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The gut microbiota affects intestinal permeability and mucosal mast cells (MMCs) responses. Activation of MMCs has been associated with absorption of dietary fat. We investigated whether the gut microbiota contributes to the fat-induced activation of MMCs in rats, and how antibiotics might affect this process.Adult male Sprague-Dawley rats were given streptomycin and penicillin for 4 days (n = 6-8) to reduce the abundance of their gut flora, or normal drinking water (controls, n = 6-8). They underwent lymph fistula surgery and after an overnight recovery were given an intraduodenal bolus of intralipid. We collected intestinal tissues and lymph fluid and assessed activation of MMCs, intestinal permeability, and fat transport parameters.Compared with controls, intestinal lymph from rats given antibiotics had reduced levels of mucosal mast cell protease II (produced by MMCs) and decreased activity of diamine oxidase (produced by enterocytes) (P.05). Rats given antibiotics had reduced intestinal permeability in response to dietary lipid compared with controls (P.01). Unexpectedly, antibiotics also reduced lymphatic transport of triacylglycerol and phospholipid (P.01), concomitant with decreased levels of mucosal apolipoproteins B, A-I, and A-IV (P.01). No differences were found in intestinal motility or luminal pancreatic lipase activity between rats given antibiotics and controls. These effects were not seen with an acute dose of antibiotics or 4 weeks after the antibiotic regimen ended.The intestinal microbiota appears to activate MMCs after the ingestion of fat in rats; this contributes to fat-induced intestinal permeability. We found that the gut microbiome promotes absorption of lipid, probably by intestinal production of apolipoproteins and secretion of chylomicrons.

Background & Aims: Activation of natural killer T cells with the synthetic ligand α-galactosylceramide (α-GalCer) induced hepatotoxicity through the tumor necrosis factor (TNF) and Fas-ligand–mediated pathway in aged mice. The aim of this study was to elucidate how α-GalCer–activated natural killer T cells function in hepatocyte proliferation and liver regeneration in partially hepatectomized (PHx) mice. Methods: Mice were injected with α-GalCer at 36 hours after 70% PHx. Hepatocyte mitosis was evaluated by either mitotic figures or proliferating cell nuclear antigen staining. The role of TNF and Fas-ligand in hepatocyte mitosis also was assessed. Results: In PHx mice injected with α-GalCer, hepatocyte mitosis was greatly enhanced at 44 hours after surgery and the increase was more obvious in aged mice than in young mice. The expression of both TNF receptor 1 and Fas-ligand in liver natural killer T cells tended to increase after α-GalCer injection in PHx mice. Treatment of mice with anti-NK1.1 Ab 3 days before and just after hepatectomy greatly inhibited the effect of α-GalCer on hepatocyte mitosis and liver regeneration. Furthermore, pretreatment of PHx mice with either anti-TNF Ab or anti-FasL Ab 1 hour before α-GalCer injection mostly abrogated the increase in hepatocyte proliferation. α-GalCer injection did not accelerate hepatocyte proliferation in Fas-mutated lpr mice after PHx. CD1d-/- mice without α-GalCer injection showed decreased hepatocyte mitosis after PHx. Conclusions: Activated natural killer T cells help hepatocyte proliferation and liver regeneration after PHx via the TNF and Fas/Fas-ligand–mediated pathway.

Background & Aims: Although nitric oxide (NO) is known to influence the recruitment of neutrophils in inflamed tissue, its role in lymphocyte-endothelial cell interactions remains poorly understood. The objectives of this study were to assess the effects of NO synthesis inhibition on T-lymphocyte migration in microvessels of rat small intestine and to define the role of adhesion molecules in this process. Methods: T lymphocytes collected from rat intestinal lymph were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein of recipient rats. The migration of T lymphocytes into normal and N G -nitro-L-arginine methyl ester (L-NAME)-treated intestinal microvessels was monitored by using an intravital microscope. Results: L-NAME significantly increased rolling and adherence of lymphocytes in postcapillary venules of Peyer's patches and submucosal venules without significantly decreasing red blood cell velocity. The subsequent appearance of lymphocytes in the initial lymphatics was also accelerated by L-NAME. Anti–α4-integrin antibody markedly inhibited the L-NAME–induced lymphocyte-endothelial cell interaction. Anti–P-selectin monoclonal antibody also significantly attenuated these adhesive interactions in both vascular regions. Conclusions: These data suggest that NO is an important modulator of lymphocyte migration in Peyer's patches and in nonlymphoid regions of the intestine. GASTROENTEROLOGY 1998;115:618-627

