Plasma ghrelin following cure of Helicobacter pylori

pain episodes. For each pain episode we calculated esophageal shortening as the difference in length between emitter and receiver during 2rain before to 30 sec after pain and the 2.5min preceding period. Acid reflux and/or high amplitude or simultaneous contractions were checked dunng the same periods Results: Recordings were technically satisfactory (> 15hs) in 6 patients. Esophageal shortening could not be measured in 2 patients due to early loss of the emitter mucosal clip. One patient was asymptomatic dunng the study. We analysed 46 pain events in 5 patients (1-16/pat). In 4 patients, 16 pain events (f-7/pat) were associated with a prolonged oral LES excursion of 7.8 • 06 ram. Esophageal shortening started 7 0 -+ 10 sec before the pain events and lasted 124-+26sec. Acid reflux was present in 5 and abnormal manometry in 10 pain events associated with esophageal shortening. Conclusion: Continuous ambulatory monitoring of esophageal shortening in man is possible. Ongoing studies will provide further information about the possible association between esophageal symptoms and abnormal contraction of the esophageal longitudinal muscle leading to prolonged esophageal shortening.

873 A Double Blind, Placebo Controlled Cross-over Study Of Oral Theophylline In Functional Chest Pain Ranjit Mudipafli, Xing Zhao, Craig Utech, Joan Kemp[ Satish Rao The treatment of functional chest pain is unsatisfactory and there is no approved drug. We have shown that IV tbeophylline improves esophageal hypersensitivity in patients with functional chest pain possibly by altering nociception (Am J Gastroenterol 2002; 97:$5). Our aim was to investigate the effects of oral theophylfine, a non-specific adenosine receptor antagonist in patients with chest pain of esophageal origin. Methods: Twenty five (M/F = 7/ 18) patients with non cardiac, non-GERD esophageal chest pain and hypersensitivity to balloon distention were randomized to receive either theophynine SR 200 mg PO bid or placebo for 8 weeks, in a double blind cross over study. The frequency, intensity and duration of chest pain were recorded for two weeks at baseline, during theophylline and placebo phases and simultaneously psychosocial and quality of life measures (SCL- 90 & SF-36) were obtained. Results: 19 patients (M/F=4/15) completed the study. During the theophylline phase, the mean frequency, intensity and duration of chest pain improved (p(0.05) when compared to baseline, (see Table = mean • sd. * = p(0.05, Baseline vs Theophylline and t = p(0.05, Theophylline vs Placebo).. In contrast, there was no change during the placebo phase. The overall change in symptoms was rated as (theophylline vs placebo) better in 11 (58%) vs 1(6%), p(0.05, worse in 4 (21%) vs 5(26%) and unchanged in 4 (21%) vs 13 (68 %), p (0.05. Six patients dropped out of the study because of compliance (n= 3) and protocol violation (n = 3). Adverse effects on theophylfine were nausea (2), insomnia (1), tremor (1), and flghtheadedness (1) and on placebo were palpitations (1) and insomnia (1), but none discontinued participation. Psychosocial and quality of life data showed that social functioning improved on theophylline (p = 0.02) but not on placebo but bodily pain, physical function, phobia, depression and anxiety scores were unchanged. Conclusions: Theophyfline was well tolerated and sigmhcantly decreased the frequency and intensity of functional chest pain in 60% of patients. Thus, a non-selective adenosine receptor antagonist such as theophylline can be useful in the treatment of visceral pain. We hope that this study may serve as an impetus for the development of gut-selective adenosine receptor antagonists for the treatment of functional chest pain.

