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United States Department of Agriculture

Agricultural Research Service

Title: Expression and Functional Analysis of Recombinant Bovine Cd14

Authors
item Wang, Yan - U MD COLLEGE PARK MD
item Zarlenga, Dante
item Paape, Max
item Dahl, Geoffrey - U ILLINOIS URBANA IL

Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 15, 2002
Publication Date: May 20, 2003
Citation: Wang, Y., Zarlenga, D.S., Paape, M.J., Dahl, G.E. 2003. Expression and functional analysis of recombinat bovine CD14. Vet. Research, vol. 34, pp.413-421.

Interpretive Summary: Bovine coliform mastitis is an inflammation of the mammary gland caused by Gram-negative bacteria, where Escherichia coli is the most common pathogen. About 80% of coliform infections will result in clinical mastitis, and 10% will result in peracute mastitis with a sudden onset of septic shock. Conventional antibiotic treatment, extensive fluid supplementation and metabolic support are not effective in relieving disease symptoms. In the US 300,000 dairy cows leave the herd each year as a result of peracute mastitis. Therefore, it is important to develop novel therapeutic regimens to control symptoms associated with acute coliform mastitis. Given the functional activity of human and mouse CD14, bovine CD14 is a potential candidate for drug development to control septic shock during coliform mastitis. Scientists in the Immunology and Disease Resistance Laboratory at the USDA, Beltsville cloned the gene that produces CD14. The expressed recombinant bovine CD14 will be used to treat cows suffering from acute septic shock. Use of this product has the potential of saving the dairy industry $1.2 billion annually.

Technical Abstract: Studies in mice and humans indicate that membrane CD14 (mCD14) on the cell surface of monocytes, macrophages and polymorphonuclear neutrophils (PMN) mediates the activation of these cells by lipopolysaccharide (LPS). The soluble CD14 (sCD14), present in the circulation, also binds to LPS and blocks LPS binding to mCD14. This study cloned and expressed both recombinant bovine mCD14 and sCD14 (rbomCD14 and rbosCD14) in a baculoviru expression system. Changes in CD18 expression on PMN and steady state levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interlukin-6 (IL-6) and IL-8, were used to measure the functional activity of rbosCD14. Whole blood (n=3 cows) treated with LPS alone caused CD18 expression to increase by 12% (P < 0.02), whereas pre-incubation of LPS with 10 or 100 microgram/ml of rbosCD14 completely inhibited the increase in CD18 expression on PMN. After treating whole blood with LPS at concentrations of 1, 100 or 100,000 ng/ml for 2 h, level of steady mRNA for TNF-alpha, IL 6 and IL-8 in leukocytes and the concentration of TNF-alpha in plasma increased. However, pre-incubation of LPS with rbosCD14 inhibited the increase in TNF-alpha mRNA, but not in IL-6 and IL-8 mRNA. Excess amount of anti-human CD14 mAb also inhibited the LPS-induced increase in TNF-alpha mRNA. Pre-incubation of LPS with rbosCD14, or rbosCD14+mAb did not affect concentration of LPS-induced TNF-alpha in plasma. Collectively, these results indicate that rbosCD14 inhibits the LPS-induced increase in CD18 expression and TNF-alpha mRNA.

Last Modified: 8/27/2014