coliJM109/p2Ais-10[58]was a recombinant strain that produced a hybrid toxin composed of A subunit of Stx-2 and B subunit of Stx-1 (Stx-2A1B). Resistance to streptomycin was spontaneously induced in GPU96MM and GPU993 by passaging in nutrient Exatecan Mesylate broth (Nissui Pharmaceutical, Tokyo, Japan) that contained 5 mg/ml streptomycin sulfate (Meiji Seika, Tokyo, Japan), and defined as GPU96MM-S and GPU993-S, respectively, to be used for the infection experiments. == Experimental animals == Commercially available chickens were maintained under conventional conditions in the animal room of the Immunology Research Institute, GHEN Corporation (Gifu, Japan), and immunized with Stx-1 and Stx-2 at 24 weeks of age. Our results therefore suggest that anti-Stx IgY antibodies may be considered as Exatecan Mesylate preventive agents for Stx-mediated diseases in EHEC infection. == Introduction == EnterohemorrhagicEscherichia coli(EHEC) causes a spectrum of human diseases, including diarrhea, hemorrhagic colitis, disordered consciousness, renal failure and hemolytic uremic syndrome (HUS)[1],[2]. EHEC colonizes the large intestine and produces Shiga toxins (Stxs) as major virulence factors that are involved in the pathogenicity of EHEC infections. EHEC produces two types of Stxs, Stx-1 and Stx-2. Stxs released into the intestinal lumen enter the systemic circulation and reach target organs, where they manifest their toxicity[3],[4]. Stxs are multimeric proteins that consist of an A subunit (32 kDa) and a pentamer of identical B subunits (7.7 kDa each). The A subunit links non-covalently to the pentamer of B subunits. The B pentamer is responsible for the toxin binding to target cell-surface glycolipid receptors such as galabiosyl ceramide (Gb2-Cer) and globotriaosyl ceramide (Gb3-Cer)[5],[6]. After binding, the toxin is internalized into the cells, and the A subunit is cleaved into A1 (28 kDa) and A2 (4 kDa) fragments. The A1 fragment exerts RNAN-glycosidase activity, which results in inhibition of protein synthesis by inactivation of 28S rRNA[7],[8]. The treatment of EHEC infection with antibiotics is controversial because of the increased risk of HUS owing to enhancement of Stx induction/release[9]. It has been reported that some antibiotics markedly enhance Stx-2 production from EHEC by inducing Stx-encoding bacteriophages[10], and release Stx-1 that Rabbit Polyclonal to COX5A is stored in the periplasm of EHEC in large amounts[11]. Based on these findings, alternative methods for therapy or prevention of Stx-mediated diseases have been investigated worldwide. Various synthetic compounds that mimic natural receptors, Gb2-Cer or Gb3-Cer, have been studied for eliminating Stxs from the intestine by binding to and/or neutralizing Stxs in the circulation as a therapeutic strategy for protecting patients from serious Stx-mediated diseases[12][18]. Clinically, Stx-2 is more potent in the development of Stx-mediated diseases than Stx-1[19],[20]. In mice, the lethal dose of Stx-2 is 400 times lower compared with Stx-1[21]. In contrast, it has been shown that the affinity of Stx-1 for the glycolipid receptor is higher than that of Stx-2[22],[23]. Therefore, the Gb2- or Gb3-conjugated compounds that have been reported as drug candidates show a good neutralizing activity against Stx-1, but are less active against Stx-2, although we recently reported that Gb2/Gb3-conjugated to phosphatidyl residues is relatively effective at neutralizing Stx-2[18]. Importantly, it has been speculated that the low affinity of Stx-2 for the receptor might be involved in the higher toxicityin vivo[22]. A potential approach for the treatment of Stx-mediated diseases may therefore be the development of agents that bind Stx-2 with high affinity, and thereby preventing the binding of Stx-2 to its natural receptor. Beside the development of compounds that mimic Stx-2 receptors, monoclonal Exatecan Mesylate antibodies against Stxs have also been investigated for the prevention of Stx-mediated diseases[24][27]. Compared with the synthetic compounds, antibodies are expected to neutralize Stx-2 effectively, owing to a different neutralizing mechanism. Antibodies have substantially higher molecular weight than the intact toxin. The antibodies might interfere with the binding of Stx to the receptor through steric hindrance and probably structural instability, regardless of the epitope recognized. This is vastly different from the inhibition mechanism of the synthetic compounds that are required to bind to the B subunit binding site. Antibodies are usually administered systemically through parenteral routes. However, EHEC produces Stx in the intestine, from where the toxin enters the bloodstream to reach the target organ[3],[4]. While we believe.