AI84 AGA ABSTRACTS

1189

CLONING OF THE MOUSE INTESTINAL SODIUM-SULFATECOTRANSPORTER GENE AND PROMOTER.Daniel Markovich, Laurent Beck, Univ of Queensland, Brisbane, Australia.

Inorganic sulfate (S) is an important nutrient anion involved in manybiological reactions in the body, including sulfation and detoxification ofendogenous and exogenous compounds. S, is important for cell matrixformation and in bone and cartilage development. The initial point of entryof S, into the bloodstream is by intestinal absorption, mediated by abrush-border membrane Na/Sj-cotransport system in enterocytes. We havepreviously cloned a rat cDNA, NaSi-I, which encodes this transportprotein. NaSi-1 protein and mRNA expression in vivo is controlled byfactors including vitamin D, dietary S, intake, thyroxine, glucocorticoidsand metabolic acidosis, suggesting NaSel may play an important role inregulating S, homeostasis. In this study, we have cloned the mouse NaSelcDNA homologue (mNaSi-l) and the mouse NaSi-1 gene (Nsil) by RT­PCR,5' and 3' -RACE and homology screening approaches. Comparisonof the rat and mouse NaSi-1 protein sequences revelaed 94% homology.Expression of mNaSi-1 protein in Xenopus oocytes resulted in the follow­ing kinetic parameters: (Vmax=49±4 pmollhour; ~=0.2±0.06 rnM) andNa+ dependence (Km=21 ±2 mM; Hill coefficient n=2.8±0.6) which arein close agreement with the properties of the intestinal brush-border mem­brane Na/S, cotransport. By RT-PCR in 24 different tissues, mNaSi-1mRNA expression was detected primarily in the duodenum, jejunum andkidney. Screening of a A FIXII mouse genomic library using full lengthmNaSe I cDNA, led to 3 genomic clones encoding the Nsil gene spanninga region of75 kb, with 15 exons and 14 introns. A total of 3229 bp of Nsi lpromoter sequence was isolated of which several regions contain steroid­thyroid hormone-responsive elements (TREs), vitamin D responsive ele­ments (VDREs) and glucocorticoid resonsive elements (GREs). The first1.2kb of the NsiI promoter showed strong transcriptional activity whenplaced in front of a luciferase reporter gene in transfected OK cells. Thisactivity was further enhanced when the vit. D receptor was co-transfectedwith the 1.2kb NsiI promoter:luciferase construct after stimulation of thecells with 1,25-dihydroxyvitamin D, suggesting that Nsil transcription isactivated by vitamin D. This is the first study to report the genomicorganization of a gene encoding the intestinal brush border membranesodium/sulfate-cotransporter. This data provides the basis for a detailedanalysis of NsiI gene regulation, and the tools required for assessing therole of NsiI in sulfate homeostasis using a mouse gene targeting strategy.

1190

CENTRAL SENSITISATION CONTRmUTES TO OESOPHAGEALHYPERSENSITIVITY IN NON-CARDIAC CHEST PAIN.Sanchoy Sarkar, Anthony Hobson, Qasim Aziz, Clifford J. Woolf, DavidG. Thompson, HOPE Hosp, Salford, United Kingdom; MA Gen Hosp,Boston.

Background: Esophageal hypersensitivity is common in Non-CardiacChest Pain (NCCP), however, its mechanism remains unknown. We havepreviously demonstrated that even 5 minutes of acid exposure in the distalhealthy esophagus can reduce the pain threshold (PT) in the proximalnon-acid-exposed esophagus (I). This hypersensitivity is called secondaryallodynia (SA)and is likely to be mediated by central sensitization (CS) ofspinal cord neurones. The role of CS in the pathophysiology of NCCPremains unknown. Aims: To determine whether (i) experimental esopha­geal acid infusion induces secondary allodynia in NCCP patients with andwithout acid reflux. ii) acid supression therapy in NCCP patients withreflux modulates the response to experimental acid infusion. Methods:Protocol I: 22 NCCP, (7 female, mean age 48) of whom 13 had evidenceof gastro-esophageal acid reflux on Ph monitoring, received either a 5minsaline or acid infusion on separate occassions in the lower esophagus.Upper esophageal PT to electrical stimulation were determined before(baseline) and then after the infusions at 30min intervals for 90min ..Protocol 2: In 6 NCCP with reflux, PT in the upper esophagus weremeasured before and after lower esophageal acid infusion, six weeks aftertaking oral Omeprazole 20mg BD. Oesophageal pH was monitored in bothstudies Results: pH. In both studies, pH in the upper esophagus remainedbetween 5-8. Protocol I: In patients without reflux, the baseline PT (48 +9 rnA) decreased by 25.5 ± 4.8% following acid but not saline infusion(p=O.03 vs saline). In patients with reflux, the baseline PT was 31 ±6mA(p=0.04 vs reflux negative patients) and did not change following acid orsaline infusions. Protocol 2. Patients reported symptomatic improvementafter treatment. Baseline PT increased from pre-treatment values to40.7+7.2 rnA (p=0.04). Following acid infusion, there was now a reduc­tion in PT (p=0.04). Conclusion: The marked secondary allodynia to acidin patients without acid reflux suggests that CS is likely to contribute to theetiology of NCCP. In contrast, reflux patients demonstrated secondaryallodynia only following acid supression.It is likely that these patients werealready "centrally sensitised" due to endogenous acid reflux and andtherefore PT did not decrease further to exogenous acid. This suggests thatCS due to acid reflux plays a role in the pathophysiology of NCCP. (I)Sarkar et al: Gastroenterology 1998; 114;(4),A830,G3411

GASTROENTEROLOGY Vol. 118, No.4

1191

IL-I0 AND TGF-B GENOTYPES IN IRRITABLE BOWEL SYN­DROME: EVIDENCE TO SUPPORT AN INFLAMMATORY COM­PONENT?Joyce Chan, Wendy M. Gonsalkorale, Christopher Perrey, Vera Previca,Ali H. Hajeer, Peter 1. Whorwell, Ian V. Hutchinson, Univ Hosp of SouthManchester, Manchester, United Kingdom; Univ of Manchester, Manches­ter, United Kingdom.

