A 3 0 6 A G A A B S T R A C T S G A S T R O E N T E R O L O G Y , V o l . 1 0 8 , N o . 4

• AMMONIA INHIBITS cAMP-ELICITED INTESTINAL EPITHELIAL C1 SECRETION: ROLE OF NON-IONIC DIFFUSION AND Na-K-2CI COTRANSPORT. J.B.Matthews, M.Prasad, A.Resnick, C.Awtrey, LSmith, B.Hrnjez. Dept. of Surgery, Beth Israel Hospital, Boston MA.

The influence of ammonia on intestinal ion transport is largely unknown despite the fact that colonocytes are normally exposed to high concentrations of this weak base, ~10-70 mM in the lumen and 1-2 mM in mesenteric venous blood. AIM: To examine the impact of ammonia on cAMP-regulated CI" secretion using the human intestinal T84 cell line as a model system. METHODS: T84 monolayers grown on permeable supports were bathed in HEPES-based buffer with 0-100mM NH4CI replacing NaC1 is0tonically. C1- secretion was measured by short-circuit current (Isc) and 36C1 fluxes. Intracellular pH (pHi) was monitored in BCECF-loaded monolayers mounted in custom cuvettes which all0wed differential apical and basolateral perfusion. RESULTS: The Isc elicited by the cAMP agonist forskolin was dose-dependently lower in monolayers exposed to apical or basolateral NH4C1 for 10 min-24 hrs. Basolateral NH4C1 was far more potent than apical (50% inhibition at ~3mM vs. ~70raM). The effect on Isc was wholly accounted for by decreased C1- secretion (e.g., 10mM basolateral NH4Clreduced Isc and net 36C1 flux to 39% and 32% of control, n=8, with no effect on flux of :2Na, n=6). NH4C1 did not affect LDH release or the Isc elicited by the Ca 2÷ agonist carbachol (n=6-8). The effect of NH4C1 was not due to altered cation composition, as determined by replacing NaCI with NMG-C1 or KC1 (n=4). The effect of NH4C1 was not due to cytosolic alkalinization; neither the weak base imidazole nor the weak acid butyrate (10raM) affected peak Isc, and butyrate did not attenuate the effects of NH4C1 (each n=4). Apical 10mM NH4C1 induced a sustained increase in pHi (from 7.24 +..06 to 7.61 + .05, n=6), consistent with NH3 entry by non-ionic diffusion. In contrast, basolateral i0mM NH4CI, which inhibited !so more profoundly than apical, induced only a transient rise in pHi followed by a rapid recovery to baseline (7.31 + .07, n=7); pHi recovery was blocked by bumetanide, consistent with NH4 ÷ entry via Na-K-2C1 cotransport Inhibition of Isc by apical NH4C1 was enhanced at pH 8.0 (which increases the NH 3 form) and attenuated at pH 6.0 (which decreases NH3), n=4. ~ 1) NH4C1 inhibits cAMP-regulated C1: secretion in T84 monolayers; 2) the effect of apical NH4C1 is largely due to entry of NH 3, whereas the enhanced basolateral potency may reflect NH4+ entry via Na-K-2C1 cotransport in addition to diffusion of NH3; 3) the effect of NH4C1 is not due to cell alkalinization. The data suggest that ammonia could serve as a novel physiological inhibitor of intestinal CI- secretion.

• EVIDENCE OF GLUTEN SENSITIVITY IN THE RECTAL MUCOSA OF FIRST-DEGREE RELATIVES OF CELIAC DISEASE PATIENTS. E. Maudfio, S. Niveloni, R. Dezi, G. Marraco, I. Doldan, H. Vazquez, S. Pedreira, E. Smecuol, R. Mazure, M. Herrera, Z. Kogan, J. Valero, L. Boerr, &C. Bai. Hospital de Gastroenterologia. Buenos Airesl Argentina.

