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The aim of this study was the investigation of the antimicrobial activity expressed by Lactobacillus plantarum strains isolated from different fermented matrices (wines, cheese, fermented sausages, and sourdoughs). A total of 106 strains of Lb. plantarum (producers) were tested against 33 undesirable microorganisms (indicators), including both moulds and bacteria. The antimicrobial activity exerted by growing cells (GC) was evaluated by the spot-on-thelawn, while the activity of cell free supernatants (CFS), neutralised CFS (nCFS) and CFS treated with proteases (pCFS) was assessed by the agar well diffusion assay. The antagonistic effect produced by GC of Lb. plantarum isolated from wines was higher than that exibited by cells isolated from other fermented matrices. Moreover, 5 CFS - all from wine strains - as well as the corresponding nCFS and pCFS were able to inhibit different bacteria and moulds. The results suggested a relationship between the origin of Lb. plantarum strains and their antimicrobial properties, while no relation was found between the intensity of inhibition and the origin of indicator strains. This fact highlights that the knowledge of conditions characterising different ecosystems can be helpful in the detection and isolation of Lb. plantarum strains to be used as protective agents. [ABSTRACT FROM AUTHOR]

Focuses on a study which investigated the impact of a Lactobacillus sakei strain on the characteristics of soppressata molisana dry sausage. Account of related studies; Methodology; Results of the study.

We have previously shown the ability of transamidated gluten (spf) to modulate both innate and adaptive intestinal immunity elicited by wheat gliadin in HLA-DQ8 transgenic mice (DQ8 mice), a model of gluten sensitivity. Herein, we evaluated the influence of spf when administered intragastrically on the immune response to native gliadin in DQ8 mice. To address the issue, we analysed three regimens of antigen administration: before immunisation (pre-treatment), during immunisation (co-treatment) and through breast milk during the lactating phase (suckling treatment). Mice were immunised mucosally by intranasal delivery of digested wheat gliadin along with cholera toxin in multiple doses. After sacrifice, isolated spleen and mesenteric lymph node (MLN) cells were challenged in vitro and the cytokine profile of culture supernatants assessed by ELISA and multiparametric assay. We found that only pre-treatment with spf was effective in down-regulating the gliadin-specific IFN-γ response and only in spleen cells. Interestingly, spf pre-treatment also induced systemic IL-6, IL-17A and TNF-α. By contrast, we found that spf pre-treatment upregulated INF-γ in MLN but also significantly decreased IL-2. In conclusion, our data provide evidence that the preventive intragastric administration of transamidated gluten is able to interfere with the classical cytokine profile induced by gliadin via mucosal immunisation in a transgenic model expressing one of the HLA molecules associated with coeliac disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Increasing evidence links the gut microbiota (GM) to Alzheimer's disease (AD) but the mechanisms through which gut bacteria influence the brain are still unclear. This study tests the hypothesis that GM and mediators of the microbiota-gut-brain axis (MGBA) are associated with the amyloid cascade in sporadic AD., Methods: We included 34 patients with cognitive impairment due to AD (CI-AD), 37 patients with cognitive impairment not due to AD (CI-NAD), and 13 cognitively unimpaired persons (CU). We studied the following systems: (1) fecal GM, with 16S rRNA sequencing; (2) a panel of putative MGBA mediators in the blood including immune and endothelial markers as bacterial products (i.e., lipopolysaccharide, LPS), cell adhesion molecules (CAMs) indicative of endothelial dysfunction (VCAM-1, PECAM-1), vascular changes (P-, E-Selectin), and upregulated after infections (NCAM, ICAM-1), as well as pro- (IL1β, IL6, TNFα, IL18) and anti- (IL10) inflammatory cytokines; (3) the amyloid cascade with amyloid PET, plasma phosphorylated tau (pTau-181, for tau pathology), neurofilament light chain (NfL, for neurodegeneration), and global cognition measured using MMSE and ADAScog. We performed 3-group comparisons of markers in the 3 systems and calculated correlation matrices for the pooled group of CI-AD and CU as well as CI-NAD and CU. Patterns of associations based on Spearman's rho were used to validate the study hypothesis., Results: CI-AD were characterized by (1) higher abundance of Clostridia&#95;UCG-014 and decreased abundance of Moryella and Blautia (p < .04); (2) elevated levels of LPS (p < .03), upregulation of CAMs, Il1β, IL6, and TNFα, and downregulation of IL10 (p < .05); (3) increased brain amyloid, plasma pTau-181, and NfL (p < 0.004) compared with the other groups. CI-NAD showed (1) higher abundance of [Eubacterium] coprostanoligenes group and Collinsella and decreased abundance of Lachnospiraceae&#95;ND3007&#95;group, [Ruminococcus]&#95;gnavus&#95;group and Oscillibacter (p < .03); (2) upregulation of PECAM-1 and TNFα (p < .03); (4) increased plasma levels of NfL (p < .02) compared with CU. Different GM genera were associated with immune and endothelial markers in both CI-NAD and CI-AD but these mediators were widely related to amyloid cascade markers only in CI-AD., Conclusions: Specific bacterial genera are associated with immune and endothelial MGBA mediators, and these are associated with amyloid cascade markers in sporadic AD. The physiological mechanisms linking the GM to the amyloid cascade should be further investigated to elucidate their potential therapeutic implications., (© 2023. The Author(s).)

