L. Maiuri

Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease

BACKGROUND Diagnosis of many immune-mediated diseases is helped by detection of antibodies directed against the pathogenetic (self or foreign) antigen. In coeliac disease (CD), we have a situation in which antiendomysial antibodies (EMA), which are not specific for the pathogenetic antigen, reach a specificity and sensitivity of detection of CD approaching 100%, whereas detection of antibodies against gliadin (AGA), the pathogenetic antigen, is far less specific and sensitive in diagnosis. No direct evidence of a relation between gluten/gliadin challenge and EMA production exists. We tried to establish whether the small intestine of CD patients is the site of EMA production and whether gliadin challenge could induce their release. METHODS Small intestine biopsy samples from treated (23) and untreated (16) CD patients and controls (18) were cultured in vitro for 24-48 h in the presence of gliadin, another alimentary antigen, or medium. EMA and AGA were detected in the supernatants of these organ culture biopsy samples by ELISA and immunofluorescence technique, respectively. FINDINGS No EMA were found in the culture supernatants of biopsy samples of 18 controls, whereas they were detected in the culture supernatants of all 16 untreated CD patients irrespective of gliadin challenge. Conversely, EMA were not detected in supernatants of biopsy samples cultured in medium only from 23 treated CD patients, but were detected in 17 of the 23 biopsy samples challenged with gliadin. INTERPRETATION Our results suggest that a more complex pathogenetic mechanism than normally accepted is involved in CD. Furthermore, our findings raise the possibility that EMA, or the antigen recognised by them, are involved directly in the pathogenesis of CD.

DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis?

Cystic fibrosis (CF) is a single‐gene disease caused by mutations in the CFTR gene, which result in disrupted chloride secretions with inspissated mucous secretions by exocrine glands. Nick‐end labelling was used to assess DNA fragmentation in 14 CF and 24 control duodenal samples, and in two CF and two control lung tissues. In CF small intestine median 46% (range: 30–82) villus enterocytes show DNA fragmentation (vs. 3% (range: 1–7) in controls P<0.001) and median 37.5% (range: 23–79) crypt enterocytes show Ki67 antigen (P<0.001). In CF airways 57% (range: 54–70) of epithelial cells show DNA fragmentation. Inappropriate high DNA fragmentation is a feature of various CF epithelia. This could have great impact in understanding the mechanisms leading to disease.

Imaging active lymphocytic infiltration in coeliac disease with iodine-123-interleukin-2 and the response to diet

Abstract. Coeliac disease is diagnosed by the presence of specific antibodies and a jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Mucosal cell-mediated immune response is considered the central event in the pathogenesis of coeliac disease, and untreated coeliac patients show specific features of T-cell activation in the small intestine. Here we describe the use of iodine-123-interleukin-2 scintigraphy in coeliac patients as a non-invasive tool for detection of lymphocytic infiltration in the small bowel and its use for therapy follow-up, and we demonstrate the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. 123I-IL2 was administered i.v. [74 MBq (2 mCi)], and gamma camera images were acquired after 1 h. Ten patients were studied with 123I-IL2 scintigraphy at diagnosis and seven were also investigated after 12–19 months of gluten-free diet. Results were expressed as target-to-background radioactivity ratios in six different bowel regions before and after the diet. At the time of diagnosis all patients showed a significantly higher bowel uptake of 123I-IL2 than normal subjects (P<0.003 in all regions). A significant correlation was found between jejunal radioactivity and the number of IL2R+ve lymphocytes per millimetre of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2=0.66; P=0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 only binds to activated T-lymphocytes infiltrating the gut mucosa. After 1 year of the diet, bowel uptake of 123I-IL2 significantly decreased in five out of six regions (P<0.03), although two patients still had a positive IL2 scintigraphy in one region. We conclude that 123I-IL2 scintigraphy is a sensitive non-invasive technique for assessing in vivo the presence of activated mononuclear cells in the bowel of patients affected by coeliac disease. Unlike jejunal biopsy, this method provides information from the whole intestine and gives a non-invasive measure of the effectiveness of the gluten-free diet.

Cytotoxic effect of prolamin-derived peptides on in vitro cultures of cell line Caco-2: Implications for coeliac disease.

The cytotoxic effects of various prolamin-derived peptides on Caco-2 cells were investigated by measuring the alterations of several parameters at different stages of cell differentiation. The PT digest of bread wheat was active in inhibiting cell proliferation (by about 50%), whereas the other digests from durum wheat, maize and bovine serum albumin (BSA) did not affect the proliferating activity of cells. Compared with the control, colony-forming ability was inhibited by 20% by treatment with cereals that are toxic in coeliac disease (bread wheat, rye, oats and barley). BSA and maize peptides are devoid of this in vitro effect. However, the decrease in alkaline phosphatase activity during Caco-2 cell differentiation was observed in the presence of bread wheat. This could be due to slowing down of the enterocytic differentiation of cells that are susceptible to interaction with toxic peptides. Therefore, long-term cultures of Caco-2 cells constitute a useful in vitro model to assess the ability of cereal proteins to damage the coeliac small intestine.

