Rita Di Benedetto

Delayed diagnosis of coeliac disease increases cancer risk.

BackgroundThe association between coeliac disease (CD) and neoplasms has been long established, but few data are available about the risk factors. The aim of this paper is to estimate the risk of developing a neoplasm among non diagnosed coeliac patients and to evaluate if this risk correlates with the age of patients at diagnosis of coeliac disease.MethodsThe study population consists of patients (n = 1968) diagnosed with CD at 20 Italian gastroenterology referral Centers between 1st January 1982 and 31st March 2005.ResultsThe SIR for all cancers resulted to be 1.3; 95% CI = 1.0–1.7 p < 0.001. The specific SIRs for non Hodgkin lymphoma was 4.7; 95% CI = 2.9–7.3 p < 0.001, for the small bowel carcinoma 25; 95% CI = 8.5–51.4 p < 0.001, for non Hodgkin lymphoma 10; 95% CI = 2.7–25 p = 0.01, finally for the stomach carcinoma 3; 95% CI = 1.3–4.9 p < 0.08. The mean age at diagnosis of CD of patients that developed sooner or later a neoplasm was 47,6 ± 10.2 years versus 28.6 ± 18.2 years of patients who did not.ConclusionCoeliac patients have an increased risk of developing cancer in relation to the age of diagnosis of CD. This risk results higher for malignancies of the gastro-intestinal sites. An accurate screening for tumors should be performed in patients diagnosed with CD in adulthood and in advancing age.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP‐mediated pathways. In this study, we investigated whether n‐3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2‐day‐old rats, which were then killed after 3 days post‐injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin‐specific mRNAs was analyzed by real‐time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA‐ and EPA‐treated animals, but the effect was more pronounced in EPA‐treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA‐treated rats. A further indicator of accelerated myelination was the increase in 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) protein levels. In EPA‐treated rats, the increased expression of myelin genes coincided with a decrease of cAMP‐response element‐binding protein (CREB)‐DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA‐treated rats was less pronounced than in controls. The down‐regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n‐3 PUFA beneficial effects on several demyelinating diseases.

Avenins from different cultivars of oats elicit response by coeliac peripheral lymphocytes

Objective. The avoidance of oats in coeliac patients is still controversial. If oats is confirmed to be safe, it would be a valuable component and offer more variation in a gluten-free diet. The aim of this work was to evaluate whether avenins from different varieties of oats show different abilities in the activation of coeliac peripheral lymphocytes. Material and methods. In order to assess whether the immunogenic effect of oats varies according to the cultivar, peripheral lymphocytes from 10 coeliac children were exposed to avenins from four different oats varieties: Lampton, Astra, Ava and Nave. Lymphocyte proliferation and interferon-gamma (IFN-γ) release in the culture medium were measured as indexes of immune activation. Results. All the varieties of oats tested were immunogenic, with Lampton and Ava avenins inducing lymphocyte activation similar to that activated by wheat gliadin, while Astra and Nave avenins showed less immunogenicity, but still with a measurable effect. Conclusions. There are still concerns about the suitability of including oats in a gluten-free diet. Coeliac patients consuming oats-containing food should be carefully monitored, until there is more evidence to show the safety of oats and varieties of low-toxicity oats.

A decapeptide from durum wheat prevents celiac peripheral blood lymphocytes from activation by gliadin peptides.

