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Role of PPAR-Г in a murine experimental colitis
A1360 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
6188
6190
VARIOUS AUTOIMMUNE ABNORMALITIES IN A PATIENT WITH CROHN'S DISEASE. Hideto Motegi, Etsuo Hoshino, Makoto Tatewaki, Daisuke Suzuki, Takaharu Ohbayashi, Tohru Ohse, Tomohide Sato, Miki Adachi, Kouta Okinaga, Tetsuo Imamura, Junji Shiga, Masayosi Kimura, Teikyo Univ Sch of Medicine, Tokyo, Japan; Sumitomo Corp Clin, Tokyo, Japan. AIM: In this paper we present a patient who developed various autoimmune abnormalities during several years after the onset of Crohn's disease. The aim of this case report is to add an evidence for the autoimmune nature of this disease. CASE REPORT: A 32 year old female was diagnosed to have Crohn's disease 7 years ago, when she developed multiple internal and external fistulae in the ileum and underwent partial ileal resection. Her younger sister had Crohn's ileocolitis. She had been well on low dose elemental diet until 3 years ago, when she developed amenorrhea. During 6 months thereafter, arthritis, hyperthyroidism (TSH: 0.09 ILIV/ml, free T4: 10.5 ng/dl, free T3:18.4 pg/ml), hyper-v-globulinemia (IgG: 4030 mg/dl), and abnormal liver function emerged one after another. She was hospitalized because of hepatic failure (albumin: 2.7 g/dl, AST: 658 IV, ALT: 696 IU, LDH: 1126 IV, total bilirubin: 2.7 mg/dl, prothrombin time: 31%, ANA: 1280x). Liver biopsy performed on the 14th hospital day revealed inflammation and fibrosis of the Glisson's capsules with piecemeal necrosis. Chest X-ray and CT showed interstitial shadows and lung biopsy revealed mild pulmonary fibrosis. Serum immune complex level was increased and compliments were decreased. Prednisolone 100 mg/day was started on the 3rd hospital day. Laboratory data including liver function tests and histology were restored and various immunological abnormalities gradually improved. She was discharged on the 42nd hospital day. But because liver function got worse again in 2 months, azathioprine 100 mg/day was administered. She had been well until 18 months ago, when she developed skin lesions resembling lichen planus which gradually changed into vitiligo vulgaris. DISCUSSION and CONCLUSION: Extraintestinal manifestations of Crohn' s disease such as skin, joint or pancreatic lesions are not rare. Sporadic cases with a single immunological disorder have been reported. But literature search yielded no report of a case of Crohn's disease who developed multiple immunological abnormalities. This case strongly suggests that Crohn's disease is an autoimmune disease.
IN SITU CHARACTERIZATION OF MACROPHAGE LINEAGE CELLS AND DENDRITIC CELLS AS ANTIGEN PRESENTING CELLS IN INTESTINAL TISSUE OF PATIENTS WITH CROHN'S DISEASE. Shiro Nakamura, Yoshio Jinno, Takayuki Matsumoto, Yoshinori Sawa, Jun-ichi Hara, Nobuhide Oshitani, Tetsuo Arakawa, Yukio Nishiguchi, Kousei Hirakawa, Haruo Otani, Anne Saaristo, Hiroshi Nagura, Dept Med III, Osaka City Univ Med Sch, Osaka, Japan; Osaka City Univ Med Sch, Osaka, Japan; Tohoku Univ Sch of Medicine, Sendai, Japan; Univrsity of Helsinki, Helsinki, Finland. Backgrounds & Aims: Crohn's disease is associated with granulomatous inflammation featuring immunological abnormalities in macrophages. We have reported that granuloma cells strongly expressed CD80/86 and may act as antigen presenting cells (APC) for disease related antigens (Hara J, et al. Lab Invest. 1997). The aim of the present study was to characterize potential antigen presenting cells and dendritic cells and their migration pathway in gastrointestinal tissues of patients with Crohn's disease. Subjects & Methods: Twenty surgical specimens of Crohn's disease were promptly fixed with 2% PLP solution and offered for the single and double color immunostaining. Mesenteric lymph nodes (MLN), resected specimens from ulcerative colitis (UC) were also treated as above. Monoclonal antibody against vascular endothelial growth factor receptor 3 (VEGFR-3) was used as a marker of lymphatic epithelium (Lymboussaki A, et al. Am J Pathol, 1998) and anti-PAL-E was used as a marker of vascular endothelium. Results: In actively inflamed intestine of Crohn' s disease, significant increase in number of dilated lymphatic vessels positive for VEGFR-3 but not for PAL-E was observed compared with that in UC or in the controls. Noncaseating granulomas were often observed adjacent or around the VEGFR-3+ lymphatic vessels. In such condition, aggregates of CD68+, HLA-DR+, CD86+ activated macrophages were often observed in the VEGFR-3+ lymphatic vessels. Most of them simultaneously expressed CDI4, CD40, CD56, CDlla, CDllb and CDllc. This phenotype is common to epithelioid cells and multinucleated giant cells in granuloma. Furthermore, about 30% of the above mentioned cells expressed CD83. Immunoelectron microscopy revealed close cell to cell contact at the surface of granuloma cells expressing CD86 to CD4 T cells. These findings were not observed in UC. Although they did not show dendritic morphology at light microscopy level, their phenotypic features were almost same as that of functional dendritic cells with professional antigen prsenting activity. Therefore, T cells activated at these location may recruit to the intestinal wall and elicit Crohn's disease specific inflammatory reactions. Conclusions: These findings suggest that macrophage lineage cells in the lymphatic vessels were activated by certain antigens inducing granuloma formation, and play important roles as functional dendritic cells to activate CD4 T cells in Crohn' s disease.