Gluten sensitivity is expressed by a wide variety of pathological features ranging from low grade of enteropathy to flat jejunal mucosa. Although, the major site of involvement is jejunum of celiac disease (CD) patients, changes may ale0 arise in rectal mucosa exposed to gluten. Recently, reotal gluten challenge was shown to be a simple, sensitive and specific tool for detemlining gluten sensitivity. First-degree relatives of CD patients may show the complete range of pathological CD features and are candidates for studying gluten sensitivity. AIMS, 1 ) To evaluate gluten sensitivity in relatives of probands using the rectal gluten challenge and, 2) to compare these findings with those obtained in the small intestinal histology, serology (CD related antibodies) and HLA status. MATERIAL: Twenty five first-degree relatives of probands were studied. Data of rectal gluten chaltenge in relatives were compared with those of 21 CO patients and 17 controls. METHODS:. A 4 h metal gluten challenge was performed with 6 g of crude gluten in saline solu!ion. Number of intraepithelial lymphocytes (IEL) in surface and crypts (expressed as number of IELJ103pm of epithelium) in pre- and post-challenge frozen rectal biopsies (pan- T cell immunocytochemistry) were quantified by computerized image analyses. An I EL response after challenge of more than 15% above pre-challenge count was indicative of gluten sensitivity. Other features of gluten sensitivity evaluated were serum antigliadin (AGA) and antiendomysium (EmA) antibodies, duodenal histology (partial or total villous atrophy and IEL count) and (he characteristic HLA haplotype OQA 0501 and B 0201. RESULTS: (X±SEM) Rectal gluten cha!lenge identified 11 first-degre e relatives with ev!dence of gluten sensitivity. Mean IE L response in gluten sensitive relatives (89% ± 15) was significantly higher than for non- sensitive relatives (-20% ± 8; p<0.001-ANOVA-) and controls (-23.1% ± 10; /o<0.0001). Moreover, no difference was assesed with CD patients (160% ± 40i p NS).

Challenge Small bowel histology CD related HLA haplotype

Atrophy IEL count >20% antibodies DGA 0501 - B 0201

Positive 2/11 2/11 2/11 3/11

(n=11) Negative 0/12 0/12 1/14 4/12 (n=14)

In cortolusion, rectal gluten challenge detected gluten sensitivity in 44% of first- degree relatives of CD patients. However, only two of these relatives had partial or complete small bowel villous atrophy. Other features of gluten sensitivity did not correlated with findings of rectal challenge. Relatives with positive rectal challenge are candidates to be strictly followed looking for evidences of active disease.

• EFFECT OF ONE YEAR TREATMENT ON BONE MINERAL DENSITY OF CELIAC DISEASE PATIENTS. ARE CALCIUM AND VITAMIN D SUPPLEMENTATION NECESSARY?. R. Mazure. H. Vazquez, O. Gonzalez, C. Mautalen, E. Mauririo, S. Pedreira. S Niveloal, E. Smecuol, L. Boerr, JC Sai. Centre de Osteopatias M6dicas, Hospital de Clinicas. Hospital de Gastroenterologia. Buenos Aires, Argentina.

Osteopenia is a well-known complication of untreated celiac disease ICD) patients. It is not known whether gluten-free diet prevents further bone deminerarization, whether dietary treatment is effective in remineralization, or whether calcium and vitamin D supplements are necessary for reversal of osteopenia in CD. AIMS'l) To determine the effect of 1 year trsatrnect on BMD and mineral metabolism of CD patients, and 2) to evaluate whether calcium and vitamin D supplementation have additional therapeutical effect. MATERIAL: Thirteen consecutive Datients (10 female, mean age 43 yr, range 21-73 yr) with newly diagnosed CD were inaluc~ed in this prospective study. All patients were instructed to follow a strict gluten-free diet and were randomized to receive diet only (n=7) or calcium (1.0 gr/day) plus vitamin D (32,000 UINceek) supplementation (n---'6). METHODS" Bone mineral density (BMD) was measured with a total body scanner for dual x-ray absorptiometry (Lunar DPX). Parameters of mineral metabolism (serum ca=clum, ionic calcium, phoscnorus, urinary calcium, alkaline phosphatase, parathyroid hormone ostao~alcin and urinary nydroXyproline) were measureo in all patients. All determinations were repeated at diagnosis and after 6 and 12 months of treatment. RESULTS: At diagnosis, 90L~ of 13 patients and 10/13 presented osteopenia (> 1 SD of normality) in the spine and total skeleton levels, respectively. At 6 months of treatment. BMD improved in 12 patient. At 1 year, 10 patients showed bone remineralization. In the 3 cases with reduced BMD after 1 year treatment (2 patients in the group with supplementation), a Eack of compliance with the gluten-free diet was shown.