Background/Aim: It has been proposed that inflammation may have apathophysiological role in some cases of irritable bowel syndrome (IBS).We have previously determined that patients with ulcerative colitis tend tobe genetically predisposed to produce lower amounts of the anti-inflam­matory cytokine interleukin-IO (IL-IO), having a reduced frequency of the-1082*G allele associated with high IL-1O production, which may increasesusceptibility to this disease '. This study aimed to test the hypothesis thatif inflammation is a factor in ms, then a reduced frequency of the highproducer IL-I 0 allele in these patients might also be expected. The possiblerole for other inflammatory cytokines, whose production is under similargenetic control was also examined. Method: DNA was extracted fromperipheral blood leukocytes of 140 ms patients (230 patients for IL-IO)and genotypes for IL-1O (-1082*G/A), transforming growth factor-S(TGF-I3) (+869*T/C - codon 10; +915*G/C - codon 25) (both anti­inflammatory), tumour necrosis factor-a (TNF-a) (-308*NG) and inter­feron-v (IFN-y) (+ 1002*T/A) (both pro-inflammatory) were determined,as previously described/. Results: The IL-1O high producer (-1082*G)allele and the TGF-I3(25) high producer (+915*G) allele were both re­duced in IBS patients compared to healthy controls (HC) [IBS v HC:IL-IO*G: 45% v 51%, OR=0.8(0.6-1.0), p<O.04; TGF-I3(25)*G: 76% v90%, OR=0.4(0.2-0.6), p<O.OOI]. In addition, there was a lower fre­quency in the homozygous high producer IL-IO and TGF-I3(25) genotypes[IBS v HC: IL-IO*G/G: 14% v 30%, p<O.ool; TGF-I3(25)*G/G: 54% v81%, p<O.ool]. Allele frequencies and genotypes for TGF-I3(10), TNF-aand IFN-y were no different from HC. Discussion: The results of this studyshow an association between IL-1O and TGF-13 genotypes and IBS, whichsuggests that high production of these anti-inflammatory cytokines mayhave a protective role against IBS, whereas those genetically predisposedto producer lower amounts may be more likely to develop the condition.These findings therefore provide additional evidence to support an inflam­matory component in IBS and need further exploration. eTissue Antigens1999; 54:386-90; 2Transplant Immunol 1999;7:127-8).

1192

T CELL ACTIVATION BY ENTERIC GLIA - A NOVEL PATH­WAY FOR THE AMPLIFICATION OF INFLAMMATORY RE­SPONSES IN THE ENTERIC NERVOUS SYSTEM (ENS).Eike Hollenbach, Anne Ruhl, Margot Zoller, Wolfgang Stremmel, Dept ofGastroenterology, Univ Hospitals, Heidelberg, Germany; Dept of Experi­mental Therapy, German Cancer Research Ctr, Heidelberg, Germany.

Enteric glia appears to playa key role in directing intestinal inflammationto the enteric nervous system. Our aim was to characterize the immuno­regulatory potential of enteroglial cells (EGC) using a stably transformedenteroglial cell line (Ruhl e.a., 1998). To investigate the phagocytoticpotential of EGC, cells were incubated with FITC-Iabeled dextran (0­loomglmL). Incorporation of dextran was visualized by fluorescence laserscanning- microscopy. To ascertain an active phagocytotic process, somecells were preincubated with colchicine (1-100 nglmL) prior to incubationwith dextran. Induction of MHC II and ICAM-I mRNA and protein wasassessed after stimulation of EGC with interferon-v (rrIFN-y; 0-1000U/mL) and/or tumor necrosis factor a (rrTNF-a; 0-50 ng/mL). After 72hrs, total cellular RNA was extracted and mRNA expression determined bysemiquantitative RT-PCR. Protein expression was quantified by FACScan.To study antigen-specific stimulation of syngeneic T-cells by EGC, cellswere stimulated with rrIFN-y and/or rrTNF-a and incubated with ovalbu­min (OVA) for 72 hrs, Subsequently, EGC were co-incubated with mes­enteric lymph node T cells of OVA-sensitized rats for a further 72 hrs.Finally, [3H]-thymidine was added for 6 hrs and eHJ-incorporation as­sessed. EGC displayed a concentration-dependent colchicine-sensitive up­take of FITC-dextran. Both at the mRNA and protein level, there was noexpression of MHC II or ICAM-I in controls or after stimulation with anyconcentration of IFN-y or TNF-a alone. In contrast, flow cytometricanalysis revealed a marked upregulation of both MHC II and ICAM-Iexpression after combined stimulation (p<O.OOOI). This was confirmed atthe mRNA level. There was no detectable lymphoproliferation of OVA­sensitized T cells after coculture with EGC preincubated with no or eithercytokine alone. Likewise, EGC incubated with cytokines but without OVAcould not induce T cell proliferation. In contrast, [3H]-incorporation wassignificantly augmented after co-incubation with EGC stimulated with bothcytokines and OVA (p<O.ool). In summary, we present evidence forphagocytosis and cytokine-inducible MHC II and ICAM-I expression inEGC which is fully functional for antigen-specific T cell activation. Hence,we suggest a role for EGC in neuroimmunological responses of theintestine which may cause enteric nerve damage in diseases such as