6189 A NOVEL INHIBITOR OF INDUCIBLE NITRIC OXIDE SYNTHASE, ONO-I714, REDUCES DEXTRAN SODIUM SULFATEINDUCED COLITIS IN MICE. Yuji Naito, Toshikazu Yoshikawa, Tomohisa Takagi, Osamu Handa, Takeshi Ishikawa, Naohisa Matsumoto, Kiichi Matsuyama, Nobuaki Vagi, Norimasa Yoshida, Motoharu Kondo, Kyoto Prefectural Univ of Medicine, Kyoto, Japan. Background: The expression of inducible nitric oxide synthase(iNOS) is upregulated in inflammatory bowel diseases (IBD), but the role of nitric oxide (NO) in colitis is poorly understood. The present study employed a novel synthetic inhibitor of iNOS, ONO-1714, in mice to evaluate the effect of NO on dextran sodium sulfate (DSS)-induced colitis. Methods: Acute colitis was induced by DSS administration at 8 % w/v in the drinking water in BALB/c female mice. ONO-1714 (IC50 for mouse macrophage: 19 nM) dissolved in physiological saline (0.03, 0.1, 0.3 mg/kg) was intraperitoneally injected everyday for drinking DSS. Length of the colon, body weight, myeloperoxidase (MPO) activity, and thiobarbituric acidreactive substances (TBARS) in homogenates of colon mucosa were measured 7 days after DSS administration. Measurement of serum NOx by Griss method and immunohistochemical detection of iNOS protein and nitrotyrosine, an index of peroxynitrite formation, was also performed. Results: Histological findings of acute inflammation was significantly reduced by the injection of ONO-1714. The decreases in body weight and length of the colon were reversed by ONO-1714 treatment. ~e increases in MPO activity and TBARS preceded the development of colitis and these increases were also significantly inhibited by ONO-1714. The treatment with ONO-1714 significantly inhibited the increase in serum NOx levels and the nitrotyrosine formation in the inflamed colonic mucosa, but did not affect the expression of iNOS mRNA. Conclusion: These results suggest that NO or nitrotyrosine derived from iNOS mediates, in part, neutrophil accumulation and lipid peroxidation during the development of DSSinduced colitis in mice, and that the selective inhibition of iNOS protein is a candidate for the therapy of IBD.
6191 THE EFFECT OF GUANABENZ ON AN EXPERIMENTAL COLITIS IN RAT. Hiroshi Nakamura, Michihiro Oya, Masahisa Onomura, Kazuhito Fukuda, Masanobu Kodama, Yuichiro Yamada, Yutaka Seino, Faculty of Medicine, Kyoto Univ, Kyoto, Japan. Inducible nitric oxide synthase (iNOs) inhibitors have a therapeutic effect on experimental colitis and expected to be a new medicine to inflammatory bowel disease. However, they are developing now. Guanabenz, which has been currently used for hypertension, includes an iNOs inhibitor aminoguanidine and inhibits NOs activity. Therefore, we hypothesized that guanabenz would ameliorate damages of experimental colitis. Materials & Methods: Male Wistar rats (n = 16) were orally administered guanabenz (10 ILMlkg/day) or distilled water every day. After 3 days, the rats were induced colitis with 0.25 ml of 10 mg trinitrobenzene suiphonic acid (TNB) /50 % ethanol. Seven days after the induction of colitis, the rats were killed to assess the effect of guanabenz. Results: Guanabenz significantly ameliorated the macro damage score (guanabenz vs. control, 4.88 ±0.74 vs. 8.38 ±0.27), the micro damage score (8.00 ±0.57 vs. 9.38 ±0.27), ulcer area (2.98 ±0.62 vs. 5.24 ±0.67 cm2), decreased myeloperoxidase activity (171.9 ±22.7 vs. 247.5 ±25.5 units/g tissue), NOs activity (502.5 ±38.0 vs. 605.2 ±27.3 cpm/g tissue), and iNOs activity (495.7 ±29.9 vs. 592.8 ±27.l cpm/g tissue). Conclusions: We considered that guanabenz prevented mucosal damage in TNB colitis by inhibition of iNOs and could be tried to use for inflammatory bowel disease immediately.