871 Combined Multichannel Intraluminal Impedance and Esophageal Manometry (MII-EM) Clarifies Function Defects During Swallowing Radn Tutuian, Lileeth Kong, Shirley Jamison, June A. Caste[1, Donald O. Castefl Background Combined manometry and muhichannel mtraluminal impedance (MII-EM) allows simultaneous assessment of bolus transit (by MI1) and pressure generation (by EM) within the esophagus. Using combined MII-EM swallows can be classified manometricafly as either normal peristaltic, ineffective or simultaneous and by MII as having complete or incomplete bolus transit. The aim of this study is to evaluate manometric factors influencing complete vs. incomplete bolus transit in patients referred for esophageal function testing. Methods Combined MII-EM studies from 150 consecutive patients referred to our laboratory were evaluated comparing results obtained with individual swallows. Manometric diagnoses included normal manometry (33%), ineffective esophageal motility (17%), achalasia (11%), distal esophageal spasm (11%), poorly relaxing LES (11%), nutcracker esophagus (10%), hypertensive LES (5%), hypotensive LES (2%) Results The 150 patients described above completed a total of 1464 liquid swallows, characterized by MII as having complete or incomplete bolus transit. Data are presented in the table below according to manometric characteristics of indivadna[ swallow. Summary Incomplete bolus transit is associated with lower contraction amplitudes in the distal part of the esophagus. Manometric ineffective swallows with incomplete bolus transit had higher LES residual pressure compared to those with complete bolus transit. Conclusion These results suggest that contraction amplitude in the distal esophagus is the major determinant of bolus transit. Esophageal function testing using combined MII-EM helps clarify the interaction between esophageal body and 1-ES that enables bolus transit V prwsure tOcm~ve LES Pres=ure 5cmabo~ LES D~tal esophil ampltude LESP.N~ t~l Prmemrl

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Natural History of Primary Eosinophilic Esophagitis: A Follow-Up of 30 Adult Patients for up to 12 Years Alex Straumann, Hans-Peter Spichtin, Hans-Uwe Simon, Mauro Pirovino, Christoph Beglinger

0001

Primary eosmophilic esophagitis (PEE) is an increasingly recognized, IL-5 driven inflammatory disorder of the esophagus. The only presenting symptom m adults is uniform attacks of dysphagia. PEE is histologically characterized by a dense infiltration of the esophageal epithelium with eosinophils. To date, the natural course of PEE has not been defined, and information regarding its potential long-term naks is still lacking. To our knowledge, this study is the first characterizing the natural course of PEE. Thirty adult patients (22 males and 8 females; mean age 40.6 years) with previously confirmed PEE were included in this study. The criteria for inclusion were: 1. typical history; 2. infiltration of the esophageal epithelium with > 24 eosinophils/hpf at the baseline examination; 3. time span > 1 year since diagnosis. GERD was excluded clinically, endoscopically and by 24-hour pH probe monitoring. After a mean follow-up time of 6.2 years (range 1.4 - 11.5), all patients underwent a foflow-up examination, consisting of a structured interview, physical examination, hematological and blood chemical analysis, determination of lymphocyte subpopulations in blood and EGD with histological examination of the esophageal, gastric and duodenal mucosa. All patients survived the study period and remained in good health. The dysphagia persisted in 28 patients and had, m 1 patient a major, and in 15, a minor negative impact on socioprofessional activities. Patients with blood eosinophiha had significantly more dysphagia attacks. The nutritional state remained stable (BMI 23.5 vs. 24.6; serum albumin 45.5 vs. 44.2). The eosinophilic infiltration of the esophagus persisted in all symptomatic patients, but the cell number decreased significantly (78.7 vs. 40.3 cells/hpi). The inflammatory process did not spread to the stomach or duodenum, but did evoke fibrosis of the esophageal lamina propria. Dysplasias or malignant esophageal conditions were not detected either endoscopically or histologically, and no lympho-proliferative diseases occurred. Based on this long-term observation of 30 adult patients, we conclude that PEE is almost always a primary-chronic disease, restricted to the esophagus It leads to persistent dysphagia and structural alterations of the esophageal tissue, but does not have an impact on the nutritional state. Furthermore, no disabling or malignant potential has been associated with this disease.

0138

Effects of Baclofen on Postprandial Fundic Tone and Lower Esophageal Sphincter Function in Man Kwang-Jae Lee, Rita Vos, Jozef Janssens, Jan Tack Background: The GABAB agonist baclofen was shown to inhibit transient lower esophageal sphincter relaxations (TLESRs) and to increase postprandial LES pressure in health and in reflux disease. Meal-induced increases in fundic volume trigger the reflex pathway underlying TLESRs, but the effect of baclofen on gastric meal accommodation is unknown. The aim of the study was to assess the effect of baclofen on postprandial fundic tone and LES function in man. Methods: Studies were performed in 7 healthy subjects (4 males: mean age 28 + 2 D's). After an overnight fast, a barostat bag was placed into the gastric fundus and a sleeve manometry catheter with pH electrode was positioned in the esophagus. With the harostat at MDP + 2 mmHg, recordings of fundic tone, esophageal manometry and esophageal pI-I were performed for 3.5 hours after oral ingestion of 40 mg baclofen or placebo in a randomized, double-blind manner. After 90 min, subjects ingested a liquid meal (200 mL, 300 kcal) Results: Ihe number of TLESP,s increased from 0.6_+0.3/h during fasting to 3.1_+ 0.5 and 2.1-+0.5/h during 1st and 2nd postprandial hour, respectively (p<0.05). Baclofen significantly reduced TLESR rate during 1st and 2nd postprandial hour (respectively, 0.6 -+ 0.3/h and 0 4 -+ 03/h; p
Abstracts