BACKGROUND & AIMS: The precise pathogenic significance of oxidative injury in the evolution of alcohol-induced liver disease is still obscure. The present report was designed to investigate whether ethanol alters the production of active oxidants and biological activities of hepatocytes. METHODS: The following parameters in rat hepatocytes were investigated by using fluorescence probes in vitro and ex vivo: (1) mitochondrial membrane potential and membrane permeability transition, (2) oxygen radicals generation, (3) membrane barrier function, and (4) glutathione level. RESULTS: Ethanol (50 mmol/L) increased oxidative stress in hepatocytes and subsequently induced an increased mitochondrial permeability transition and a decreased membrane potential. These ethanol-induced alterations were attenuated by an inhibitor of alcohol dehydrogenase and an intracellular oxidant scavenger, whereas they were enhanced by diethyl maleic acid, a glutathione depletor. Ethanol plus diethyl maleic acid but not ethanol alone increased the number of hepatocytes with membrane barrier dysfunction. A continuous infusion of ethanol (50 mmol/L) increased oxidative stress and decreased mitochondrial membrane potential in the pericentral area of isolated perfused rat liver. CONCLUSIONS: Active oxidants generated during ethanol metabolism increase mitochondrial permeability transition and modulate mitochondrial energy synthesis in hepatocytes. Reduction of glutathione level enhances mitochondrial dysfunction and impairs membrane barrier function of hepatocytes. (Gastroenterology 1997 Apr;112(4):1331-43)

BACKGROUND & AIMS: Although dietary fat is known to modulate immune functions, there is a paucity of data concerning the effects of fat absorption on migration of lymphocytes in intestinal lymphoid tissue. The objective of this study was to assess the influence of fat absorption on T-lymphocyte migration in Peyer's patches. METHODS: T lymphocytes collected from rat intestinal lymph were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein of recipient rats. Olive oil or octanoic acid was placed into the duodenum, and Peyer's patch microcirculation was observed by intravital fluorescence microscopy. RESULTS: Rolling and adherence of lymphocytes in postcapillary venules were noted in both fasted and fed rats. However, lymphocyte adherence was significantly greater in olive oil-fed rats than in fasted rats. Olive oil also significantly increased the transendothelial migration of T lymphocytes. Octanoic acid stimulated lymphocyte rolling but did not affect lymphocyte adherence. Olive oil enhanced the expression of both alpha4-integrin and L-selectin on lymphocytes, whereas octanoic acid only increased the expression of L-selectin. CONCLUSIONS: Lymphocyte rolling and adherence are enhanced by olive oil feeding, possibly through an interaction between activated adhesion molecules on lymphocytes and counter- receptors on endothelial cells. (Gastroenterology 1997 Mar;112(3):813-25)

