Carolina Gomez-Llorente

Probiotic Mechanisms of Action

Probiotics are live microorganisms that provide health benefits to the host when ingested in adequate amounts. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria. Probiotics have demonstrated significant potential as therapeutic options for a variety of diseases, but the mechanisms responsible for these effects have not been fully elucidated yet. Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host. Accumulating evidence demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors and nucleotide-binding oligomerization domain-containing protein-like receptors, which modulate key signaling pathways, such as nuclear factor-ĸB and mitogen-activated protein kinase, to enhance or suppress activation and influence downstream pathways. This recognition is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage. A clear understanding of these mechanisms will allow for appropriate probiotic strain selection for specific applications and may uncover novel probiotic functions. The goal of this systematic review was to explore probiotic modes of action focusing on how gut microbes influence the host.

The Role of Probiotic Lactic Acid Bacteria and Bifidobacteria in the Prevention and Treatment of Inflammatory Bowel Disease and Other Related Diseases: A Systematic Review of Randomized Human Clinical Trials

Inflammatory bowel disease (IBD), which includes Crohns disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the small intestine and colon caused by a dysregulated immune response to host intestinal microbiota in genetically susceptible subjects. A number of fermented dairy products contain lactic acid bacteria (LAB) and bifidobacteria, some of which have been characterized as probiotics that can modify the gut microbiota and may be beneficial for the treatment and the prevention of IBD. The objective of this review was to carry out a systematic search of LAB and bifidobacteria probiotics and IBD, using the PubMed and Scopus databases, defined by a specific equation using MeSH terms and limited to human clinical trials. The use of probiotics and/or synbiotics has positive effects in the treatment and maintenance of UC, whereas in CD clear effectiveness has only been shown for synbiotics. Furthermore, in other associated IBD pathologies, such as pouchitis and cholangitis, LAB and bifidobacteria probiotics can provide a benefit through the improvement of clinical symptoms. However, more studies are needed to understand their mechanisms of action and in this way to understand the effect of probiotics prior to their use as coadjuvants in the therapy and prevention of IBD conditions.

Role of Toll-like receptors in the development of immunotolerance mediated by probiotics.

Commensal bacteria are important in intestinal homeostasis and appear to play a role in early tolerance to foreign antigens. The requirement for homeostatic balance between tolerance and immunity poses a unique regulatory challenge to mucosal immune systems. Dysregulation of this balance can contribute to the pathogenesis of numerous inflammatory conditions such as inflammatory bowel diseases. The primary response to these bacteria is triggered by pattern recognition receptors (PRR), which bind pathogen-associated molecular patterns (PAMP). PRR comprise Toll-like receptors (TLR), nucleotide-binding oligomerization domains, adhesion molecules and lectins. Probiotics are living commensal micro-organisms of the intestinal tract with clinically documented health effects in human subjects. They are known to affect the gastrointestinal tract and the associated immune system and to have numerous effects on intestinal function and immune responses, including immunotolerance. This last effect appears to be mediated via regulatory T-cell activation by intestinal dendritic cells and the low activation of T-helper 1 and 2 (Th1 and Th2) cell inflammatory responses. However, the precise mechanisms of probiotic activity remain poorly understood. The aim of the present work was to review the function of TLR in the development of immunotolerance and examine the specific role of probiotics in the regulation of tolerance to antigens.

