Three Novel Candidate Probiotic Strains with Prophylactic Properties in a Murine Model of Cow's Milk Allergy.

Food allergies can have significant effects on morbidity and on quality of life. Therefore, the development of efficient approaches to reduce the risk of developing food allergies is of considerable interest. The aim of this study was to identify and select probiotic strains with preventive properties against allergies using a combination of in vitro and in vivo approaches. To that end, 31 strains of bifidobacteria and lactic acid bacteria were screened for their immunomodulatory properties in two cellular models, namely, human peripheral blood mononuclear cells (PBMCs) and T helper 2 (Th2)-skewed murine splenocytes. Six strains inducing a high interleukin-10 (IL-10)/IL-12p70 ratio and a low secretion of IL-4 on the two cellular models were selected, and their protective impact was tested in vivo in a murine model of food allergy to ß-lactoglobulin. Three strains showed a protective impact on sensitization, with a decrease in allergen-specific IgE, and on allergy, with a decrease in mast cell degranulation. Analysis of the impact of these three strains on the T helper balance revealed different mechanisms of action. The Lactobacillus salivarius LA307 strain proved to block Th1 and Th2 responses, while the Bifidobacterium longum subsp. infantis LA308 strain induced a pro-Th1 profile and the Lactobacillus rhamnosus LA305 strain induced pro-Th1 and regulatory responses. These results demonstrate that a combination of in vitro and in vivo screening is effective in probiotic strain selection and allowed identification of three novel probiotic strains that are active against sensitization in mice.

Effects of a Lacticaseibacillus Mix on Behavioural, Biochemical, and Gut Microbial Outcomes of Male Mice following Chronic Restraint Stress.

The effect of supplementation with Lactobacillus strains to prevent the consequences of chronic stress on anxiety in mouse strains sensitive to stress and the consequences on gut microbiota have been relatively unexplored. Thus, we administered a Lacticaseibacillus casei LA205 and Lacticaseibacillus paracasei LA903 mix to male BALB/cByJrj mice two weeks before and during 21-day chronic restraint stress (CRS) (non-stressed/solvent (NS-PBS), non-stressed/probiotics (NS-Probio), CRS/solvent (S-PBS), CRS/probiotics (S-Probio)). CRS resulted in lower body weight and coat state alteration, which were attenuated by the probiotic mix. S-Probio mice showed less stress-associated anxiety-like behaviours than their NS counterpart, while no difference was seen in PBS mice. Serum corticosterone levels were significantly higher in the S-Probio group than in other groups. In the hippocampus, mRNA expression of dopamine and serotonin transporters was lower in S-Probio than in S-PBS mice. Few differences in bacterial genera proportions were detected, with a lower relative abundance of Alistipes in S-Probio vs. S-PBS. CRS was accompanied by a decrease in the proportion of caecal acetate in S-PBS mice vs. NS-PBS, but not in the intervention groups. These data show that the probiotic mix could contribute to better coping with chronic stress, although the precise bacterial mechanism is still under investigation.

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders.

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

Inhibition of intestinal FXR activity as a possible mechanism for the beneficial effects of a probiotic mix supplementation on lipid metabolism alterations and weight gain in mice fed a high fat diet.

Supplementation with probiotics has emerged as a promising therapeutic tool to manage metabolic diseases. We investigated the effects of a mix of Bifidobacterium animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 on high-fat (HF) diet -induced metabolic disease in mice. Supplementation with the probiotic mix in HF diet-fed mice (HF-Pr2) reduced weight and fat mass gains, decreased hepatic lipid accumulation, and lowered plasma triglyceride peak during an oral lipid tolerance test. At the molecular level, the probiotic mix protected against HF-induced rise in mRNA levels of genes related to lipid uptake, metabolism, and storage in the liver and white adipose tissues, and strongly decreased mRNA levels of genes related to inflammation in the white adipose tissue and to oxidative stress in the liver. Regarding intestinal homeostasis, the probiotic mix did not prevent HF-induced gut permeability but slightly modified microbiota composition without correcting the dysbiosis induced by the HF diet. Probiotic supplementation also modified the cecal bile acid (BA) profile, leading to an increase in the Farnesoid-X-Receptor (FXR) antagonist/agonist ratio between BA species. In agreement, HF-Pr2 mice exhibited a strong inhibition of FXR signaling pathway in the ileum, which was associated with lipid metabolism protection. This is consistent with recent reports proposing that inhibition of intestinal FXR activity could be a potent mechanism to overcome metabolic disorders. Altogether, our results demonstrate that the probiotic mix evaluated, when administered preventively to HF diet-fed mice could limit obesity and associated lipid metabolism disorders, likely through the inhibition of FXR signaling in the intestinal tract.

