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}.

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}.

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.

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.

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.

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.

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.

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.

Agglutinating activity of wheat gliadin peptide fractions in coeliac disease.

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.

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.

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.

Teratogenicity study of ammonium glycyrrhizinate in the Sprague-Dawley rat.

Ammonium glycyrrhizinate (AG), a commercially used salt of glycyrrhizic acid, was administered in the drinking-water to Sprague-Dawley rats on days 7-17 of pregnancy. The actual intakes were 0, 21.33 +/- 1.22, 238.75 +/- 17.50 and 679.94 +/- 69.87 mg AG/kg body weight/day for groups 0, 1, 2 and 3, respectively. AG caused polydipsia in the dams. Foetuses from the treated litters did not present an increase in external malformations, a decrease in weight or a decrease in the degree of ossification. However, there was a slight but significant increase in embryolethality and in the prevalence of external haemorrhages. Skeletal examination revealed a dose-related increase in minor anomalies, especially in the sternebral variants. Renal ectopy also increased significantly at the highest dose. These results indicate that the possible embryotoxicity of aromatizing compounds should be considered.

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: 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: 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: 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.

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.

Gluten-sensitive enteropathy.

Peptides originating from wheat gluten during digestion in the alimentary tract are known to cause primary intolerances in genetically-predisposed individuals. Other cereals such as rye, barley and probably oats are also toxic for coeliac patients, whereas rice and maize are considered to be non-toxic. The mechanism by which prolamine-derived peptides produce jejunal lesions is not fully understood. Most investigators favour a dysregulated immune response to gliadins as the underlying abnormality in coeliac disease, but according to other authors a non-immuno-mediated cytotoxic activity of gliadin peptides on the small intestine seems to be the primary cause of intestinal mucosal damage in coeliac patients. This paper is a critical appraisal of current theories on the pathogenic mechanism underlying this disease. Moreover, many in vitro systems needed to investigate the cereal toxicity are described.