubator and let it grow for another 20-24 hours. There should be a bacterial lawn on the plate the next day, indicating good growth. For Agrobacterium, start a secondary culture in about 5-10 ml LB with 20 &mu;M acetosyringone and let it grow overnight to 20 hours at 250 r.p.m., 30&deg;C.</li>
</ol>
Part 3: Preparing plants for the assay
DAY 2:
<ol>
<li>One day before the assay the plants should be moved to a room at 22-24&deg;C, ~35-40% RH and constant light.</li>
<li>Mark circles on leaves that are at least 1.5 cm in diameter using a thick black marker. Two to four circles can be marked per leaf. The circles should be well spaced and preferably not cross a large vein. Choose well-expanded leaves. Avoid older leaves or leaves that are tough or thick to the touch and avoid marking circles on the lower portions of the leaf.</li>
</ol>
Marking circles a day prior to the experiment is not essential to the protocol. However, it saves time if there are large numbers of plants being assayed, and makes it easier to achieve precise timing between the induction of PTI and challenge inoculations.
Part 4: Induction of PTI
DAY 3:
<ol>
<li>Harvest the cells from the plate in 10 mM MgCl2 for P. fluorescens and P. putida. For Agrobacterium, centrifuge the culture to collect cells and resuspend in 10mM MgCl2 + 10 mM MES pH 5.6. Repeat the centrifugation and resuspend in the MgCl2 -MES solution. Measure the optical density (O.D.) of the cells at 600 nM. Adjust the O.D.s to the final required values as described in Table 2. Pseudomonas should be finally resuspended in 10 mM MgCl2 and Agrobacterium in the MgCl2 &#x2013;MES solution. A total volume of 25 ml is sufficient to inoculate about 40-50 spots.</li>
<li>Infiltrate the PTI inducers into the pre-marked circles on the leaves using a 1 ml needleless syringe. Write down the order in which the plants were infiltrated and the exact time when the infiltration was begun.</li>
</ol>
Part 5: Challenge inoculation
<ol>
<li>Prepare P. syringae pv. tomato DC3000, P.s.t. DC3000 &Delta;hopQ1-1 and P.s. pv. tabaci for the challenge as described in part 4.1 and Table 2.</li>
<li>Seven hours after the inducer infiltration, perform the challenge infiltration in the same order of plants as the induction was done. Generally, a point on the periphery of the first inoculation circle can be used as the center of the second inoculation circle. If there are multiple spots on a leaf then make sure the infiltrations do not overlap.</li>
<li>Plate serial dilutions of all the cultures used in the assay on KBM or LB plates containing the appropriate antibiotics to determine the exact C.F.U. number.</li>
</ol>
Part 6: Scoring for breakdown of PTI
Day 5:
<ol>
<li>Look for the appearance of cell death due to ETI in the spots that were challenged with P.s.t. DC3000. Cell death inside the overlapping area of infiltration indicates a breakdown of PTI and should be scored as a positive phenotype.</li>
</ol>
Day 7:
<ol start="2">
<li>Look for the appearance of cell death due to disease in the spots that were challenged with P.s.t. DC3000 &Delta;hopQ1-1 or P.s. pv. tabaci. Score for a positive phenotype in the same manner as described in part 6.1.</li>
</ol>
When the control plants (that should not be compromised for PTI) begin to show cell death in the overlapping area of infiltration, stop making any further observations.
Part 7: Representative results
Figure 1 shows the outcome of an assay when P. fluorescens was used as the inducer and P.s.t. DC3000 as the challenge.
<img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. P. fluorescens was infiltrated onto N. benthamiana leaves (black circle) to induce PTI and 7 hours later, the spot was challenged with P.s.t DC3000 (white circle). Plants silenced for FLS2 showed a breakdown of PTI in the region where P. fluorescens was infiltrated (A), as seen by cell death. Control plants that were not silenced showed no cell death in the overlapping area due to induction of PTI (B). Red arrows indicate lack of or presence of cell death in the overlapping area of infiltration. Photographs were taken 2 days after the infiltrations. Please <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">click here</a> to see a larger version of figure 1.
<table border="1">
<tbody>
<tr>
<td>PTI inducer</td>
<td>Cell death-eliciting challenge </td>
<td>Nature of cell death </td>
<td>Code </td>
</tr>
<tr>
<td>Pseudomonas fluorescens 55</td>
<td>Pseudomonas syringae pv tomato (P.s.t.) DC30006</td>
<td>ETI</td>
<td>Pf/DC</td>
</tr>
<tr>
<td>P. putida KT2440</td>
<td>P.s.t. DC3000</td>
<td>ETI</td>
<td>Pp/DC</td>
</tr>
<tr>
<td>P. fluorescens 55</td>
<td>P.s.t. DC3000 &Delta;hopQ1-16</td>
<td>Disease</td>
<td>Pf/Q1-1</td>
</tr>
<tr>
<td>Agrobacterium tumefaciens GV2260</td>
<td>P. syringae pv. tabaci 11528R</td>
<td>Disease</td>
<td>Agro/Ptab</td>
</tr>
</tbody>
</table>
Table 1: Combinations of PTI inducing and cell death-eliciting microbes used in the cell death assay for PTI.
<table border="1">
<tbody>
<tr>
<td>Bacterial strain</td>
<td>Selection medium</td>
<td>Final O.D. used in experiment</td>
<td>Corresponding C.F.U./ml </td>
</tr>
<tr>
<td>P. fluorescens 55</td>
<td>KBM Ampicillin (100 ug/ml)</td>
<td>0.5</td>
<td>1 x 109</td>
</tr>
<tr>
<td>P. putida KT2440</td>
<td>KBM Ampicillin (100 ug/ml)</td>
<td>0.5</td>
<td>1 x 108</td>
</tr>
<tr>
<td>A. tumefaciens GV2260</td>
<td>LB Rifampicin (100 ug/ml)</td>
<td>0.5</td>
<td>5 x 108</td>
</tr>
<tr>
<td>P.s.t. DC3000</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>0.02</td>
<td>2 x 107</td>
</tr>
<tr>
<td>P.s.t. DC3000 &Delta;hopQ1-1</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>100 fold dilution of 0.1</td>
<td>1 x 106</td>
</tr>
<tr>
<td>P. syringae pv. tabaci 11528R</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>100 fold dilution of 0.1</td>
<td>1 x 106</td>
</tr>
</tbody>
</table>
Table 2:Culture conditions and inoculum levels used in the assay. The O.D. and corresponding C.F.U. levels may vary between different brand of spectrophotometers.

