was stripped off by heat-denaturation leaving the basement membrane basement membrane exposed on the surface of remaining tissue Primary squamous oesophageal epithelial cells (Sq cells) were isolated by explant tissue culture of normal oesophageal nmcosa. These cells were subsequently propagated in a low Ca + + serum-free tissue culture maintenance medium Sq cells were allowed to adhere to the luminal basement membrane of either Sq BM orCLO BM. Sq cells cultured in this orientation are at an airdiqind interface, which allow prolderation, stratification and dift?rentiation in response to the high Ca + + medium and interaction with the ECM and basement membrane. Results: The model was cultured for three weeks and histology confirmed sqnamous epithelium colonises sqnamous BM Laminin, an abundant ECM component was characterised by immunohistochemical staining. Conclusions: Tile role of the basement membrane in CLO on Sq cell proliferation and diflerentiation can be examined using this model

M2069

Effects of Bile Acids and Acidic Exposure on IL-8 Expression in Human Esophageal Epithelial Cells Eiko lmaraoto, Norimasa Yoshida, Kazuhiko Uchiyama, Masashi Kuchide, Hiroshi Higashihara, Takeshi lshikawa, Tomohisa Takagi, Satoshi Kokura, Hiroshi Ichikawa, Yuji Naito, Atsushi Kawabe, Yutaka Shimada, Toshikazu Yoshikawa

BACKGROUND & AIMS We have reported that IL-8 expression increases in the esophageal mucosa of patients with gastmesophageal reflux disease (GERD). In patients with GERD, the degree of esophageal mucosal iniury correlates with not only the concentration of bile acids but also the pH. In the present study, we investigated the effects of bile acids and acidic exposure on IL-8 expression in human esophageal epithelial cells (HEEC). METHODS: HEEC were stimulated with bile acids (cholic acid = CA, cbenodeoxycholic acid = CD, tauro- cholic acid = TCa~ or taurochenodeoxycholic acid = TCD) in neutral-pH or acidic pH media. 1L-8 protein in culture medium was measured by enzyme-linked immunosorbent assay' (ELISA), and mRNA levels of IL-8 were assessed by reverse-transcriptase polymemse chain reaction (RT-PCR) method. In addition, we exammed luciferase activities in HEEC which were trar~sfected with hiciferase expression vectors containing a minimally essential promoter region of tile IL-8 gene. RESULTS: Unconjugated bile acids, CA and CD induced 1L-8 protein production from HEEC in a dose- and titan-dependent manner. Expression of 1L-8 mRNA increased 3 hours after stimulation of CA. Conjugated bile acids increased IL-8 protein production less than unconjugated bile acids. Acidic exposure of HEEC did not induce the increase in IL-8 expression. However, conjugated bile acids in acidic media resulted in remarkable increase of IL-8 production compared to those in neutral-pH media. Stimulation with CA enhanced markedly luciterase activhies m HEEC. Mutation of the NF-KB binding site abrogated completely the induction of hiciferase activities, whereas that of AP-1 or NF- lL6 binding site partially reduced the induction. CONCLUSIONS: These results indicate that bile acids, especially in acidic condition, induce IL-8 expression in esophageal epithelial cells, via the activation of AP-1 and NF-IL6 as well as NF-KB, leadmg to IL-8 gene transcrip- tion. Reflux of duodenal contents with gastric acids into the esophagus may occur esophageal inflammation by inducing IL-8 expression

M2070

Transition of chemokine expression in esophageal mucosa after the treatment of GERD patients with proton pomp inhibitors Kazuhiko Uchiyama, Norimasa Yoshida, Masaaki Kuroda, Yutaka Isozaki, Kazuhiro Katada, Hiroshi Higashihara, TomoNsa Takagi, Yuji Naito, Toshiki Takemura, Toshikazu Yoshikawa