BACKGROUND & AIMS: Evidence increasingly indicates that nitric oxide plays an important role in antitumor mechanisms. The aim of this study was to investigate the role of NO in the mechanisms regulating the proliferation and death of hepatoma cells cocultured with Kupffer cells. METHODS: Kupffer cells were isolated from male Wistar rats and cocultured with rat hepatoma AH70 cells. Proliferation was determined by calculating the number of total and 5-bromodeoxyuridine-positive AH70 cells. Apoptosis was assessed by electron-microscopic and fluorescence-microscopic observations and in situ nick end labeling method. Immunofluorescence and in situ hybridization studies were performed to investigate the induction of inducible NO synthase (iNOS). RESULTS: Kupffer cells reduced proliferation and induced apoptosis of AH70 cells, which were attenuated by the NO synthesis inhibitors NG- monomethyl-L-arginine and aminoguanidine. Increased inductions of iNOS messenger RNA and iNOS were observed in Kupffer cells cocultured with AH70 cells. Addition of monoclonal antibody directed against either rat CD18 or intercellular adhesion molecule 1 also attenuated the increased NO production of Kupffer cells and the alterations of AH70 cells. CONCLUSIONS: Kupffer cell-derived NO suppresses proliferation and induces apoptosis of hepatoma cells. The CD18 intercellular adhesion molecule 1-dependent adhesive interaction with hepatoma cells triggers NO production by Kupffer cells. (Gastroenterology 1996 Oct;111(4):1058-70)

Background & Aims: Nitric oxide is now recognized to regulate immune responses and cell viability in various organs. The present study was designed to clarify whether NO released from Kupffer cells modulates the lymphokine-activated killer (LAK) activity of interleukin 2 (IL-2)-treated splenocytes. Methods: Splenocytes and Kupffer cells were isolated from male Wistar rats and cocultured for 48 hours in the presence of lipopolysaccharide (1 μg/mL). The splenocyte LAK activity and expression of IL-2 receptor were determined. Results: Kupffer cells with lipopolysaccharide reduced the IL-2 receptor expression and LAK activity of splenocytes. The addition of either N G -monomethyl-l-arginine, an inhibitor of NO synthesis, or aminoguanidine, an inhibitor of inducible NO synthase, to the medium reversed the suppression of IL-2 receptor expression and LAK activity by lipopolysaccharide-stimulated Kupffer cells. 8-bromoguanosine 3′,5′-cyclic monophosphate and NO donors decreased the splenocyte LAK activity and IL-2 receptor expression. Treatment with lipopolysaccharide increased the inducible NO synthase activity as well as the nitrite and nitrate levels in the culture medium of Kupffer cells but not in splenocytes. Conclusions: The results of this study suggest that NO produced by the inducible NO synthase of Kupffer cells in response to lipopolysaccharide modulates the IL-2 receptor expression and LAK activity of splenocytes.

Background/Aims: In the small intestine, lymphocytes migrate through Peyer's patches. The distribution of lymphatic microvessels in rat Peyer's patches and lymphocyte traffic through them were studied. Methods: Vital dyes were injected via a micropipette into the Peyer's patches tissue to fill lymphatic microvessels and to stain lymphocytes in lymphatic microvessels. Results: Direct microscopic observation revealed a dense plexus of lymphatic microvessels in the perifollicular and interfollicular areas. Injection of the dyes into the germinal center failed to delineate lymphatic microvessels. The lymphatic microvessels in the perifollicular area were filled with lymphocytes. Most lymphocytes in the perifollicular lymphatics stayed in the lymphatic microvessels. Some lymphocytes became detached and drained into lymphatic microvessels in the interfollicular areas. Lymphocytes then moved toward the submucosal lymphatics beneath the villi around the Peyer's patches. The interfollicular lymphatics did not display contractile activity but had valves. Opening and closing of valves was synchronized with the respiration and the back and forth flow of lymphocytes. Conclusions: There are numerous lymphocytes in a dense lymphatic network in the perifollicular and interfollicular areas of Peyer's patches. This welldeveloped lymphatic network has the potential capacity for storage of lymphocytes and modulation of lymphocyte migration.