Is adipose tissue metabolically different at different sites

This review focuses on metabolic differences of adipose tissue at different sites of the body, with emphasis in pediatrics. Adipose tissue is composed of various cell types, which include adipocytes and other cells of the stromal vascular fraction such as preadipocytes, blood cells, endothelial cells and macrophages. Mammals have two main types of adipose tissue: white adipose tissue (WAT), and brown adipose tissue (BAT), each of which possesses unique cell autonomous properties. WAT and BAT differ at the functional, as well as the morphological and molecular levels. WAT accumulates surplus energy mainly in the form of triacylglycerols and BAT dissipates energy directly as heat. Recently, functional BAT in humans has been located in the neck, supraclavicular, mediastinal and interscapular areas. WAT is distributed throughout the body in the form of two major types: subcutaneous adipose tissue (SWAT) and the intra-abdominal visceral adipose tissue (VWAT). VWAT tissue is associated with insulin resistance, diabetes mellitus, dyslipidaemia, hypertension, atherosclerosis, hepatic steatosis, and overall mortality whereas SWAT and BAT have intrinsic beneficial metabolic properties. Subcutaneous and visceral adipocytes derive from different progenitor cells that exhibit a different gene expression pattern. SWAT responds better to the antilipolytic effects of insulin and other hormones, secrets more adiponectin and less inflammatory cytokines, and is differentially affected by molecules involved in signal transduction as well as drugs compared with VWAT. Current research is investigating various approaches of BAT and SWAT transplantation, including new sources of adipocyte progenitors. This may be important for the potential treatment of childhood obesity.

Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review

Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue.

Cell-Free Culture Supernatant of Bifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged with Salmonella typhi through TLR Activation

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways.

Human intestinal dendritic cells decrease cytokine release against Salmonella infection in the presence of Lactobacillus paracasei upon TLR activation.

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods.

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.

Three Main Factors Define Changes in Fecal Microbiota Associated With Feeding Modality in Infants

Objectives: There are many differences in the fecal infant microbiota associated with various feeding methods. The aim of this study was to examine the major differences in the fecal microbiota of breast-fed (BF) and formula-fed (FF) infants and to describe the principal bacterial components that would explain the variability in the predominant bacterial families and genus clusters. Methods: Fecal samples from 58 infants, 31 of whom were exclusively BF and 27 of whom were exclusively FF with a standard formula in agreement with the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition recommendations, were analyzed by fluorescent in situ hybridization combined with flow cytometry. Principal component analysis was used to maximize the information gained for the predominant bacterial families and genus clusters using a minimal number of bacterial groups. Results: The predominant detected group was Bifidobacterium, followed by Enterobacteriaceae and Bacteroides in both BF and FF infants. The Lactobacillus group was the only independent variable associated with FF infants. We also found that 3 principal components were sufficient to describe the association between the bacterial group, genus, and species studied in BF and FF infants; however, these components differed between BF and FF infants. For the former, the 3 factors found were Bifidobacterium/Enterobacteriaceae, Lactobacillus/Bacteroides, and Clostridium coccoides/Atopobium; for the latter, Bifidobacterium/Enterobacteriaceae, Bacteroides and C coccoides were observed. Conclusions: There is a clear clustering of components of infant microbiota based on the feeding method.

Pyrosequencing Analysis Reveals Changes in Intestinal Microbiota of Healthy Adults Who Received a Daily Dose of Immunomodulatory Probiotic Strains

The colon microbiota plays a crucial role in human gastrointestinal health. Current attempts to manipulate the colon microbiota composition are aimed at finding remedies for various diseases. We have recently described the immunomodulatory effects of three probiotic strains (Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034, and Bifidobacterium breve CNCM I-4035). The goal of the present study was to analyze the compositions of the fecal microbiota of healthy adults who received one of these strains using high-throughput 16S ribosomal RNA gene sequencing. Bacteroides was the most abundant genus in the groups that received L. rhamnosus CNCM I-4036 or L. paracasei CNCM I-4034. The Shannon indices were significantly increased in these two groups. Our results also revealed a significant increase in the Lactobacillus genus after the intervention with L. rhamnosus CNCM I-4036. The initially different colon microbiota became homogeneous in the subjects who received L. rhamnosus CNCM I-4036. While some orders that were initially present disappeared after the administration of L. rhamnosus CNCM I-4036, other orders, such as Sphingobacteriales, Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes, were detected after the intervention. In summary, our results show that the intake of these three bacterial strains induced changes in the colon microbiota.