FUNCTIONS OF SHIGA LIKE TOXIN RECEPTORS IN IMMUNE RESPONSES

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Shiga-like toxins (SLT's, also called verotoxins or VT's) produced by E. coli sertotype 0157;H7 and other serotypes have been implicated as causative agents of hemorrhagic colitis, thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. The receptors for the SLT's are globotriaosyl ceramide or Gb3 (CD77) and possibly other glycosphingolipids which cotnain Galalpha1-4Gal residues. Gb3 is expressed on a wide variety of human cell types including germinal center B lymphocytes. The Gb3- binding SLT B subunits have been shown to mimic the 63 kD subunit of the human alpha-interferon receptor (IFNAR) and the pan B cell marker CD19 in their amino acid sequences. Experimental evidence to date supports a role for Gb3/IFNAR interaction in alpha-IFN signaling in some cell types and CD19/Gb3 interaction as a mechanism for homotypic adhesion and homing of B lymphocytes. The majority of human cells which express Gb3, including B lymphocytes, are capable of functioning as antigen-presenting cells. Therefore, SLT's may be a means by which enterohemorrhagic E. coli suppress antibody responses and other functions of the immune system during infection. The long term objectives of the proposed research are to determine the cellular functions of Gb3 and other Galalpha1-4Gal-containing glycolipids and their endogenous protein ligands and to determine how the targeting of Gb3 and other Galalpha1-4Gal-containing glycolipids and their endogenous protein ligands and to determine how the targeting of Gb3+ cells by SLT affects immune responses and athe pathogenesis of SLT-producing E. coli infection. Specific aims are to; 1. Define cell functions which involve Gb3 expression in human B cell lines and potential antigen-presenting cells. SLT's will be used as probes to further define Gb3 expression on B cell lines and to probe the effect of cytokines, antibodies to surface markers, mitogens and other inducers of differentiation or activation on Gb3 expression and SLT-induced cytotoxicity in B cells and other potential antigen presenting cells. Any differential effects of these agents on wild-type Gb3+ cells versus mutant cells selected to be Gb3 and Gb3-binding proteins in human B cell lines. Human B cell lines which are CD19+(e.g. Daudi) and CD19-(e.g. U266) will be injected into SCID mice to investigate a possible role for CD19/Gb3 interaction in homing to sites of high Gb3 expression. 3. Examine the potential of developing a mouse model for the cytotoxic effects of SLT and cellular functions of Gb3 with regard to cells of the immune system. The Gb3 expression and susceptibility to SLT-induced cytotoxicity of murine cell lines corresponding to different stages of B cell development as well as other murine cells capable of antigen presentation will be compared to the Gb3 composition of corresponding human cells. The responses elicited in mice by various antigens administered with SLT B subunits will determine whether the binding of B subunits to Gb3+ cells is immunosuppressive.