M. De Vincenzi

Effects of Gliadin-Derived Peptides from Bread and Durum Wheats on Small Intestine Cultures from Rat Fetus and Coeliac Children

Summary: Peptic-tryptic-cotazym (PTC) digests were obtained, simulating in vivo protein digestion, from albumin, globulin, gliadin and glutenin preparations from hexapiod (bread) wheat as well as from diploid (monococcum) and tetraploid (durum) wheat gliadins. The digest from bread wheat gliadins reversibly inhibited in vitro development and morphogenesis of small intestine from 17-day-old rat fetuses, whereas all the other digests (obtained both from nongliadin fractions and from gliadins from other wheat species) were inactive.The PTC-digest from bread wheat gliadins was also able to prevent recovery of and to damage the in vitro cultured small intestinal mucosa from patients with active coeliac disease (gluteninduced entheropathy). The PTC-digest from durum wheat gliadins caused a much less adverse effect on this human pathologic tissue culture system.Speculation: When tested with different in vitro systems, bread wheat gliadin peptides active in coeliac disease displayed several peculiar biologic activities including an immunogenic character in susceptible individuals (Z), a probable immunomediated cytotoxic activity on small intestinal mucosa specimens from coeliac patients (6-10, 13-15, 17), and a probable direct cytotoxic activity on developing rat fetal intestine (5). In fact, these peptides induce the following: (I) proliferation of peripheral blood lymphocytes from coeliac patients as well as from many first degree relatives of coeliacs and from only a few normal controls; (2) damage of the in vitro cultured small intestinal mucosa of patients with active coeliac disease; and (3) reversible inhibition of the in vitro development of fetal rat intestine.Although no one of the mentioned in vitro systems can be considered a model fully representative of coeliac disease, it is likely that each one of them highlights a different important feature underlying the appearance of the small intestine lesion in coeliac patients ingesting bread wheat gliadin peptides. Peptide mixtures obtained by enzymic digestion of the gliadin fraction from hexaploid (bread) wheats significantly differ from those from tetraploid (durum) wheat gliadins for their higher toxicity toward cultures of intestine from rat fetuses or coeliac children. However, gliadin peptides from both bread and durum wheats are capable of inducing proliferation of peripheral blood lymphocytes from coeliac patients thus suggesting a similar immunogenic character (2).We suggest that durum wheat gliadins peptides are in vitro less toxic than bread wheat gliadins peptides for the small intestinal mucosa of coeliacs as they have less direct cytotoxic effect on the enterocyte; moreover, durum wheat products (e.g., spaghetti), as compared to bread wheat products (e.g., bread and biscuits), migh present a lower risk for patients suffering for coeliac disease or other wheat intolerances.

Constituents of aromatic plants : II. Estragole

Estragole (ES) is a natural constituent of a number of plants (e.g. tarragon, sweet basil and sweet fennel) and their essential oils have been widely used in foodstuffs as flavouring agents. Several studies with oral, i.p. or s.c. administration to CD-1 and B6C3F1 mice have shown the carcinogenicity of ES. The 1-hydroxy metabolites are stronger hepatocarcinogens than the parent compound. Controversial results are reported for the mutagenicity of ES. However, the formation of hepatic DNA adducts in vivo and in vitro by metabolites of ES has been demonstrated.

Agglutinating activity of gliadin-derived peptides from bread wheat: Implications for coeliac disease pathogenesis

The PT-digest of bread wheat gliadin was very active in agglutinating undifferentiated human K562(S) cells. This activity was quantitatively, but not qualitatively, similar to that of Con A or WGA. Moreover, Con A-induced cell agglutination was inhibited by mannan and mannose, WGA-induced agglutination by NAG only, and cell agglutination induced by bread wheat gliadin peptides was inhibited by each of these three saccharides. Not only was mannan the most active saccharide in preventing cell agglutination induced by bread wheat gliadin peptides, but it was also able to dissociate agglutinated cells. As compared to the PT- digest of whole bread wheat gliadin, the digest obtained from purified A-gliadin was tenfold more active. The PT-digest of durum wheat gliadin did not show any agglutinating activity.

Gliadin induced changes in the expression of MHC-class II antigens by human small intestinal epithelium. Organ culture studies with coeliac disease mucosa.

Jejunal biopsies from 16 treated coeliac disease patients and from nine controls were cultured with and without a peptic-tryptic digest of gliadin. Cultures with a peptic-tryptic digest of maize prolamins were also undertaken. Frozen sections of baseline and cultured mucosa were stained by immunofluorescence with an anti-HLA-DR monoclonal antibody. Before culture the villous epithelium from both controls and treated coeliac disease expressed DR molecules while the crypt epithelium did not. When biopsies from treated coeliac disease were cultured with gliadin the expression of DR was enhanced in the crypt epithelium in eight of 14 cultures and in 11 of 14 was reduced or absent on the villous epithelium. No change was observed in control cultures. We conclude that gliadin is capable of inducing HLA-DR on the crypt epithelium of in vitro cultured coeliac disease mucosa, providing indirect evidence that gliadin may activate cell mediated immune mechanisms within the small bowel mucosa. This model could prove useful in identifying the immunogenic sequence(s) of gliadins and related prolamins.