Introduction Anti-adhesion molecule therapy is useful for inflammatory bowel diseases. Smoking has been reported to have a harmful effect on Crohn's disease, but it is still controversial in ulcerative colitis. Although effect of nicotine on adhesion molecules of cardiovascular system has been well recognized, it has not been studied in the intestine. We investigated effect of nicotine on murine colitis induced by dextran sodium sulfate (DSS) and compared expression of adhesion molecules in vitro in several conditions including preconditioning exposure. Involvement of nicotinic acetylcholine receptor (nAChR) was also examined. Method In in vivo study C57BL/6J mice were treated with 3% DSS and 0.1mg/ ml nicotine in drinking water for 7 days. Disease activity of colitis was assessed by DAI score. mRNA expressions of ICAM-1, VCAM-1 and MAdCAM-1 in colonic tissues were measured by qPCR. Microvascular endothelial cell line, bEnd3, was used for in vitro study. The cultured cells (2x105/ml) were treated with 5ng/ml TNF-α and 200μg/ml nicotine for 12 hours. mRNA expressions of ICAM-1, VCAM-1 and MAdCAM-1 in cells were measured by qPCR. Various concentrations of nicotine (0.01-1000μM) were tested. In pre-treatment protocol, cells were treated with nicotine for 3 days, and after 12 hours interval without nicotine, exposed to TNF-α and nicotine for 12 hours as treatment. In some experiments cells were cultured with TNF-α and nicotine under the presence of 0.01μM mecamylamine (Meca, nonselective nAChR antagonist) or 0.01μM methyllycaconitine (MLA, selective α7nAChR antagonist). Result In in vivo study, nicotine treatment decreased DAI score induced by DSS. Although nicotine alone did not affect expression of ICAM-1, VCAM-1 and MAdCAM-1 in control mice, addition of nicotine to DSS mice significantly attenuated the increased expression of VCAM-1 and MAdCAM-1 induced by DSS, and tended to inhibit ICAM-1. In in vitro study, expression of ICAM-1, VCAM-1 and MAdCAM-1 was increased by TNF-α. Increased expression of MAdCAM-1 was significantly attenuated by simultaneous administration of nicotine. In dose-response study, degree of ICAM-1 inhibition by nicotine was only observed at high doses, whereas that of MAdCAM-1 was observed in a low dose already. Interestingly pretreatment with nicotine before TNF-α decreased inhibitory effect of nicotine. These inhibitory effects tended to be attenuated by Meca or MLA, but not

Background & Aims: Although the recirculation of lymphocytes through the intestinal mucosa is important for the specific immune defense, the homing of lamina propria lymphocytes (LPLs) has not been clearly understood. The aim of this study is to compare, under an intravital microscope, the dynamic process of lymphocyte-endothelium recognition and binding in the murine intestinal mucosa of T lymphocytes from the lamina propria of intestine to that of T lymphocytes from the spleen. Methods: LPLs isolated from nonlymphoid areas of the small intestine and spleen (SPL) were fluorescence-labeled and injected into a jugular vein of recipient mice. Microvessels of the villus mucosa and ileal Peyer's patches were observed under an intravital fluorescence microscope, and the effects of anti–adhesion-molecule antibodies on lymphocyte-endothelial interaction were investigated. Results: LPLs accumulated abundantly in the microvessels of villus tips but not in the submucosal venules or postcapillary venules of Peyer's patches, where SPLs migrated selectively. The accumulation of LPLs in the villus tips was almost completely inhibited by anti–β7-integrin and was significantly inhibited by anti–mucosal addressin cell-adhesion molecule 1 (MAdCAM-1) and anti–α4-integrin. Significant MAdCAM-1 expression was observed in the microvessels of the villus mucosa. Some SPLs adhered to the nonlymphoid mucosa, but most soon detached. Conclusions: It was shown in vivo for the first time that T lymphocytes from the lamina propria but not from the spleen adhere selectively, mostly via α4β7 and MAdCAM-1, to the microvessels of villus tips of the intestine, but not to the postcapillary venules of Peyer's patches. GASTROENTEROLOGY 2002;122:734-744

