The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) Preparation of Cell Line:
<ol>
	<li>Developing the RBL-2H3.1 cell line expressing equine Fc&#949;RI&#945;:
	<ol>
		<li>Using basic tissue culture techniques for monolayer cell lines, transfect parental RBL-2H3.1 cells using the pEE6 plasmid, carrying the equine Fc&#949;RI&#945; gene (GenBank: Y18204.1) 28. Add 2 &#956;g &#956;l-1 of the plasmid DNA to 0.8 ml of cells at a density of 1.2 x107 cells ml-1. Electroporate the cells at 250 V 960 &#956;F using a 0.4 cm electrocuvette then immediately incubate on ice for 10 min.</li>
		<li>Select the transformed cells using media containing 0.4 g of geneticin G418 sulphate, then sort the remaining living cells through FACS by tagging them with a fluorescent IgE antibody. Use the resulting RBL-2H3.1 expressing equine Fc&#949;RI&#945; cell line for the investigation 2, 3.&#160;</li>
	</ol>
	</li>
	<li>Pre-assay antibody sensitization:
	<ol>
		<li>Harvest the RBL-2H3.1 expressing equine Fc&#949;RI&#945; cells from a confluent Petri dish. Wash then re-suspend the cells in culture media to a cell density of 5x105 cell ml-1.</li>
		<li>Add the IgE of interest to the suspended cells to a final concentration of 1 ng ml-1, then plate 100 &#956;l of cells onto a 96 well plate at columns 1-6 and incubate at 37 &#176;C + 5% CO2 + 90% relative humidity for 16 hr. After the incubation time, and before performing the release assay, check the wells under a microscope for well confluency and cell adherence.&#160;</li>
	</ol>
	</li>
</ol>
2) Release Assay:
<ol>
	<li>Washing the cells:
	<ol>
		<li>Warm release buffer (25 mM PIPES, 120 mM sodium chloride, 5 mM potassium chloride, 0.04 mM magnesium chloride, and 1 mM calcium chloride) at 37 &#176;C to allow for gentle cell washing.</li>
		<li>Wash cells by flicking the plate to remove cell media and adding 100 &#956;l warm, 37 &#176;C, release buffer. Repeat twice.</li>
	</ol>
	</li>
	<li>Antigen challenge:
	<ol>
		<li>Prepare a serial dilution of the antigen (NIP-HSA or DNP-HSA) of 0 ng ml-1, 0.1 ng ml-1, 1 ng ml-1, 10 ng ml-1, 100 ng ml-1, 1,000 ng ml-1, 10,000 ng ml-1 in release buffer and warm at 37 &#176;C.</li>
		<li>After the second cell wash, discard the media and replaced with 100 &#956;l of the antigen solutions. Ensure that wells in the same row (A1-6 for example) have the same antigen concentration added to them.</li>
		<li>Set up a negative control in row A by adding 0 ng ml-1 antigen. Add increasing antigen concentration down the rows (B-G) followed by triton-x buffer (5% Triton X-100) in row H cells to lyse the cells to be used as a positive control. Incubate at 37 &#176;C for 20 min&#160;to allow the cells to release its mediators.&#160;</li>
	</ol>
	</li>
	<li>Setting up individual well controls:
	<ol>
		<li>After the incubation, transfer 50 &#956;l of cell supernatant to the other half of the plate (wells A1-6 to wells A7-12, etc.). Discard the remaining 50 &#956;l of supernatant and replace with 50 &#956;l of triton-x buffer to allow the measurement of the quantity of released mediators in each well in columns 7-12 as a percentage of the total mediators inside the cells in column 1-6.</li>
	</ol>
	</li>
	<li>Enzyme substrate:</li>
	<li>Add 50 &#956;l of &#946;-hexosaminidase substrate (50 mM 4-nitrophenyl N-acetyl-&#946;-D-glucosaminide prepared in DMSO diluted down to 2 mM by adding it to citrate buffer 0.2 M citric acid and 0.2 M sodium acetate, pH 4.5) to all the wells to facilitated the conversion of the substrate to 4-nitrophenol by the &#946;-hexosaminidase enzyme. Incubate the plates at 37 &#176;C for 2 hr.</li>
</ol>
<img alt="Figure 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width="400" />
<ol start="5">
	<li>Terminating the reaction:
	<ol>
		<li>Stop the reaction by the adding 150 &#956;l Tris buffer (1 M Tris-HCl, pH 9) to each well as the high pH of the buffer stops the reaction and turns the 4-nitrophenol into a yellow color.</li>
	</ol>
	</li>
	<li>Reading and analyzing the results:
	<ol>
		<li>Read the plate using a plate spectrophotometer at 405 nm to measure the absorbance of the yellow color. Calculated the percentage of released &#946;-hexosaminidase using the following formula:</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" />
<ol start="2">
	<li>Apply this formula to each well, after which an average is taken for each row. A1 and A7 represent the location of the wells in the 96 well plate. Plot a graph of percentage of &#946;-hexosaminidase release (which corresponds to total mediator release) against antigen concentration 2, 3.</li>
</ol>

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The authors declare that they have no competing financial interests in this paper.

In summary the results of this investigation showed that when RBL-2H3.1 cells expressing equine Fc&#949;RI&#945; are sensitized with equine IgE and challenged by an antigen, they give a peak mediator release of 36.68% &#177; 4.88% of the total amount of mediator inside the cells, compared to the RBL-2H3.1 parental cells not expressing equine Fc&#949;RI&#945;.
Thus this assay provides a useful tool for investigating and studying equine allergic responses in vitro. Its allows the determ...