Synergistic Effects of Licorice Root and Walnut Leaf Extracts on Gastrointestinal Candidiasis, Inflammation and Gut Microbiota Composition in Mice.

Candida albicans is an opportunistic pathogen that causes gastrointestinal (GI) candidiasis closely associated with intestinal inflammation and dysbiosis. Drug resistance, side effects of available antifungal agents, and the high recurrence of candidiasis highlight the need for new treatments. We investigated the effects of hydroethanolic extracts of licorice root (LRE) and walnut leaf (WLE) on GI colonization by C. albicans, colon inflammation, and gut microbiota composition in C57BL/6 female mice. Oral administration of LRE and WLE alone or in combination once daily for 12 days before C. albicans infection and then for 5 days after infection significantly reduced the level of C. albicans in the feces of gastrointestinal infected mice as well as colonization of the GI tract, both extracts showing robust antifungal activity. Although total bacterial content was unaffected by the extracts (individually or combined), the abundance of protective bacteria, such as Bifidobacterium spp. and Faecalibacterium prausnitzii, increased with the combination, in contrast to that of certain pathobiont bacteria, which decreased. Interestingly, the combination induced a more robust decrease in the expression of proinflammatory genes than either extract alone. The anti-inflammatory activity of the combination was further supported by the reciprocal increase in the expression of anti-inflammatory cytokines and the significant decrease in enzymes involved in the synthesis of proinflammatory eicosanoids and oxidative stress. These findings suggest that LRE and WLE have synergistic effects and that the LRE/WLE combination could be a good candidate for limiting GI candidiasis and associated inflammation, likely by modulating the composition of the gut microbiota. IMPORTANCE The adverse effects and emergence of resistance of currently available antifungals and the high recurrence of candidiasis prompt the need for alternative and complementary strategies. We demonstrated that oral administration of hydroethanolic extracts of licorice root (LRE) and walnut leaf (WLE) separately or in combination significantly reduced the colonization of the gastrointestinal (GI) tract by C. albicans, highlighting a robust antifungal activity of these plant extracts. Interestingly, our data indicate a correlation between LRE and WLE consumption, in particular the combination, and a shift within the gut microbiome toward a protective profile, a decrease in colonic inflammation and prooxidant enzymes, suggesting a synergistic effect. This study highlights the significant prebiotic potential of the LRE/WLE combination and suggests that the health benefits are due, at least in part, to their ability to modulate the gut microbiota, reduce inflammation and oxidative stress, and protect against opportunistic infection.

Serpin-positive Bifidobacterium breve CNCM I-5644 improves intestinal permeability in two models of irritable bowel syndrome.

Probiotic supplementation can help to mitigate the pathogenesis of irritable bowel syndrome (IBS) by reinforcing the intestinal barrier, and reducing both inflammation and proteolytic activity. Here, a combination of in vitro tests was performed on 33 Bifidobacterium strains as probiotic candidates for IBS. In addition to the classical tests performed, the detection of the serine protease inhibitor (serpin) enzyme capable of decreasing the high proteolytic activity found in IBS patients was included. Three serpin-positive strains were selected: Bifidobacterium breve CNCM I-5644, Bifidobacterium longum subsp. infantis CNCM I-5645 and B. longum CNCM I-5646 for their immunomodulation properties and protection of intestinal epithelial integrity in vitro. Furthermore, we found that B. breve CNCM I-5644 strain prevented intestinal hyperpermeability by upregulating Cingulin and Tight Junction Protein 1 mRNA levels and reducing pro-inflammatory markers. The ability of CNCM I-5644 strain to restore intestinal hyperpermeability (FITC-dextran) was shown in the murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS). This effect of this strain was corroborated in a second model of IBS, the neonatal maternal separation model in mice. Altogether, these data suggest that serpin-positive B. breve CNCM I-5644 may partially prevent disorders associated with increased barrier permeability such as IBS.

Oral Administration of Lactobacillus helveticus LA401 and Lactobacillus gasseri LA806 Combination Attenuates Oesophageal and Gastrointestinal Candidiasis and Consequent Gut Inflammation in Mice.