Resistance to cancer therapies remains a clinical challenge and an unsolved problem. In a previous study, we characterized a new colon cancer cell line, namely HT500, derived from human HT29 cells and resistant to clinically relevant levels of ionizing radiation (IR). Here, we explored the effects of two natural flavonoids, quercetin (Q) and fisetin (F), well-known senolytic agents that inhibit genotoxic stress by selectively removing senescent cells. We hypothesized that the biochemical mechanisms responsible for the radiosensitising effects of these natural senolytics could intercept multiple biochemical pathways of signal transduction correlated to cell death resistance. Radioresistant HT500 cells modulate autophagic flux differently than HT29 cells and secrete pro-inflammatory cytokines (IL-8), commonly associated with senescence-related secretory phenotypes (SASP). Q and F inhibit PI 3 K/AKT and ERK pathways, which promote p16 INK4 stability and resistance to apoptosis, but they also activate AMPK and ULK kinases in response to autophagic stress at an early stage. In summary, the combination of natural senolytics and IR activates two forms of cell death: apoptosis correlated to the inhibition of ERKs and lethal autophagy dependent on AMPK kinase. Our study confirms that senescence and autophagy partially overlap, share common modulatory pathways, and reveal how senolytic flavonoids can play an important role in these processes.

Objective: To compare the effect of an isocaloric multifactorial diet with a diet rich in monounsaturated fatty acids (MUFA) and similar macronutrient composition on pancreatic fat (PF) and postprandial insulin response in type 2 diabetes (T2D)., Research Design and Methods: According to a randomized controlled parallel-group design, 39 individuals with T2D, 35-75 years old, in satisfactory blood glucose control, were assigned to an 8 week isocaloric intervention with a multifactorial diet rich in MUFA, polyunsaturated fatty acids, fiber, polyphenols, and vitamins (n = 18) or a MUFA-rich diet (n = 21). Before/after the intervention, PF content was measured by the proton-density fat fraction using a three-dimensional mDIXON MRI sequence, and plasma insulin and glucose concentrations were measured over a 4 h test meal with a similar composition as the assigned diet., Results: After 8 weeks, PF significantly decreased after the multifactorial diet (from 15.7 ± 6.5% to 14.1 ± 6.3%; P = 0.024), while it did not change after the MUFA diet (from 17.1 ± 10.1% to 18.6 ± 10.6%; P = 0.139) with a significant difference between diets (P = 0.014). Postprandial glucose response was similar in the two groups. Early postprandial insulin response (incremental postprandial areas under the curve [iAUC0-120]) significantly increased with the multifactorial diet (from 36,340 ± 34,954 to 44,138 ± 31,878 pmol/L/min; P = 0.037), while it did not change significantly in the MUFA diet (from 31,754 ± 18,446 to 26,976 ± 12,265 pmol/L/min; P = 0.178), with a significant difference between diets (P = 0.023). Changes in PF inversely correlated with changes in early postprandial insulin response (r = -0.383; P = 0.023)., Conclusions: In patients with T2D, an isocaloric multifactorial diet, including several beneficial dietary components, markedly reduced PF. This reduction was associated with an improved postprandial insulin response., (© 2022 by the American Diabetes Association.)

