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Background: Studies on childhood obesity mainly focus on the genetic component and on the lifestyle that may be associated with the development of obesity. However, the study of perinatal factors in their programming effect toward future obesity in children or adults is somewhat more recent, and there are still mechanisms to be disentangled. Summary: In this narrative review, a comprehensive route based on the influence of some early factors in life in the contribution to later obesity development is presented. Maternal pre-pregnancy BMI and gestational weight gain have been pointed out as independent determinants of infant later adiposity. Lifestyle interventions could have an impact on pregnant mothers through epigenetic mechanisms capable of redirecting the genetic expression of their children toward a future healthy weight and body composition and dietary-related microbiome modifications in mothers and newborns might also be related. After birth, infant feeding during the first months of life is directly associated with its body composition and nutritional status. From this point of view, all the expert committees in the world are committed to promote exclusive breastfeeding up to 6 months of age and to continue at least until the first year of life together with complementary feeding based on healthy dietary patterns such as Mediterranean Diet. Key Messages: To develop future effective programs to tackle early obesity, it is necessary not only by controlling lifestyle behaviors like infant feeding but also understanding the role of other mechanisms like the effect of perinatal factors such as maternal diet during pregnancy, epigenetics, or microbiome.

In general terms, fetal growth restriction (FGR) is considered the impossibility of achieving the genetically determined potential size. In the vast majority of cases, it is related to uteroplacental insufficiency. Although its origin remains unknown and causes are only known in 30% of cases, it is believed to be related to an interaction of environmental and genetic factors with either a fetal or maternal origin. One hypothesis is that alterations in the gastrointestinal microbiota composition, and thus alteration in the immune response, could play a role in FGR development. We performed an observational, prospective study in a subpopulation affected with FGR to elucidate the implications of this microbiota on the FGR condition. A total of 63 fetuses with FGR diagnosed in the third trimester as defined by the Delphi consensus, and 63 fetuses with fetal growth appropriate for gestational age will be recruited. Obstetric and nutritional information will be registered by means of specific questionnaires. We will collect maternal fecal samples between 30 to 36 weeks, intrapartum samples (maternal feces, maternal and cord blood) and postpartum samples (meconium and new-born feces at 6 weeks of life). Samples will be analyzed in the Department of Biochemistry and Molecular Biology II, Nutrition and Food Technology Institute of the University of Granada (UGR), for the determination of the gastrointestinal microbiota composition and its relationship with inflammatory biomarkers. This study will contribute to a better understanding of the influence of gastrointestinal microbiota and related inflammatory biomarkers in the development of FGR. Trial registration: NCT04047966. Registered August 7, 2019, during the recruitment stage. Retrospectively registered. Ongoing research., Instituto de Salud Calos III, Fondos de Desarrollo Regional Europeos (FEDER) PI17/01215

Asthma is a multifactorial condition where patients with identical clinical diagnoses do not have the same clinical history or respond to treatment. This clinical heterogeneity is reflected in the definition of two main endotypes. We aimed to explore the metabolic and microbiota signatures that characterize the clinical allergic asthma phenotype in obese children. Methods: We used a multi-omics approach combining clinical data, plasma and fecal inflammatory biomarkers, metagenomics, and metabolomics data in a cohort of allergic asthmatic children. Results: We observed that the obese allergic asthmatic phenotype was markedly associated with higher levels of leptin and lower relative proportions of plasma acetate and a member from the Clostridiales order. Moreover, allergic children with a worse asthma outcome showed higher levels of large unstained cells, fecal D lactate and D/L lactate ratio, and with a higher relative proportion of plasma creatinine and an unclassified family member from the RF39 order belonging to the Mollicutes class. Otherwise, children with persistent asthma presented lower levels of plasma citrate and dimethylsulfone. Conclusion: Our integrative approach shows the molecular heterogeneity of the allergic asthma phenotype while highlighting the use of omics technologies to examine the clinical phenotype at a more holistic level., Fundación Progreso y Salud Project PI-0373-2014, Redes temáticas de investigación cooperativa RETIC Red SAMID RD12/0026/0015

The aim of this systematic review is to evaluate whether the use of probiotics has any effect on the components of metabolic syndrome (MetS) before patients develop type 2 diabetes. A qualitative systematic review, following the Cochrane methodology, and a comprehensive literature search of randomized controlled trials (RCTs) were conducted in PubMed and Scopus from inception until 4 July 2019. According to our inclusion criteria, nine clinical studies were finally analyzed, corresponding to six RCTs. Probiotics intake in patients with MetS resulted in improvements in body mass index, blood pressure, glucose metabolism, and lipid profile in some studies. Regarding inflammatory biomarkers, probiotics also positively affected the soluble vascular cell adhesion molecule 1 (sVCAM-1), interleukine-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), vascular endothelial growth factor (VEGF), and thrombomodulin. Despite the diversity of the published studies, the intake of probiotics for patients with MetS may offer a discrete improvement in some of the clinical characteristics of the MetS and a decrease in inflammatory biomarkers. Nevertheless, these beneficial effects seem to be marginal compared to drug therapy and a healthy lifestyle and clinically non-relevant.

