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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<li>Eyre, P., Lewis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+systemic+anaphylaxis+in+the+horse%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">Acute systemic anaphylaxis in the horse.</a> Br. J. Pharmacol. 48 (3), 426-437 (1973).</li>
<li>Bruggink, M. Global betting stable, but some countries suffer recession. , International Federation of Horseracing Authorities. Available from:
 
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<li>Wagner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IgE+in+horses%3A+occurrence+in+health+and+disease%5BTitle%5D)+AND+%22Vet+ImmunolImmunopathol%22%5BJournal%5D)">IgE in horses: occurrence in health and disease.</a> Vet ImmunolImmunopathol. 132 (1), 21-23 (2009).</li>
<li>Hellberg, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equine+insect+bite+hypersensitivity%3A+immunoblot+analysis+of+IgE+and+IgG+subclass+responses+to+Culicoidesnubeculosus+salivary+gland+extract%5BTitle%5D)+AND+%22Vet.+Immunol.+Immunopathol%22%5BJournal%5D)">Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoidesnubeculosus salivary gland extract.</a> Vet. Immunol. Immunopathol. 113 (1-2), 99-112 (2006).</li>
<li>Schaffartzik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equine+insect+bite+hypersensitivity%3A+what+do+we+know%5BTitle%5D)+AND+%22Vet+ImmunolImmunopathol%22%5BJournal%5D)">Equine insect bite hypersensitivity: what do we know.</a> Vet ImmunolImmunopathol. 147 (3-4), 113-126 (2012).</li>
<li>Künzle, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IgE-bearing+cells+in+bronchoalveolar+lavage+fluid+and+allergen-specific+IgE+levels+in+sera+from+RAO-affected+horses%5BTitle%5D)+AND+%22J+Vet+Med+A+PhysiolPatholClin+Med%22%5BJournal%5D)">IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses.</a> J Vet Med A PhysiolPatholClin Med. 54 (1), 40-47 (2007).</li>
<li>Tahon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+allergy+tests+compared+to+intradermal+testing+in+horses+with+recurrent+airway+obstruction%5BTitle%5D)+AND+%22Vet+ImmunolImmunopathol%22%5BJournal%5D)">In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction.</a> Vet ImmunolImmunopathol. (1-2), 85-93 (2009).</li>
<li>Wagner, B., Radbruch, A., Rohwer, J., Leibold, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monoclonal+anti-equine+IgE+antibodies+with+specificity+for+different+epitopes+on+the+immunoglobulin+heavy+chain+of+native+IgE%5BTitle%5D)+AND+%22Vet+ImmunolImmunopathol%22%5BJournal%5D)">Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE.</a> Vet ImmunolImmunopathol. 92 (1-2), 45-60 (2003).</li>
<li>Wilson, A. D., Harwood, L., Torsteinsdottir, S., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+monoclonal+antibodies+specific+for+native+equine+IgE+and+their+application+to+monitor+total+serum+IgE+responses+in+Icelandic+and+non-Icelandic+horses+with+insect+bite+dermal+hypersensitivity%5BTitle%5D)+AND+%22Vet+ImmunolImmunopathol%22%5BJournal%5D)">Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity.</a> Vet ImmunolImmunopathol. 112 (3-4), 156-170 (2006).</li>
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</ol>