Constituents of aromatic plants: I. Methyleugenol

Methyleugenol (ME) is a natural constituent of the essential oils of a number of plants widely used in foodstuffs as flavouring agents. Its occurrence, routes of intake, and available information on toxicity, kinetics and metabolism are reviewed. In view of the carcinogenic potential of ME, the need to check its presence in food products with effective analytical methods is pointed out.

An in Vitro Animal Model for the Study of Cereal Components Toxic in Celiac Disease

ABSTRACT: Peptic-tryptic-Cotazym (PTC) digests were obtained, simulating in vivo protein digestion, from rice, maize, rye, oats, barley, and sorghum prolamines and tested on small intestine cultures from rat fetus. The PTC digests of the prolamine fractions from rice and maize, even when tested at a concentration as high as 0.5 mg/ml, did not affect in vitro differentiation and maturation of fetal rat jejunum that took place in vitro in a way comparable to what happens in vivo. On the contrary, the PTC digests of prolamines from rye, oats, barley, and sorghum were very active in slowing down in vitro development of fetal rat intestine. These results further strengthen earlier findings and all together show that there is a strong correlation between toxicity results of cereal and/or cereal components assessed with clinical trials or in vitro systems based on bioptic specimens of intestinal mucosa from celiac patients and with the culture of rat fetal intestine. Therefore, the rat fetal intestine culture is considered to be an adequate model for screening and investigating cereal peptides which are toxic for the celiac small intestinal mucosa.

Intestinal Mucosa of Celiacs in Remission Is Unable To Abolish Toxicity of Gliadin Peptides on in Vitro Developing Fetal Rat Intestine And Cultured Atrophic Celiac Mucosa

ABSTRACT: Subfraction 2R of fraction 9 from a peptic-tryptic-pancreatic digest of wheat gliadin is known to be toxic in vivo to celiac patients. We have found that fractions 9 and 2R inhibit the in vitro development of fetal rat intestine and the increase of enterocyte height occurring in organ culture of atrophic celiac mucosa (0.1–0.5 mg/ml medium). Other peptide fractions of the gliadin digest are devoid of such in vitro effects. Subfraction 2R, after incubation with morphologically normal small intestinal mucosa of celiacs in remission and ultrafiltration, was still very active in both culture systems at low concentration (0.1 mg/ml); on the contrary, subfraction 2R was inactivated after incubation with normal mucosa. These results are compatible with the hypothesis that there is a mucosal defect in handling gliadin peptides in celiac disease, and suggest that there is either a primary (or secondary) enzyme deficiency or some other mechanism operating in the intestinal mucosa of celiac patients in remission.

Gliadin activates mucosal cell mediated immunity in cultured rectal mucosa from coeliac patients and a subset of their siblings

Background—CD3 and γδ cells in the rectal mucosa increase after local instillation of gluten in children with coeliac disease and in half of their siblings. Aim—To establish an in vitro system for assessing immunological changes induced by gluten in the rectum. Patients and Methods—Rectal biopsy specimens obtained from 13 treated coeliac children, nine of their siblings, and nine controls were cultured in vitro with a peptic-tryptic digest of gliadin or ovalbumin. CD3 and CD25 cells were counted, and the expression of adhesion molecules evaluated. Results—In the lamina propria of coeliac biopsy samples cultured with gliadin, but not in those from controls, the expression of vascular cell adhesion molecule 1 (VCAM-1) was enhanced, and the number of CD25 cells was significantly higher than in those cultured in medium alone; the density of intraepithelial CD3 cells was also significantly higher. No differences were noted in coeliac biopsy specimens cultured with ovalbumin. A discriminant analysis allowed correct classification of all controls and all coeliacs but one, but three of nine siblings were allocated to the coeliac group. Conclusions—Our data confirm that gliadin is able to activate cell mediated immunity in the rectal mucosa in coeliac patients and in a subset of their first degree relatives.

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.

Inhibition of the cellular metabolism of Caco-2 cells by prolamin peptides from cereals toxic for coeliacs.

Peptic-tryptic (PT) digests of prolamins derived from several cereals were tested on differentiated Caco-2 cells to study the effect on cellular metabolism, particularly on DNA, RNA, protein and glycoprotein synthesis. Cell viability was evaluated after treatment with the same cereals. Whereas PT digests from bovine serum albumin and both durum wheat types (diploid and tetraploid) did not exert any effect, bread wheat, oats, barley and rye exerted an inhibitory effect close to 80% for DNA and RNA synthesis and close to 60% for (glyco)protein synthesis. Cell viability evaluated by MTT tests did not show any differences between treated and untreated cells. These observations, and previous results, suggest that, whereas prolamin-derived peptides from bread wheat, barley, rye and oats did not cause an immediate cytotoxic effect, they, were however, responsible for cell damage impairing cell metabolism.