Background: We have already reported that the IL~8 mRNA expression can be detected in esophageal mucosa of gastroesophageal reflux disease (GERD) patients and expression level of IL-8 mRNA correlates ruth endoscopic esophagitis grading, in this study', we assessed the mRNA expression of a CC chemokine, monocyte chemoattractant protein -i (MCP-1), from the biopsy matenals and compared with endoseopic findings and subjective symptoms. In addition, to evaluate the benefit of proton pump inhibitors (PPI), we also examined the expression of IL-8 and MCPA mRNA before and after PPI therapy, sMethods: Biopsy samples wel~ obtained from 65 patients. None had recewed PPI, steroids, and non-steroidal anti- inflammatory drugs during the prior 4 weeks. Endoscopic diagnosis was determined accord- ing to the LOs Angeles (LA) classification: LA-A (n = 15), LA-B (n = 15), LA-C (n = 10), and normal esophageal mucosa in patients suffered from heart burn (n= 15) As the normal control, 10 patients having neither endoscopic esophageal abnormality nor subjective S)~T~p- toms were defined. In those 65 patients, 10 patients treated with PPI were estimated by endoscoptc examination before and after medication of 8 weeks. Total cellular RNA from the biopsy samples was extracted with RNAzol using a single-step isolation method. The RNA was reversetran~seribed into complementary DNA. Real-time PCR was carried out with the Ligt'~t Cycler instrument (Roche Molecular Biochemicals, Marmheim, Genvany) using the DNA binding dye SYBR Green 1 tot the detection of PCR products. Results: Expression level of lL-8 and MCP-1 mRNA in patients with GERD were significantly" increased compared to that expressed in normal control group. Though there were significant correlations between endoscopic esophagitis grading and expression level of IL-8 mRNA (p<O.O1), there was little correlation between expression level of MCPd mRNA and endoscopic esophagitis grading. After PPI therapy, IL-8 mRNA expression went dowm to lower level that was determined in normal control group, which was associated with decrease of neutrophfl accumulation. However, expression lel,el of MCP-1 mRNA was almost the same level as betbre PPI therapy. Conclusion: These results indicate that 1L-8 is a sensitive index of esophageal acute inflanm~ation wbich rapidly nommlize after PPI therapy, while MCP-I may reflect long term inflammation with the infifimtion of mononuclear cells in esophageal nmcosa,

M2071

Hill Grade, Gender, NSAID Use, Body Mass Index and Helicobacter pylori Status Are Independent Predictors of Endoscopic Findings of Reflux Disease Benson T. Massey, Safoum S. Haghighi

BACKGROUND/AIMS: The Hill grade of tile gastroesophageal junction (GEJ), a measure of the anatomic integrity of the "flap valve," is a known determinant of the presence of esophagitis. We assessed whether other endoscopic and demographic factors independently- and additionally coutribute to esophagitis. METHODS: We studied 200 consecutive patients (Mean 52 yr [18-94 yr], 102 F) undergoing elective upper endoscopy. Patients with prior gastric surgery or acute GI bleeding were excluded. Patients were assessed tbr the presence of esophagitis by the Los Angeles (L4) classification system as well as the presence of other features of reflux disease (Barrett's, stricture, ulcer). The Hill grade of the gastroesophageal junction was also assessed, as were gastric biopsies fbr evidence of Helieobacter pylori (HP) infection. Endoscopic intake history and hospital medical records were reviewed to obtain information on height, weight, smoking status, NSAID use (including aspirin 81 mg and over-the-counter use), and acid suppreaswe therapy use (1%X). RESULTS: 37% of patients had endoscopic evidence of reflux disease and 20% had e;ndence of HP. 35% had lost the normal flap valve configuration of the GE junction (Hill 3 and 4). In univariate analysis higher Hill grades were associated with higher LA grades of esophagitis (p< 001) and the presence of any findings of GERD (p<.O01). Male sex (p= .008), NSA1D use (p = .009), and age (p = .014) were positively associated with esophagitis, whereas body" mass index (BMI) was negatively associated (p= .009). Male sex (p<.O01) and age (p= .022) were positively associated with the presence of an)' endoscopic features of GERD, while HP infection was negatively associated (p= .036). Smoking and RX were not associated with reflux findings. Higher Hill grade was significantly associated with increasing age (p<.O01), RX (p< 001), and NSAID use (p = .023), but did not vary by gender, BMI, smoking status or HP status. In multivariate analyses, Hill grade, gender, NSAID use, and BM1 were independent predictors of LA grade, while Hill grade, gender, and HP infection were independent predict- ors of any endoscopic GERD findings. CONCLUSIONS: Gender, NSAID use, BMI, and HP infection contribute to the presence and/or severity of endoscopically identified reflux disease independent of the loss of the flap valve. Any effects of increased age on reflux findings are mostly mediated through the loss of the flap valve with age. Supported in part by N1H POl 03191-03.

