Massimo De Vincenzi

Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

ABSTRACT Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.

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.

Wheat gliadin induces apoptosis of intestinal cells via an autocrine mechanism involving Fas^Fas ligand pathway

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro‐apoptotic activity exerted by gliadin‐derived peptides in Caco‐2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest‐induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas‐mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity.

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.

Effect of a gluten-free diet on the risk of enteropathy-associated T-cell lymphoma in celiac disease.

Patients with celiac disease have an increased rate of enteropathy-associated T-cell lymphoma, but conflicting data are available about the protective role of a gluten-free diet with regard to the development of this malignancy. We followed 1,757 celiac patients for a total period of 31,801 person-years, collecting data about the frequency of gluten intake and the incidence of the enteropathy-associated T-cell lymphoma. Out of the nine celiac patients who developed an intestinal lymphoma [standard morbidity ratio of 6.42 (95% CIxa0=xa02.9–12.2; Pxa0<xa00.001)], only two kept a strict gluten-free diet after the diagnosis of celiac diasese and developed the malignancy after the peridiagnosis period of 3xa0years, dropping therefore the standard morbidity ratio to 0.22 (95%CIxa0=xa00.02–0.88; Pxa0<xa00.001). The risk of developing an intestinal lymphoma for the celiac patients that used to have dietary gluten was significant (X2xa0=xa04.8 Pxa0=xa00.01). These results show that a strict gluten-free diet is protective towards the development of enteropathy-associated T-cell lymphoma.

Quorum sensing in sourdough Lactobacillus plantarum DC400: Induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells

This work aimed at showing the effect of pheromone plantaricin A (PlnA) by Lactobacillus plantarum DC400 towards other sourdough lactic acid bacteria and the potential of PlnA to protect the function of the human intestinal barrier. Growth and survival of sourdough lactic acid bacteria were differently affected by co‐cultivation with L. plantarum DC400. Compared to mono‐cultures, Lactobacillus sanfranciscensis DPPMA174 and Pediococcus pentosaceus 2XA3 showed growth inhibition and decreased viability when co‐cultured with L. plantarum DC400. L. sanfranciscensis DPPMA174 induced the highest synthesis of PlnA. Survival of strain DPPMA174 only slightly varied by comparing the addition of PlnA to the culture medium and the co‐cultivation with L. plantarum DC400. Compared to mono‐culture, the proteome of L. sanfranciscensis DPPMA174 grown in co‐culture with L. plantarum DC400 showed the variation of expression of 58 proteins (47 over expressed and 11 repressed). Thirty‐four of them were also over expressed or repressed during growth of DPPMA174 with PlnA. Fifty‐one of the above 58 proteins were identified. They had a central role in stress response, amino acid, energy and nucleotide metabolisms, membrane transport, regulation of transcription, and cell redox homeostasis. PlnA markedly increased the viability of human Caco‐2/TC7 cells and the transepithelial electrical resistance.

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.

In vitro toxicity testing of alcohol-soluble proteins from diploid wheat Triticum monococcum in celiac disease.

Peptic-tryptic digests of alcohol-soluble proteins from flours of 10 accessions of Trificum monococcum with contrasting storage protein compositions and bread-making characteristics were found unable to agglutinate K562(S) cells even at a peptide concentration as high as 14 g/L, agglutination being strongly correlated with toxicity in celiac disease. When fractionated by affinity chromatography on Sepharose-6B coupled with mannan, peptic-tryptic digests separated into three fractions. Fraction C peptides were shown to agglutinate K562(S) cells, whereas peptides in fractions A and B and in the mixed fraction B + C were inactive, suggesting that fraction B contains protective peptides that interfere with toxic peptides in fraction C in their agglutinating activity. These results offer an opportunity to study the biochemical and genetic bases of wheat toxicity at the diploid level. Moreover, the reduced toxicity, if any, of Triticum monococcum in the celiac disease, along with the good grain characteristics of some monococcum accessions, greatly increases the economical prospects of this wheat species.

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.

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.