Background: Non-alcoholic liver steatosis (NAS) results from an imbalance between hepatic lipid storage, disposal, and partitioning. A multifactorial diet high in fiber, monounsaturated fatty acids (MUFAs), n-6 and n-3 polyunsaturated fatty acids (PUFAs), polyphenols, and vitamins D, E, and C reduces NAS in people with type 2 diabetes (T2D) by 40% compared to a MUFA-rich diet. We evaluated whether dietary effects on NAS are mediated by changes in hepatic de novo lipogenesis (DNL), stearoyl-CoA desaturase (SCD1) activity, and/or β-oxidation., Methods: According to a randomized parallel group study design, 37 individuals with T2D completed an 8-week isocaloric intervention with a MUFA diet ( n = 20) or multifactorial diet ( n = 17). Before and after the intervention, liver fat content was evaluated by proton magnetic resonance spectroscopy, serum triglyceride fatty acid concentrations measured by gas chromatography, plasma β-hydroxybutyrate by enzymatic method, and DNL and SCD-1 activity assessed by calculating the palmitic acid/linoleic acid (C16:0/C18:2 n6) and palmitoleic acid/palmitic acid (C16:1/C16:0) ratios, respectively., Results: Compared to baseline, mean ± SD DNL significantly decreased after the multifactorial diet (2.2 ± 0.8 vs. 1.5 ± 0.5, p = 0.0001) but did not change after the MUFA diet (1.9 ± 1.1 vs. 1.9 ± 0.9, p = 0.949), with a significant difference between the two interventions ( p = 0.004). The mean SCD-1 activity also decreased after the multifactorial diet (0.13 ± 0.05 vs. 0.10 ± 0.03; p = 0.001), but with no significant difference between interventions ( p = 0.205). Fasting plasma β-hydroxybutyrate concentrations did not change significantly after the MUFA or multifactorial diet. Changes in the DNL index significantly and positively correlated with changes in liver fat (r = 0.426; p = 0.009)., Conclusions: A diet rich in multiple beneficial dietary components (fiber, polyphenols, MUFAs, PUFAs, and other antioxidants) compared to a diet rich only in MUFAs further reduces liver fat accumulation through the inhibition of DNL. Registered under ClinicalTrials.gov no. NCT03380416.

Common beans ( Phaseolus vulgaris L.) are an important source of nutrients with beneficial effects on human health. However, they contain lectins, that limit the direct use of flour in food preparations without thermal treatment, and phytic acid, that reduces mineral cation bioavailability. The objectives of this research were: to obtain biofortified snacks and a cream using an untreated common bean flour devoid of active lectins ( lec - ) and with reduced content of phytic acid ( lpa ) and to evaluate the sensorial appreciation for these products. The main results of the present work were: the products with the lpa lec - flour did not retain residual hemagglutinating activity due to lectins; they showed higher residual α-amylase inhibitor activity (from 2.2 to 135 times), reduced in vitro predicted glycemic index (about 5 units reduction) and increased iron bioavailability compared to the products with wild type flour; products with common bean flour were less appreciated than the reference ones without this flour, but the presence of an intense umami taste can be a positive attribute. Results confirmed that the use of the lpa lec - flour has important advantages in the preparation of safe and nutritionally improved products, and provide useful information to identify target consumers, such as children and elderly people.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