Identifying antagonist peptides able to inhibit the abnormal immune response triggered by gliadin peptides in celiac disease (CD) is an alternative therapeutic strategy for CD. The aim of this study was to evaluate the antagonist effect of 10mer, a decapeptide (sequence QQPQDAVQPF) from alcohol-soluble protein fraction of durum wheat, assessing its ability to prevent celiac peripheral blood lymphocytes from activation by gliadin peptides. Peripheral blood mononuclear cells (PBMC) were obtained from DQ2-positive untreated coeliac children and from healthy controls and incubated with the peptic-tryptic digest of bread wheat gliadin (GLP) and peptide 62–75 from α-gliadin both alone and with 10mer simultaneously. PBMC proliferation, release of pro-inflammatory Th1 cytokines interferon-γ and tumor necrosis factor-α, release of immunoregulatory cytokine IL-10, and analysis of CD25 expression as indexes of lymphocytes activation were carried out. Enhanced lymphocytes activation was seen after exposure to GLP and p62–75, whereas the simultaneous incubation with 10mer inhibits the lymphocytes response. These data indicate that a peptide naturally occurring in durum wheat exerts in vitro an antagonist effect against gliadin toxicity and could have a protective effect in CD disease.

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells

n−3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 μM of either C20:4n−6 (ARA), or C20:5n−3 (EPA), or C22:6n−3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n−6 and C22:5n−3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n−3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.

Prevention by a decapeptide from durum wheat of in vitro gliadin peptide-induced apoptosis in small-bowel mucosa from coeliac patients

TO THE EDITOR: Villous atrophy is the main distinctive feature of coeliac small-bowel mucosa. Enterocyte apoptosis is the pivotal mechanism in determining the villous atrophy [1], and therefore prevention of this event could be a therapeutic strategy for treatment of coeliac disease (CD). A decapeptide from the alcohol-soluble protein fraction of durum wheat (10mer, sequence QQPQDAVQPF) has been previously reported to protect some cell lines from gliadin-induced programmed death [2,3]. We tested the ability of 10mer to prevent gliadin-induced enterocyte apoptosis in small-bowel mucosa from coeliac patients. In order to do this, small-bowel mucosa specimens from five untreated coeliac patients (mean age 13 years, range 5.3 17.1 years) were incubated with the alcohol-soluble protein fraction from whole cereal flour of bread wheat (Triticum aestivum , variety S. Pastore) peptic-tryptic digest [4] and 10mer, both alone and simultaneously. Briefly, the specimens obtained from three patients were sliced into two parts. One portion was exposed in vitro to peptic-tryptic gliadin digest (GLP) (1 mg/ml) in RPMI medium supplemented with foetal bovine serum (FBS) 10% and 1% antibiotic/antimycotic (all reagents obtained from Invitrogen, Carlsbad, Calif., USA) for 24 h on a stainless steel grid in a centre-well organ culture dish (Falcon BD, Franklin Lakes, N.J., USA). The other portion was incubated with 10mer (0.5 mg/ml) and GLP (1 mg/ml) for 24 h, under the same conditions as those described above. As negative controls, the mucosa specimens from two patients were incubated with medium alone. The sequence of peptide 10mer (MW: 1157 D) was identified in the alcohol-soluble protein fraction of durum wheat (T. durum , variety Adamello) by De Vincenzi et al. [2]. Peptide 10mer was synthesized (Primm Company, Milan, Italy) by the solid-phase method using the Applied Biosystem model 431A and purified up to 99% by reverse-phase high-performance liquid chromatography on the Varian 5020 system. The incubation was carried out in a modular incubator chamber at 378C, with a mixture of 95% O2/5% CO2. After the incubation, the specimens were embedded in optimal cutting temperature (OCT), Bioptica, Milano, Italy) and tissue sections of 5 mm were obtained at cryostat. Informed consent was obtained from all the patients and the Ethics Committee of Istituto Superiore di Sanità approved the study. DNA fragmentation as the index of cell apoptosis was assayed on tissue sections by terminal deoxynucleotidyl transferase (TdT) mediated dUTP digoxigenin nick-end labelling (TUNEL) using a commercial kit, in accordance with the manufacturer’s instructions (Roche, Basel, Switzerland). Two-colour immunofluorescence staining was used to determine whether the TUNEL / cells were epithelial cells or CD3 / T lymphocytes. The experiments were carried out by developing the TUNEL reaction in green fluorescence and incubating the tissue sections with anti-CD3 mAb (1:300; Dako Company, Copenhagen, Denmark) followed by red anti-mouse IgG (1:100; Molecular Probes, Carlsbad, Calif., USA). Images were merged using Adobe Photoshop software.