Efficacy and Safety of Rikkunshito, a Japanese Herbal Medicine, on the Treatment of Functional Dyspepsia: A Multi-Center, Double-Blind, Randomized, Placebo-Controlled Study Hidekazu Suzuki, Juntaro Matsuzaki, Yasushi Fukushima, Fumio Suzaki, Kunio Kasugai, Toshihiro Nishizawa, Yuji Naito, Toshihiko Hayakawa, Takeshi Kamiya, Takashi Ando, Hideo Yoshida, Yoshifumi Tokura, Hiroshi Nagata, Masao Kobayakawa, Mikiji Mori, Kimihiko Kato, Hiroshi Hosoda, Toru Takebayashi, Soichiro Miura, Naomi Uemura, Takashi Joh, Toshifumi Hibi, Jan F. Tack

Background: Cronkhite-Canada Syndrome (CCS) is an uncommon gastrointestinal polyposis syndrome that exists mostly in Japan characterized by dermatologic manifestations associated with chronic diarrhea, malnutrition, and protein wasting that can be fatal in as high as 50% if untreated or if treatment is delayed or inadequate. Due to its rarity, its subjects cannot be studied in clinical trials. Here, we report patient characteristics derived from a national database in an effort to identify prognostic factors and to improve treatment outcomes. Method: Retrospective analysis of 150 patients with CCS from a national database including 80 institutions specializing in gastroenterology. Clinical features including endoscopic findings, treatment, prognosis, and response to medical therapy were included. This study was supported by Intractable Disease, Health and Labor Sciences research groups for the Japanese Ministry of Health, Labor and Welfare. Results: Patient's age was 68.1±9.7 and treatment duration was 6.0±4.6years. CCS-related polyposis endoscopically confirmed to be throughout the gastrointestinal tract. Of the 140 patients who received steroids, 5 failed to respond, 2 underwent colectomy due to massive bleeding, 2 received cyclosporine, and 1 received biologics. Improvement of diarrhea followed by recovery of hypoalbuminemia was observed usually within 3 months after the start of medical therapy, with weight gain and improvement of dermatologic manifestations occurring at 5-6.5 months, and improvement of endoscopic appearance by 7.7 months. In 17%, no improvement of polyposis occurred despite clinical response. CCS-related gastric and colonic polyps progressed to advanced gastric and colon cancer in 7 of 107 and 15 of 120, respectively, suggesting the importance of endoscopic follow-up. Among those who discontinued steroid therapy, 7.4% developed advanced cancer, compared with 2.7% in those who maintained clinical remission with steroids. No patient who received nutritional support alone experienced clinical remission. An induction dose of steroid of >0.5mg/kg, followed, in some cases, with steroidmaintenance appeared beneficial for preventing the risk of recurrence of CCS-related polyps, reducing cancer risk, underscoring the importance of maintaining endoscopic remission. As for prognosis, 108 patients were alive and 22 had expired. The causes of death were colon cancer (1), gastric cancer (2), other malignancies (5), and other diseases, but none died of malnutrition. Conclusion: Contrary to earlier reports, the prognosis of CCS has greatly improved through improved medical treatment, although the course of CCS is relentlessly progressive with high cancer risk. A sufficiently high induction steroid dose, long-term steroidmaintenance, and aggressive nutritional support appear to improve the natural history of CCS.

Background: Lymphocyte infiltration into the intestinal mucosa is an important pathogenic feature of inflammatory bowel diseases (IBD). In case of dermatitis, lymphatics play a role as drainage route of lymphocytes from skin and an inhibition of lymphangiogenesis keeps lymphocytes remaining in the local area leading to exacerbate inflammation. However, the role of lymphatics in enterocolitis is not well understood. In this study, we examined changes in lymphatic densities and expression of lymphangiogenic factors in patients with IBD and in animal model of colitis, and investigated a role of lymphangiogenic using inhibitor of VEGF (vascular endothelial growth factor)-R3. Method: Colonic biopsies were obtained from 40 patients with ulcerative colitis (UC), 17 patients with Crohn's disease (CD), and controls. Messenger RNA expressions of lymphangiogenic factors, VEGFC & D, VEGFR3, podoplanin, Prox-1, and LYVE1 were determined using real time RT-PCR. Expression of CD34 and LYVE1 was studied immunohistochemically. In dextran sulfate sodium (DSS)-induced mice colitis, a VEGF-R3 kinase inhibitor (10mg/kg) was administered every day and the effects of lymphangiogenetic inhibition on activity of colitis and lymphatic density were determined in the colonic mucosa. Result: In the colonic mucosa of patients with UC and CD, the expressions of VEGFC, VEGFR3, podoplanin, and LYVE1 were significantly increased than the mucosa of control patients. The expression levels of podoplanin and LYVE1 were well correlated to endoscopic Matt's score in UC patients. By immunohistochemistry, the lymphatic densities in the colonic mucosa of IBD patients also increased compared to control patients. However, interestingly those densities were not significantly increased in the severely inflamed mucosa of IBD patients compared with those in the quiescent mucosa, suggesting that lymphangiogenesis and lymphatic drainage are not well organized in the severely inflamed mucosa. In murine colitis model, VEGF-R3 inhibition group showed significant aggravation of colitis such as shortening of colonic length and increase in the lymphocyte infiltration in the submucosa compared with DSS-treatment alone. Lymphatic densities decreased in the submucosa of VEGF-R3 inhibition group. Conclusions: The results of our study revealed that lymphangiogenic factors and lymphatic density are increased in both UC and CD patients. However, the density of lymphatic did not increase as inflammation advanced. Aggravation of colitis by lymphangiogenic receptor inhibition suggests that lymphatic networks may play some protective roles as exit of immune cells from intestinal mucosa, and that some mechanisms which block lympangiogenesis play aggravating role in severe IBD patients. In the severely inflamed conditions lymphatic drainage through lymphangiogenesis is not well functioning in IBD.

Background Ulcerative colitis (UC) is an intractable disease. Lymphocytes migration to colonic mucosa through endotherial venule like vessel is considered to be largely involved in pathophysiology of UC, and anti-adhesion molecule therapy is one of the established therapy. Although smoking has been reported to have a beneficial effect on UC, pathophysiology of nicotine from the point of lymphocytes trafficking is not studied. We investigated the involvement of nicotine in the colonic inflammation in the following three studies: 1) in vitro effect of nicotine on vascular endothelial cells, 2) in vivo effect of nicotine on inflamed mucosa of murine colitis model, and 3) intravital observation of lymphocyte migration to microvessels. Method 1) Microvascular endothelial cell line, bEnd3, was used. The bEnd3 cells (2x105) were cultured for 3days, and were treated with 5ng/ml TNFα and/or 200μg/ ml nicotine for 5 hours. Degree of mRNA expression of ICAM-1, VCAM-1 and MAdCAM1 was determined by quantitative RT-PCR. 2) C57BL/6J mice were used. For induction of colitis, mice were treated with 3% dextran sodium sulfate (DSS, 40kDa) in drinking water for 7 days. In some group, mice were treated with 0.1mg/ml nicotine in drinking water for 7 days. Disease activity of colitis was assessed by DAI score. We measured mRNA expressions of VCAM-1, ICAM-1 and MAdCAM-1 in the colonic tissue. 3) C57BL/6J mice were treated with 0.1mg/ml nicotine in drinking water 1 day after 3% DSS treatment for 7 days. Mice were injected FITC-labeld lymphocytes (3x107 dissoleved in 0.3ml) obtained from spleen of another mouse, via cervical vein. Microcirculation in the colonic mucosa was observed with a fluorescence microscope. Lymphocytes that moved outside the vessel were counted as transmigrated cells. Result In in vitro study, expression of ICAM-1, VCAM-1 andMAdCAM1 was increased by TNFα. Increase in expression of ICAM-1 by TNFα was not changed by nicotine. However, increase in expression of VCAM-1 and MAdCAM-1 was significantly attenuated by nicotine. In in vivo study, nicotine improved DAI score. Nicotine alone treatment did not affect expression of ICAM-1, VCAM-1 and MAdCAM-1, and DSS treatment significantly increased them. Addition of nicotine treatment to DSS significantly attenuated the increased expression of VCAM-1 and MAdCAM-1 induced by DSS, and tended to inhibit ICAM-1, although not statistically significant. By intravital observation, DSS increased the number of lymphocytes adhering to vascular endothelium and migrating out of vessel. Increase in lymphocytes migration was attenuated by nicotine. Conclusion Microvascular endothelial expression of adhesion molecules, especially VCAM-1 and MAdCAM-1 was significantly inihibited by exposure to nicotine. Down-regulation of vascular adhesion molecules by nicotine may play one of attenuative roles in colonic inflammation.

Background & Aims: The basic mechanisms of food allergies are still unknown. The aims of this study were to investigate whether endothelins (ETs) in the intestinal mucosa are involved in the pathogenesis of intestinal anaphylaxis. Methods: Sprague–Dawley rats were sensitized to chicken egg albumin (EA) by intraperitoneal injection. Fourteen days after sensitization, EA was administered in the jejunal segments to induce intestinal anaphylaxis. Net water outflux and histamine release into loops and serum concentrations of rat mast cell protease II (RMCP-II) were determined. ET-1 and ET-3 concentrations in the jejunal mucosa were determined, and expression of the corresponding messenger RNAs was examined by competitive polymerase chain reaction. Results: In sensitized animals, challenge with intraluminal antigen caused a significant increase in net water outflux and histamine release together with an elevation of serum RMCP-II concentrations. Mucosal concentrations of ET-1 and ET-3 and expression of their messenger RNAs were significantly increased in sensitized animals after EA challenge. Treatment with an ET A -receptor antagonist, but not an ET B -receptor antagonist, attenuated the increase in net water outflux, histamine release, and serum RMCP-II concentrations in rats with EA-induced intestinal anaphylaxis. Conclusions: Release of ETs in the intestinal mucosa increased in sensitized animals after EA challenge. ETs may play a significant role in the development of intestinal anaphylaxis via an ET A receptor. GASTROENTEROLOGY 1998;115:348-356

Background & Aims : Although recirculation of lymphocytes through Peyer's patches is important for specific immune defense, the intraorgan migration of lymphocyte subpopulations has not been clearly understood. The aim of this study was to compare the spatial distributions of labeled lymphocytes among various subpopulations in rat Peyer's patches. Methods : Lymphocytes collected from intestinal lymph were separated into CD4+, CD8+, and T and B cells, labeled with a fluorochrome carboxyfluorescein diacetate succinimidyl ester, and injected into the jugular vein. Peyer's patches of recipient rats were observed by intravital fluorescence microscopy. Results : No significant difference was found in the percentage of lymphocytes in transit or in the rolling velocity among different subpopulations. Lymphocytes sticking to the venules increased in number at 10–20 minutes, with preferential adherence of CD4+ cells to venules of 25–50 μm and preferential adherence of B cells to the venules of a wider size range. After 30 minutes, extravasated lymphocytes moved into the interstitium. B cells migrated from venules more quickly than CD4+ cells. CD8+ cells showed an intermediate pattern between CD4+ and B cells in sticking and migratory behaviors. Subsequently, CD4+ and CD8 cells preferentially appeared in parafollicular microlymphatics. Conclusions : Significant differences were observed among lymphocyte subpopulations in terms of spatial distribution of lymphocytes sticking to venules, migration into the interstitium, and their lymphatic transport.

