AGAAbstracts
`
`was visualized by autoradiography. Experiment (2) Human promonocytic THP-1 cells were
`pretreated with LAN (1~100 μM) and then incubated with lipopolysaccharide (LPS). Produc-
`tion of TNFα was determined by ELISA and phosphorylation and degradation of IκBα and
`phosphorylation of ERK were evaluated by Western blotting. Results: (1) Administration
`of DSS caused inflammation and damage in the colonic mucosa and increased MPO activity
`and expression of TNFα mRNA. Treatment with LAN inhibited increase in MPO activity
`and overexpression of TNFα mRNA induced by DSS by 70% and 49%, respectively. Double-
`immunostaining demonstrated that monocytes/macrophages were main source of TNFα.
`The uptake site of 3H-LAN in the normal colon mucosa were few, while in inflamed colonic
`mucosa, 3H-LAN was extensively accumulated in inflammatory cells including macrophages
`and polymorphonuclear cells. (2) LPS induced production of TNFα, which was inhibited
`by pretreatment with LAN. LPS induced phosphorylation and degradation of IκBα and
`phosphorylation of ERK within 60 min. LAN inhibited phosphorylation and degradation of
`IκBα and phosphorylation of ERK induced by LPS. Conclusion: These results suggest that
`LAN suppresses colonic mucosal inflammation induced by DSS via reduction of TNFα
`expression in inflammatory cells and this reduction by LAN is due to inhibition of activation
`of NFκB and ERK signaling pathways.
`
`M1698
`
`Anti-Inflammation and Repair Induced By Bone Marrow-Derived Mesenchymal
`Stem Cells for Dextran Sulfate Sodium-Induced Colitis in Rats
`Fumio Tanaka, Kazunari Tominaga, Masahiro Ochi, Tetsuya Tanigawa, E.J. Sasaki,
`Masatsugu Shiba, Toshio Watanabe, Yasuhiro Fujiwara, Nobuhide Oshitani, Kazuhide
`Higuchi, Tetsuo Arakawa
`
`Background: Bone marrow-derived cells including a small amount of mesenchymal stem
`cells (MSCs) had therapeutic effects for clinical human and experimental animal colitis. Its
`detailed mechanism(s) may be partly mediated by mucosal regeneration, since MSCs have
`potential for differentiation to several parts of cells. But MSCs were thought to have other
`functions such as anti-inflammation as well as mucosal regeneration, because anti-inflammat-
`ory system is involved in the repair of colitis. We examined the therapeutic efficacy and
`anti-inflammatory effects of bone marrow-derived MSCs for dextran sulfate sodium (DSS)-
`induced acute colitis in rats. Materials & Methods: Experimental colitis was induced by
`orally administration of 0, 1, 2, or 4% DSS in drinking water for 7 days in inbred male
`Lewis rats. Bone marrow was extruded from tibias and femurs. Then, its mononuclear cells
`were isolated and cultured in low-glucose DMEM containing 10% fetal calf serum for MSCs
`outgrowth. On 0, 2, and 4 days after the administration of DSS, MSCs (5 × 106 cells) were
`injected via tail vein. We checked the volumes of food and water intake, stool condition,
`and body weight everyday. On day 7, total colon was excised and each colonic mRNA
`expression of inflammatory cytokines such as TNF-α, IL-1β, IL-10, and COX2 was measured
`by real time RT-PCR method. Results: We confirmed the MSC’s characterization by both
`the immunostaining for vimentin and α-smooth muscle actin and the cell surface markers
`such as CD90, the bone marrow progenitor cell marker, but not CD45, HLA-DR, CD11b,
`nor CD31 using flow cytometric technique. Optimal dose of DSS for the rats used was
`confirmed at 4% by the assessing for loss of body weight and appetite, bloody fluid stool,
`and the shortening of colon length. MSC treatment improved the bloody stool and body
`weight loss, and significantly inhibited the shortening of colon length. At the rectum of
`MSC-treated rats, expressions of local inflammatory cytokines such as TNF-α and IL-1β
`were markedly decreased to about 40 and 15%. Local COX2 expression was also suppressed
`to 15%. IL-10, an anti-inflammatory cytokine, expression was also decreased to 25%. At
`the distal colon site (slightly oral side of rectum), similar tendency was observed about the
`expressions of cytokines in the MSC-treated colons. Conclusion: These findings suggested
`that MSC could have the therapeutic efficacy for the experimental colitis via anti-inflammat-
`ory functions.