6192 ROLE OF PPAR·r IN A MURINE EXPERIMENTAL COLITIS. Tsutomu Nishiyama, Keiichi Mitsuyama, Atsushi Toyonaga, Kosuke Takagi, Nobuo Tomiyasu, Yutaka Matsui, Asuka Suzuki, Michio Sata, Kurume Univ Sch of Medicine, Kurume, Japan. BACKGROUND: Peroxisome proliferator-activated receptor-')I(PPAR-y) is a newly identified nuclear hormone receptor, yet its endogenous ligands and functions are still undefined. Recently, the high level of expression of PPAR-yin colonic mucosa and its in vitro anti-inflammatory effects have suggested the important role of this receptor in the regulation of intestinal inflammation. Nothing is known, however, about the role of PPAR-yin animal models of experimental colitis. We hypothesized that PPAR-yis involved in the pathogenesis of experimental colitis through its ability to control the inflammatory process within the intestinal wall. METHODS:
AGAA1361
April 2000
8-week-old female Balb/c mice, weighing 25-30 g, were housed in standard, wire-mesh cages. The mice were fed laboratory-pelleted formula and colitis was induced by drinking water supplemented with 4% dextran sulfate sodium (DSS; molecular mass = 40 kDa) ad libitum for 13 days. To evaluate the anti-inflammatory effect of PPAR-y, animals received daily feed containing troglitazone (200 mg/kg/day, n = 10), an agonist of PPAR-y, or vehicle (n = 10) from 2 days before the induction of colitis. Body weight, occult blood of feces, gross bleeding per rectum and survival rate were checked every day. On day 13 the severity of colitis was quantified by clinical signs, colonic length, and blinded histologic grading. RESUL TS:/n vivo treatment with troglitazone resulted in marked improvements in various inflammatory indicators of DSS colitis, including mortality, weight loss, stool consistency, rectal bleeding, colon length and histologic scores. CONCLUSIONS: PPAR-yactivation protected against colonic inflammation induced in mice by DSS. These results demonstrate that PPAR-yis a potential clinical candidate as a pharmacotherapeutic for the treatment of intestinal tissue damage in patients with inflammatory bowel disease.
6193 MUCOSAL IL-18 ACTIVITY IN INFLAMMATORY BOWEL DISEASE. Kazuo Nobata, Kenji Ina, Masaaki Shimada, Mitsuhiro Noshiro, Tomoyuki Tsuzuki, Masahiro Ohsuga, Yuji Nishio, Akira Imada, Takafumi Ando, Kazuo Kusugami, Nagoya Univ Sch of Medicine, Nagoya, Japan. Background and Aims: A novel cytokine, interleukin (IL)-18 has been reported to induce IFN-yproduction; however, its relevance to the mucosal immunological abnormality remains to be clarified. Our purpose was to investigate the role of IL-18 in inflammatory bowel disease (IBD) patients. Material and method: Colonic mucosal tissues were obtained from colonoscopic biopsies or surgical specimens in ulcerative colitis (UC, n= 15), Crohn's disease (CD, n=lO) and control patients (colon cancer; n=9, colon polyp; n=4), and cultured in 48 hours on a culture insert. The localization of IL-18 mRNA was determined by in situ hybridization using specific RNA probes. Lamina propria mononuclear cells (LPMC) were isolated from surgical specimens by enzymatic method and cultured for 24 hours in the presence of anti-CD3 antibody and recombinant (r)I1·18 (0, I, 10, 100 ng/ml). The contents of IL-18 and IFN-y(pg/mg protein) in the culture supernatants were measured by ELISA. Results: Compared with control subjects (median 491, range 277-1610), both UC (median 1379, range 488-3306) and CD (median 2132, range 3619-5661) patients showed higher levels of IL-18 activity. In situ hybridization detected enhanced signals of IL-18 mRNA in epithelial cells and infiltrating macrophages in IBD specimens. IFN-ywas produced from LPMC by stimulation of rIL-18 in a dose-dependent manner and this production was found to be increased in CD. Conclusion: Mucosal IL-18 activity was increased in IBD patients and rIL-18 upregulated the IFN-yproduction from LPMC. These data suggest that enhanced IL-18 activity may be associated with abnormal local immune responses in IBD patients.