Candida albicans is an opportunistic pathogen that causes mucosal gastrointestinal (GI) candidiasis tightly associated with gut inflammatory status. The emergence of drug resistance, the side effects of currently available antifungals and the high frequency of recurrent candidiasis indicate that new and improved therapeutics are needed. Probiotics have been suggested as a useful alternative for the management of candidiasis. We demonstrated that oral administration of Lactobacillus gasseri LA806 alone or combined with Lactobacillus helveticus LA401 in Candida albicans-infected mice decrease the Candida colonization of the oesophageal and GI tract, highlighting a protective role for these strains in C. albicans colonization. Interestingly, the probiotic combination significantly modulates the composition of gut microbiota towards a protective profile and consequently dampens inflammatory and oxidative status in the colon. Moreover, we showed that L. helveticus LA401 and/or L. gasseri LA806 orient macrophages towards a fungicidal phenotype characterized by a C-type lectin receptors signature composed of Dectin-1 and Mannose receptor. Our findings suggest that the use of the LA401 and LA806 combination might be a promising strategy to manage GI candidiasis and the inflammation it causes by inducing the intrinsic antifungal activities of macrophages. Thus, the probiotic combination is a good candidate for managing GI candidiasis by inducing fungicidal functions in macrophages while preserving the GI integrity by modulating the microbiota and inflammation.

Multiple Selection Criteria for Probiotic Strains with High Potential for Obesity Management.

Since alterations of the gut microbiota have been shown to play a major role in obesity, probiotics have attracted attention. Our aim was to identify probiotic candidates for the management of obesity using a combination of in vitro and in vivo approaches. We evaluated in vitro the ability of 23 strains to limit lipid accumulation in adipocytes and to enhance the secretion of satiety-promoting gut peptide in enteroendocrine cells. Following the in vitro screening, selected strains were further investigated in vivo, single, or as mixtures, using a murine model of diet-induced obesity. Strain Bifidobacterium longum PI10 administrated alone and the mixture of B. animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 limited body weight gain and reduced obesity-associated metabolic dysfunction and inflammation. These protective effects were associated with changes in the hypothalamic gene expression of leptin and leptin receptor as well as with changes in the composition of gut microbiota and the profile of bile acids. This study provides crucial clues to identify new potential probiotics as effective therapeutic approaches in the management of obesity, while also providing some insights into their mechanisms of action.

A Probiotic Mixture Induces Anxiolytic- and Antidepressive-Like Effects in Fischer and Maternally Deprived Long Evans Rats.

A role of the gut microbiota in psychiatric disorders is supported by a growing body of literature. The effects of a probiotic mixture of four bacterial strains were studied in two models of anxiety and depression, naturally stress-sensitive Fischer rats and Long Evans rats subjected to maternal deprivation. Rats chronically received either the probiotic mixture (1.109 CFU/day) or the vehicle. Anxiety- and depressive-like behaviors were evaluated in several tests. Brain monoamine levels and gut RNA expression of tight junction proteins (Tjp) and inflammatory markers were quantified. The gut microbiota was analyzed in feces by 16S rRNA gene sequencing. Untargeted metabolite analysis reflecting primary metabolism was performed in the cecal content and in serum. Fischer rats treated with the probiotic mixture manifested a decrease in anxiety-like behaviors, in the immobility time in the forced swimming test, as well as in levels of dopamine and its major metabolites, and those of serotonin metabolites in the hippocampus and striatum. In maternally deprived Long Evans rats treated with the probiotic mixture, the number of entries into the central area in the open-field test was increased, reflecting an anxiolytic effect. The probiotic mixture increased Tjp1 and decreased Ifnγ mRNA levels in the ileum of maternally deprived rats. In both models, probiotic supplementation changed the proportions of several Operational Taxonomic Units (OTU) in the gut microbiota, and the levels of certain cecal and serum metabolites were correlated with behavioral changes. Chronic administration of the tested probiotic mixture can therefore beneficially affect anxiety- and depressive-like behaviors in rats, possibly owing to changes in the levels of certain metabolites, such as 21-deoxycortisol, and changes in brain monoamines.

Anti-inflammatory potential of the probiotic dietary supplement Lactibiane Tolérance: in vitro and in vivo considerations.

