MODULATION OF HOST INNATE IMMUNITY BY HEALTH-PROMOTING BACTERIA AND DIETARY COMPOUNDS

In its widest meaning, the probiotic approach consists in exogenous administration of microbial cells (or cell components) aimed at benefiting the host\u2019s health, both in terms of maintenance of homeostasis and also as alternative strategy for the prevention and/or treatment of infectious diseases. More recently, it has been demonstrated that an important way through which probiotics can exert their beneficial effects is the ability to interact with host\u2019s immune system, both at local and systemic level, thus having efficacy also in body niches different from the gut. Starting from these observations, in the first part of the PhD research activity we screened several bacterial strains for their potential use as probiotics for the pharyngeal mucosa. We tested the ability of bacteria employed in food industry and newly isolated from the pharynx of healthy volunteers to adhere to the human pharyngeal epithelium, and to antagonize the oro-pharyngeal pathogen S. pyogenes on FaDu cells and HaCat keratinocytes. Two oral bacterial strains, Streptococcus salivarius ST3, and the dairy starter Lactobacillus helveticus MIMLh5 were selected and compared with the oral commercial probiotic S. salivarius strain K12. These strains were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. The in vitro immunological characterization performed on FaDu cells revealed that ST3 and MIMLh5 were all able to significantly reduce the activation of the nuclear-factor (NF)-\u3baB, both at baseline and in presence of the pro-inflammatory stimulus interleukin (IL)-1\u3b2, whereas showing different cytokine profile under the above mentioned conditions. We subsequently decided to characterized the effects of the combined use of strain ST3 and MIMLh5. We found that strains MIMLh5 and ST3 activated innate immunity by inducing in U937 human macrophages the expression of cyclooxygenase (COX)-2, a balanced IL10/Tumor-Necrosis Factor (TNF)-\u3b1 ratio, and we demonstrated that Toll-like receptor 2 (TLR-2) participates in the recognition of both strains. We also observed that these microorganisms grow efficiently when co-coltured in milk, suggesting that the preparation of a milk-based fermented product containing both strains can be a practical solution for the administration of these bacteria. Considered the ability of L. helveticus MIMLh5 to trigger immune responses also on murine bone marrow-derived dendritic cells, in the second part of the research we focused our attention on the possible molecular determinants involved in the immunostimulating activity of this strain. We studied MIMLh5 surface layer protein (SlpA) and we found that the bacterium and SlpA exerted anti-inflammatory effects on the intestinal epithelial Caco-2 cell line by reducing the activation of NF-\u3baB. On the contrary, MIMLh5 and SlpA acted as stimulators of the innate immune system by triggering the expression of pro-inflammatory factors TNF-\u3b1 and COX-2 in the human macrophage cell line U937 via recognition through TLR-2, whereas the protein had no effect on the anti-inflammatory IL10. When we tested MIMLh5 bacterial cells depleted from the protein, we observed a reduced pro-inflammatory activity, suggesting that SlpA plays a major role in mediating the immunostimulatory attitude of the bacterium, which could help to induce host\u2019s defenses against and responses towards infections. In the third part of the research we analyzed the effects on immune system of food compounds from vegetal origin. To this aim, we evaluated the immunomodulatory potential of different fractions extracted from wild blueberries (WB) powder. We observed that only the anthocyanin (ACN) fraction was effective in reducing the activation of NF-\u3baB on Caco-2 cells, whereas both the soluble and the phenolic fractions had no significant effects. Consequently, we used only the anthocyanin fraction for the subsequent characterization on U937 macrophages. We found that the presence of ACNs decreased the induction of TNF-\u3b1 triggered by lipopolysaccharide (LPS) from Escherichia coli on U937, particularly when the cells were pretretated with ACNs and afterwards treated with LPS. These data suggest that ACNs from WB might have a protective role towards inflammation and that, probably, the described anti-oxidant features of these compound might be partially mediated by direct effects on immune system. In conclusion, this PhD work evidenced the noticeable abilities of bacteria and dietary compounds to modulate host immune system responses. Particularly, this study suggests that the use of selected food-grade bacteria, bacterial components or dietary compounds has a promising potential for the maintenance of host health and the prevention of diseases