6194 THROMBOEMBOLISM IN INFLAMMATORY BOWEL DISEASE - PRELIMINARY RESULTS OF A CASE-CONTROL STUDY. Gottfried Novacek, Wolfgang Miehsler, Eva Valic, Wolfgang Tillinger, Walter Reinisch, Thomas Feichtenschlager, Susanna Linsbichler, Christophoros Konnaris, Christian Wolf, Harald Vogelsang, Univ of Vienna, Vienna, Austria; Lainz Hosp, Vienna, Austria; Rudolfstiftung Hosp, Vienna, Austria. Background: It has been reported that patients with inflammatory bowel disease (IBD) are at increased risk of thromboembolic complications (TE). However, the relative risk of TE in patients with IBD in comparison with control subjects is not known until now. Aims: To assess the prevalence and the relative risk of TE in patients with IBD in comparison with control subjects. Methods: Until now 513 IBD patients (Crohn s disease 411, ulcerative colitis 102; men 226, women 287; age (x±SD): 38± 13 yrs.) and 474 age- and sex-matched control subjects (men 209, women 265; age: 37± II yrs.) who underwent a preventive occupational health examination due to ionizing radiation exposure were asked for TE in the history. Only those TE were accepted which were documented by radiological procedures. Results: 31 IBD patients (6%) (Crohn s disease 21, ulcerative colitis 10; women 16, men 15) and 7 control subjects (1.5%) (woman I, men 6) had TE in the history (p
6195 THE EVALUATION OF BOWEL BLOOD FLOW IN CROHN'S DISEASE BY FLASH ECHO IMAGING. Hideharu Okanobu, Kenjiro Nakamura, Ken Haruma, Jiro Hata, Shyunji Matsumura, Shigeto Yoshida, Hiroaki Kusunoki, Shinnji Tanaka, Ysuhiko Kitadai, Masaharu Yoshihara, Kohji Sumii, Goro Kajiyama, Hiroshima, Japan. Background: Although the alteration " of bowel blood perfusion has been considered to play an important role in the pathogenesis of Crohn' s disease, non-invasive modality to evaluate the bowel blood perfusion has not been established. We have reported the canine study on the Flash Echo Imaging (FEI), which is the intermittent harmonic imaging under the administration of microbubble contrast agent (Levovist), as the noninvasive modality for the evaluation of gastrointestinal blood perfusion (Gastroenterology A947, 1999). The AIM of this study was to evaluate the bowel blood flow of human subjects by FEI. Patients and Methods: Three patients with Crohn's disease were enrolled in this study. An SSA-390A ultrasound system (Toshiba, Japan) with 2.3MHz convex array probe was used for FEI. After the intravenous injection of Levovist, second harmonics (4.6MHz) emitted from microbubble were obtained to enhance the B-mode images. The intermittent scan with 3 shots for each Flash was performed every 4 seconds. Since microbubbles are fragile with respect to the ultrasound beams, scanning by multiple emission of the sonographic beam with a very short interval (30 msec.) provides the enhanced image (I st frame) and the plain image (3 rd frame). We subtracted the 3rd frame from the lst frame to obtain the blood flow images, and measured the mean brightness of the range of interest in the subtracted images to draw the time intensity curve (TIC). Sonographic results were compared with the disease activity desided by other parameters including endoscopy. Results: The results are shown in Fig. 1. The TIC of case2, who had more active bowel lesion and less preferable clinical course than those of other 2 cases, showed higher level of Peak Intensity and prolonged T112. Conclusion: The FEI provides the detailed information about the bowel blood perfusion in Crohn' s disease, which leads to the better understanding of disease pathogenesis and the better therapeutic strategies.
6196 YOGHURT ENEMAS FOR CONTROL OF INTRACTABLE CLOSTRIDIUM DIFFICILE COLITIS. Cathal OKeeffe, Suzanne Corcoran, Tara McBride, Diarmuid ODonoghue, St Vincent's Univ Hosp, Dublin, Ireland; Acad Univ Hosp, Dublin, Ireland. Clostridium difficile is a common and potentially fatal cause of antibiotic associated enterocolitis in hospitalised patients. Relapsing and resistant Clostridium difficile colitis (CdC) has enormous implications in terms of patient comfort, nursing care, infection control and health service costs. Oral probiotics, faecal enemas and iv pooled immunoglobulin (Ig) have all been advocated in cases resistant to antibiotics. We report here the use of rectal administration of probiotics (bioactive yogurt) in a patient with intractable CdC and our subsequent experience in a small cohort of patients. A 57 year old woman admitted with an intra-cerebral bleed remained in a persistant vegetative state and dependent on full nursing care. CdC developed after treatment with cephalosporins and severe diarrhoea continued despite courses of Metronidazole and Vancomycin. Enteral probiotics were of no avail but iv pooled Ig led to an immediate but short-lived response. The administration of 50 mls of commercially available bioactive yoghurt 3 times a day by rectal tube resulted in a gradual but complete resolution of diarrhoea and clearance of Clostridium difficile toxin from the stool. This response was maintained for the duration of treatment (3 months) but toxin positive diarrhoea recurred after discontinuation. Reintroduction of the enemas led to clinical remission. Six additional patients with relapsing CdC were treated in a similar manner: 4 responded well, I could not tolerate the treatment and I died from an unrelated cause without resolution of symptoms. Conciusion:Rectal administration of probiotics would appear to be an effective management of resistant CdC. Although it may not result in the clearance of infection it provides a cheap,simple and effective means of symptom control for a condition that can be distressing for patients and carers alike.
