Methodology for the measurement of HIF-1α in bovine leukocytes and assessment of the molecule as a biomarker for bovine respiratory disease outcome [thesis]

Sarah Gestier
Bovine respiratory disease (BRD) is a multi-factorial inflammatory respiratory disease complex and is a significant problem for the beef industry. While the infectious agents involved are known and vaccines exist for some, the host factors determining outcome of infections are still only partially understood. The aim of this project was to explore the usefulness of an inflammation regulatory molecule as a biomarker for susceptibility/resistance to the development of BRD in beef cattle. The
more » ... ef cattle. The transcription factor HIF-1α is recognised for its importance in the development and coordination of an immune response to hypoxia and inflammation caused by infectious agents. For this reason, HIF-1α was identified as an attractive biomarker candidate for BRD susceptibility. The initial objective of this study was to develop methods by which the level of HIF-1α stabilisation in response to hypoxia could be measured in key immune cells. Once developed, the second objective of the study was to use these methodologies to determine whether there was significant positive or negative correlation between the regulation of HIF-1α and the development of clinical BRD. The experimental hypothesis was that there is a significant difference in HIF-1α regulation between animals with clinical BRD compared with other clinically healthy animals, and thus in response to hypoxia there would be a difference in the lymphocyte and monocyte HIF-1α expression between these two groups of cattle. This project developed a methodology for hypoxic culture of bovine lymphocytes and monocytes to induce HIF-1α stabilisation. Tube cultures for lymphocyte analysis were cultured with 1.25 x10 6 cells in 250 µl serum-free medium suspension treated with 200 µM cobalt chloride and 5 µg/ml LPS; with a non-stimulated control tube containing only the cells suspended in serum-free medium. Chamber slides for monocyte analysis were cultured containing 2.5 x 10 6 cells in 500µl serum-free medium with or without stimulants. Tubes and chamber slides were cultured for 18 hours in 5% CO 2 at 37°C. Tube cultured cells underwent post-culture fluorescent staining using a fluorochromeconjugated HIF-1α antibody for measurement of HIF-1α fluorescence in B lymphocytes and T lymphocytes using flow cytometry. T lymphocytes were labelled using a fluorochrome-conjugated anti-CD3 antibody and B lymphocytes labelled using a fluorochrome-conjugated anti-CD20 antibody. An additional protocol was developed to measure monocyte HIF-1α expression using immunocytochemical staining of chamber slide cultures. This alternative approach was necessitated by the finding of poor monocyte survival during hypoxic culture and the the pre-flow cytometry staining procedures. These methodologies for the stabilisation and measurement of HIF-1α in bovine lymphocytes and monocytes were then applied to a sample population of 88 feedlot cattle, 34 of which had been treated in the feedlot for clinical BRD (cases), while the remainder was untreated, clinically healthy controls. The groups were compared using two-sample t-tests for both v
doi:10.14264/uql.2014.443 fatcat:szqfkytz5jgyhozbaszvxoda4y