Methodological issues in the study of intestinal microbiota in irritable bowel syndrome

Irritable bowel syndrome (IBS) is an intestinal functional disorder with the highest prevalence in the industrialized world. The intestinal microbiota (IM) plays a role in the pathogenesis of IBS and is not merely a consequence of this disorder. Previous research efforts have not revealed unequivocal microbiological signatures of IBS, and the experimental results are contradictory. The experimental methodologies adopted to investigate the complex intestinal ecosystem drastically impact the quality and significance of the results. Therefore, to consider the methodological aspects of the research on IM in IBS, we reviewed 29 relevant original research articles identified through a PubMed search using three combinations of keywords: "irritable bowel syndrome + microflora", "irritable bowel syndrome + microbiota" and "irritable bowel syndrome + microbiome". For each study, we reviewed the quality and significance of the scientific evidence obtained with respect to the experimental method adopted. The data obtained from each study were compared with all considered publications to identify potential inconsistencies and explain contradictory results. The analytical revision of the studies referenced in the present review has contributed to the identification of microbial groups whose relative abundance significantly alters IBS, suggesting that these microbial groups could be IM signatures for this syndrome. The identification of microbial biomarkers in the IM can be advantageous for the development of new diagnostic tools and novel therapeutic strategies for the treatment of different subtypes of IBS

Quantitative recovery of viable Lactobacillus paracasei CNCM I-1572 (L. casei DG®) after gastrointestinal passage in healthy adults

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG\uae; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life

Impact of a multistrain probiotic formulation with high bifidobacterial content on the fecal bacterial community and short-chain fatty acid levels of healthy adults

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio

Health-Promoting Properties of Lactobacillus helveticus

Lactobacillus helveticus is an important industrial thermophilic starter that is predominantly employed in the fermentation of milk for the manufacture of several cheeses. In addition to its technological importance, a growing body of scientific evidence shows that strains belonging to the L. helveticus species have health-promoting properties. In this review, we synthesize the results of numerous primary literature papers concerning the ability of L. helveticus strains to positively influence human health. Several in vitro studies showed that L. helveticus possesses many common probiotic properties, such as the ability to survive gastrointestinal transit, adhere to epithelial cells, and antagonize pathogens. In vivo studies in murine models showed that L. helveticus could prevent gastrointestinal infections, enhance protection against pathogens, modulate host immune responses, and affect the composition of the intestinal microbiota. Interventional studies and clinical trials have also demonstrated a number of health-promoting properties of L. helveticus. Finally, several studies suggested that specific enzymatic activities of L. helveticus could indirectly benefit the human host by enhancing the bioavailability of nutrients, removing allergens and other undesired molecules from food, and producing bioactive peptides through the digestion of food proteins. In conclusion, this review demonstrates that in light of the scientific literature presented, L. helveticus can be included among the bacterial species that are generally considered to be probiotic

Association between aortic calcification, cardiovascular events, and mortality in kidney and pancreas-kidney transplant recipients