GASTROENTEROLOGY Vol. 118, No.4
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6190
VARIOUS AUTOIMMUNE ABNORMALITIES IN A PATIENT WITH CROHN'S DISEASE. Hideto Motegi, Etsuo Hoshino, Makoto Tatewaki, Daisuke Suzuki, Takaharu Ohbayashi, Tohru Ohse, Tomohide Sato, Miki Adachi, Kouta Okinaga, Tetsuo Imamura, Junji Shiga, Masayosi Kimura, Teikyo Univ Sch of Medicine, Tokyo, Japan; Sumitomo Corp Clin, Tokyo, Japan. AIM: In this paper we present a patient who developed various autoimmune abnormalities during several years after the onset of Crohn's disease. The aim of this case report is to add an evidence for the autoimmune nature of this disease. CASE REPORT: A 32 year old female was diagnosed to have Crohn's disease 7 years ago, when she developed multiple internal and external fistulae in the ileum and underwent partial ileal resection. Her younger sister had Crohn's ileocolitis. She had been well on low dose elemental diet until 3 years ago, when she developed amenorrhea. During 6 months thereafter, arthritis, hyperthyroidism (TSH: 0.09 ILIV/ml, free T4: 10.5 ng/dl, free T3:18.4 pg/ml), hyper-v-globulinemia (IgG: 4030 mg/dl), and abnormal liver function emerged one after another. She was hospitalized because of hepatic failure (albumin: 2.7 g/dl, AST: 658 IV, ALT: 696 IU, LDH: 1126 IV, total bilirubin: 2.7 mg/dl, prothrombin time: 31%, ANA: 1280x). Liver biopsy performed on the 14th hospital day revealed inflammation and fibrosis of the Glisson's capsules with piecemeal necrosis. Chest X-ray and CT showed interstitial shadows and lung biopsy revealed mild pulmonary fibrosis. Serum immune complex level was increased and compliments were decreased. Prednisolone 100 mg/day was started on the 3rd hospital day. Laboratory data including liver function tests and histology were restored and various immunological abnormalities gradually improved. She was discharged on the 42nd hospital day. But because liver function got worse again in 2 months, azathioprine 100 mg/day was administered. She had been well until 18 months ago, when she developed skin lesions resembling lichen planus which gradually changed into vitiligo vulgaris. DISCUSSION and CONCLUSION: Extraintestinal manifestations of Crohn' s disease such as skin, joint or pancreatic lesions are not rare. Sporadic cases with a single immunological disorder have been reported. But literature search yielded no report of a case of Crohn's disease who developed multiple immunological abnormalities. This case strongly suggests that Crohn's disease is an autoimmune disease.
IN SITU CHARACTERIZATION OF MACROPHAGE LINEAGE CELLS AND DENDRITIC CELLS AS ANTIGEN PRESENTING CELLS IN INTESTINAL TISSUE OF PATIENTS WITH CROHN'S DISEASE. Shiro Nakamura, Yoshio Jinno, Takayuki Matsumoto, Yoshinori Sawa, Jun-ichi Hara, Nobuhide Oshitani, Tetsuo Arakawa, Yukio Nishiguchi, Kousei Hirakawa, Haruo Otani, Anne Saaristo, Hiroshi Nagura, Dept Med III, Osaka City Univ Med Sch, Osaka, Japan; Osaka City Univ Med Sch, Osaka, Japan; Tohoku Univ Sch of Medicine, Sendai, Japan; Univrsity of Helsinki, Helsinki, Finland. Backgrounds & Aims: Crohn's disease is associated with granulomatous inflammation featuring immunological abnormalities in macrophages. We have reported that granuloma cells strongly expressed CD80/86 and may act as antigen presenting cells (APC) for disease related antigens (Hara J, et al. Lab Invest. 1997). The aim of the present study was to characterize potential antigen presenting cells and dendritic cells and their migration pathway in gastrointestinal tissues of patients with Crohn's disease. Subjects & Methods: Twenty surgical specimens of Crohn's disease were promptly fixed with 2% PLP solution and offered for the single and double color immunostaining. Mesenteric lymph nodes (MLN), resected specimens from ulcerative colitis (UC) were also treated as above. Monoclonal antibody against vascular endothelial growth factor receptor 3 (VEGFR-3) was used as a marker of lymphatic epithelium (Lymboussaki A, et al. Am J Pathol, 1998) and anti-PAL-E was used as a marker of vascular endothelium. Results: In actively inflamed intestine of Crohn' s disease, significant increase in number of dilated lymphatic vessels positive for VEGFR-3 but not for PAL-E was observed compared with that in UC or in the controls. Noncaseating granulomas were often observed adjacent or around the VEGFR-3+ lymphatic vessels. In such condition, aggregates of CD68+, HLA-DR+, CD86+ activated macrophages were often observed in the VEGFR-3+ lymphatic vessels. Most of them simultaneously expressed CDI4, CD40, CD56, CDlla, CDllb and CDllc. This phenotype is common to epithelioid cells and multinucleated giant cells in granuloma. Furthermore, about 30% of the above mentioned cells expressed CD83. Immunoelectron microscopy revealed close cell to cell contact at the surface of granuloma cells expressing CD86 to CD4 T cells. These findings were not observed in UC. Although they did not show dendritic morphology at light microscopy level, their phenotypic features were almost same as that of functional dendritic cells with professional antigen prsenting activity. Therefore, T cells activated at these location may recruit to the intestinal wall and elicit Crohn's disease specific inflammatory reactions. Conclusions: These findings suggest that macrophage lineage cells in the lymphatic vessels were activated by certain antigens inducing granuloma formation, and play important roles as functional dendritic cells to activate CD4 T cells in Crohn' s disease.