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17(14) 5(4) 3.3(,0.9)

874

872

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875 Plasma Ghrelin following Cure of Helicobacter pylori Dennis A. Freshwater, Paul O'Hare, Harpal S. Randeva, Chuka U Nwokohi Background: In the western world, the incidence of oesophageal adenocarcinoma has increased over the last 30 years coinciding with a decrease in the prevalence of Helicobacter pylori. Trends of increasing oesophageal adenocarcinoma can be linked causally to increasing gastroesophageal reflux disease (GORD) which can be linked to an increasingly obese population. However there is no plausible biological mechanism of association between Helicobacter pylori, obesity and GORD. Ghrelin, a peptide produced in the stomach, which regulates appetite, food intake and body composition as studied in Heflcobacter pylori positive asymptomatic subjects. Methods: Plasma ghrelin, leptin and gastrin were measured for 6 hours after an overnight fast, before and after cure of Helicohacter pylon in ten subjects. Twenty-four hour intragastric acidity was also assessed. Results: After cure median (95%

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Confidence Intervals) integrated plasma ghreliu increased from 1160.5 (765.5-1451) pg/ ral.hr to 1910.4 (1675.6-2395.6) pg/ml.hr (P = 0.002 Wilcoxun Rank Sum Test), a 75% increase. This was associated with a 14% increase in 24 hour intragastric acidity (p = 0.006) and non-significant ~hanges in leptin and gastrin. There was a significant positive correlation between plasma ghrelni and intragastric acidity (rs 0.44, p = 0.05, Spearman rank correlation) Conclusions: After Helicobacter pylon cure, plasma ghrelin increases profoundly in asymptomatic subjects. This could lead to increased appetite and weight-gain and contribute to the increasing obesity seen in western populations where Helicobacter pylon prevalence is low. This plausible biological mechanism links Helicobacter pylori, through increasing obesity and GORD to the increase in oesophageal adenocarcinoma observed in the western world.

(Affymetrix). The results from this analysis detected a few genes that had peak expression at Ell stage of development. We identified Insulin like growth factor binding protein 5 (1GFBP5) that displayed significant up regulation at E 11. The expression of IGFBP5 was 10 fold higher at Ell than all the later stages and at least 20 fold higher than in adults. We also utilized whole cell lysates from pooled livers at several stages of development and adult livers to investigate any changes in total IGFBP5 protein. We detected peak protein levels at El2 and 14 followed by a gradual decrease in the total protein levels after E16 and the levels continued to fall into adulthood with adult liver showing some protein, lmmunnhistochemistry also detected IGFBP5 as early as El0. Adult liver displayed minimal immunoreactivity substantiating low preponderance of this protein. We then utilized total cell lysates from several human, mouse and rat hepatoma and hepatoblastoma cell lines to examine the levels of IGFBPS. Sigmfieant increases in total IGFBP5 levels were observed in all hepatoma cells and not in hepatoblastoma cells. Thus peak gene and protein expression of [GFBP5 during liver development coincides with cellular events such as active stem cell self-renewal, lineage specification and proliferation. These properties are also shared by liver tumors that also show an upregufated IGFBP5 levels. Thus, we have thus identified a novel role of IGFBP5 in liver development and carcinogenesis.