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 determination of the quantity of mediator released by cells of mast cell/basophil lineage and thus can be expected to advance research in equine allergy, whether assessing allergy causing agents, or researching IgE/receptor blocking agents.
This assay was modified to study equine allergy from previous publications that used it to study human and canine allergies 4529 for the same purposes. The engineered cell lines expressing human and canine Fc&#949;RI&#945; were employed to study the efficacy and safety of allergy blocking agents such as vaccines targeting the inhibition of IgE/receptor interaction 30. The investigation revealed an anaphylactogenic side effect of immunogens comprising the entire C&#949;3 domain, which was also proposed by others as a vaccine for attenuating IgE-mediated allergic responses in dogs 24. Based on our investigation, the molecular basis for this potentially serious complication could be rationalized on the basis of recognition of epitopes in the IgE antibody by the anti-IgE immune serum containing anti-IgE antibodies which target epitopes in C&#949;3 and are not obscured when IgE is complexed to its receptor. This observation could not have been made in the absence of this assay system and is in striking contrast to the investigation by 24, who also investigated the anti-allergy intervention strategy based on in vivo immunization with C&#949;3 domains, but have not discussed the safety of the strategy due to the absence of such an assay.
The critical steps of this protocol is to ensure that the majority of the RBL-2H3.1 cells are of the secreting phenotype; having the cells in culture greater than 10 weeks causes them to start to drift towards the non-secreting phenotype 27 regardless of whether they expressed the cloned Fc&#949;RI&#945; of interest or not. Therefore it is always essential to run mouse IgE positive controls alongside the experiment (Figure 1), and to maintain a large frozen stock of secreting cells.
Furthermore, the pH of all buffers must be accurate or they risk giving false negative results; extending the shelf-life of buffers alters their pH, and thus any buffer older than 2 months needs to be discarded.
The technique shows the interaction of a chemical with the RBL-2H3.1 cells, and how it promotes, or suppresses, their mediator release in vitro. This is a good indication of what might happen in vivo, thus this protocol is an essential step when researching potential anti-allergy therapeutic strategies. The technique does not, however, indicate which part of the antibody/receptor complex the molecule of interest interacts with.
Other research work, such as 20 have indicated that equine RAO is not related to IgE mediation, which is why some discrepancy can be seen with other publishers such as Moran et al.31 when using total equine serum to test to Ca2+ influx from RAO suffering horses using unspecific&#160;reaginic antibodies, for which different antibodies would be responsible for the cellular Ca2+ influx.
This assay is specific for IgE mediated hypersensitivities, which can also be applied to other organisms, such as humans and dogs 29, and specifically used to test the safety of anti-allergy intervention strategies, such as synthetic/chimeric anti-allergy antibodies 30.&#160;

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><tbody><tr><td>RBL-2H3.1 Expressing Equine Fc&amp;epsilon;RI&amp;alpha;</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-&amp;beta;-D-glucosaminide</td><td>Sigma</td><td>N9376</td><td>Stock solution called &amp;beta;-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 drift towards the non-secreting phenotype 27. The parental cells supported a peak mediator release of 51.54% &#177; 4.79%, while the cells expressing the equine Fc&#949;RI&#945; receptor had a peak mediator release of 45.99% &#177; 5.76%. These same cell lines where then sensitized with equine IgE anti NIP-HSA and challenged with the NIP-HSA antigen (Figure 1 B). Parental cells did not undergo mediator release, since the equine IgE does not bind to the endogenous rat receptor. On the other hand, as expected, RBL-2H3.1 cells expressing the equine Fc&#949;RI&#945; receptor underwent mediator release when sensitized with equine IgE anti NIP-HSA and challenged with the NIP-HSA antigen, giving a peak release of 36.68% &#177; 4.88%.
These results confirm the efficiency of this release assay protocol in the investigation of mediator release via equine IgE receptor. It shows that this cell line is a useful diagnostic tool in the quest to asses novel therapeutic intervention strategies in equine allergy in vitro reducing the need for animal experimentation. The same protocol is also applicable to RBL-2H3.1 cells transfected with the human and canine Fc&#949;RI&#945; receptors.
This protocol was also used to test the safety of a potential immunization strategy that attempts to neutralize canine serum IgE antibodies 30. From the results in (Figure 2) it can be determined that the immunization strategy is not safe. The anti-canine-IgE serum successfully bound to the canine IgE as was originally intended in order to neutralize serum IgE and prevent it from binding onto its receptor. But this binding was unspecific, and thus the anti-IgE serum cross linked the receptor on the cells&#8217; surface, resulting in mediator release, potentially resulting in an anaphylactic shock if this immunization strategy was used as an anti-allergy vaccine.
<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>
Table 1: Table of Material and Equipment:
<img alt="Figure 1" fo:content-width="5.5in" src="/files/ftp_upload/52222/52222fig1highres.jpg" width="700" /> 
Figure 1: Release assay results: Graph A shows the release assay preformed on RBL-2H3.1 cells not expressing equine Fc&#949;RI&#945; and expressing the receptor using mouse IgE anti DNP-HSA. The mouse IgE binds to the endogenous rat receptor resulting in mediator release when challenged with the DNP-HSA antigen, thus confirming that both cell lines are viable and support IgE-mediated antigen induced mediator release 2, 3. Graph B shows the release assay preformed on RBL-2H3.1 parental cells which not express equine Fc&#949;RI&#945; and those expressing equine Fc&#949;RI&#945;, using equine IgE anti NIP-HSA for cell sensitization. The equine IgE binds to the equine receptor, but not the endogenous rat receptor, to result in mediator release, this is confirmed since the parental cells did not release mediator mediators, while the cells expressing the equine receptor support equine IgE-mediated, antigen induced, mediator release 2, 3.
<img alt="Figure 2" fo:content-width="6.5in" src="/files/ftp_upload/52222/52222fig2highres.jpg" width="650" /> 
Figure 2: Immunization release assay results: The graph shows the release assay preformed on RBL-2H3.1 cells expressing canine Fc&#949;RI&#945;, using canine IgE anti NIP-HSA for cell sensitisation, and immunized rat anti-canine-IgE serum as the antigen. The control used was pre-immunized serum, and the positive control was NIP-HSA antigen. The canine IgE binds to the canine receptor. Furthermore, the anti-IgE serum binds to the receptor bound canine IgE and cross links it, and the receptor, on the cells&#8217; surface resulting in mediator release. This, in effect, proves that this particular immunization strategy has the potential to cause an anaphylactic shock reaction, and is thus considered not safe 30.