6189 A NOVEL INHIBITOR OF INDUCIBLE NITRIC OXIDE SYNTHASE, ONO-I714, REDUCES DEXTRAN SODIUM SULFATEINDUCED COLITIS IN MICE. Yuji Naito, Toshikazu Yoshikawa, Tomohisa Takagi, Osamu Handa, Takeshi Ishikawa, Naohisa Matsumoto, Kiichi Matsuyama, Nobuaki Vagi, Norimasa Yoshida, Motoharu Kondo, Kyoto Prefectural Univ of Medicine, Kyoto, Japan. Background: The expression of inducible nitric oxide synthase(iNOS) is upregulated in inflammatory bowel diseases (IBD), but the role of nitric oxide (NO) in colitis is poorly understood. The present study employed a novel synthetic inhibitor of iNOS, ONO-1714, in mice to evaluate the effect of NO on dextran sodium sulfate (DSS)-induced colitis. Methods: Acute colitis was induced by DSS administration at 8 % w/v in the drinking water in BALB/c female mice. ONO-1714 (IC50 for mouse macrophage: 19 nM) dissolved in physiological saline (0.03, 0.1, 0.3 mg/kg) was intraperitoneally injected everyday for drinking DSS. Length of the colon, body weight, myeloperoxidase (MPO) activity, and thiobarbituric acidreactive substances (TBARS) in homogenates of colon mucosa were measured 7 days after DSS administration. Measurement of serum NOx by Griss method and immunohistochemical detection of iNOS protein and nitrotyrosine, an index of peroxynitrite formation, was also performed. Results: Histological findings of acute inflammation was significantly reduced by the injection of ONO-1714. The decreases in body weight and length of the colon were reversed by ONO-1714 treatment. ~e increases in MPO activity and TBARS preceded the development of colitis and these increases were also significantly inhibited by ONO-1714. The treatment with ONO-1714 significantly inhibited the increase in serum NOx levels and the nitrotyrosine formation in the inflamed colonic mucosa, but did not affect the expression of iNOS mRNA. Conclusion: These results suggest that NO or nitrotyrosine derived from iNOS mediates, in part, neutrophil accumulation and lipid peroxidation during the development of DSSinduced colitis in mice, and that the selective inhibition of iNOS protein is a candidate for the therapy of IBD.
6191 THE EFFECT OF GUANABENZ ON AN EXPERIMENTAL COLITIS IN RAT. Hiroshi Nakamura, Michihiro Oya, Masahisa Onomura, Kazuhito Fukuda, Masanobu Kodama, Yuichiro Yamada, Yutaka Seino, Faculty of Medicine, Kyoto Univ, Kyoto, Japan. Inducible nitric oxide synthase (iNOs) inhibitors have a therapeutic effect on experimental colitis and expected to be a new medicine to inflammatory bowel disease. However, they are developing now. Guanabenz, which has been currently used for hypertension, includes an iNOs inhibitor aminoguanidine and inhibits NOs activity. Therefore, we hypothesized that guanabenz would ameliorate damages of experimental colitis. Materials & Methods: Male Wistar rats (n = 16) were orally administered guanabenz (10 ILMlkg/day) or distilled water every day. After 3 days, the rats were induced colitis with 0.25 ml of 10 mg trinitrobenzene suiphonic acid (TNB) /50 % ethanol. Seven days after the induction of colitis, the rats were killed to assess the effect of guanabenz. Results: Guanabenz significantly ameliorated the macro damage score (guanabenz vs. control, 4.88 ±0.74 vs. 8.38 ±0.27), the micro damage score (8.00 ±0.57 vs. 9.38 ±0.27), ulcer area (2.98 ±0.62 vs. 5.24 ±0.67 cm2), decreased myeloperoxidase activity (171.9 ±22.7 vs. 247.5 ±25.5 units/g tissue), NOs activity (502.5 ±38.0 vs. 605.2 ±27.3 cpm/g tissue), and iNOs activity (495.7 ±29.9 vs. 592.8 ±27.l cpm/g tissue). Conclusions: We considered that guanabenz prevented mucosal damage in TNB colitis by inhibition of iNOs and could be tried to use for inflammatory bowel disease immediately.