BMD (gr/cm =) Groups Spine (L2-L4) Total skeleton

Before 1 yr Before 1 yr without supplement 1.108±0.086 * 1.183==0,077 1.043±0.055 * 1.133±0.031

with supplement 0,915±0.072 0.947±0.057 0.937=6.040 0.969±0.042

all patlenta 1.018±0.061 * 1.074:1:0.058 0.984±0.038 * 1.050±O.035

(X==SEM) * Before vs. 1 yr p<O.05 (Friedman and WiIcoxon tests) The treatment of CD produced a significant remineraUzation of bones at 12 months (6.1% in total

skeleton mass and 6.4% in the lumbar seine levels). Kinetic of remineralization was almost twice more effective in patients with diet only (8.1% vs 3.8% and 8.0% vs~:=5% at total skeleton and lumbar spine, respectlyely) and this was probably related to gluten indi~retions. Secondary hyperparathyroidisn~ was biochemically evident in 7 patients. Treatment of CD produced a normalization of these features Bone remineralization was independent of age or menopause, in conclusion, a strict glutsn-free die1 in CD prompts significant bone remineralization and treatment does not seem to require calcium and vitamin O supplementation.

• IMMUNE CELL INTERACTIONS IN T CELL-INDUCED EPITHELIAL PATHOPHYSIOLOGY. D.M. McKay, G. Kovadk, K. Croitoru, M.H. Perdue. Intestinal Disease Res'Prog, McMaster Univ, Hamilton, Canada.

Inflamed intestine in IBD and animal models of inflammation contains large numbers of immune cells in close proximity to the epithelium. Interactions of T ceUs with monocytes/macrophages (M~) are essential for immune responses; but the importance of such interactions in causing epithelial dysfunction/injury is unclear. In this study, we examined the effect of T cell activation in the presence or absence of M~. on epithelial electr01yte transport and barrier functions. Peripheral blood mononuclear cens (PBM, ~75% T ceils, 15% MO) were isolated from normal humans and T" cell-enriched populations (To, <2% CD14 ÷ MO) ~ere obtained by removing adherent cells (plastic plating 2x4h at 37°C). Confluent monolayers of T84 epithelial cells were co-cultured for 48h with either PBM (106) or Tc (0.75x106); anti-CD3 was used m activate T cells. T84 monolayers were then mounted in Ussing chambers a~d changes in ion transport and permeability measured. Co-culture with PBM resulted in: 1) decreased secretory capacity indicated by reduced short-circuit current (Isc) responses to carbachol (CCh) and forskolin (FSK); and 2) increased epithe]ial permeability indicated by decreased transepithelial resistance (TER), and increased fluxes of 3H-mannitol and ~ICr-EDTA. Surprisingly, co-culture with Tc alone did not significantly alter epithelial physiology. Nor did M~ alone (0.15x10 e) reproduce the abnormalities. Restoration of tile abnormalities was only achieved by treating MO with Tc conditioned media before co-culture.

% of Control Response PBM Tc Mq~ Tc media+Mq~

AIsc to CCh 31_+7"~ 121_+20 74_+7 30_+6** Alsc to FSK 46_+10"* 91_+4 98_+5 47_+6** TER 28_+7** 81_+9 65_+8* 19_+3"* (mean_+SEM; n=6; *p<0.05, *~'p<0.01 to control values at 100%) These data suggest that T cells release soluble factor(s) that activate M~. and that MO mediators act on the epithelium, either directly or synergistically with T cell factors, to alter physiology. We conclude that experiments investigating immune cell interactions are essential to the understanding of intestinal pathophysiology in giat inflammatory conditions.