Friday, April 3

“Human Bronchioepithelial Cell Culture Model to Test the Antimicrobial Role of Cholesteryl
Esters against Pseudomonas aeruginosa”

Abebayehu Yilma
MBRS-RISE MS to PhD Scholar
MS Biology

Human Bronchioepithelial Cell Culture Model to Test the Antimicrobial Role of Cholesteryl Esters against Pseudomonas aeruginosa Infection Faculty Mentor: Edith Porter, MD

Respiratory infectious diseases continue to be among the leading causes of illness and death worldwide. Previous research in our laboratory showed that cholesteryl esters contribute to the inherent antibacterial activity of nasal fluid. Here, we wish to develop an in vitro system to investigate the regulation of cholesteryl esters production in human bronchioepithelial cells (HBE) and to assess the bactericidal activity of cholesteryl esters against Pseudomonas aeruginosa. HBE cells were cultured in Keratinocyte-Serum Free Medium (KSFM) supplemented with 50 ?g/ml bovine pituitary extract, 5 ng/ml recombinant human epidermal growth factor, and 10 ng/ml cholera toxin. Lipid extracts were prepared from cell culture supernatants and subjected to reverse phase HPLC. RNA was extracted from HBE cells before and after 6 hour incubation with Pseudomonas aeruginosa and LPS, a component of the bacterial cell wall that is recognized by epithelial TLR4. RNA was reverse transcribed and subjected to real time PCR and SYBR GREEN chemistry with probes for SOAT-1, a key enzyme for biosynthesis of cholesteryl esters, and GAPDH, a housekeeping enzyme. Our data show measurable amounts of cholesteryl esters in cell culture supernatants. Furthermore, real time PCR analysis indicates up-regulation of SOAT-1 mRNA after stimulation. This work may lead to better treatment of infectious diseases through manipulation of cholesteryl ester production. [210 words] References Bergsson, G, J., Arnfinnsson, O., Steingrimsson, H,. Thormar, H. 2001. Killing of Gram-positive cocci by fatty acids and monoglycerides. A.P.M.I.S. 109: 670-678. Chang, Y, T., Chang, Y, C., Huh, Y, H., Cadigan, M, K. (1993) Molecular cloning and functional expression of human Acyl-Coenzyme A:Cholesterol Acyltranferase cDNA in mutant Chinese hamster ovary cells. The Journal of Biological Chemistry. 268: 20747-20755. Do, T, Q., Moshkani, S., Castillo, P., Anunta, S., Pogosyan, A., Cheung, A., Marbois, B., Faull, K.F., Ernst, W., Chiang, S.M., Fujii, G., Clarke, C.F., Foster, K., and Porter, E. (2008) Lipids Including Cholesteryl Linoleate and Cholesteryl Arachidonate Contribute to the Inherent Antibacterial Activity of Human Nasal Fluid. Journal of Immunology. 181:4177-87. Thormar, H., Hilmarsson, H. (2007) The role of microbial lipids in host defense against pathogens and their potential as therapeutic agents. Chemistry and Physics of Lipids. 150: 1-11. Wertz, P, W., Drake, D, R., Brogden, K, A., Dawson, D, V. (2008) Antimicrobial lipids at the skin surface. Journal of Lipid Research. 49 (1): 4-11.


Research website: http://instructional1.calstatela.edu/eporter/#research
Department website: /