6192 ROLE OF PPAR·r IN A MURINE EXPERIMENTAL COLITIS. Tsutomu Nishiyama, Keiichi Mitsuyama, Atsushi Toyonaga, Kosuke Takagi, Nobuo Tomiyasu, Yutaka Matsui, Asuka Suzuki, Michio Sata, Kurume Univ Sch of Medicine, Kurume, Japan. BACKGROUND: Peroxisome proliferator-activated receptor-')I(PPAR-y) is a newly identified nuclear hormone receptor, yet its endogenous ligands and functions are still undefined. Recently, the high level of expression of PPAR-yin colonic mucosa and its in vitro anti-inflammatory effects have suggested the important role of this receptor in the regulation of intestinal inflammation. Nothing is known, however, about the role of PPAR-yin animal models of experimental colitis. We hypothesized that PPAR-yis involved in the pathogenesis of experimental colitis through its ability to control the inflammatory process within the intestinal wall. METHODS:
AGAA1361
April 2000
8-week-old female Balb/c mice, weighing 25-30 g, were housed in standard, wire-mesh cages. The mice were fed laboratory-pelleted formula and colitis was induced by drinking water supplemented with 4% dextran sulfate sodium (DSS; molecular mass = 40 kDa) ad libitum for 13 days. To evaluate the anti-inflammatory effect of PPAR-y, animals received daily feed containing troglitazone (200 mg/kg/day, n = 10), an agonist of PPAR-y, or vehicle (n = 10) from 2 days before the induction of colitis. Body weight, occult blood of feces, gross bleeding per rectum and survival rate were checked every day. On day 13 the severity of colitis was quantified by clinical signs, colonic length, and blinded histologic grading. RESUL TS:/n vivo treatment with troglitazone resulted in marked improvements in various inflammatory indicators of DSS colitis, including mortality, weight loss, stool consistency, rectal bleeding, colon length and histologic scores. CONCLUSIONS: PPAR-yactivation protected against colonic inflammation induced in mice by DSS. These results demonstrate that PPAR-yis a potential clinical candidate as a pharmacotherapeutic for the treatment of intestinal tissue damage in patients with inflammatory bowel disease.
6193 MUCOSAL IL-18 ACTIVITY IN INFLAMMATORY BOWEL DISEASE. Kazuo Nobata, Kenji Ina, Masaaki Shimada, Mitsuhiro Noshiro, Tomoyuki Tsuzuki, Masahiro Ohsuga, Yuji Nishio, Akira Imada, Takafumi Ando, Kazuo Kusugami, Nagoya Univ Sch of Medicine, Nagoya, Japan. Background and Aims: A novel cytokine, interleukin (IL)-18 has been reported to induce IFN-yproduction; however, its relevance to the mucosal immunological abnormality remains to be clarified. Our purpose was to investigate the role of IL-18 in inflammatory bowel disease (IBD) patients. Material and method: Colonic mucosal tissues were obtained from colonoscopic biopsies or surgical specimens in ulcerative colitis (UC, n= 15), Crohn's disease (CD, n=lO) and control patients (colon cancer; n=9, colon polyp; n=4), and cultured in 48 hours on a culture insert. The localization of IL-18 mRNA was determined by in situ hybridization using specific RNA probes. Lamina propria mononuclear cells (LPMC) were isolated from surgical specimens by enzymatic method and cultured for 24 hours in the presence of anti-CD3 antibody and recombinant (r)I1·18 (0, I, 10, 100 ng/ml). The contents of IL-18 and IFN-y(pg/mg protein) in the culture supernatants were measured by ELISA. Results: Compared with control subjects (median 491, range 277-1610), both UC (median 1379, range 488-3306) and CD (median 2132, range 3619-5661) patients showed higher levels of IL-18 activity. In situ hybridization detected enhanced signals of IL-18 mRNA in epithelial cells and infiltrating macrophages in IBD specimens. IFN-ywas produced from LPMC by stimulation of rIL-18 in a dose-dependent manner and this production was found to be increased in CD. Conclusion: Mucosal IL-18 activity was increased in IBD patients and rIL-18 upregulated the IFN-yproduction from LPMC. These data suggest that enhanced IL-18 activity may be associated with abnormal local immune responses in IBD patients.
