Constraints on Sub-GeV Dark-Matter-Electron Scattering from the DarkSide-50 Experiment.

We present new constraints on sub-GeV dark-matter particles scattering off electrons based on 6780.0 kg d of data collected with the DarkSide-50 dual-phase argon time projection chamber. This analysis uses electroluminescence signals due to ionized electrons extracted from the liquid argon target. The detector has a very high trigger probability for these signals, allowing for an analysis threshold of three extracted electrons, or approximately 0.05 keVee. We calculate the expected recoil spectra for dark matter-electron scattering in argon and, under the assumption of momentum-independent scattering, improve upon existing limits from XENON10 for dark-matter particles with masses between 30 and 100 MeV/c^{2}.

Low-Mass Dark Matter Search with the DarkSide-50 Experiment.

We present the results of a search for dark matter weakly interacting massive particles (WIMPs) in the mass range below 20 GeV/c^{2} using a target of low-radioactivity argon with a 6786.0 kg d exposure. The data were obtained using the DarkSide-50 apparatus at Laboratori Nazionali del Gran Sasso. The analysis is based on the ionization signal, for which the DarkSide-50 time projection chamber is fully efficient at 0.1 keVee. The observed rate in the detector at 0.5 keVee is about 1.5 event/keVee/kg/d and is almost entirely accounted for by known background sources. We obtain a 90% C.L. exclusion limit above 1.8 GeV/c^{2} for the spin-independent cross section of dark matter WIMPs on nucleons, extending the exclusion region for dark matter below previous limits in the range 1.8-6 GeV/c^{2}.

Constituents of aromatic plants: carvacrol.

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.

Constituents of aromatic plants: elemicin.

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.

Constituents of aromatic plants: teucrin A.

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.

Constituents of aromatic plants: eucalyptol.

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.

Immunologic evidence of no harmful effect of oats in celiac disease.

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.

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.

Induction of apoptosis in caco-2 cells by wheat gliadin peptides.

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.

31-43 amino acid sequence of the alpha-gliadin induces anti-endomysial antibody production during in vitro challenge.

BACKGROUND: Wheat gliadin is the culprit antigen of coeliac disease (CD). Two short sequences of NH2-terminal portion of gliadin seem to be responsible for CD. Antiendomysial antibodies (EMA), highly sensitive and specific for CD, are detectable in the culture media from treated CD patients, after in vitro challenge with peptic-tryptic (PT) digest of gliadin. In this study we detected EMA production after in vitro challenge with 31-43 peptide. We used 56-68 peptide, lacking toxic sequences, as a negative control. METHODS: Duodenal samples from 11 treated CD patients and 9 control patients were cultured with 31-43 and 56-68 peptides and PT gliadin. Indirect immunofluorescence analysis was used for EMA detection. RESULTS: EMA were detected in culture media of 10 of 11 specimens challenged with PT-gliadin and in the media of all specimens challenged with 31-43 peptide. No EMA were detectable in any treated patients cultured with 56-68 peptide or with medium alone. No EMA were observed in cultures of control specimens. DISCUSSION: The ability of the 31-43 sequence of the alpha-gliadin to induce EMA production suggests its involvement in the pathogenesis of CD. Furthermore, it may be a more useful antigenic substance than PT gliadin for both in vitro and in vivo studies of CD.

In vitro screening of food peptides toxic for coeliac and other gluten-sensitive patients: a review.

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.

Tyrosol, the major olive oil biophenol, protects against oxidized-LDL-induced injury in Caco-2 cells.

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.

Bioactive antinutritional peptides derived from cereal prolamins: a review.

Alcohol-soluble endosperm proteins (prolamins) from some cereals (e.g. wheat, barley, and rye) give origin upon proteolytic digestion to biologically-active antinutritional peptides able to adversely affect in vivo the intestinal mucosa of coeliac patients, whereas prolamins from other cereals (e.g. maize and rice) do not. These antinutritional peptides are also able to: (a) prevent in vitro recovery of atrophic coeliac mucosa; (b) to inhibit differentiation of isolated rat fetal and chick fetal intestines; and (c) to interact with undifferentiated cells either agglutinating them or affecting their proliferation and metabolism. Studies performed with A-gliadin, a highly purified bread wheat prolamin fraction, and its fragments obtained either by chemical cleavage of A-gliadin or by synthesis from aminoacids, clearly pointed out to a few small sequences very rich in glutamine and proline residues as the biologically-active agents. Several protective substances, including mannan and N,N',N"-triacetylchitotriose, have been identified as being able to prevent the effects of these peptides in vitro, but the evidence of their in vivo activity is still missing. The present paper provides a synthetic overview of the available data concerning this highly complex matter and offers a critical appraisal of present hypotheses on the action mechanism of biologically-active peptides derived from cereal prolamins.

In vitro cytotoxic effect of wheat gliadin-derived peptides on the Caco-2 intestinal cell line is associated with intracellular oxidative imbalance: implications for coeliac disease.

Coeliac disease (CD) is an inflammatory disorder of the upper small intestine in which gluten acts as an essential factor in its pathogenesis. Although it is generally accepted that cereal protein activation of the immune system is involved in CD progression, a non-immunomediated cytotoxic activity of gliadin-derived peptides on the jejunal/duodenal tract cannot be excluded. In this work, considering that (a) little has been reported about the intracellular metabolic events associated with gliadin toxicity, and (b) an important role for free radicals in a number of gastrointestinal disease has been demonstrated, we investigated the in vitro effects of gliadin-derived peptides on redox metabolism of Caco-2 intestinal cells during a kinetic study in which cells were exposed to peptic-tryptic digest of bread wheat up to 48 h. We found that the antiproliferative effects displayed by gliadin exposure was associated with intracellular oxidative imbalance, characterised by an increased presence of lipid peroxides, an augmented oxidised (GSSG)/reduced (GSH) glutathione ratio and a loss in protein-bound sulfhydryl groups. Significant structural perturbations of the cell plasma membrane were also detected. Additional experiments performed by using the specific GSH-depleting agent buthionine sulfoximine provide evidence that the extent of gliadin-induced cell growth arrest critically depends upon the 'basal' redox profile of the enterocytes. On the whole, these findings seem to suggest that, besides the adoption of a strictly gluten-free diet, the possibility for an adjuvant therapy with antioxidants may be considered for CD patients.

Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B.

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

BACKGROUND: CD3 and gamma delta 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.

Structural specificities and significance for coeliac disease of wheat gliadin peptides able to agglutinate or to prevent agglutination of K562(S) cells.

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 small peptide from durum wheat gliadin prevents cell agglutination induced by prolamin-peptides toxic in coeliac disease.

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.