BACKGROUND & AIMS: Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. In inflammatory bowel disease (IBD), where major modifications of the intestinal microflora have been reported, there is an increasing interest in modulating the flora with probiotic products. This work addresses the anti-inflammatory potential of Lactibiane Tolérance, a probiotic dietary supplement (mixture of four strains) using in vitro and in vivo approaches. METHODS: Comparison of the four individual strains and the commercial product reconstituted from them was conducted by in vitro tests (cytokine release after 24h stimulation of human peripheral blood mononuclear cells (PBMC)). The potential immunomodulatory characteristics of Lactibiane Tolérance were determined in vivo in an acute mice model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Assessment of colitis included blinded histological and macroscopic scores. RESULTS: We showed that Lactibiane Tolérance has anti-inflammatory properties in vitro by stimulating IL-10 production and in vivo by conferring a significant protective effect in the TNBS-induced colitis model (more than 50% decrease of colitis symptoms, P<0.01). CONCLUSIONS: These results demonstrate that a probiotic dietary supplement, Lactibiane Tolérance, can significantly prevent the initial injury of TNBS and could stimulate the initiation of clinical trials in IBD.

Multispecies probiotic protects gut barrier function in experimental models.

AIM: To investigate the effect of the probiotic combination Lactibiane Tolerance(®) (LT) on epithelial barrier function in vitro and in vivo. METHODS: The effect of the multispecies probiotic LT was assessed on several models of epithelial barrier function both in vitro (in basal and inflammatory conditions) and in vivo [visceral hypersensitivity induced by chronic stress or by colonic perfusion of a fecal supernatant (FSN) from patients with irritable bowel syndrome (IBS)]. In vitro, we measured the permeability of confluent T84 cell monolayers incubated with or without LT by evaluating the paracellular flux of macromolecules, in basal conditions and after stimulation with lipopolysaccharide (LPS) or with conditioned medium of colonic biopsies from IBS patients (IBS-CM). In vivo, male C57/Bl6 mice received orally NaCl or LT for 15 d and were submitted to water avoidance stress (WAS) before evaluating visceral sensitivity by measuring the myoelectrical activity of the abdominal muscle and the paracellular permeability with (51)Cr-EDTA. Permeability and sensitivity were also measured after colonic instillation of FSN. Tight-junctions were assessed by immunoblotting and TLR-4 expression was evaluated by immunohistochemistry RESULTS: Incubation of T84 cell monolayers with LT in basal conditions had no significant effect on permeability (P > 0.05 vs culture medium). By contrast, addition of LT bacterial bodies (LT) completely prevented the LPS-induced increase in paracellular permeability (P < 0.01 vs LPS 10 ng/mL (LPS 10); P < 0.01 vs LPS 100 ng/mL (LPS 100), P > 0.05 vs culture medium). The effect was dose dependent as addition of 10(9) LT bacterial bodies induced a stronger decrease in absorbance than 10(6) LT (10(9) LT + LPS 10: -20.1% ± 13.4, P < 0.01 vs LPS 10; 10(6) LT + LPS 10: -11.6% ± 6.2, P < 0.01 vs LPS 10; 10(9) LT + LPS 100: -14.4% ± 5.5, P < 0.01 vs LPS 100; 10(6) LT + LPS 100: -11.6% ± 7.3, P < 0.05 vs LPS 100). Moreover, the increase in paracellular permeability induced by culturing T84 cells with conditioned medium of colonic biopsies from IBS patients (IBS-CM) was completely inhibited in the presence of 10(9) LT (P < 0.01 vs IBS-CM). LT also significantly prevented the epithelial disruption induced by intracolonic infusion of fecal supernatant from IBS patients (P < 0.01 vs IBS FSN) or water avoidance stress P < 0.01 vs WAS) in C57/Bl6 mice and increased the expression of occludin in vitro and in vivo, as assessed by immnunoblotting. The WAS-induced effect on visceral sensitivity was prevented by LT treatment since values obtained for all steps of colorectal distension were significantly (P < 0.01) different from the WAS group. Finally, LT down-regulated the response mediated through TLR-4 in vitro (decrease in tumor necrosis factor α secretion in response to LPS: -65.8% for 10(9) LT and -52.5% for 10(6) LT, P < 0.01 vs LPS) and in vivo (inhibition of WAS induced an increase in TLR-4 expression in the LT treated mice colon, P < 0.01 vs WAS). CONCLUSION: The probiotic LT mix prevented the disruption to the epithelial barrier induced by LPS, stress or colonic soluble factors from IBS patients and prevented visceral hypersensitivity.

Genetic variants in BCMO1 and CD36 are associated with plasma lutein concentrations and macular pigment optical density in humans.