40. SELECTIVE BINDING TO MANNAN OF GLIADIN PEPTIDES WHICH AGGLUTINATE UNDIFFERENTIATED K 562 S CELLS AND INHIBIT IN VITRO DEVELOPMENT OF FETAL RAT INTESTINE

Mannan and N,N′-diacetyl-chitobiose and N,N′,N″-triacetyl-chitotriose prevent the agglutinating activity on K 562(5) cells and the damaging in vitro effect on fetal rat intestine and atrophic coeliac mucosa of mixtures of gliadin peptides and pure A-gliadin peptides (S.Auricchio et al., J. Ped. Gastroenterol. and Nutr. 4,923, 1985). We separated by cromatography on mannan-Sepharose 4-B of peptic-tryptic(PT) digest of gliadins: 1) A small fraction (C) which was very active in agglutinating cells and inhibiting the in vitro development of fetal rat intestine (20γ/ml medium). Both effects were prevented by the sugars. 2) A much larger fraction (B) which is devoid of both activities. These results show that only a few peptides of a gliadin digest, which are specifically bound by mannan, are active in the two in vitro systems.

41 AMINES PREVENT THE IN VITRO TOXICITY OF GLIADIN PEPTIDES (gp) ON CULTURES OF FETAL RAT INTESTINE AND INHIBIT THE AGGLUTINATING ACTIVITY ON K 562 (S) CELLS

All proteins and peptides which in vitro and/or in vivo are toxic for the celiac small intestine are able to damage the in vitro developing fetal rat intestine and to agglutinate K 562 (S) cells (S.Auricchio et al.,J.Ped.Gastroenterol, and Nutr.4, 923, 1985). The minimal concentration of peptic-tryptic gp agglutinating all the cells (MAC) was found to be 73 mg/l. Various amines were able to inhibit the agglutinating activity of gp. The minimal concentration(mM) of the amines completely preventing cell agglutination induced by a gp concentration four fold the MAC was: 1.3 for putrescine, 0.8 for spermidine, 1.2 for spermine, 3 for glycinethylester, 2.3 for histamine, 1.6 for serotonine and 2.5 for monodansylcadaverine. 10 mM spermine or spermidine were unable to protect the cells from the agglutinating activity of Wheat Germ Agglutinin and Concanavalin A.Spermidine 0.35 mM significantly protected the in vitro developing fetal rat intestine from the toxic activity of gp tested at a concentration of 0.1 mg/ml culture medium in 11 experiments. Spermidine and other amines are therefore able to prevent the activity of gp in these in vitro systems; this effect might be related to a possible regulatory role of amines in endocytosis and/ or in brush border membrane functions (A.Elgavish et al., Biochim.Biophys.Acta 777, 1,1984).

A-GLIADIN RELATED SYNTHETIC PEPTIDES AGGLUTINATE K 562 S CELLS AND AFFECT IN VITRO DEVELOPING FETAL RAT INTESTINE AND ATROPHIC COELIAC MUCOSA

Peptides from wheat gliadlns, A-gliadin and prolamins from cereals toxic for coeliac patients agglutinate K 562(S) cells; they also damage in vitro cultured fetal rat intestine and atrophic coeliac mucosa. The largest common sequences among the in vitro active A-gliadin peptides were -Pro-Ser-Gln-Gln-and -(Gln)3 -Pro-. The following peptides all containing the aminoacid sequence -(Gln)3-Pro have been synthesized: the pentapeptide Tyr-(Gln) -Pro, its dimer and tetramer and the eptapeptide Gln-Pro-Tyr-(Gln)3-Pro in their free and N-acetylated forms and the Pyroglutamic derivate of the heptapeptlde (Pyr7). Pyr 7 agglutinated cells and inhibited the in vitro development of fetal rat intestine (mediums concentration 0.5-2mg/ml);it was non toxic on the in vitro cultured coeliac atrophic mucosa. The N-acetylated form of the pentapeptides tetramer (1mg/ml) also damaged the atrophic coeliac mucosa in 4 cultured biopsies. These results suggest that the sequence -(Gln)3-Pro when part of a larger peptide may be toxic in vitro for the atrophic coeliac mucosa.

38. A-GLIADIN RELATED SYNTHETIC PEPTIDES AGGLUTINATE UNDIFFERENTIATED K TO 562 S CELLS AND AFFECT IN VITRO DEVELOPING FETAL RAT INTESTINE AND ULTURED ATROPHIC COELIAC MUCOSA

Peptides from wheat gliadins, A-gliadin and prolamins from cereals toxic for coeliac patients agglutinate K 562(5) cells; they also damage in vitro cultured fetal rat intestine and atrophic coeliac mucosa. The largest common sequences among the in vitro active A-gliadin peptides were -Pro-Ser-Gln-Gln and -(Gln)3-Pro-. The following peptides all containing the aminoacid sequence -(Gln)3-Pro have been synthesized: the pentapeptide Tyr-(Gln)3-Pro, its dimer and tetramer and the epta-peptide Gln-Pro-Tyr-(Gln)3-Pro in their free and N-acetylated forms and the Pyro-glutamic derivate of the heptapeptide (Pyr 7). Pyr 7 agglutinated cells and inhibited the in vitro development of fetal rat intestine (mediums concentration 0.5-2 mg/ml); it was non toxic on the in vitro cultured coeliac atrophic mucosa. The N-acetylated form of the pentapeptides tetramer (1 mg/ml) also damaged the atrophic coeliac mucosa in 3 cultured biopsies. These results suggest that the sequence -(Gln)3-Pro when part of a larger peptide may be toxic in vitro for the atrophic coeliac mucosa.