The resemblance of physiological traits of cell lines with their target/original tissue is an important prerequisite for the choice of the in vitro model. Although cytoprotective defenses, activated by the nuclear factor erythroid 2-related factor2 (Nrf2), have a preeminent importance in intestinal protection, nevertheless their levels inin vitro models have been never compared with those of their original tissue. Basal level of Nrf2-mediated defenses in murine enterocyte cells (Mode-K) and in human colon adenocarcinoma cells -at differentiated (DCaco2) or confluent stage (CCaco2)- were compared with those found in mouse or human duodenum. The pro-oxidant and cytotoxic effects of peptic-tryptic digest of gluten prepared from wheat bread (PT-b), einkorn (PT-e) or durum wheat (PT-d) were evaluated in Mode-k and DCaco2 by combining enzymatic, immune-enzymatic and real-time PCR assay. The results presented reveal that Mode-k cells resemble cytoprotective defenses of human/murine duodenum and are more susceptible to pro-oxidant, cytotoxic and pro-inflammatory effect of gliadin digest (in comparison with Caco2). Prolamins digests from the considered wheat exhibit different inhibitory effect on Nrf2-mediated defenses (PT-b > PT-e > PT-d). These data indicate, for the first time, that Mode-k are a reliable model for investigating wheat prolamins toxicity and for evaluating the signaling pathway of gluten-associated disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Probiotics play a key role in the modulation of the gut immune system in health and disease and their action is mediated by molecules exposed on the microorganism surface or secreted probiotic-derived factors. In particular, Lactobacillus gasseri OLL2809, a probiotic microorganism isolated from human feces, has the potential to modulate various immune responses. The dendritic cells (DCs) are considered the main players in orchestrating the immune response, and their contact with intestinal microbiota is crucial for the development and homeostasis of gut immunity. To gain a perspective on the molecular mechanisms involved in the maturation process of DCs and investigate factors that could modulate these processes, a differential proteomic analysis was performed on the secretome of immature DCs, mature DCs (mDCs, induced by lipopolysaccharide (LPS)), and immature DCs challenged with L. gasseri OLL2809 before treatment with LPS (LGmDCs). The maturation process of DCs was associated to profound changes in the protein secretome and probiotic pre-treatment led to a dramatic modulation of several secreted proteins of mDC, not only classical immune mediators (i.e., cytokines, complement factors, T cell Receptor ligands) but also proteins involved in the contractile and desmosome machineries. The latter data highlight a novel mechanism by which L. gasseri can modulate the maturation process of DCs, reinforcing the concept of a protective anti-inflammatory role ascribed to this probiotic strain., Competing Interests: The authors state no conflict of interest.

In this study, for the first time, we examined some of the physico-chemical properties of the cell surface of A kkermansia muciniphila DSM 22959, comparing it with those of Lactobacillus rhamnosus GG-one of the most extensively studied probiotic microorganisms. In particular, hydrophobicity, auto-aggregation, co-aggregation, and biofilm formation were investigated. In addition, antibiotic susceptibility, co-culture, and antimicrobial activity of the two strains were compared . Hydrophobicity was evaluated using xylene and toluene, showing that A. muciniphila DSM 22959 possessed moderate hydrophobicity. A. muciniphila showed a faster and higher auto-aggregation ability than Lb. rhamnosus GG, but a lower aptitude in biofilm formation. In the co-aggregation test, the best performance was obtained by Lb. rhamnosus GG. Regarding the susceptibility to antibiotics, the differences between the two strains were remarkable, with A. muciniphila DSM 22959 showing resistance to half of the antibiotic tested. Interesting results were also obtained with regard to the stimulating effect of Lb. rhamnosus GG on the growth of A. muciniphila when co-cultured., Competing Interests: The authors declare no conflict of interest.

