Title page for ETD etd-08192010-172631

Type of Document Master's Thesis
Author Frellstedt, Linda
Author's Email Address lfrellst@vt.edu
URN etd-08192010-172631
Title Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro
Degree Master of Science
Department Biomedical and Veterinary Sciences
Advisory Committee
Advisor Name Title
Furr, Martin O. Committee Chair
Barrett, Jennifer G. Committee Member
McKenzie, Harold C. III Committee Member
  • Endotoxin
  • endotoxin tolerance
  • interleukin-12
  • interleukin-10
  • equine
Date of Defense 2010-06-23
Availability restricted
Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of ‘endotoxin tolerance’ (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET.

Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR.

ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells.

This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model.

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