`
`M1699
`
`Induction of Regulatory T Cell Capacities By the Sphingosine-1-Phosphate
`Analogue Fty720 in TNBS-Colitis
`Carolin Daniel, Nico Sartory, Heinfried H. Radeke, Gerd G. Geisslinger, Juergen M. Stein
`
`Background & Aims: The sphingosine-1-phopshate analogue FTY720 is known to alter
`migration and homing of lymphocytes via sphingosine-1-phosphate receptor interactions.
`We studied the effect of FTY720 in acute and established trinitrobenzene sulfonic acid
`(TNBS)-colitis focused on the induction of regulatory T cell capacities. Methods: A rectal
`enema of TNBS [100 mg/kg body weight (BW)] was applied to male Balb/c mice, and
`FTY720 [1 or 3 mg/kg] was administered intraperitoneally from day 0-3 or from day 3-5
`following the instillation of the haptenating agent. The study is conforming to the Guiding
`principles in the care and use of animals and was performed under approval of the ethical
`committee of Darmstadt/Germany (F134/03). Colon tissue was analyzed macroscopically
`and microscopically, and IL-10, transforming growth factor β (TGFβ) and FoxP3 expression
`were determined in colon protein extracts. Results: Treatment with FTY720 reduced the
`histopathologic severity of TNBS-colitis abrogating macroscopic and microscopic intestinal
`inflammation. Additionally, treatment with FTY720 resulted in a significant induction of
`IL-10, TGFβ and FoxP3 (see Table). Conclusion: FTY720 exhibits beneficial prophylactic
`as well as therapeutic effects in TNBS-colitis. Moreover, the induction of IL-10, TGFβ and
`FoxP3 raised evidence for a tolerance inducing activity also contributing to the beneficial
`capacities of FTY720 offering new auspicious therapeutic strategies for the treatment of
`inflammatory bowel disease.
`Results FTY720 TNBS-colitis
`
`A-351
`
`AGA Abstracts
`
`OXZ on the skin of abdomen on days 0 followed by intrarectal administration of the low
`concentration of OXZ on day 7. OXZ colitis was assessed with the disease activity score
`(DAS) on weight loss, diarrhea and hemorrhage until day 10. Macroscopic examination of
`the colitis with colonic damage score (CDS) was performed, and then the colon was used
`for myeloperoxidase (MPO) activity analysis. RESULT: Exposure of the primed mice to
`intrarectal OXZ challenge developed a colitis marked by weight loss, hemorrhagic diarrhea,
`ulceration, erosion etc. associated with about 13-fold increased MPO activities in the colon.
`However, OXZ failed to induce the colitis in C57BL/6 mice which are used for Th1-
`mediated disease models. In addition, pretreatment with calcineurin inhibitor to suppress
`Th1 responses deteriorated OXZ colitis. Therefore, oxazolone colitis is Th2-mediated and
`has similar histologic features to UC. Although oral administration of 5-ASA (100mg/kg)
`had no significant therapeutic effect on OXZ colitis, prednisolone (10mg/kg p.o.) significantly
`alleviate the disease state. Noteably, the central stimulation of vagus nerves with 2-deoxy-
`d-glucose (200mg/kg i.p.) significantly (P<0.05) improved OXZ colitis [DAS: 5.9±1.8 vs
`2.3±0.8; CDS: 4.4±1.3 vs 1.5±0.3; MPO: 3562.5±1034.2 vs 1399.0±353.4 units/g wwt (OXZ
`colitis control vs mice pretreated with 2-deoxy-d-glucose)]. Furthermore, subcutaneous
`administration of nicotine (3.2 mg/kg) significantly (P<0.05) suppressed OXZ colitis [DAS:
`10.5±1.6 vs 5.1±1.8; CDS: 7.6±1.3 vs 3.7±1.3; MPO: 5258.6±1466.9 vs 2585.7±234.7
`units/g wwt (OXZ colitis control vs mice pretreated with nicotine)]. CONCLUSION: Nicotinic
`acetylcholine receptors work on the cholinergic anti-inflammatory and immune pathway to
`alleviate Th2-mediated OXZ colitis.