Enzymatic transamidation of wheat gliadin by microbial transglutaminase inhibits IFN-γ secretion by intestinal T cell lines from celiac disease (CD) patients. Here, we analysed its effects on intestinal biopsies from CD patients and studied the underlying mechanisms in HLA-DQ8 transgenic (tg) mice, a model of T-cell mediated gluten sensitivity. In vitro challenge with a soluble form of transamidated gliadin (spf) upregulated IL-10 transcript levels in human biopsy samples. Furthermore, the ratio of IL-10/IFN-γ transcripts was significantly increased following treatment with spf. In DQ8 tg mice, recall responses in vitro in the presence of dendritic cells pulsed with transamidated gliadin showed that gliadin-specific CD4 + T cells did not produce IFN-γ at any tested dose. On the contrary, spf-specific CD4 + T cells still secreted IFN-γ, but they also produced significant levels of IL-10 with both native and transamidated gliadin. Interestingly, this anti-inflammatory activity was restricted to a specific reverse-phase high-pressure liquid chromatography (RP-HPLC) fraction encompassing α-gliadins. These findings suggested an ability of transamidated gliadin to revert, as well as to prevent, the inflammatory phenotype triggered by native gliadin. This property was intrinsically associated with specific components of the α-gliadin fraction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group (P<0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari's volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Enterocytes are actively involved in the defense against pathogens and they limit penetration of commensal microbes into tissues. They also have an important role in gut immunity as enterocytes confer mucosal dendritic cell specialisation. On the other hand, the microbiota is directly involved in the development and modulation of the intestinal immune system. Particularly, lactobacilli and bifidobacteria play a primary role in shaping the immune response. We further explored this issue by evaluating whether functional differences in Caco-2 cells could characterise faecal populations of lactobacilli (155 samples) and bifidobacteria (110 samples) isolated from three dietary cohorts (omnivores, ovo-lacto-vegetarians and vegans) recruited at four Italian centres (Turin, Parma, Bologna and Bari). According to our findings, tested bacteria were unable to modulate expression of IL-8, IL-10, TGF-β or thymic stromal lymphopoietin (TSLP) cytokines in unstimulated Caco-2 cells. Conversely, in phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) stimulated Caco-2 cells, lactobacilli from the omnivorous group and all bifidobacteria significantly down-regulated IL-8. Notably, both genera also lowered the TSLP expression in stimulated Caco-2 cells, regardless of the diet regimen. By further examining these data on the basis of geographical origin, we found that lactobacilli from the vegetarian group recruited in Bari, significantly up-regulated this cytokine. In conclusion, we highlighted a peculiar immune-modulatory activity profile for lactobacilli on enterocytes undergoing a stimulatory signal, which was associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the inflammatory potential of members of the Lactobacillus genus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aims/hypothesis: The aim of this work was to investigate hepatic lipid metabolic processes possibly involved in the reduction of liver fat content (LF) observed in patients with type 2 diabetes after an isoenergetic diet enriched in monounsaturated fatty acids (MUFAs)., Methods: This is an ancillary analysis of a published study. In a parallel-group design, 30 men and eight women, aged 35-70 years, with type 2 diabetes and whose blood glucose was controlled satisfactorily (HbA 1c  < 7.5% [58 mmol/mol]) by diet or diet plus metformin, were randomised by MINIM software to follow either a high-carbohydrate/high-fibre/low-glycaemic index diet (CHO/fibre diet, n = 20) or a high-MUFA diet (MUFA diet, n = 18) for 8 weeks. The assigned diets were known for the participants and blinded for people doing measurements. Before and after intervention, LF was measured by 1 H-MRS (primary outcome) and indirect indices of de novo lipogenesis (DNL) (serum triacylglycerol palmitic:linoleic acid ratio), stearoyl-CoA desaturase activity (SCD-1) (serum triacylglycerol palmitoleic:palmitic acid ratio) and hepatic β-oxidation of fatty acids (β-hydroxybutyrate plasma concentrations) were measured., Results: LF was reduced by 30% after the MUFA diet, as already reported. Postprandial β-hydroxybutyrate incremental AUC (iAUC) was significantly less suppressed after the MUFA diet (n = 16) (-2504 ± 4488 μmol/l × 360 min vs baseline -9021 ± 6489 μmol/l × 360 min) while it was unchanged after the CHO/fibre diet (n = 17) (-8168 ± 9827 μmol/l × 360 min vs baseline -7206 ± 10,005 μmol/l × 360 min, p = 0.962) (mean ± SD, p = 0.043). In the participants assigned to the MUFA diet, the change in postprandial β-hydroxybutyrate iAUC was inversely associated with the change in LF (r = -0.642, p = 0.010). DNL and SCD-1 indirect indices did not change significantly after either of the dietary interventions., Conclusions/interpretation: Postprandial hepatic oxidation of fatty acids is a metabolic process possibly involved in the reduction of LF by a MUFA-rich diet in patients with type 2 diabetes., Trial Registration: ClinicalTrials.gov NCT01025856 FUNDING : The study was funded by Ministero Istruzione Università e Ricerca and Italian Minister of Health.