and activity of myeloperoxidase. Results: While healthy controls gained weight during the experiment (+8.1%±1.9), untreated colitis animals significantly lost weight (-6.8%±3.0). Animals treated with MSA or gp96 showed a diminished relative loss of body weight (2.4%±2.3, and -0.4%±2.0, respectively). Endoscopic analysis of inflammation (MEICS score) revealed strong sings of inflammation in untreated colitis animals (3.8±0.8) compared to healthy controls (0.3±0.3). Also MSA treated animals showed strong changes (2.8±0.1), while gp96 treatment restored macroscopic signs of inflammation (1.5±0.8). Strong histological signs of inflammation were seen in untreated animals suffering from colitis (3.4±0.2), compared to healthy controls (0.0±0.0). Treatment with gp96 resulted in ameliorated histological signs of inflammation (2.2±0.3). Relative spleen weight of untreated colitis animals was found to be significantly increased (8.1±4.0) compared to healthy controls (1.5±0.8). While treatment with MSA even increased the effect (7.3±1.3), gp96 treatment restored relative spleen weight (2.6±0.8). Gene expression profiles of colonic specimen revealed lower levels of TNFα (0.7-fold) and IFN γ (0.5-fold) in gp96 treated animals than in untreated colitis animals. Myeloperoxidase activity was used as a quantitative assessment of neutrophil infiltration. While healthy controls showed the lowest activity (0.05±0.00), it was increased in untreated (0.13±0.04) and MSA treated (0.09±0.02) colitis animals. gp96 treated animals (0.04±0.01) showed activity comparable to healthy controls. Conclusion: Our observations show that gp96 treatment ameliorates experimental transfer colitis. gp96 might affect naive T cells epigenetically, exerting a memory effect. A deeper understanding of the underlying regulatorymechanisms will essentially contribute to reveal new therapeutic treatment options.

Background: GLP-2 is a proglucagon-derived peptides released from intestinal L-cells together with GLP-1. GLP-2 is an intestinotropic growth factor and is now going to be applied to the therapy for nonfunctioning gastrointestinal tract. However, regulatory mechanisms of GLP-2 secretion remain unknown because of its very short half-life time in a venular concentration. We hypothesized that GLP-2 secretion is regulated by food intake just like GLP-1. We have established measuring concentration of GLP-2 in intestinal lymph. Aim of this study is to elucidate which luminal nutrients stimulate GLP-2 secretion In Vivo and to clarify how sweet taste receptor is involved in that process In Vitro. Methods: In Vivo study; Male Wistar rats weighing around 250g were used. A plastic tube was inserted into the thoracic duct to collect lymph. A single bolus of sweet agent (glucose, sucrose, and sucralose) alone or mixed with dietary lipids (fish oil, olive oil, etc.) was administered into the duodenum. Lymph was collected during 2hrs before and after a bolus injection of nutrients. In Vitro study; NCI-H716 cells, human cell line that secretes GLP-1, were seeded into 96well culture plate precoated with Matrigel and preserved for 2 days. Then cells were incubated with various nutrients for 1h and each supernatant was collected with an anti-proteolytic solution, including EDTA, aprotinin, and heparin. Sweet agents (glucose, sucrose, saccharin, and sucralose) were added alone (10 to 500μM) or added with an equimolar concentration of alpha linolenic acid(LA). In some experiments, lactisole, a T1R3 inhibitor, was added to the culture. GLP-2 concentration was measured by ELISA (Yanaihara, Fujinomiya Japan) both In Vivo and In Vitro study. Results: 1) Lymphatic output of GLP-2 was significantly increased In Vivo by sweet agents (range: 5.75-6.31ng/h) or by dietary lipids (8.80-9.02) compared with controls (3.92±0.80). Mixture of sweet agent and lipid further increased GLP-2 output (7.40-24.0) compared to the sum of GLP-2 output by each component. 2) Each sweet agent brought dose-dependent increase in GLP-2 secretion (1.55 to 2.19-fold from control) from cells In Vitro. Increased GLP-2 secretion by sweet agent was attenuated by addition of lactisole. Meanwhile, lactisole itself did not affect GLP-2 secretion (1.07fold). 3) LA also induced GLP-2 secretion in a dose dependent fashion (up to1.39-fold), but increased GLP-2 secretion by LA was not attenuated by lactisole. Conclusions: We demonstrated carbohydrates and sweeteners induced GLP-2 secretion possibly through sweet taste receptor, which may be different from that of fatty acids. It requires further experimental support to see whether this endocrine mechanism is identical to GLP-1, but these results help us to understand that luminal nutrients may regulate secretion of GLP-2 by several pathways.