`
`M1696
`
`Sp304, An Analog of Uroguanylin, Ameliorates Inflammation in a Model of
`Experimental Colitis
`Refaat Hegazi, Fengling Li, Gabriel Calilao, Antonia Sepulveda, Kunwar Shailubhai, Scott
`Plevy
`
`Introduction: Guanylyl cyclase C (GC-C) agonists (uroguanylin (UG) and guanylin), regulate
`water and ion homeostasis in a variety of tissues and organs, including the gastrointestinal
`(GI) tract, via cyclic GMP (cGMP). GC-C and its agonists are expressed by intestinal epithelial
`cells (IEC). The cGMP pathway mediates anti-inflammatory effects of cellular molecules such
`as nitric oxide and heme oxygenase-1, and therapies that induce cGMP (phosphodiesterase-4
`inhibitors) demonstrate efficacy in murine models of IBD. Accordingly, we reasoned that
`agonists of GC-C, when orally administered may demonstrate anti-inflammatory effects in
`murine IBD. Aim: The present study investigates the immunomodulatory effects of a GC-
`C agonist, SP304 (Callisto Pharmaceuticals, Inc., NY) in TNBS-induced murine colitis.
`Methods: GC-C, guanylin and UG mRNA expression was studied by RT-PCR in intestinal
`tissue from 12-week old IL-10 deficient (-/-) mice. To induce colitis, 0.1 ml of 2.5% tri-
`nitro benzene sulfonic acid (TNBS) in 50% ethanol was administered via catheter into the
`colonic lumen of 8 week old female in BALB-c mice (day 0). Mice were administered the
`GC-C agonist SP304 at 10 (n = 6), 50 μg/day (n = 5) or vehicle PBS for 7 days (n = 8 each)
`by oral gavage starting at day 0. On day 7, mice were sacrificed. H&E stained colonic tissue
`sections were assessed for colitis by a pathologist blinded to treatment group. Intestinal
`explants were cultured for 24 hours and levels of IL-12 p40, IL-12 p70, IL-23 and TNF
`protein secretion were measured in culture supernatants by ELISA. Results: GC-C, guanylin
`and UG mRNA are expressed in intestinal tissue of IL-10-/- mice. Intestinal guanylin and
`UG mRNA expression is induced during interventions in IL-10-/- mice that ameliorate colitis.
`In TNBS-induced colitis, histological assessment of colonic tissue demonstrated significant
`improvement of the colitis in the SP304-treated mice compared with the vehicle treated
`mice (Table). Intestinal explant cultures from SP304 treated mice express less IL-12 p40,
`IL-12 p70, IL-23 and TNF than the vehicle treated control group. Conclusions: Histological
`improvement in IL-10-/- mice correlates with upregulation of UG and guanylin mRNA.
`Treatment with SP304 exhibited anti-inflammatory effects in TNBS-induced colitis in mice.
`Importantly, amelioration of colitis was associated with downregulation of proinflammatory
`cytokines such as TNF.
`
`*P<0.05
`
`M1697
`Lansoprazole, a Proton Pump Inhibitor, Suppresses Induction of TNF-α in
`Experimental Colitis in Rats-New Molecular Implication for Therapy for
`Intestinal Mucosal Inflammation Beyond Acid Suppression
`Tetsuya Tanigawa, Toshio Watanabe, E.J. Sasaki, Masatsugu Shiba, Kazunari Tominaga,
`Yasuhiro Fujiwara, Nobuhide Oshitani, Masahiko Nakamura, Kazuhide Higuchi, Tetsuo
`Arakawa
`
`Background and Aim: Enteric bacteria play a crucial role in the pathogenesis of inflammatory
`bowel diseases which are characterized by leukocytic infiltration and overexpression of
`cytokines in intestinal mucosa. Lansoprazole (LAN), a proton pump inhibitor, has been
`shown to exert anti-inflammatory effect other than inhibitory effect on acid secretion. We
`have previously demonstrated that LAN inhibits development of dextran sodium sulfate
`(DSS)-induced experimental colitis in rats. In this study we investigated molecular mechan-
`isms underlying preventive effect of LAN on colitis. Methods: Experiment (1) Experimental
`colitis was induced in male Wistar rats by administration of 3% DSS solution for 3 days.
`The rats were also orally given LAN (30 mg/kg BW) or vehicle from day 1 of experiment
`for 3 days. Colonic tissue was subjected to measurement of myeloperoxidase (MPO) activity
`(a marker of neutrophil infiltration), assay of mRNA level of tumor necrosis factor-α (TNFα)
`by real-time RT-PCR, and double-immunostaining with antibodies against TNFα and mono-
`cytes/macrophages. To clarify the uptake sites of LAN in the colonic tissues, 3H-labeled
`LAN (0.5 mCi/100g BW) was given through intraaortic catheter and localization of 3H-LAN
`
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