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Title: MEASUREMENT OF BOVINE INFLAMMATORY CYTOKINES BY RT-PCR USING AN EX-VIVO WHOLE BLOOD ASSAY: RELEVANCE TO ENDOTOXIN LEVELS IN ANIMAL PHARMACEUTICALS

Author
item SCOTT, M - US FDA CVM
item Paape, Max
item MYERS, M - US FDA CVM

Submitted to: American Dairy Science Association Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 8/15/2002
Publication Date: 9/12/2002
Citation: Scott, M.L., Paape, M.J., Myers, M.J. 2002. Measurement of bovine inflammatory cytokines by rt-pcr using an ex-vivo whole blood assay: relevance to endotoxin levels in animal pharmaceuticals [abstract]. American Dairy Science Association.

Interpretive Summary:

Technical Abstract: FDA requires that sterile products meet the guidelines for pyrogen levels. However, due to a lack of adequate information in food animals a very conservative approach has been set for animal drugs. The objective was to develop an in vivo method to generate data to help refine these guidelines. Blood collected from ten Holstein cows were cultured at 37C in ultraculture media for 0, 1, 3, 6, 12, 24, 48, and 72 hours with 1 ug/ml LPS. RNA was extracted and expression by RT-PCR used to analyze cytokine genes. Time course studies indicated interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFa) hit a plateau between 1 and 3 hours. Blood from nine Holstein cows were cultured for 3hrs with LPS concentrations of 0, 1ug, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, and 100fg. Surprisingly, IL-6 at 100fg were 7x higher than no LPS, providing evidence that very low concentrations of endotoxin can provoke an inflammatory response. These values are greater than 10-fold more sensitive than current FDA approved pyrogen testing kits. In conclusion, the novel bioassay developed within are very sensitive and provide evidence that inflammatory responses can be triggered by very small quantities of LPS.