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., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Background: Probiotics species appear to differentially regulate the intestinal immune response. Moreover, we have shown that different immune-modulatory abilities can be found among probiotic strains belonging to the same species. In this study, we further addressed this issue while studying L. gasseri, a species that induces relevant immune activities in human patients., Results: We determined the ability of two strains of L. gasseri, OLL2809 and L13-Ia, to alter cell surface antigen expression, cytokine production and nuclear erythroid 2-related factor 2 (Nrf2)-mediated cytoprotection in murine bone marrow-derived dendritic cells (DCs) and MODE-K cells, which represent an enterocyte model. Differential effects of L. gasseri strains were observed on the expression of surface markers in mature DCs; nevertheless, both strains dramatically induced production of IL-12, TNF-α and IL-10. Distinctive responses to OLL2809 and L13-Ia were also shown in MODE-K cells by analyzing the expression of MHC II molecules and the secretion of IL-6; however, both L. gasseri strains raised intracellular glutathione. Treatment of immature DCs with culture medium from MODE-K monolayers improved cytoprotection and modified the process of DC maturation by down-regulating the expression of co-stimulatory markers and by altering the cytokine profile. Notably, bacteria-conditioned MODE-K cell medium suppressed the expression of the examined cytokines, whereas cytoprotective defenses were significantly enhanced only in DCs exposed to OLL2809-conditioned medium. These effects were essentially mediated by secreted bacterial metabolites., Conclusions: We have demonstrated that L. gasseri strains possess distinctive abilities to modulate in vitro DCs and enterocytes. In particular, our results highlight the potential of metabolites secreted by L. gasseri to influence enterocyte-DC crosstalk. Regulation of cellular mechanisms of innate immunity by selected probiotic strains may contribute to the beneficial effects of these bacteria in gut homeostasis.

CD is an immune-mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tTGase plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with K-C2H5 by mTGase yielded spf and K-gliadins fractions. By Western blot, we found that these modifications were associated with strongly reduced immune cross-reactivity. With the use of DQ8 tg mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin-specific spleen cells challenged with spf and K-gliadins in vitro (n=12; median values: 813 vs. 29 and 99; control vs. spf and K-gliadins, P=0.012 for spf, and P=0.003 for K-gliadins). For spf, we also observed an increase in the IL-10/IFNγ protein ratio (n=12; median values: 0.3 vs. 4.7; control vs. spf, P=0.005). In intestinal biopsies from CD patients challenged in vitro with gliadins (n=10), we demonstrated further that K-gliadins dramatically reduced the levels of antigen-specific IFNγ mRNA in all specimens responsive to native gliadins (four of 10; P<0.05). As cytotoxic effects have been described for gliadins, we also studied GST and caspase-3 activities using the enterocytic Caco-2 cell line. We found that neither activities were modified by flour transamidation. Our results indicate that K-C2H5 cross-linking via mTGase specifically affects gliadin immunogenicity, reversing the inducible inflammatory response in models of gluten sensitivity without affecting other aspects of the biological activity of gliadins.