Essential oils (EOs) are a mixture of natural, volatile, and aromatic compounds obtained from plants. In recent years, several studies have shown that some of their benefits can be attributed to their antimicrobial, antioxidant, anti-inflammatory, and also immunomodulatory properties. Therefore, EOs have been proposed as a natural alternative to antibiotics or for use in combination with antibiotics against multidrug-resistant bacteria in animal feed and food preservation. Most of the results come from in vitro and in vivo studies; however, very little is known about their use in clinical studies. A systematic and comprehensive literature search was conducted in PubMed, Embase®, and Scopus from December 2014 to April 2019 using di erent combinations of the following keywords: essential oils, volatile oils, antimicrobial, antioxidant, immunomodulation, and microbiota. Some EOs have demonstrated their efficacy against several foodborne pathogens in vitro and model food systems; namely, the inhibition of S. aureus, V. cholerae, and C. albicans has been observed. EOs have shown remarkable antioxidant activities when used at a dose range of 0.01 to 10 mg/mL in cell models, which can be attributed to their richness in phenolic compounds. Moreover, selected EOs exhibit immunomodulatory activities that have been mainly attributed to their ability to modify the secretion of cytokines., Part of the research currently in progress in the authors’ laboratory is funded by the company PAYMSA, S. A. through the grant #4501 managed by the Fundacion General Empresa-Universidad de Granada.

Background: S100A4 is a metastasis-associated protein also reported as a promising marker for dysfunctional white adipose tissue (WAT) and insulin resistance (IR) in adult and adolescent populations. Objective: We aimed to evaluate the association between the protein S100A4 and obesity and IR in children and during pubertal development. Design and methods: The study design consisted of three cross-sectional populations of 249, 11 and 19 prepubertal children respectively (named study population 1, 2 and 3), and a longitudinal population of 53 girls undergoing sexual maturation (study population 4). All subjects were classified into experimental groups according to their sex, obesity and IR status. All study populations counted on anthropometry, glucose, and lipid metabolism, inflammation and cardiovascular biomarkers as well as S100A4 plasma levels measured. The study population 1 was intended as the discovery population in which to elucidate the relationship between Obesity-IR and S100A4 plasma levels in prepubertal children. The cross-sectional populations 2 and 3 further counted on WAT gene expression data for investigating the molecular basis of this association. Instead, the longitudinal study population 4 presented blood whole-genome DNA methylation data at each temporal record, allowing deepening into the Obesity-IR-S1004 relationship during puberty as well as deciphering plausible epigenetic mechanisms altering S100A4 plasma levels. Results: S100A4 plasma levels were strongly associated with several metabolic and anthropometric outcomes, namely IR, in prepubertal non-diabetic obese children. We also found highly significant positive associations during the course of puberty between the increase in S100A4 levels and the increase in HOMA-IR (P = 0.0003, FDR = 0.005) and insulin levels (P = 0.0003, FDR = 0.005). Methylation in two-enhancer related CpG sites of the S100A4 region (cg07245635 and cg10447638) was associated with IR biomarkers at the prepubertal stage and with longitudinal changes in these measurements. We further reported an association between visceral WAT (vWAT) S100A4 expression and HOMA-IR, insulin levels and BMI Z-Score, but not with circulating S100A4. Conclusions: We report for the first time the association of S100A4 with IR and WAT dysfunction in prepubertal populations as well as how the change in plasma S100A4 levels accompanies longitudinal trajectories of IR in children during pubertal development. Moreover, we propose epigenetic changes in two methylation sites and an altered S100A4 vWAT expression as plausible molecular mechanisms underlying this disturbance in obesity.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This paper will be part of Augusto Anguita-Ruiz’s doctorate, which is being performed under the “Nutrition and Food Sciences Program” at the University of Granada., Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-40482-0., Tenomodulin (TNMD) is a type II transmembrane glycoprotein that has been recently linked to obesity, and it is highly expressed in obese adipose tissue. Several sex-dependent associations have been observed between single-nucleotide polymorphisms (SNPs) of the TNMD gene, which is located in the X-chromosome, and obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome in adults. On the other hand, results are lacking for children. We aimed (i) to study the association between TNMD genetic variants and metabolic complications related to childhood obesity and (ii) to investigate the function of TNMD in human adipocytes. We conducted a case-control, multicenter study in 915 Spanish children and demonstrated significant positive associations between TNMD genetic variants and BMI z-score, waist circumference, fasting glucose, and insulin resistance in boys, highlighting the SNP rs4828038. Additionally, we showed a BMI-adjusted inverse association with waist circumference in girls. Second, in vitro experiments revealed that TNMD is involved in adipogenesis, along with glucose and lipid metabolism in differentiated adipocytes, and these effects may be mediated through AMPK activation. Hence, these results suggest that TNMD genetic variants could be potentially useful as early life risk indicators for obesity and T2DM. In addition, we support the fact that TNMD exhibits significant metabolic functions in adipocytes., This work was supported by Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I + D + I), Instituto de Salud Carlos III-Fondo de Investigación Sanitaria (Projects numbers PI020826, PI051968, PI1102042, PI1600871), RETIC (Red SAMID RD12/0026/0015), Fondo Europeo De Desarrollo Regional (FEDER) and the Junta de Andalucía (project number CTS-6770); Secretaría General de Universidades, Investigación y Tecnología. Consejería de Economía, Innovación y Ciencia).

