Use of GRAS bacteria for the modulation of immune functions in cellular and mouse models.

Intestinal bacteria play a pivotal role in shaping gut immunity. Epidemiologic evidence also suggests that Lactobacilli and Bifidobacteria are key players inthis context. Furthermore, literature data reported that the immune modulatory activity of generally recognized as safe (GRAS) bacteria could be associated with the secretion of specific molecules, like enzymes or other unidentified substances. Based on these findings, the general objective of this Doctoral Thesis was to evaluate the modulation of the immune response by using different bacterial populations, isolated from the human intestine, or already availableas GRAS stains, according with the American FDA designation. Both in vitro(Caco-2cell line and mouse dendritic cells) and in vivo (HLA-DQ8 transgenic mice) models were adopted to address this issue. In the first study, the immunomodulatory effects of various probiotic strains of L. paracaseion dendritic cells were determined. We found that bacterial metabolites from only some of tested strains showed an anti-inflammatory activity. In a second study,another GRAS bacterial strain, Streptomyces mobaraensis, was assessed. In particular, newculture conditions were developed to optimize the secretion of microbial transglutaminase (mTG), previously found to be able to block the inflammatory response induced by gluten in Coeliac disease (CD) patients. Importantly, we found that wheat flour treated with sterile-filtered supernatant of S.mobaraensis culture, resulted also effective in modulatingthe immune response to gluten. Furthermore, bread manufactured with treated flour had only minor changes in the baking parameters. In other two studies, we specifically focused on the relationship between the dietary habit and immunomodulatory abilities exerted in vitro by intestinal Bifidobacteria and Lactobacilli, isolated from individuals following omnivorous, vegetarian and vegan diets. Interestingly, both lactobacilli and Bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. In conclusion, these data highlighted different applications of GRAS bacteria and their metabolites with possible implications for the management of inflammatory diseases like CD

Effects of Two-Step Transamidation of Wheat Semolina on the Technological Properties of Gluten

Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to alleviate symptoms and to normalize the intestinal mucosa. We previously found that transamidation reaction by microbial transglutaminase (mTG) was effective in down-regulating the gliadin-specific immune response in CD patients. In this study, the two-step transamidation protocol was adopted to treat commercial wheat semolina on a pilot scale. The effectiveness of the enzymatic reaction was tested by means of consolidated biochemical and immunological methods on isolated prolamins. We found that water-insoluble gliadin and glutenin yields decreased in wheat semolina to 5.9% ± 0.3% and 11.6% ± 0.1%, respectively, after a two-step transamidation reaction. Using DQ8 transgenic mice as a model of gluten sensitivity, we observed a dramatic reduction in IFN-γ production in spleen cells challenged in vitro with the residual insoluble gliadin from transamidated semolina (N = 6; median values: 850 vs. 102; control vs. transamidated semolina, p < 0.05). The technological properties of treated wheat semolina were then tested by manufacturing classical pasta (spaghetti). Notably, the spaghetti manufactured with transamidated semolina had only minor changes in its features before and after cooking. In conclusion, the two-step transamidation reaction modified the immunogenic epitopes of gliadins also on a pilot-scale level without influencing the main technological properties of semolina. Our data shed further light on a detoxification strategy alternative to the current gluten-free diet and may have important implications for the management of CD patients

Brain Nrf2 pathway, autophagy, and synaptic function proteins are modulated by a short-term fructose feeding in young and adult rats

Objectives: A strong rise of the fructose content in the human diet occurred in the last decade, as corn syrup is widely used as a sweetener for beverages and processed food. Since young people make a widespread consumption of added sugars, we evaluated the effects of a two weeks fructose-rich diet on brain redox homeostasis, autophagy and synaptic plasticity in the cortex of young and adults rats, in order to highlight the early risks to which brain is exposed. Methods and Results: Short-term fructose feeding was associated with an imbalance of redox homeostasis, as lower amount of Nuclear factor (erythroid derived 2)-like 2, lower activity of Glucose 6-phosphate dehydrogenase and Glutathione reductase, together with lower Glutathione/Oxidized Glutathione ratio, were found in fructose-fed young and adult rats. Fructose-rich diet was also associated with the activation of autophagy, as higher levels of Beclin, LC3 II and P62 were detected in cortex of fructose-fed rats. A diet associated decrease of synaptophysin, synapsin I, and synaptotagmin I, was found in fructose-fed young and adult rats. Interestingly, BDNF amount was significantly lower only in fructose-fed adult rats, while the level of its receptor TrkB decreased in both groups of treated rats. A further marker of brain functioning, Acetylcholinesterase activity, was found increased only in fructose-fed young animals. Conclusion: Overall, our findings suggest that young rats may severely suffer from the deleterious influence of fructose on brain health as the adults and provide experimental data suggesting the need of targeted nutritional strategies to reduce its amount in foods

Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis

Background: Mucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes. Results: We report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN- in spleen and mesenteric lympho nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form. Conclusion: Our results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine delivery approac

Application of response surface methodology for the optimization of supercritical CO2 extraction of oil from olive paste: yield, content of bioactive molecules and biological effects in vivo

none8noneLenucci MS, Durante M, Ferramosca A, Zara V, Piro G, Bergamo P, Treppiccione L, Mita GLenucci, Ms; Durante, M; Ferramosca, A; Zara, V; Piro, G; Bergamo, P; Treppiccione, L; Mita,

Effect of Lactobacillus paracasei Culture Filtrates and Artichoke Polyphenols on Cytokine Production by Dendritic Cells

The most recent trend in research on probiotic bacteria aims at the exploitation of bioactive bacterial compounds that are responsible for health-promoting effects and suitable for medical applications. Therefore, the main purpose of this study was to ascertain if the immunomodulatory effects of L. paracasei strains on dendritic cells (DCs) were caused by bacterial metabolites released in the culture medium. For that reason, bacterial strains were grown in two media generally used for the culture of DCs, and the effects of culture filtrates on the maturation of DCs and cytokine production were evaluated. Moreover, to reveal potential synergistic effects on the immunomodulation of DCs, an artichoke phenolic extract (APE) was added to the media before bacterial growth. The experiments pointed out an interesting anti-inflammatory activity of a culture filtrate obtained after growing a probiotic L. paracasei strain in one of the media supplemented with APE. Therefore, this culture filtrate—which combines the anti-inflammatory activity and the other well-known health-promoting properties of artichoke phenolic compounds—could represent the basis for future particular exploitations