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Background & Aims: Celiac disease is a gluten sensitive disorder that occurs in genetically susceptible individuals. The present study deals with variation in the immune response of mucosal T-cells from celiac children to prolamins extracted from nine landraces of farro wheat (Triticum turgidum ssp. dicoccum) with contrasting storage protein compositions., Methods: The prolamin fraction from nine 'dicoccum' wheat landraces was subjected to peptic-tryptic digestion and supplied to T-cells from mucosal explants of four celiac patients. Immune reactions in terms of cell proliferation and INF-gamma secretion by intestinal T lymphocytes were then determined., Results: T-cell lines exposed to digested prolamins from landraces L5563, L5558 and L5540 showed negligible proliferative responses and released INF-gamma amounts similar to that of untreated control cells. By contrast, landraces Ersa 6, Ersa 8, Leonessa 4 and Leonessa 5 proved to be very active in triggering the immune responses, whereas landraces Filosini and Prometeo exhibited an intermediate behavior. One-dimensional fractionations by A-PAGE or SDS-PAGE revealed distinctive prolamin patterns amongst the landraces analysed., Conclusions: 'Dicoccum' wheat represents a heterogeneous species showing a wide variation in both prolamin composition and T-cell immunological activation, some 'dicoccum' landraces being poor in or devoid of noxious gluten proteins.

Background: Celiac disease (CD) is a Th1-driven autoimmune permanent enteropathy that is triggered by dietary gluten. Molecules able to shift the immune response from a Th1- to a Th2-type response have been suggested as therapeutic agents for Th1 autoimmune diseases., Objective: We sought to investigate the possibility that a decapeptide from durum wheat (p10mer, QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may promote a shift from a Th1- to a Th2-type immune response in gluten-specific intestinal T cells of CD patients., Design: Intestinal T lymphocyte lines derived from 8 children with CD were incubated with gliadin peptides both alone and simultaneously with p10mer. Cell proliferation and the production of interferon-gamma and interleukin-10 by these T cells were measured., Results: The incubation of celiac intestinal T cells with deamidated gliadin peptides resulted in a significant (P < 0.008) increase in cell proliferation and interferon-gamma release, whereas the simultaneous exposure to p10mer totally abolished the cell proliferation and cytokine release. Moreover, incubation with p10mer maintained an elevated release of interleukin-10, whereas exposure of the cells to culture medium only did not. The replacement of the residues of aspartic acid in position 5 or those of alanine in position 6 in the sequence of p10mer resulted in peptides with no activity in the activation experiments., Conclusion: In vitro, p10mer showed the ability to shift the pathogenic immune response of a CD patient from a Th1- to a Th2-type response.

Carvacrol is a component of numerous aromatic plants. Up to now, no toxicological data were available. Carvacrol show a weak activity in the mutagenicity studies. Moreover, in the metabolism study, carvacrol has shown to be excreted with urine after 24 h in large quantities or unchanged or as glucoronide and sulphate conjugates. The available data do not allow the assessment of the NOEL. Further toxicological studies are needed.

No results of short-term or chronic toxicity studies have been found. Elemicin did induce UDS in hepatocytes from male rats. Studies on carcinogenicity were negative, but the 1'-hydroxy-metabolite of elemicin gave positive and negative results. The total intake of elemicin from essential oil seems to be limited. The main source of intake appears to be nutmeg. Further studies are needed to evaluate if the intake of elemicin may represent a health risk.

Under the Italian Presidency of the Council of the European Union (July 2003-December 2003) an agreement has been reached by the European Parliament and the Council on the approval of the proposal of Directive of the European Parliament and the Council amending the Directive 2001/83/EC as regards traditional herbal medicinal products. Once implemented in the E.U. Member States, this new Directive will remove the constraints that have made it difficult granting marketing authorisations of herbal substances and preparations as traditional medicinal products under the pre-existing Community legislation. The main features (i.e. traditional herbal medicine definition, simplified registration procedure, provisions for Community herbal monographs and Community list of herbal substances and preparations and establishment of the Committee for Herbal Medicinal Products) of this new Community legislation are analysed and discussed in the present paper together with some expected positive public health impacts.

Teucrium chamaedrys L. (Labiatae), a herb used to combat obesity, can occasionally cause hepatitis. All mutagenicity tests done were negative. After 13 weeks of administration by oral route in Sprague Dawley rats T. chamaedrys proved to be well tolerated at 0.056 g kg(-1) day(-1) (i.e. 0.4 mg kg(-1) day(-1) of teucrin A). At this dose the compound induced minor effects on body weight of both males and females and slight, reversible liver changes, confined to females, which mainly consisted of hepatocellular hypertrophy. This modification, in absence of other morphological findings can be considered an adaptative metabolic, rather than toxic effect.

A reading time and an ERP experiment conducted in Italian investigated the parser's responses to a syntactic violation (subject-verb number agreement) and to a semantic violation (subject-verb selectional restriction), examining the time course of comprehension processes until sentence end. The reading-time data showed that the syntactic violation was detected earlier than the semantic one and that the two violations differed in the time-course. The ERP data fully supported the reading time data: Syntactic anomalies elicited a left anterior negativity (LAN) and a P600. Semantic anomalies elicited a N400 centred on the parietal sites which started 90 ms later (latency 430 ms) than the LAN. Furthermore, the N400 evoked by the words that followed the target word continued and increased until sentence end. The results are discussed with respect to the hypotheses that the parser constructs distinct syntactic and semantic analyses of a sentence and that this characteristic holds cross-linguistically. The appropriateness of different methodologies to the study of sentence processing is also evaluated.

The subacute toxicity studies reported up to now in rats and mice suggested that mice were less susceptible than rats to the toxicity of eucalyptol. In fact, after gavage, it was found toxic in male rats at doses higher than 600 mg/kg while no effect was seen in mice up to 1200 mg/kg. However, the limitations and the quality of the study do not allow the extrapolation of a 'no effect level'. Several reports in rat and brushtail possum show the formation of hydroxylated bicycled products of eucalyptol as main metabolites. Moreover, metabolites which require ring opening have been also detected. Following the accidental exposure of human beings, death was reported in two cases after ingestion of 3.5-5 ml of essential eucalyptus oil, but a number of recoveries have also been described for much higher amounts of oil.

Background: It was recently shown that antiendomysial antibodies (EMAs), which are highly sensitive and specific for celiac disease, are produced by intestinal mucosa. Furthermore, EMAs were detected previously in supernatant fluid from cultured duodenal mucosa specimens collected from untreated celiac disease patients and in culture media of biopsy specimens collected from treated celiac disease patients after an in vitro challenge with gliadin. Moreover, it was recently shown in vivo that oats are not toxic to celiac disease patients, suggesting the safety of oats in a gluten free-diet., Objective: The objective was to better define the controversial role of oats in celiac disease to determine whether oats can be safely included in a gluten-free diet., Design: We used an in vitro model to test whether oats induce EMA production in supernatant fluid from cultured duodenal mucosa specimens collected from 13 treated celiac disease patients. The biopsy specimens were cultured with and without peptic-tryptic digest (PT) of gliadin and avenin (from oats) and in medium alone. Samples from 5 of the 13 patients were cultured with the C fraction of PT-avenin. Indirect immunofluorescence was used to detect EMAs., Results: EMAs were detected in specimens from all 13 patients after the challenge with gliadin but not after culture in medium alone. By contrast, no EMAs were detected in any of the specimens cultured with PT-avenin and its C fraction., Conclusions: Because the in vitro challenge with PT-avenin and its C fraction did not induce EMA production in treated celiac disease patients, it appears that oats have no harmful effect on celiac disease. Therefore, oats can be safely included in a gluten-free diet.

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.

Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H(2)N-gln-gln-pro-gln-asp-ala-val-gln-pro-phe-COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the '1157.5' Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the '1157.5' Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide '1157.5' Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the '1157.5' Da peptide is capable of preventing such an effect.

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.

Experimental and clinical evidence suggest that oxidative stress causes cellular damage, leading to functional alterations of the tissue. Free radicals may thus play an important role in the pathogenesis of a number of human diseases. Among pro-oxidant agents, oxidized LDL lead to the production of cytotoxic reactive species, e.g., lipoperoxides, causing tissue injury and various subsequent pathologies including intestinal diseases. Thus, to analyze the oxidative damage induced by oxidized LDL to intestinal mucosa, we evaluated morphological and functional changes induced in the human colon adenocarcinoma cell line, Caco-2. In addition, we examined the protective effects exerted by tyrosol, 2-(4-hydroxyphenyl)ethanol, the major phenolic compound present in olive oil. Caco-2 cell treatment (24 and/or 48 h) with oxidized LDL (0.2 g/L) resulted in cytostatic and cytotoxic effects characterized by a series of morphological and functional alterations: membrane damage, modifications of cytoskeleton network, microtubular disorganization, loss of cell-cell and cell-substrate contacts, cell detachment and cell death. The oxidized LDL-induced alterations in Caco-2 cells were almost completely prevented by tyrosol which was added 2 h before and present during the treatments. Our results suggest that some biophenols, such as those contained in olive oil, may counteract the reactive oxygen metabolite-mediated cellular damage and related diseases, by improving in vivo antioxidant defenses.

