Olimpia Vincentini

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.

Synthesis of Isoflavone Aglycones and Equol in Soy Milks Fermented by Food-Related Lactic Acid Bacteria and Their Effect on Human Intestinal Caco-2 Cells

One hundred and three strains of lactic acid bacteria, isolated from various food ecosystems, were assayed for β-glucosidase activity toward p-nitrophenyl-β-D-glucopyranoside substrate. Lactobacillus plantarum DPPMA24W and DPPMASL33, Lactobacillus fermentum DPPMA114, and Lactobacillus rhamnosus DPPMAAZ1 showed the highest activities and were selected as the mixed starter to ferment various soy milk preparations, which mainly differed for chemical composition, protein dispersibility index, and size dimension. The soy milk made with organically farmed soybeans (OFS) was selected as the best preparation. All selected strains grew well in OFS soy milk, reaching almost the same values of cell density (ca. 8.5 log cfu/mL). After 96 h of fermentation with the selected mixed starter, OFS soy milk contained 57.0 μM daidzein, 140.3 μM genistein, 20.4 μM glycitein, and 37.3 μM equol. Fermented and nonfermented OFS soy milks were used for the in vitro assays on intestinal human Caco-2/TC7 cells. Fermented OFS soy milk markedly inhibited the inflammatory status of Caco-2/TC7 cells as induced by treatment with interferon-γ (IFN-γ) (1000 U/mL) and lipopolysaccharide (LPS) (100 ng/mL), maintained the integrity of the tight junctions, even if subjected to negative stimulation by IFN-γ, and markedly inhibited the synthesis of IL-8, after treatment with interleukin-1β (2 ng/mL). As shown by using chemical standards, these effects were due to the concomitant activities of isoflavone aglycones and, especially, equol, which were synthesized in the fermented OFS soy milk preparation.

Environmental factors of celiac disease: Cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. dicoccum and T. aestivum ssp. spelta

Background and Aim:  In the present paper, the toxicity of prolamines derived from three cereals with a different genome was investigated in human colon cancer Caco‐2/TC7 and human myelogenous leukemia K562(S) cells. The purpose of this study was to investigate if species from ancient wheat could be considered as healthy food crops devoid or poor in cytotoxic prolamines for celiac disease.

Exploitation of the health-promoting and sensory properties of organic pomegranate (Punica granatum L.) juice through lactic acid fermentation

Two strains (POM1 and C2) or LP09 of Lactobacillus plantarum, which were previously isolated from tomatoes and carrots, and another commercial strain of L. plantarum (LP09), were selected to singly ferment (30 °C for 120 h) pomegranate juice (PJ) under standardized protocol. PJs were further stored at 4 °C for 30 days. Filtered PJ, not added of starters (unstarted PJ), was used as the control. After fermentation, all starters grew to ca. 9.0 Log CFU/mL. Viable cells of strain LP09 sharply decreased during storage. The other two strains survived to ca. 7.0 and 8.0 Log CFU/mL. Lactic acid bacteria consumed glucose, fructose, malic acid, and branched chain and aromatic amino acids. The concentration of free fatty acids increased for all started PJs. Compared to unstarted PJ, color and browning indexes of fermented PJs were preferable. The concentration of total polyphenolic compounds and antioxidant activity were the highest for started PJs, with some differences that depended on the starter used. Fermentation increased the concentration of ellagic acid, and enhanced the antimicrobial activity. Fermented PJs scavenged the reactive oxygen species generated by H2O2 and modulated the synthesis of immune-mediators from peripheral blood mononuclear cells (PBMC). Unstarted and fermented PJs inhibited the growth of K562 tumor cells. The sensory attributes of fermented PJs were preferred. The fermentation of pomegranate juice would represent a novel technology option, which joins health-promoting, sensory and preservative features to exploit the potential of pomegranate fruits.

Metabolism of furazolidone: alternative pathways and modes of toxicity in different cell lines

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.

Diversity of oat varieties in eliciting the early inflammatory events in celiac disease

AbstractPurpose Celiac disease (CD) is an autoimmune enteropathy, triggered by dietary gluten. The only treatment is a strict gluten-free diet. Oats are included in the list of gluten-free ingredients by European Regulation, but the safety of oats in CD is still a matter of debate. The present study examined the capability of different oat cultivars of activating the gliadin-induced transglutaminase-2 (TG2)-dependent events in some in vitro models of CD. In addition, we compared this capability with the electrophoresis pattern of peptic–tryptic digests of the proteins of the oat cultivars.MethodsK562(S) cells agglutination, transepithelial electrical resistance of T84-cell monolayers, intracellular levels of TG2 and phosphorylated form of protein 42–44 in T84 cells were the early gliadin-dependent events studied.ResultsThe results showed that the Nave oat cultivar elicited these events, whereas Irina and Potenza varieties did not. The ability of a cultivar to activate the above-described events was associated with the electrophoretic pattern of oat proteins and their reactivity to anti-gliadin antibodies.ConclusionWe found significant differences among oat cultivars in eliciting the TG2-mediated events of CD inflammation. Therefore, the safety of an oat cultivar in CD might be screened in vitro by means of biochemical and biological assays, before starting a clinical trial to definitely assess its safety.