Free radical release in C6 glial cells enriched in hexacosanoic acid: implication for X-linked adrenoleukodystrophy pathogenesis.

Free radicals have been implicated in the etiopathology of some neurological and demyelinating diseases. To evaluate their involvement in the cerebral form of X-linked adrenoleukodystrophy (cerALD) disorder, characterised by very long chain fatty acid (VLCFA) accumulation, we utilised an in vitro model using rat C6 glial cells, enriched in hexacosenoic acid (C26:0, HA). Modified cells were incubated in presence of oxidative stressors, such as bacterial endotoxin lipopolisaccharides (LPS) and human oxidised low-density lipoprotein (ox-LDL), and the production of proinflammatory cytokines, nitrite, nitrate and superoxide was determined in the supernatants. The results show that modified cells produce higher amounts of nitric oxide (NO) products and superoxide compared to native C6 cells, supporting the role of free radicals as important pathophysiological modulator of the neuroinflammatory response in ALD. This hypothesis suggests that the cerebral damage in ALD could be due to intracellular signalling activated by interaction of exogenous factors with the particular membrane fatty acid composition.

Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 μM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.

Micronutrient-enriched rapeseed oils reduce cardiovascular disease risk factors in rats fed a high-fat diet.

Many epidemiological studies have demonstrated that vegetable food consumption is associated with a reduced risk of cardiovascular diseases. The beneficial effects have been attributed to the content of bioactive molecules present in large quantities in plant food. The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils (optimised oils) obtained using different crushing and refining procedures and characterised by different quantities and qualities of micronutrients, could have any beneficial effect on lipid profile and antioxidant status of plasma and liver. Sprague-Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimised rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimised oils all had a hypolipidaemic effect. In the group fed the highest levels of micronutrients, the reduction in plasma and hepatic triglycerides reached 25% and 17%, respectively, that of cholesterol 20% and 14%, respectively. In plasma, the ferric antioxidant capacity, superoxide dismutase, glutathione peroxidase and reduced glutathione significantly increased and lipid peroxidation decreased in parallel with the enhancement of micronutrients. The same trend was observed in the liver, except for glutathione peroxidase which was not affected by optimised oils. These results indicate that a regular intake of optimised rapeseed oils can help to improve lipid status and prevent oxidative stress, providing evidence that optimised oils could be a functional food with potentially important cardioprotective properties.

Ethyl-eicosapentaenoic acid ameliorates the clinical course of experimental allergic encephalomyelitis induced in dark agouti rats

Eicosapentaenoic acid (EPA), a fatty acid present in high amount in fish, modulates immune response and stimulates myelin gene expression. In the present paper, we investigated the effects of EPA in an established animal model for multiple sclerosis (MS): experimental autoimmune encephalomyelitis (EAE) induced in dark agouti rats. Diets supplemented either with 0.2% or 0.4% of EPA were administrated daily from the day of induction until the end of experiment. One group of rats received diet supplemented with 0.2% of EPA 10 days before induction. The control group (immunized rats) was fed with chow diet. The animals were analyzed at two different stages of the disease: during the acute phase (14 d.p.i.) and during the recovery phase (32 d.p.i.). We showed a delayed onset of clinical severity of disease in all groups of rats fed EPA-supplemented diets. This effect was associated to an increased expression of myelin proteins and an improved integrity of the myelin sheath as well as an up-regulation of FoxP3 expression in the central nervous system during the acute phase of EAE. No significant changes in T cell subsets were noted at the periphery. On the contrary, during the recovery phase of EAE, in animals assuming EPA-supplemented diet, an increase of CD4(+)CD25(+) and CD4(+)CD25(+)FoxP3(+) in peripheral lymphocytes was noted. Our results indicate that EPA-supplemented diets may provide benefits to MS patients.