A lifelong gluten-free diet (GFD) is mandatory for celiac disease (CD) but has poor compliance, justifying novel strategies. We found that wheat flour transamidation inhibited IFN-γ secretion by intestinal T cells from CD patients. Herein, the primary endpoint was to evaluate the ability of transamidated gluten to maintain GFD CD patients in clinical remission. Secondary endpoints were efficacy in prevention of the inflammatory response and safety at the kidney level, where reaction products are metabolized. In a randomized single blinded, controlled 90-day trial, 47 GFD CD patients received 3.7 g/day of gluten from nontransamidated (12) or transamidated (35) flour. On day 15, 75% and 37% of patients in the control and experimental groups, respectively, showed clinical relapse (P = 0.04) whereas intestinal permeability was mainly altered in the control group (50% versus 20%, P = 0.06). On day 90, 0 controls and 14 patients in the experimental group completed the challenge with no variation of antitransglutaminase IgA (P = 0.63), Marsh-Oberhuber grading (P = 0.08), or intestinal IFN-γ mRNA (P > 0.05). Creatinine clearance did not vary after 90 days of treatment (P = 0.46). In conclusion, transamidated gluten reduced the number of clinical relapses in challenged patients with no changes of baseline values for serological/mucosal CD markers and an unaltered kidney function.

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.

The present work was aimed at investigating whether Bacillus subtilis spores, widely used in probiotic as well as pharmaceutical preparations for mild gastrointestinal disorders, can suppress enteric infections. To address this issue, we developed a mouse model of infection using the mouse enteropathogen Citrobacter rodentium, a member of a family of human and animal pathogens which includes the clinically significant enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli strains. This group of pathogens causes transmissible colonic hyperplasia by using attaching and effacing (A/E) lesions to colonize the host colon. Because of its similarities to human enteropathogens, C. rodentium is now widely used as an in vivo model for gastrointestinal infections. Swiss NIH mice were orally administered B. subtilis spores one day before infection with C. rodentium. Mice were sacrificed on day 15 after infection, and distal colon, liver and mesenteric lymph nodes were removed for bacteria counts, morphology, immunohistology and IFNgamma mRNA analysis. We observed that spore predosing was effective in significantly decreasing infection and enteropathy in suckling mice infected with a dose of C. rodentium sufficient to cause colon colonization, crypt hyperplasia and high mortality rates. Moreover, in mice predosed with spores, the number of CD4(+) cells and IFNgamma transcript levels remained high. These results thus indicate that our newly established model of C. rodentium infection is a suitable system for analyzing the effects of probiotic bacteria on enteroinfections and that B. subtilis spores are efficient at reducing C. rodentium infection in mice, leaving unaltered the immune response against the pathogen.

In a search for bacteria having putative probiotic activity, we screened a collection of food-isolated microorganisms for the ability to survive at low pH in the presence of bile salts and for the production of antimicrobial compounds active against a number of animal pathogens. Among these, we found a strain that we classified as a member of Lactobacillus fermentum sp., and we further investigated its features. This organism was able to adhere to human enterocyte-like (Caco-2) cells with high efficiency as compared to that of a well known indicator strain. Chromatographic analysis indicated that at least two small (less than 3 kDa) factors were involved in mediating the in vitro interaction of L. fermentum with Caco-2 cells. Adhesion activity could be abolished by mild treatment of the bacterial cells in buffer and rescued by incubating them with either the same buffer after its use in the treatment or with chromatographic fractions containing each of the two factors, which indicated that these factors were loosely associated with the cell wall and that each of them was sufficient to warrant the adhesiveness of L. fermentum to Caco-2 cells. These data are suggestive of a novel mechanism of bacterial adhesion to epithelial cells.