M2072

Acid Reflux is Common at the Proximal Margin of Lower Esophageal Sphincter (LES) but Gastric Acid does not Come Up at 5 can above the Proximal Margin of LES in Healthy Subjects Katsuhiko iwakiri, Yoshinori Hayashi, Makoto Kotoyori, Toshiaki Sugiura, Akihiko Kawakami, Choitsu Sakamoto

Background and Aim: Transient LES relaxation (TLESR) is a major mechanism of inducing gastroesophagea] reflux. However, there is no difference in the rate of TLESR between healthy subjects and patients with reflux esophagitis, in fact, the only difl?rence between these groups is the rate of acid reflux during TLESR In healthy subjects, we noticed that the rate of acid reflux during TLESR is more frequent at the proximal margm of LES, although it is low at 5 cm above the proximal margin of LES. In this study, we investigated the hypothesis that the rate of acid reflux during TLESR in healthy subjects at the proxmral margin of LES is similar to that in patients with reflux esophagitis but we found that, gastric acid does not come up at 5 cm above the proximal margin of LES in healthy subjects. Method: 7 Hpykiri- negative health), subjects (3 males and 4 females, mean age 568 years) and 7 H pylori- negative patients with reflux esophagitis (3 males and 4 females, mean age 57.8 years) of grade C of Los Angeles classification were studied Manometry was carried out with a perfused assembly which incorporated side holes in the pharynx, the upper esophageal sphincter, the esophagus, and 9 side holes (lcm apart) which straddled the distal esophagus, the esophagogastric junction and the proximal stomach. 21 pressures and 2 esophageal pH measurements, at 2 cm above the proximal margin of LES and 7 cm above the proximal margin of LES, were done 1 hour before and 3 hours after a meal in the sitting position The energy level of the solid meal was 692 Kcal and consisted of 33 % fat. Results: There was no difference in the rate of TLESR between healthy subjects (4.6(0 9), No./hour, mean(SD)) and patients with reflux esophagitis (4.3(1.1)). The rate of acid reflux (a pH drop of at least 1 pH unit) during TLESR in patients with reflux esophagitis (345 %) was significantly (p < 0.05) higher than that in healthy subjects (13.6 %) at 7 cm above the proxire.al margin of kES. However, there was no difference in the rate of acid reflux dumlg TLESR between healthy subjects (66.9 %) and patients with reflux esophagitis (70.2 %) at 2 cm above the proximal margin of LES. Conclusions: These findings indicate that the difference between heal@ subjects and patients with reflux esophagitis, after refluxing into the esophagus occurs, is whether gastric acid comes up at 7 cm above the proximal margin of LES or not.

M2073

The Sleeve Sensor Underdetects Transient Lower Esophageal Sphincter Relaxations(TLESRs) Stefanla Carmagnola, Jac Oors, Andre J P. Smout

Background and aim: despite the availability of well-defined criteria (Am J Physiol 1995;268:G129-G 133) the detection of TLESRs with a sleeve sensor device remains problem- atic especially when LES pressure is low. The aim of this study was to investigate whether high-resolution (Hi-Res) manometry of the high pressure zone (HPZ) represents a valid ahemative for prolonged monitoring of TLESRs. Methods: 5-hour HPZ manometry was carried out in 15 healthy subjects (7 meiV8 women, aged 19-50 yr) using a 21-channel manometric assembly with incorporated 6-cm long reverse perfused sleeve sensor and sideholes spaced at 1 cm. For positioning the catheter the transmucosal potential difference (TMPD) between esophagus and stomach was recorded continuously to ensure that the

A-409 AGA Abstracts