title: Induction of cytolytic anti-Gal antibodies in alpha-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria.
abstract: Naturally occurring antibodies against [Gal alpha-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional alpha-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT(+) enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT(+) bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 10(10) live GalT(+) Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT(+) target cells, with extensive cytolysis observed consistently at serum IgM titers of >/=1:320. Absorption with synthetic [Gal alpha-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT(+) bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.
authors.affiliation: Department of Microbiology and Immunology, The Brody School of Medicine at East Carolina University, Greenville, North Carolina 27858, USA.
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Posekany
authors.forename: H Keith
authors.initials: HK
authors.lastname: Pittman
authors.forename: John F
authors.initials: JF
authors.lastname: Bradfield
authors.forename: Carl E
authors.initials: CE
authors.lastname: Haisch
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Verbanac
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D003602:Cytotoxicity, Immunologic; D004187:Disaccharides / Q000276:immunology*; D004926:Escherichia coli / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000276:immunology*; D005243:Feces / Q000382:microbiology; D005700:Galactosyltransferases / Q000502:physiology*; D007132:Immunoglobulin Isotypes / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout
chemical_list: D000906:Antibodies; D004187:Disaccharides; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; C055075:galactosyl-(1-3)galactose; D005700:Galactosyltransferases