<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>

The cell death-based assay can be used to determine the involvement of a gene in PTI. For instance, loss-of-function mutants for a gene of interest can be tested using this assay. Out of the 4 combinations of inducer and challenge inoculations given in Table 1, it is possible that only one combination may result in a phenotype in your plant background. We have observed that plants silenced for FLS2 show a breakdown of PTI in the Pf/DC and Pf/Q1-1 combinations of inducer and challenge inoculations (Table 1).
It is essential that the environmental conditions for the assay are as close as possible to those described in Part 3.1. Low humidity causes cell death to proceed too quickly, making the assay difficult to score. If the conditions described in our protocol do not work in your lab, then try altering the level of inducer or challenge inoculation. Another parameter that can be modified is the time gap between induction and challenge, although we have found that shorter time gaps caused the assay to break down too quickly in control plants.
We recommend that at least 4-5 plants be tested in an experiment and a positive phenotype should be repeated in at least 3-4 independent experiments.

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP300-100</td>
<td>Prepare as a1M stock, adjust pH and filter sterilize. Store at room temperature. </td>
</tr>
<tr>
<td>Life Science UV/Vis Spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>DU 730</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop 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

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

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

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

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Immunology and Infection

27.1K Views.  Boyce Thompson Institute for Plant Research. Гибель клеток основе анализа для ПТИ в Nicotiana растений benthamiana описано.

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.

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

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.

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.

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.

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

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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.

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.

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.

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.

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.

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