Allergy has been known for millennia. An asthma treatment was described in the Ancient Egyptian medical text known as the Ebers Papyrus (~1550 BCE) and discussed herbal remedies to treat it 7.
Today allergy is classified as a type I hypersensitivity response, where the T helper cell type 2 (TH2) arm of the immune system steers the production of immunoglobulin E (IgE) antibodies in response to environmental antigens called allergens. These are diverse substances that commonly interact with cells in the immune system and stimulate the synthesis and secretion of pro-inflammatory cytokines, including interleukin-4 and interleukin-13 8, 9 as do particles in cigarette smoke or diesel exhaust particles which enhance IgE synthesis 10.
The rise in allergic manifestations in industrialized countries in the last 50 years has been attributed to a combination of the effect of environmental pollutants and a trend to a more sanitized environment, which combine to shift the immune response towards a profile predominated by TH2 cytokines, as proposed by the &#8216;Hygiene Hypothesis&#8217; 11.
As mentioned above, humans are not the only mammals afflicted by allergy. Notably horses and dogs can also develop classic allergic responses and a study by 12 has shown that, as in humans, equine allergy is attributed to genetic and environmental factors. As a consequence, these animals present good models for studying the interplay between genetic and environmental causes of allergy, its progression from sensitization to disease, and possible intervention strategies once clinical manifestations have set in
In 1887, St&#246;mmer was the first person to describe the similarity between human and equine asthma 13, the effect of histamine on the equine cardiovascular system is very similar to that of humans 14. Horses are also the cornerstone of the horse racing industry, which is worth US$72 billion with a betting turnover of US$115 billion annually 15.
Most contemporary racehorses are descendants of the small number of Arabian horses bred by Lady Anne Blunt from 1878 onwards. Modern racehorses are commonly inbred to select for performance abilities. They are prone to genetic disorders, one of which is their susceptibility to mount allergic responses. They also have 1000 times higher serum IgE levels than even the most severely allergic humans 16. Horse allergic responses are usually manifested as insect bite hypersensitivity (IBH) 17, 18. IBH results in dermatitis due to bites form insects in the genus Culicoides. Another form of equine allergic disease is recurrent airway obstructions (RAO), this is manifested in the lungs and airways. It is characterized by wheezing and labored breathing. RAO commonly occurs in response to mould spores, and high allergen-specific IgE levels have been recorded in horses suffering from RAO in one study 19 although another investigation has not confirmed this 20.
Studies on equine allergy revolved around the attempt at monitoring and neutralizing equine IgE by developing anti-equine IgE monoclonal antibodies (mAbs) 21, 22. Furthermore the study by 23 discusses the production of the extracellular domains of the equine high-affinity Fc receptor&#8217;s &#945; chain (Fc&#949;RI&#945;) receptor in an attempt to detect and quantitate equine serum IgE. A related study by Ledin24 discusses a new approach aimed at neutralizing serum IgE by priming the immune system using a self/non-self immunogen. All these studies, however, lacked an effective assay to test the safety and efficacy of their protocols. In this article, we now present such an assay system applicable to the study of diagnostic and therapeutic strategies relevant to the equine system, where &#946;-hexosaminidase release, as an indicator of cell mediator degranulation, was assessed on RBL-2H3.1 cells expressing equine Fc&#949;RI&#945;. This protocol is based on previous publications 25, 4, 5, 2, 3 describing the engineering of RBL cells transfected with the gene encoding the IgE binding domain of the high-affinity receptor for IgE from different species. The protocol explains how to perform a &#946;-hexosaminidase release assay, the results of which are presented as the mean &#177; standard deviation of triplicate experiments.
The release assay was first developed by Siraganian and Hook 25 to study human allergy. The lab group led by Dr. Reuben Siraganian also developed the RBL cell line. These RBL cells were developed to express the human Fc&#949;RI&#945; and the protocol was published by 4. The final piece of the assay came with the development of the pSV plasmid in the paper by Neuberger 26 which described the production of an IgE antibodies by cloning its heavy chain gene downstream of a mouse gene for an IgE variable region that targets the hapten 4-hydroxy-3-nitro-phenacetyl (NP), the resulting chimeric antibody was fully functional. The ability to develop any IgE targeting the same hapten, while also cloning its receptor on the surface of RBL cells resulted in the standardization of the assay making it a useful protocol to measure the degranulation of basophil cells.
The assay does have pros and cons. The pros of the assay is its adaptability to be used in any mammalian system, our lab has thus used it to test for the degranulation in the human, canine and equine systems, and this is achievable simply by synthesizing the organism&#39;s IgE and cloning its receptor onto the surface of the RBL cells.
On the other hand, the cons of the assay is that the RBL cells are very sensitive to thermal, mechanical and PH changes, making them give a variation of degranulation levels within the same assay. It is thus strongly advised that the assays are always repeated in triplicates and then an average is taken from them. Furthermore, the RBL cells tend to shift toward a non-releasing phenotype if they are left in tissue culture for an extended times (&#62;10 weeks) 27, making their maintenance cumbersome. They are also prone to infections by mycoplasma bacteria, which are not visible from a light microscope and do not change the cell&#39;s morphology, but would drastically change their degranulation levels. Thus regular mycoplasma tests are needed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The parental RBL-2H3.1 cells and those transfected with the equine Fc&#949;RI&#945; receptor gene were first sensitized with mouse IgE anti DNP-HSA and challenged with the DNP-HSA antigen. Mouse IgE binds to the endogenous rat receptor in both cell lines and thus acts as a control to test the release viability of both cell lines to release mediators (Figure 1 A). This is an important check and should be carried out routinely since upon extended (&#62; 10 weeks) passage in cell culture, RBL-2H3.1 cells dr...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Research

JoVE Journal

Immunology and Infection

12.3K Views.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Video: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...