Date of Award

Spring 5-15-2014

Degree Type

Thesis

Degree Name

MS Microbiology

Department

Biology

Advisor

Constantine Bitsaktsis

Committee Member

Daniel B. Nichols

Committee Member

Allan D. Blake

Committee Member

Jane L. Ko

Keywords

cytokines, Cholera Toxin, adjuvant, CTB

Abstract

Francisella tularensis is an intracellular pathogen that has been classified as a category “A” bioterrorism agent by the Centers For Disease Control. To date, there is no approved vaccine to provide protection against this pathogen. Previous in vivo studies with mice have shown that a mucosally targeted vaccine preparation of inactivated F. tularensis (iFt) adjuvanted with Cholera toxin “B” (CTB), successfully granted full protection against a less virulent strain (FT LVS) of the bacterium and provided partial protection against a more virulent strain (SchuS4). However, the mechanisms of this protection are not fully understood. In this present study, an in vitro system was utilized to further elucidate the mechanisms that drive protection against lethal F. tularensis challenge in iFt+CTB mucosally immunized mice. Specifically, the focus was directed towards determining the effects of iFt+CTB on macrophages, the common host of F. tularensis, and their ability to present antigen to naïve T-cells, express costimulatory molecules, and produce pro-inflammatory cytokines. We found that RAW264.7 cells, a murine macrophage cell line, responded to treatment with iFt+CTB by an increased secretion of the pro-inflammatory cytokines IL-6 and TNF-α. It was also determined that treatment with iFt+CTB up-regulated the expression of TLR4 on the macrophage cell surface. The effects of iFt+CTB treatment were shown to increase the expression of both costimulatory molecules B7.1 and B7.2 on the macrophage cell surface. Furthermore, we found that iFt+CTB enhanced the ability of macrophages to present antigen to a FT-specific T-cell hybridoma cell line. These findings allow us to elucidate in part, the mechanisms of protection against F. tularensis challenge in iFt+CTB immunized mice.

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