6194 THROMBOEMBOLISM IN INFLAMMATORY BOWEL DISEASE - PRELIMINARY RESULTS OF A CASE-CONTROL STUDY. Gottfried Novacek, Wolfgang Miehsler, Eva Valic, Wolfgang Tillinger, Walter Reinisch, Thomas Feichtenschlager, Susanna Linsbichler, Christophoros Konnaris, Christian Wolf, Harald Vogelsang, Univ of Vienna, Vienna, Austria; Lainz Hosp, Vienna, Austria; Rudolfstiftung Hosp, Vienna, Austria. Background: It has been reported that patients with inflammatory bowel disease (IBD) are at increased risk of thromboembolic complications (TE). However, the relative risk of TE in patients with IBD in comparison with control subjects is not known until now. Aims: To assess the prevalence and the relative risk of TE in patients with IBD in comparison with control subjects. Methods: Until now 513 IBD patients (Crohn s disease 411, ulcerative colitis 102; men 226, women 287; age (x±SD): 38± 13 yrs.) and 474 age- and sex-matched control subjects (men 209, women 265; age: 37± II yrs.) who underwent a preventive occupational health examination due to ionizing radiation exposure were asked for TE in the history. Only those TE were accepted which were documented by radiological procedures. Results: 31 IBD patients (6%) (Crohn s disease 21, ulcerative colitis 10; women 16, men 15) and 7 control subjects (1.5%) (woman I, men 6) had TE in the history (p
6195 THE EVALUATION OF BOWEL BLOOD FLOW IN CROHN'S DISEASE BY FLASH ECHO IMAGING. Hideharu Okanobu, Kenjiro Nakamura, Ken Haruma, Jiro Hata, Shyunji Matsumura, Shigeto Yoshida, Hiroaki Kusunoki, Shinnji Tanaka, Ysuhiko Kitadai, Masaharu Yoshihara, Kohji Sumii, Goro Kajiyama, Hiroshima, Japan. Background: Although the alteration " of bowel blood perfusion has been considered to play an important role in the pathogenesis of Crohn' s disease, non-invasive modality to evaluate the bowel blood perfusion has not been established. We have reported the canine study on the Flash Echo Imaging (FEI), which is the intermittent harmonic imaging under the administration of microbubble contrast agent (Levovist), as the noninvasive modality for the evaluation of gastrointestinal blood perfusion (Gastroenterology A947, 1999). The AIM of this study was to evaluate the bowel blood flow of human subjects by FEI. Patients and Methods: Three patients with Crohn's disease were enrolled in this study. An SSA-390A ultrasound system (Toshiba, Japan) with 2.3MHz convex array probe was used for FEI. After the intravenous injection of Levovist, second harmonics (4.6MHz) emitted from microbubble were obtained to enhance the B-mode images. The intermittent scan with 3 shots for each Flash was performed every 4 seconds. Since microbubbles are fragile with respect to the ultrasound beams, scanning by multiple emission of the sonographic beam with a very short interval (30 msec.) provides the enhanced image (I st frame) and the plain image (3 rd frame). We subtracted the 3rd frame from the lst frame to obtain the blood flow images, and measured the mean brightness of the range of interest in the subtracted images to draw the time intensity curve (TIC). Sonographic results were compared with the disease activity desided by other parameters including endoscopy. Results: The results are shown in Fig. 1. The TIC of case2, who had more active bowel lesion and less preferable clinical course than those of other 2 cases, showed higher level of Peak Intensity and prolonged T112. Conclusion: The FEI provides the detailed information about the bowel blood perfusion in Crohn' s disease, which leads to the better understanding of disease pathogenesis and the better therapeutic strategies.
6196 YOGHURT ENEMAS FOR CONTROL OF INTRACTABLE CLOSTRIDIUM DIFFICILE COLITIS. Cathal OKeeffe, Suzanne Corcoran, Tara McBride, Diarmuid ODonoghue, St Vincent's Univ Hosp, Dublin, Ireland; Acad Univ Hosp, Dublin, Ireland. Clostridium difficile is a common and potentially fatal cause of antibiotic associated enterocolitis in hospitalised patients. Relapsing and resistant Clostridium difficile colitis (CdC) has enormous implications in terms of patient comfort, nursing care, infection control and health service costs. Oral probiotics, faecal enemas and iv pooled immunoglobulin (Ig) have all been advocated in cases resistant to antibiotics. We report here the use of rectal administration of probiotics (bioactive yogurt) in a patient with intractable CdC and our subsequent experience in a small cohort of patients. A 57 year old woman admitted with an intra-cerebral bleed remained in a persistant vegetative state and dependent on full nursing care. CdC developed after treatment with cephalosporins and severe diarrhoea continued despite courses of Metronidazole and Vancomycin. Enteral probiotics were of no avail but iv pooled Ig led to an immediate but short-lived response. The administration of 50 mls of commercially available bioactive yoghurt 3 times a day by rectal tube resulted in a gradual but complete resolution of diarrhoea and clearance of Clostridium difficile toxin from the stool. This response was maintained for the duration of treatment (3 months) but toxin positive diarrhoea recurred after discontinuation. Reintroduction of the enemas led to clinical remission. Six additional patients with relapsing CdC were treated in a similar manner: 4 responded well, I could not tolerate the treatment and I died from an unrelated cause without resolution of symptoms. Conciusion:Rectal administration of probiotics would appear to be an effective management of resistant CdC. Although it may not result in the clearance of infection it provides a cheap,simple and effective means of symptom control for a condition that can be distressing for patients and carers alike.