$941 Hesl Is Required for the Development of Intrahepatic Bile Ducts Yuzo Kodama, Jun Hatakeyama, Ryoichiro Kageyama, Makoto Hijikata, Tsutomu Chiba, Kunitada Shimotohno { Background/Aims]Notch signaling is an evolutionally conserved mechanism that regulates the determination of cell fates during the development of various tissues and organs. In human, the defect of Jagged1 gene encoding a ligand for Notch receptors causes Alagille syndrome, an autosumal dominant disorder characterized by an impaired differentiation of intrahepatic bile ducts (1HBD). Heterozygons knock-out of bothJaggedl and Notch2 in mice also induced abnormalities similar to Alagille syndrome in the liver. These findings suggested that Notch signaling plays a crucial role iu the mammalian IHBD development. However, a precise role for Notch signaling in IHBD development is still unclear. In this study, we aimed to elucidate the role of Notch signaling in IHBD development. { Methods] The expression patterns of Notch signaling elements during the mouse liver development were serially analyzed by RT-PCR, western blotting, and immunohistochemistry. The development of IHBD in Hesl-null mice was also studied. [ Results]Expression of Jaggedl, Notch2, and Hesl proteins were observed in the liver at [ate fetal to neonatal period. Jaggedl protein was detected in the portal veins, hepatic arteries, and penportal connective t~sue. Both Notch2 and Hesl proteins were found only in the biliary epithelial cells that form tubular structures during the remodehng process of the ductal plate, while they were absent in the nontubular part of the ductal plate. Thus, Jagged1 and Notch2 were expressed at the same period in the different but adjacent ceils to each other. Biliary epithelial cells producing Notch2 were also positive for Hesl. These results suggested that Notch signaling induced the expression of Hesl in the developing blliary epithelial cells. Moreover, an impaired development of IHBD was also found in the liver of Hesl-null mice as was reported in mice having heterozygous knock-out of both Jagged1 and Notch2. In Hesl-null mice, remodeling of the ductal plate and tubular formation of bihary epithelial cells were not observed, implying that Hesl plays an important role in the tubular formation of the ductal plate. [ ConclnsionJin the late fetal to neonatal mouse liver, Notch signaling pathway, consisting of Jagged 1, Notch2, and Hes 1, was supposed to be activated through the epithelial-mesenchymal interactions between biliary epithelial cells and periportal mesenchyme or endothelinm. Activation of Hes 1 in the biliary epithelial cells was appeared to be essential for the remodeling of the ductal plate and its tubular formation.

$944 Follistatin 315 Accelerates Proliferation of Hepatocytes After Peak DNA Synthesis in Hepatectomized Rat Kazuhito Takamura, Kunihiro Tsuchida, Hidenori Miyake, Seiki Tashiro, Hiromu Suginn Purpose: Activins consist of homo- and heterodimers of activinb subuints. Activin A (bAbA) acts as an autocrine inhibitor of cell growth, and also induces apoptosis in parenchymal liver cells. The biological activities of activins are mediated by heteromeric receptor complexes consisting of two different types of receptors, type I and II receptors; both are essential for signal transduction. We previously reported that proliferating cell nuclear antigen (PCNA) positive nuclei and mitosis of hepatocytes reached peaks 24 h after 70% hepatectomy, and that activin receptor type IB and activin receptor type IIA mRNAs increased and reached peak levels 48 to 72 h after hepatectomy. In only faparotomy, both types of receptor mRNA expression showed little change during the study period. In remnant liver, strong staining for activin receptor type IIA was detected 72 h after hepatectomy. It is known that follistatin binds to activin and inhibit its biological effects; thus to examine the significance of timedependent increases in hepatocyte activin receptors, we induced intravenous administration of exogenous follistatin for regenerating hver in rats. Methods: The left median and lateral lobes, about 70% of the liver, were removed in all rats that were then divided into two groups. The foflistatin treated group, at 72 h or immediately after hepatectomy, had follistatin 315 had infused intravenously under diethylether anesthesia. The sham group, had saline was infused instead of fullistatin. Rats were sacrificed at 120 h after the operations, lobes were removed, immediately weighed and immunostained for PCNA. Result: In rats treated with follistatin 315 immediately after operation, No significant difference compared with saline treated rats was seen in wet liver weights. In rats treated with follistatin 315 at 72 h after operation, wet liver weight was sigmfieantly greater, and there were more PCNA positive nndei of hepatocytes than in the saline treated group. Conclusions: We found that intravenous administration of follistatin at 72 h after operation result in more potent stimulation of DNA synthesis than in saline treated rats. We also found harmonized changes of both in hepatic activin A level and, mainly, in hepatocyte activin receptor levels, resulting in a dampened of the proliferative response after the peak of DNA synthesis after hepatectomy. These results will help to elucidate the mechanisms of liver regeneration.