Early tissue transglutaminase-mediated response underlies K562(S)-cell gliadin-dependent agglutination.

Intoduction:K562(S) agglutination has been used as a rapid and economic tool for the in vitro screening of the toxicity of cereal fractions and prolamins in celiac disease (CD). A strict correlation has been reported between the toxicity of cereals and cereal fractions for celiac patients and their ability to agglutinate K562(S) cells. Whether this specificity of K562(S)-cell agglutination is caused by the activation of the same pathogenic events triggered by toxic cereal fractions in CD intestine or simply represents a bystander event of gluten toxicity is, however, unknown.Methods:K562(S) cells were incubated in vitro with the peptic-tryptic digest of wheat gliadin.Results:The agglutination of K562(S) cells by wheat gliadin peptides is orchestrated by a cascade of very early events occurring at the K562(S)-cell surface similar to those occurring at the intestinal epithelial surface. They involve a rapid increase in intracellular calcium levels that activate tissue transglutaminase (TG2), leading to a rapid actin reorganization that is pivotal in driving cell agglutination. These specific effects of toxic cereals are phenocopied by the gliadin-derived peptide p31–43, which orchestrates the activation of innate response to gliadin in CD.Discussion:Our study provides the rationale for the extensive use of K562(S)-cell agglutination as a valuable tool for screening cereal toxicity.

Toxic, Immunostimulatory and Antagonist Gluten Peptides in Celiac Disease

Celiac disease (CD) is an increasingly diagnosed, permanent autoimmune enteropathy, triggered, in susceptible individuals, by the ingestion of gluten, the alcohol - soluble protein fraction of some cereals, such as wheat, rye and barley. The main protein of wheat gluten is called gliadin, the similar proteins of rye and barley are secalin and hordein, respectively. Approximately 96% of CD patients express the HLA molecule DQ2, while the remainder mostly express the less common haplotype DQ8, reflecting the pivotal role of these molecules in the pathogenesis of CD. Because of their aminoacid sequence and tri-dimensional structure, gluten peptides selectively bind to these HLA alleles present on the surface of antigen presenting cells and then they are presented to the T lymphocytes in intestinal mucosa, thus starting the inflammatory immune response. CD is defined by the characteristic histological changes of small bowel mucosa: villous atrophy, crypts hyperplasia and T cells infiltration of the lamina propria, along with the increase of the number of intra-epithelial lymphocytes. The withdrawal of the gluten- containing food from the diet determines a complete recovery of the intestinal mucosa, whereas the reintroduction causes a relapse of the disease. This review focuses on the description of gluten peptides that elicit the mucosal immune response via the activation of innate and adaptive immunity in CD. It also describes the antagonist gluten peptides, obtained by artificial modification of gluten T epitopes or naturally occurring in the alcohol protein fraction of a cultivar of durum wheat, able to immuno-modulate the pathogenic immune response of CD.

Functional alterations induced by the food contaminant furazolidone on the human tumoral intestinal cell line Caco-2

Caco-2 cells, which are derived from a human colon carcinoma and are able to differentiate in culture, have been used to study the effect of furazolidone (FZ), a chemical belonging to the nitrofuran family which is frequently used for the prevention of animal infections. Its potentially toxic residues could remain in some food products of animal origin and affect human health. Toxicity has been measured by different parameters, either in undifferentiated cells (day 7 of culture), or on differentiated cells (day 21 of culture). Our results indicate that FZ may seriously affect the proliferating portion of the intestinal mucosa, while the differentiated cells appear to be more resistant. However, the slight effect recorded on the aspecific and specific functions of the differentiated cells may suggest that the specialized portion of the intestine can also be compromised by the drug. Caco 2 cells seem a good model for a deeper investigation of the mechanism involved in the toxic action of FZ.

Papillary cancer of thyroid in celiac disease.

Background Celiac disease (CD) has been established as being associated with several thyroid diseases. However, occurrence of thyroid epithelial cancer in celiac patients has been rarely described. Goal We describe the prevalence of thyroid carcinoma detected in a cohort of Italian celiac patients, with the aim to carry out a prospective analysis of the risk of celiac patients to develop thyroid carcinoma. Methods The study population included all CD patients diagnosed at the Collaborating Centers of the Italian Registry of CD between January 1, 1982 and December 31, 2006. Upon diagnosis of CD and at every subsequent clinical control, the Collaborating Centers filled in a validated form for each CD patient reporting information about demographic data, possible occurrence of a thyroid disease, and adherence to a gluten-free diet. Results Of the 1757 celiac patients enrolled, 6 developed a papillary thyroid carcinoma during the follow-up period (mean: 18.1 y). The Standardized Incidence Ratio resulted 2.55 (95% confidence interval=0.93-5.55; P<0.01). The mean age of diagnosis of CD in patients who developed thyroid carcinoma was rather low (40 y) and not statistically different from that of those who did not develop thyroid carcinoma. The number of gluten-containing diet per month did not correlate with the development of a thyroid carcinoma. Conclusions There is a 2.5-fold increased risk of papillary cancer of thyroid for celiac patients. A prompt diagnosis of CD and a strict adherence to gluten-free diet do not seem to protect from the development of this malignancy.