Document Type

Thesis - University Access Only

Award Date

2012

Degree Name

Master of Science (MS)

Department / School

Animal Science

First Advisor

Jeffrey A. Clapper

Abstract

Lipopolysaccharide (LPS} is a cell wall component of Gram negative bacteria that can activate immune cells, leading to a release of cytokines. Uncontrolled cytokine production can lead to endotoxemia, an often fatal inflammatory condition. Genistein is a non-steroidal phytoestrogen with tyrosine kinase inhibitory activities. Genistein has been shown to have anti-inflammatory effects in humans and mice, specifically by attenuating tumor necrosis factor alpha (TNF-a) production by peripheral blood mononuclear cells (PBMC}. To determine if genistein decreases TNF-a production from porcine PBMC the following experiments were performed. Blood was collected from 5 month old barrows and PBMC were isolated. Different concentrations of genistein and LPS (E. coli 0111:B4) were used to treat the PBMC, in triplicate. Plates were incubated the specified amount of time at 37° C in 5% CO2. Supernatants were collected and frozen at -80° C, then media concentrations of TNF-a were determined by E LISA. Fold changes in media concentration of TNF-a were analyzed by one-way Analysis of V ariance with JMP 8.0. No difference (P>0.05) was found between mean fold change from baseline of TNF-a in all L PS doses, however, all doses of LPS provided adequate TNF-a stimulation, so an intermediate (10 ng) LPS dose was selected for use in further validation steps. To determine the optimal cell number needed to induce TNF-a release from PBMC stimulated with 10 ng LPS, varying concentrations of cell numbers were used, ranging from 0.125 x 106 to 4 x 106 cells per well. Mean fold changes from baseline of TNF-a were greater (P< 0.01) in the 0.5 million cells/well treatment compared to 0.125, 1, 2, and 4 million cells/well treatments and no difference (P>0.05) was detected between the 0. 5 and 0. 25 million cells/well treatments. Therefore, the 0.5 million PBMC treatment was selected for further use in validation to provide adequate PBMC RNA for future experiments. Peripheral blood mononuclear cells were stimulated with 10 ng LPS for varying amounts of time, ranging from 2 to 16 hours of incubation, to determine optimal incubation time. No difference (P>0. 05) existed in mean fold change of TNF-a among PBMC incubated for 6, 8, 10, and 16 hours. Mean fold change from baseline of TNF-a was greater (P< 0. 01) in the 12 h incubation than all other times, however, the 10 h incubation was selected for the remainder of the experiments. Increasing amounts of genistein, ranging from 1 μM to 40 μM, were used to determine the optimal dose required to decrease TNF-a production in PBMC stimulated with 10 ng LPS. Genistein decreased (P< 0. 01) mean fold change from baseline of TNF-a in the 40 μM and 20 μM doses, however, 10 μM genistein did not decrease (P>0.05) the mean fold change from baseline of TNF-a in PBMC treated with 10 ng LPS. The 40 μM dose of genistein decreased (P< 0. 01) media concentrations of TNF-a to a greater extent than 10 μM and 20 μ M genistein doses.

Library of Congress Subject Headings

Swine -- Immunology
Genistein
Cytokines
Endotoxins

Publisher

South Dakota State University

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Rights Statement

In Copyright