$942 Generation of Hepatocytes from Mouse Embryonic Stem Cells Kuai Xiao Ling, Xiao Shu Dong St.

$945

Objectives: Embryonic stem (ES) cells are pfuripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study was to examine the differentiating potential of mouse ES cells into hepatocytes. Methods: RA, HGF and beta-NGF were added to the cell culture containing ES and mouse embryonic hepatocytes. The ES cells also cocuhured with fetal hepatocyte line- BNL.CL2. We also transplanted the green fluorescent protein (GFP) positive ES cells into rats liver though the portal vein to study it's differentiation in vivo. The induction of hepatocytes was confirmed morphologically as well as biochemically through immunohistochemical assays of the expressions of alphal-antitrypsin and alphafetoprotein and RT-PCR tests of the existence of albumin and transthyretin mRNA produced only by functioning hepatocytes. Results: Fifteen days after the addition of HGF and betaNGF to the cell culture, many epithelioid cells were noticed. The expressions of alphaantitrypsin, alpha-fetoprotein, albumin and transthyretin mRNA were also detected, indicating a successful cell differentiation from ES cells into functiunmg hepatocytes. However, in the presence of RA alone, only the transthyretin mRNA was positive, whereas all other expressions pertaining to functioning hepatocytes could not be detected. When ES cells cocnhured with BNL.CL2 cells for 48 hours, bepatocyte-like cells could be detected by the immunohistochemical and molecular expressions. Transplanted GFP-positive ES cells were found in rats' liver slices by GFP fluorescence, and no development of teratomas was observed. Though immnnohistochemistry, the transplamed GFP-positive ES cells seemed to retain an albumin- producing ability. Conclusions: Mouse embryonic stem cells can be differentiated into functioning bepatocytes in vitro and vivo.It may be a new source for hepatocyte transplantation.

New Insight in the Biologic Support of Rat Hepatocytes in Primary Culture Silvia Tomat, Francesco P. Russo, Giorgio Kechagias, Maria Teresa Cnnconi, Pier Paolo Parnigotto, Remo Naccarato, Patrizia Burra Introduction: Maintenance of differem hepatocyte function in monolayer culture is generally short-lived. A biological support influencing cell development, migration, proliferation, shape and metabolic function would be very useful The aim of the study was to obtain an homologous acellular matrix (HAM) in order to support rat hepatocytes in primary culture. Materials and methods: HAM was prepared the day before each experiment by multiples rat liver slices taken in sterile water, deoxycholic acid sodium salt solution and then digested with DNase. Experiments were performed on 25 male Sprague-Dawley rats. Each animals was anesthetized. The abdomen was opened and the liver was perfnsed in situ through vena porta with collagenase. Isolated cells were seeded on 60 polystyrene wells, 60 collagen coated wells and 60 HAM coated wells. The number of viable cells was assayed by the thiazolyl blue (MTT) assay based on mltochondrial reduction reaction. Hepatocytes were detected by using an antialbumin rat antibody. Albumin and urea nitrogen concentration was also measured by a diagnostic kit. Rat hepatocytes were then examined by scanning electron microscopy (SEM). Results: 8xlO6[sup6] hepatocytes were obtained by each rat perfused liver. Cell viability was about 90%. Cells stained positive with the amialbumin rat antibody were 95% of the isolated cells. Hepatocytes seeded on HAM showed a significantly high viability compared to what observed on polystyrene and on collagen (p<0,05). Albumin and urea nitrogen concentration were significantly higher on HAM compared with polystyrene and collagen (p<0,05). Hepatocytes seeded on HAM and examined with SEM were organized on clusters, well anchored to HAM and morphologically stable. Conclusion: It seems that rat hepatocytes maintain for more than one week their synthetic and metabolic functions when seeded on homologous acellular matrix.

$943 Insulin like Growth Factor Binding Protein-5 Plays an Important Role in Early Liver Development and Liver Cancer Sunny Hnssain, Dufabb Monga, Tamara Sneddnn, Xinping Tan, Satdarshan Pal 5. Monga Several commonalities exist between the processes of development and carcinogenesis. In order to identify novel pathways that might be playing a role in these events, we utilized gene array analysis from RNA obtained fromdeveloping livers at different stages of gestational development. RNA was isolated from pooled livers from Ell, E14 and E]8 prenatal stages as well as adult livers and subjected to micro array analysis using U74A mouse chip

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Abstracts