1. Lipopolysaccharide-Sensitized Transgenic Mice Constitutively Expressing Human Serum Amyloid P Component are Protected from Stx2 by Polymeric Heterobifunctional Ligands Jared M. Jacobson 1, Pavel I. Kitov 1, George L. Mulvey 2, Thomas P. Griener 2, David R. Bundle 1, and Glen D. Armstrong 2 Departments of Chemistry, University of Alberta 1 and Microbiology, Immunology, and Infectious Diseases, University of Calgary 2, Canada. Materials & Methods A.Preparation of PolyBAIT-P k NAc: PolyBAIT-P k NAc was synthesized (Figure 1) via the step-wise coupling of three individual monosaccharides to form the P k Nac trisaccharide component for binding to Shiga toxins. The glucose moiety at the reducing end was made to incorporate the pyruvate functional group for binding to HuSAP as well as the incorporation of the linker for conjugation to the polymer. B. Stx2 HuSAP Mouse Challenge Experiments: Groups of mice were I.P. injected with 225 pg/g body weight Synsorb-P k -purified Stx2 (< 0.001 EU/ μ g protein) combined with 300 ng/g body weight E. coli O55 LPS (Sigma) or 225 pg/g body weight Stx2 combined with 300 ng/g body weight LPS and then immediately injected (I.V.) with either PolyBAIT-P k or PolyBAIT-P k NAc. Animals were monitored every 2-4 hours and euthanized by CO 2 asphyxia if signs of toxaemia (lethargy) became apparent. References 1.Kitov, P. I., G. L. Mulvey, T. P. Griener, T. Lipinski, D. Solomon, E. Paszkiewicz, J. M. Jacobson, J. M. Sadowska, M. Suzuki, K. Yamamura, G. D. Armstrong, and D. R. Bundle. 2008. In vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the Escherichia coli O157 AB5 toxins. Proc Natl Acad Sci U S A 105:16837-42. 2.Kimura, T., S. Tani, Y. Matsumoto Yi, and T. Takeda. 2001. Serum amyloid P component is the Shiga toxin 2-neutralizing factor in human blood. J Biol Chem 276:41576-9. 3.Griener, T. P., J. G. Strecker, R. M. Humphries, G. L. Mulvey, C. Fuentealba, R. E. Hancock, and G. D. Armstrong. Lipopolysaccharide renders transgenic mice expressing human serum amyloid P component sensitive to Shiga toxin 2. PLoS One 6:e21457. 4.Kale, R. R., C. M. McGannon, C. Fuller-Schaefer, D. M. Hatch, M. J. Flagler, S. D. Gamage, A. A. Weiss, and S. S. Iyer. 2008. Differentiation between structurally homologous Shiga 1 and Shiga 2 toxins by using synthetic glycoconjugates. Angew Chem Int Ed Engl 47:1265-8. P-228 Conclusion These results suggest that PolyBAIT-P k NAc might reduce the occurrence of the hemolytic-uremic syndrome (HUS) in children suffering from enterohemorrhagic E. coli-mediated hemorrhagic colitis. Introduction, Objective & Results Previously we reported 1 that PolyBAIT-P k, a linear acrylamide polymer containing tethered heterobifunctional groups composed of the Shiga toxin 1 (Stx1) Gb 3 P k -trisaccharide (See insert) receptor and a human serum amyloid P component (HuSAP) ligand, 1,3-cyclic pyruvate ketal of glycerol, induced face-to-face binding of the Stx1 B pentamer with HuSAP. As a consequence, PolyBAIT-P k effectively inhibited Stx1 from binding to Gb 3 receptor sequences immobilized in 96 well microtiter plates or on Vero cells thereby preventing its cytotoxicity. PolyBAIT-P k also protected HuSAP transgenic mice from Stx1-mediated toxemia. However, because HuSAP alone neutralizes Stx2 2, we were unable to determine the in vivo efficacy of PolyBAIT-P k constructs in Stx2-challenged HuSAP transgenic mice. Since these earlier reports, we demonstrated that HuSAP transgenic mice are rendered susceptible to Stx2 if they also receive an injection of purified lipopolysaccharide (LPS) 3. We have now exploited the LPS-sensitized HuSAP transgenic mouse system to demonstrate that PolyBAIT-P k also protects mice from Stx2 (Figure 2). Moreover, a PolyBAIT derivative containing the P k trisaccharide in which the terminal  -galactose was replaced by N- acetylgalactosamine (P k NAc) (Insert and Figure 1), as reported by Kale R. R., et al. 4, appeared to be more effective (Figure 2) than the original PolyBAIT-P k at protecting LPS-sensitized HuSAP transgenic mice from Stx2. Figure 1 PolyBAIT-P k NAc Figure 2 Stx2/LPS/HuSAP - (n = 15) Stx2/LPS/HuSAP + (n = 21) Stx2/LPS/HuSAP + / PolyBAIT-P k /100  g/g (n = 15) Stx2/LPS/HuSAP + /PolyBAIT-P k NAc/100  g/g (n = 11) Stx2/LPS/HuSAP + / PolyBAIT-P k NAc/32  g/g (n = 10) Stx2/LPS/HuSAP + / PolyBAIT-P k NAc/10  g/g (n = 5) Stx2/LPS/HuSAP + / PolyBAIT-P k NAc/3  g/g (n = 5)