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 FcÃŽÂµRI at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.4K 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 FcεRI

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Establishment of a 2D Monolayer from 3D Bovine Ileal Enteroid Culture

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Author Spotlight: Advanced Enteroid Model for Studying Host-Pathogen Interactions 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Biology

Studying Enteric Glial-Epithelial Interactions in Colic-Related Inflammation

Equine Enteric Glial Culture and Application to the Study of a Neural Inflammatory Mechanism in Equine Colic

Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and hospitalization time, but in many cases, prove to be life-threatening. A unique population of intestinal cells, enteric glia, are increasingly acknowledged for their roles in sensing the gastrointestinal environment and communicating with surrounding cell types. Interactions between enteric glia and intestinal epithelia may prove critical in establishing how equine enteric glia can alter the mucosal barrier to modulate inflammation in health and colic.
To study this interaction, we present a method of establishing primary equine enteric glial cultures from equine jejunum and exposing the cultures to inflammatory conditions known to be present in colic. Primary enteric glial cultures were obtained from adult horses euthanized for reasons unrelated to colic. Intestinal villi and lamina propria were micro-dissected to expose the submucosa. The isolated submucosa underwent enzymatic digestion with collagenase, protease, and bovine serum albumin for 2-3 h. Next, mechanical digestion involving centrifugation, pipetting, and cell strainers (40-100 &#181;m), yielded a pellet used for plating on 0.05 mg/mL poly-L-lysine-coated wells at a concentration of ~400,000 cells/300 &#181;L of media.
Following confluence and first passage, the enteric glial cells were then exposed to equine recombinant IL-1&#946; (0, 10, 25 ng) for 24 h. To model epithelial-glial interactions at the time of colic, medium conditioned by either control or treated enteric glia was added directly to confluent equine jejunal monolayers while measuring transepithelial electrical resistance (TEER) using a dual-electrode EndOhm chamber. These data demonstrate just one of many potential impactful applications of equine enteric glial culture.

Author Spotlight: Isolation and Characterization of Equine Submucosal Enteric Glia &#8212; Implications for Preventing Postoperative Complications in Colic Surgery

Enteric glia are becoming increasingly recognized for their roles in intestinal homeostasis and disease processes, including postoperative complications. Equine patients recovering from emergency exploratory laparotomy suffer from a high risk of inflammatory postoperative conditions, highlighting the importance of establishing repeatable equine enteric glial primary cell culture for study.

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

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