BACKGROUND: Cardiovascular (CV) disease is the leading cause of death in kidney and simultaneous pancreas-kidney (SPK) transplant recipients. Assessing abdominal aortic calcification (AAC), using lateral spine x-rays and the Kaupilla 24-point AAC (0-24) score, may identify transplant recipients at higher CV risk. METHODS: Between the years 2000 and 2015, 413 kidney and 213 SPK first transplant recipients were scored for AAC at time of transplant and then followed for CV events (coronary heart, cerebrovascular, or peripheral vascular disease), graft-loss, and all-cause mortality. RESULTS: The mean age was 44 ± 12 years (SD) with 275 (44%) having AAC (26% moderate: 1-7 and 18% high: ≥8). After a median of 65 months (IQR 29-107 months), 46 recipients experienced CV events, 59 died, and 80 suffered graft loss. For each point increase in AAC, the unadjusted hazard ratios (HR) for CV events and mortality were 1.11 (95% CI 1.07-1.15) and 1.11 (1.08-1.15). These were similar after adjusting for age, gender, smoking, transplant type, dialysis vintage, and diabetes: aHR 1.07 (95% CI 1.02-1.12) and 1.09 (1.04-1.13). For recipients with high versus no AAC, the unadjusted and fully-adjusted HRs for CV events were 5.90 (2.90-12.02) and 3.51 (1.54-8.00), for deaths 5.39 (3.00-9.68) and 3.38 (1.71-6.70), and for graft loss 1.30 (0.75-2.28) and 1.94 (1.04-3.27) in age and smoking history-adjusted analyses. CONCLUSION: Kidney and SPK transplant recipients with high AAC have 3-fold higher CV and mortality risk and poorer graft outcomes than recipients without AAC. AAC scoring may be useful in assessing and targeted risk-lowering strategies

Probiotics action on gliadin sequences relevant to gluten sensitivity

The Celiac disease in genetically predisposed individuals is mainly induced by specific repetitive sequences in gliadins (PQPYP). This autoimmune disease stems from the interaction between toxic sequences and lamina propria cells, that is relevant also to other forms of gluten sensitivity. Specific endo-esoprolinase were isolated from lactic acid bacteria, suggesting possible practical applications. The ability of some probiotics at removing "toxic" celiac sequences was investigated, at first by assessing the presence and level of endo- and eso-prolinase activity in some of the most popular probiotic bacteria. Significant activities were detected in Lactobacillus and Bifidum species, as well as in the probiotic Escherichia coli Niessle 1917. On the basis of prolinase data, we investigated by mass spectroscopy the removal of "toxic" sequences in gliadin. A complete disappearance of these sequences was observed only with Escherichia coli Niessle 1917. Among the Bifidus and Lactobacillus species, only B. bifidum MIMBb23SG and L. acidophilus LA5 showed a significant decrease in the "toxic" sequences. All together, this study suggests a potential use of lactic bacteria to lower gluten response in sensitive individuals, including celiacs and gluten-sensitive

Evidence of dysbiosis in the intestinal microbial ecosystem of children and adolescents with primary hyperlipidemia and the potential role of regular hazelnut intake

Hyperlipidemia starts at a pediatric age and represents an unquestionable risk factor for cardiovascular disease. Modulation of the intestinal microbial ecosystem (IME), in principle, can ameliorate lipid profiles. In this study, we characterized the IME of children and adolescents with primary hyperlipidemia by analyzing fecal samples through 16S rRNA gene profiling (n\ua0=\ua015) and short chain fatty acid (SCFA) quantification (n\ua0=\ua032). The same analyses were also carried out on age-matched normolipidemic controls (n\ua0=\ua015). Moreover, we evaluated the modulatory effect of regular hazelnut intake (approximately 0.43 g of hazelnuts with skin per kg of body weight) on the IME of 15 children and adolescents with hyperlipidemia for eight weeks. We found alterations of numerous operational taxonomic units potentially associated with SCFA-producing bacteria and reductions in the fecal levels of acetate, butyrate and propionate in hyperlipidemic subjects. Furthermore, we observed that an eight-week hazelnut intervention may induce limited changes in fecal microbiota composition but can significantly modulate the fecal levels of predominant intestinal SCFAs, such as acetate. Finally, correlation analyses indicated that changes in lipidemic parameters are linked to modifications of the abundance of specific bacterial taxa, such as the families Lachnospiraceae and Ruminococcaceae and the genera Akkermansia, Bacteroides, Roseburia, and Faecalibacterium. This study suggests that children and adolescents with primary hyperlipidemia possess an altered IME. The promising results presented here support the need for future dietary interventions aimed at positively modulating the IME of hyperlipidemic subjects

A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response

The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-\u3baB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-\u3b1), in a dose-dependent manner. After stimulation of cells with IL-1\u3b2, active NF-\u3baB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-\u3b3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-\u3b1 and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties

Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem