Name: determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages
Uploaded: 2013-05-30T13:00:00
Description: Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

We thank Paula Cobos and Dr. Evros Vassiliou for lab assistance and providing the mouse macrophages, respectively. We also thank our lab manager Richard Criasia for guidance and help with materials. Finally, the authors thank Ramanpreet Kaur for help with video production.

1. Tissue Culture
<ol>
 <li>Use Mus musculus macrophages (BALB/c mice) for cholera toxin determinations. Initially culture all of M. musculus cells following vendor's instructions.</li>
 <li>Propagate BALB/c mice cells in Dulbecco's Modified Eagle's Medium with L-glutamine completed with 10% fetal bovine serum, and 1% antibiotic/antimycotic or RPMI 1640 base media completed with 10% fetal bovine serum, 5% L-glutamine, and 1% antibiotic/antimycotic reagent.</li>
 <li>Grow cells in 75 cm2 cor...

<ol>
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Suggestion of Concentration of Fatty Acids and Cholera Toxin
While the exact mechanism of how fatty acids enhance mucosal immunity is unknown, several studies have attempted to investigate their beneficial effects. Our study aims to provide methodology to determine the maximum concentration of fatty acids that cells can tolerate as well as the maximum concentration of cholera toxin that cells can tolerate without a significant influence on cell survival.
To determ...

The health benefits of fatty acids, such as oleic, linoleic and linolenic acids have been and continue to be documented. For example, oleic acid helps facilitate penetration of lipophilic drugs in the body1,2, reduces coronary heart disease by 24% when substituted for saturated fatty acids3, and is used to treat metabolic diseases such as Adrenoleukodystrophy4 which is an X-linked genetic disorder of fatty acid metabolism. While a necessary precursor for arachidonic acid in mammals, linoleic acid (unlike oleic acid) is not synthesized by the body and must be obtained through outside sources such as by flax seed consumption.5 Studies show several beneficial health effects of linoleic acid such as: anti-aging properties for the skin;6 anti-inflammatory properties;7 reduced proliferation of colorectal and prostate carcinoma cells;8 and the ability to fight obesity and promotion of cardiovascular health.9 Linolenic acid plays a role in reducing periodontal inflammation,10 and modulating thromboxane and prostacyclin biosynthesis.11
Arpita12 studied the influence of bile fatty acids and cholesterol on V. cholerae's expression of virulence factors and motility. Yamasaki13 indicated that methanol extract from red chili peppers, and other naturally extracted compounds, can potentially decrease cholera toxin production. It is conceivable to consider the use of food products that are rich in the above fatty acids (such as flax seeds) in the prevention and alleviation of infectious disease such as cholera through providing mucosal immunity. We conducted investigations to solubilize fatty acids and to determine, using cell proliferation assays, the maximum concentration of fatty acids that human intestinal epithelial cells can tolerate without detrimental effects on cell viability. We hypothesized that oleic, linoleic and linolenic acids provide a beneficial effect on cell viability at lower concentrations, but that at higher concentrations they will be toxic to the cells. We also solubilized the cholera toxin and determined the maximum concentration of cholera toxin that BALB/C mouse macrophages can tolerate without a significant decrease in cell viability. We hypothesize a toxic effect of cholera toxin on cell viability even at very low level. The method of solubilizing a cholera toxin and using it to determine the maximum amount of the toxin that the cells can tolerate without a significant decrease in survivability provides an advantage for future studies. For example, a combination of the above methodologies can be used to determine whether fatty acids provide cells with mucosal immunity against cholera infections. To the best of our knowledge, this rational and methodology has not been explored.
 We discuss how our preliminary data can be used in later investigations to determine if oleic, linoleic and linolenic acids provide cells with mucosal immunity against cholera infection.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells/Reagent</td>
		 </tr>
		 <tr>
			<td>Mus musculus macrophages</td>
			<td>ATCC</td>
			<td>ATCC RAW 264.7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle's Medium</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal bovine serum </td>
			<td>Bio-west</td>
			<td>S0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotic/antimycotic </td>
			<td>Hyclone</td>
			<td>SV3007901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human intestinal epithelial cells </td>
			<td>ATCC</td>
			<td>ATTC CCL-241</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HybriCare media </td>
			<td>ATCC</td>
			<td>46-X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-1376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-2376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>BD Falcon 96-Well Cell Culture Plates</td>
			<td>BD Biosciences </td>
			<td>351172</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer with Dynex Revelations 4.22 software</td>
			<td>Dynex</td>
			<td>91000101</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

Determination of the Optimum Concentration of Fatty Acids
The optimum concentration for fatty acids is defined as the maximum concentration at which cell growth is comparable to or exceeds that of control cells, with relatively low variability in results. To determine the optimum concentration of oleic, linoleic and linolenic acids cells were initially treated with varying concentrations of each fatty acid in large increments and later with smaller increments. Figure 1 shows the ...

Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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