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Title:   Author  item
                                            Droleskey, Robert

                                                         - *Bob*item
                                            Anderson, Robinitem
                                            Callaway, Todditem
                                            ANDERSON, TIMOTHY - FORMER ARSitem
                                            Bischoff, Kennethitem
                                            Sheffield, Cynthiaitem
                                            Harvey, Rogeritem
                                            Nisbet, David
  Submitted to: Poisonous Plants Symposium Proceedings
                                    Publication Type: Book / Chapter
                                    Publication Acceptance Date: 2/25/2001
                                    Publication  Date: 11/20/2003
                                    Citation: Droleskey, R.E., Anderson, R.C., Callaway, T.R., Anderson, T.J., Bischoff, K.M., Sheffield, C.L., Harvey, R.B., Nisbet, D.J. 2003. Modulation of Escherichia coli shiga toxin activity on vero cells by the use of the secondary plant compound swainsonine. In: Acamovic, T., Stewart, C.S., Pennycott, T.W., editors. Poisonous Plants and Related Toxins. Cambridge, MA: CABI Publishing. p. 198-203.Interpretive Summary: Escherichia coli O157:H7 is a dangerous food poisoning bacterium that produces toxins called "shiga toxins". In humans, these "shiga toxins" can cause bloody diarrhea and can damage kidney cells thus causing a potentially deadly condition known as hemolytic uremic syndrome (HUS). The "shiga toxins" damage human tissues by binding to special receptors  known as Gb3 receptors. We explored the possibility that the locoweed toxin, swainsonine, could be used at safe levels to protect susceptible kidney cells by preventing the production of the Gb3 receptors and thus prevent binding by the "shiga toxins". We found that with as low as 0.3 micrograms swainsonine (a microgram is one one-millionth of a gram), the toxic effects of the "shiga toxins" on laboratory grown kidney cells were reduced by up to 100%. As expected, swainsonine by itself was not toxic at this concentration. These results demonstrate that  swainsonine was able to protect susceptible cells from being killed by the "shiga toxins" and may ultimately be able to be used as a drug to treat the life threatening effects of Escherichia coli O157:H7 infections.
                                Technical Abstract: Escherichia coli O157:H7 produce Shiga toxins, Stx 1 and Stx 2, which are responsible for much of the diarrhea and enterocolitis resulting from infection. The primary receptor on the epithelial cell surface for Stx is the glycolipid receptor globotriaosylceramide, Gb3. Earlier work had shown that tunicamycin, an inhibitor of N-linked glycoprotein  synthesis, inhibited expression of GB3 on the surface of HeLa cells, a result which reduced cell cytotoxicity by Shiga toxin from Shigella dysenteriae. We explored the possibility that the known alpha-D- mannosidase II inhibitor, swainsonine, could also regulate the expression of GB3 and/or a second low affinity Stx binding site. Suspensions of Stx 1 and 2 were tested for toxin activity against Vero cells using a LDH cytotoxicity assay procedure. Vero cells were seeded into the wells of a 96 well microtiter plate at 10e4 cells per well. For  wells in which the protective effects of swainsonine were evaluated, the cells were co-incubated with swainsonine during the 24 h establishment period. After establishment, the cells were incubated overnight with toxin, toxin plus swainsonine, or the appropriate control. LDH activity was assayed 24 h after the Stx addition. Incubation of the Vero cells with 0.3 ug swainsonine per well reduced the cytotoxic effect of Stx 1 or 2 (2.5 ng/well) by 49% and 100%, respectively, compared to that of controls incubated without swainsonine. Swainsonine by itself (5 ug/well) was not toxic to the Vero cells. These results demonstrate a protective effect from co-incubation of Vero cells with swainsonine.


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