Experience gained through investigations on coeliac disease makes it possible to propose a screening method based on agglutination of isolated K562(S) cells to evaluate the occurrence in food protein of amino acid sequences that are able to adversely affect coeliac and related gluten-sensitive patients. The method consists of in vitro sequential peptic and tryptic digestion of food protein fractions under optimal pH, temperature and time conditions and in vitro incubation of the digest with K562(S) cells; the toxic potential is detected as an agglutination of K 562 (S) cells after a short incubation. Other in vitro test systems, including atrophic coeliac intestinal mucosa and rat fetal intestine, can be used to confirm the results obtained with the isolated cells. A fractionation step of the proteolytic digest on a sepharose-mannan column before exposure of the in vitro systems to the separated peptide fractions adds to the sensitivity of the method. This screening method is not only very useful to investigate action mechanisms in coeliac disease, but also to assess the safety of genetically-modified plant foods and novel foods for gluten-sensitive patients.

Two peptides corresponding to bread wheat A-gliadin fragments 31-43 and 44-55, well known for their ability to damage the coeliac disease intestinal mucosa both in vitro and in vivo, have been confirmed to be very active in inducing in vitro agglutination of K 562 (S) cells. Removal of six amino acid residues from the carboxy-terminal end of the 31-43 peptide, or of five amino acid residues from the amino terminal end of the 44-55 peptide, resulted in a lower, but still very significant, cell agglutination activity. The peptide consisting of ten amino acid residues with a molecular mass of 1157.5 Da, isolated from durum wheat gliadin, was able to prevent agglutination of K 562 (S) cells induced not only by prolamine peptic-tryptic digests from all the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oats), but also by the 31-43 and 44-55 peptides. The ability to protect K 562 (S) cells from agglutination was exhibited to the fullest extent also by all the peptides derived from the 1157.5-Da peptide by five progressive deletions of the terminal carboxylic residue, whereas the sixth consecutive deletion yielded a completely inactive peptide. A similar total loss of activity was observed upon addition of a glycine residue to the amino terminal residue of the 1157.5-Da peptide and all the above-mentioned active peptides derived from it. The remarkable sequence homologies existing between peptides able to induce [Gln-Gln-Gln-Pro and -Pro-Ser-Gln-Gln-] or to prevent [H2N-Gln-Gln-Pro-Gln-Asp-COOH] induction of cell agglutination strongly suggest that all these peptides compete for identical or structurally related binding sites on the cell surface.

A peptide (m.w. 1157.5 Da) able to prevent the agglutination of K562(S) cells induced by the peptic-tryptic prolamine digests of the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oat) was characterized as one of the components of the peptic-tryptic digest of durum wheat gliadin. This peptide was synthesized in a high degree of purity with the solid phase method with the Applied Biosystem 431A. An amino acid sequence was identified in the 1157.5 Da peptide as being related to the largest common sequences previously detected in a series of bread wheat toxic peptides by other authors.

The K 562 (S) cell agglutinating activity of peptides obtained from in vitro digestion of bread wheat gliadins has been shown to be associated with a small fraction (coded as Fraction C), that can be easily separated by affinity chromatography of the whole digest on a sepharose 6-B-mannan or sepharose 6-B-oligomers of N-acetyl-glucosamine. Although the whole gliadin digests from 12 durum wheat varieties were unable to agglutinate K 562 (S) cells, all these digests were found to contain an active Fraction C. The lack of agglutinating activity of the whole durum wheat gliadin digests has been shown to be associated with the presence in these digests of another peptide fraction (coded as Fraction B) that is eluted much earlier from the sepharose 6-B-mannan column and is able to inhibit the cell agglutinating activity of Fraction C. Such an active Fraction B is not present in bread wheat gliadin peptides, although peptides with the same elution profile as Fraction B have been detected.

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.