UNLABELLED: Lutein is recovered at high concentration in the human macula lutea. Recent studies suggest that this micronutrient might be implicated in prevention of age-related macular degeneration. OBJECTIVE: to identify genes which affect blood and retina lutein concentrations among candidate genes (intestinal sterol transporters and carotenoid oxygenases). DESIGN: a comparative plus an observational study. PARTICIPANTS: twenty-nine healthy subjects for the comparative study and 622 subjects for the observational study. INTERVENTION AND METHODS: all the participants were genotyped for single nucleotide polymorphisms (SNPs) in the candidate genes. Fasting plasma lutein concentrations were measured in all the participants and after 6 months' supplementation, with either a lutein-rich supplement or a placebo, in the 29 subjects who participated in the comparative study. Macular pigment optical density (MPOD), which is a measure of macula concentration of lutein, was measured before and after the dietary intervention in the 29 subjects. Associations between SNPs and plasma lutein and MPOD were assessed by partial least square (PLS) regression followed by univariate analysis. Observed associations between SNPs and plasma lutein were verified by haplotype-based association analysis in the cohort of 622 subjects. MAIN OUTCOME MEASURES: plasma lutein levels and MPOD. RESULTS: six SNPs in four genes (ABCG8, BCMO1, CD36, and NPC1L1) explained 25% and 38% of the plasma and MPOD variance, respectively. Subjects with TT at the BCMO1 rs7501331 locus had lower (P < 0.05) plasma lutein than CT subjects. Subjects with CC at the CD36 rs13230419 locus had lower (P < 0.05) plasma lutein than subjects who carried a T allele. The association between CD36 and plasma lutein was confirmed in the cohort of 622 subjects. Subjects with TT at the BCMO1 rs7501331 locus had a higher (P < 0.05) MPOD, and subjects with GG at rs1761667 CD36 locus had a higher (P < 0.05) MPOD than those with an A allele. CONCLUSIONS: these results suggest that BCMO1 and CD36 are implicated in plasma and retina concentrations of lutein and that genetic variants in these genes can modulate blood and retina concentrations of lutein.

Antioxidants intake and dry eye syndrome: a crossover, placebo-controlled, randomized trial.

PURPOSE: To assess whether an orally administered antioxidant dietary supplement could improve the objective clinical signs and alleviate the subjective symptoms of dry eye syndrome. METHODS: Twenty-four subjects diagnosed with dry eye syndrome were randomized in a crossover, double-blind, controlled, randomized study to receive a placebo or an antioxidants combination (Oxybiane) for 12 weeks. In all subjects, break-up time (BUT) test, Schirmer test, ocular symptoms (sore eyes, burning, itching, sensation of foreign object in the eye, photophobia, sticky eyes, and redness), visual comfort, and general well-being were evaluated weekly. RESULTS: After 12 weeks of supplementation with Oxybiane, both the BUT scores (27.3%+/-8.4% with Oxybiane versus 3.61%+/-4.3% with the placebo, p=0.017) and the Schirmer scores (26.9%+/-14.2% with Oxybiane versus -4.7%+/-3.4% with the placebo, p=0.037) were significantly increased. A significantly improvement was also observed considering subjective clinical symptoms such as burning (p=0.031), itching (p=0.027), sensation of foreign body in eye (p=0.030), and redness (p=0.043). CONCLUSIONS. Supplementation with oral antioxidants can improve both tear stability and quantity but also subjective clinical signs.

Beta-galactosidase production by Streptococcus thermophilus is higher in the small intestine than in the caecum of human-microbiota-associated mice after lactose supplementation.

Transit kinetics and survival rates of a bacterial species from yoghurt (i.e. Streptococcus thermophilus strain FBI3) were examined in different digestive compartments of gnotoxenic and human-microbiota-associated mice. The production of the lactose-hydrolysing enzyme (i.e. beta-galactosidase) was also investigated within the digestive tract, using a chromosomal reporter system based on luciferase genes from Photorhabdus luminescens under the control of the plac promoter. In both mice models, S. thermophilus cells transited within 2 h from the stomach to the caecum-colon compartment of the digestive tract where they displayed a survival rate of nearly 100 %. In gnotoxenic mice, luciferase activity was found to increase in the second half of the small intestine and in the caecum-colon compartment when lactose was added to the drinking water provided to the animals. In human-microbiota-associated mice drinking lactose, luciferase activity was similarly increased in the second half of the small intestine but was drastically reduced in the caecum-colon compartment. This feature could be ascribed to the presence of the resident human microbiota.