- ABo DQ8 Mice is Affected by Dietary Gluten

Abstracts Hong Wang, Marna Williams. Genentech, Inc., South S.F., CA rhuMAb Beta7 is a humanized anti-human integrin β7 monoclonal antibody that binds the integrins αEβ7 and α4β7. Cells that express these integrins have been associated with inflammatory diseases of the gut such as ulcerative colitis. To investigate safety, pharmacokinetics (PK), and pharmacodynamics (PD) of rhuMAb Beta7, studies were conducted in cynomolgus monkeys. Elevation of peripheral blood CD45RA-β7high CD4+ lymphocytes (phenotypically similar to gut-homing cells) was used as a PD biomarker. In these studies, peripheral blood CD45RA-β7high CD4+ lymphocytes increased approximately 3 to 6-fold over baseline levels following administration of rhuMAb Beta7. In contrast, animals dosed with vehicle showed no substantial changes peripheral blood CD45RA-β7high CD4+ lymphocytes. CD45RA-β7low CD4+ lymphocytes (phenotypically similar to peripheral-homing cells) showed no substantial differences in cynomolgus monkeys dosed with vehicle or rhuMAb β7. In addition, β7 receptors on peripheral blood T cells appeared to be fully saturated following the first of 4 weekly doses of 5 or 25 mg/kg rhuMAb Beta7. The PD observed in cynomolgus monkeys appeared to be related to PK because the observed reversibility and return to baseline of the PD effects correlated with clearance of rhuMAb Beta7. These results are consistent with the proposed mechanism of action of rhuMAb β7: inhibition of homing of β7 positive lymphocytes to the gut, through blocked α4β7 binding to its ligands. This mechanism is expected to lead to accumulation of CD45RAβ7high CD4+ lymphocytes in the peripheral circulation. Based on the preclinical data, rhuMAb Beta7 exhibits encouraging PK, PD, and safety profiles that support clinical development for potential treatment of inflammatory bowel disease. doi:10.1016/j.clim.2008.03.338

Sa.118. Molecular Regulation of IL-23 p19 Gene Expression in Macrophages and Murine Inflammatory Bowel Disease Shehzad Sheikh, Katsuyoshi Matsuoka, Fengling Li, Scott Plevy. Univeristy of North Carolina, Chapel Hill, NC Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. The molecular regulation of IL-23 p19 remains largely unknown. IFN-γ inhibits LPS-induced IL-23 p19 mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence (CNS) was identified in the proximal promoter of the IL-23 p19 gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine IL-23 p19 luciferase reporter plasmid, LPS induces (15 fold), and IFN-γ inhibits LPSinduced p19 promoter activity (70%) in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter

S119 activity. To understand the contribution of specific IRFs, IRF1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/- BMMs demonstrate increased LPSinduced IL-23 expression and decreased IFN-γ mediated p19 inhibition. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free (GF) and conventionalized (CNV) wild type (WT) and colitis-prone IL10-/- mice. Enteric bacteria induce greater intestinal IL-23 in IL-10-/- mice compared to WT mice when transitioned from GF to CNV. In conclusion, IRF-1 is a negative regulator of IL-23 p19 gene expression in macrophages. Future studies will describe the molecular regulation of IL-23 by enteric microbiota and cytokines in vivo. doi:10.1016/j.clim.2008.03.339

Sa.119. Development of Colitis in NOD IL10-/- ABo DQ8 Mice is Affected by Dietary Gluten Eric Marietta, Shadi Rashtak, Mark Pittelkow, Chella David, Joseph Murray. Mayo Clinic, Rochester, MN Background/Aim: Transgenic mice that express HLA-DQ8 on a background deficient in the expression of endogenous mouse MHC II (Abo) are gluten sensitive, and those that express DQ8 on an NOD background in the absence of endogenous MHC II will develop a blistering disease once sensitized to gluten. IL10 is a crucial cytokine in maintaining the homeostasis of the intestine, such that IL10-/- mice develop colitis spontaneously. Objective: To determine the effect of IL10 upon the development of disease in the gluten sensitive NOD Abo DQ8 mice. Methods: NOD Abo DQ8 mice were crossed with IL10-/- mice. 68 mice were weaned and maintained upon a gluten containing chow (GCC) and 62 upon a gluten free chow (GFC). The development of diarrhea was visually assessed and the large intestines evaluated for microscopic colitis by hematoxilyn and eosin staining (HE). Results: When raised and maintained upon GCC, a significant percentage of NOD IL10-/-Abo DQ8 mice developed diarrhea at four months of age (20%). This increased to 70% by seven months of age. However, mice that were weaned and maintained upon GFC did not develop diarrhea until 14 months of age. Even then though, only 33% developed diarrhea. HE staining of the colons of the diarrheic mice demonstrated that these mice had developed microscopic colitis characteristic of colitis that develops in IL10-/- mice. Conclusion: A gluten free diet significantly delays the development of diarrhea/colitis caused by the IL10-/construct in the NOD IL10-/- Abo DQ8 mice. doi:10.1016/j.clim.2008.03.340

Sa.120. Altered Innate Immunity Contributes to the Development of Colitis in PI3K p110δ Mutant Mice Jennifer Uno, Kavitha Rao, Katsuyoshi Matsuoka, Fengling Li, Ryan Sartor, Scott Plevy. University of North Carolina at Chapel Hill, Chapel Hill, NC