Funding Financial support has been provided by Centro para el Desarrollo Tecnologico Industrial (CDTI) in the framework of the project "Investigacion industrial de nuevos ingredientes, alimentos, tecnologias y seguridad en el ambito de alergias e intolerancias alimentarias" (IDI20170872) . Part of the research currently in progress in the authors' laboratory is funded by the company PAYMSA through the grant #4501 managed by the Fundacion General EmpresaUniversidad de Granada., Carolina Gomez-Llorente, Julio Plaza-Diaz and Angel Gil are part of the “UGR Plan Propio de Investigaci´on 2016” and the “Excellence actions: Unit of Excellence on Exercise and Health (UCEES), University of Granada”., The present study was conducted to evaluate the in vitro antimicrobial effect of three essential oils (cinnamon, white thyme, and clove) on selected intestinal pathogen bacteria, probiotic strains, and commensal bacteria. Additionally, their effects on inflammatory gene expression were also evaluated in a well-known intestinal human cellular model (Caco-2 cells). Cinnamon showed the highest antipathogenic microbial activity, followed by white thyme and clove. In the case of the probiotic strains, the inhibitory effect was observed at 0.1% (v/v), although growth recovery was seen after 24 h of incubation. Besides, cinnamon down-regulated the expression of genes related to the Toll-like receptor, nitric oxide, and apoptosis pathways. Similarly, secretion of interleukin 2 was inhibited by cinnamon. Results support the potential use of essential oils in the food industry for their antimicrobial and immunomodulatory effects; however, the underlying mechanisms are unknown and warrant further research., Centro para el Desarrollo Tecnologico Industrial (CDTI) IDI20170872, company PAYMSA 4501

Background Obesity is characterized by increased fat mass and is associated with the development of insulin resistance syndrome (IRS), usually known as metabolic syndrome. The alteration of the intestinal microbiota composition has a role in the development of IRS associated with obesity, and probiotics, which are live microorganisms that confer a health benefit to the host, contribute to restore intestinal microbiota homeostasis and lower peripheral tissue insulin resistance. We aim to evaluate the effects of the probiotic strain Lactobacillus reuteri (L. reuteri) V3401 on the composition of intestinal microbiota, markers of insulin resistance and biomarkers of inflammation, cardiovascular risk, and hepatic steatosis in patients with overweight and obesity exhibiting IRS. Methods/design We describe a randomized, double-blind, crossover, placebo-controlled, and single-centre trial. Sixty participants (aged 18 to 65 years) diagnosed with IRS will be randomized in a 1:1 ratio to receive either a daily dose of placebo or 5 × 109 colony-forming units of L. reuteri V3401. The study will consist of two intervention periods of 12 weeks separated by a washout period of 6 weeks and preceded by another washout period of 2 weeks. The primary outcome will be the change in plasma lipopolysaccharide (LPS) levels at 12 weeks. Secondary outcomes will include anthropometric parameters, lipid profile, glucose metabolism, microbiota composition, hepatic steatosis, and inflammatory and cardiovascular biomarkers. Blood and stool samples will be collected at baseline, at the midpoint (only stool samples) and immediately after each intervention period. Luminex technology will be used to measure interleukins. For statistical analysis, a mixed ANOVA model will be employed to calculate changes in the outcome variables. Discussion This is the first time that L. reuteri V3401 will be evaluated in patients with IRS. Therefore, this study will provide valuable scientific information about the effects of this strain in metabolic syndrome patients. Trial registration The trial has been retrospectively registered in ClinicalTrials.gov on the 23rd November 2016 (ID: NCT02972567), during the recruitment phase. Electronic supplementary material The online version of this article (10.1186/s12906-018-2371-x) contains supplementary material, which is available to authorized users.

Oxidative stress is defined as molecular damage derived from impaired redox signaling and equilibrium as a consequence of an imbalance between prooxidants and antioxidants. Disorders of the mitochondrial electron transport chain, overgeneration of reactive oxygen species, and lipoperoxides or alterations in antioxidant defenses are responsible for the increase in oxidative stress. Oxidative stress induces reactive oxygen species sensing, transduction of signals through specific pathways, and upregulation of target genes, for example those coding antioxidant defenses and specifically coding some inflammatory mediators. Moreover, by damaging cell structures, oxidative stress can trigger or potentiate an inflammatory response driven by immune cells and mediators. Furthermore, inflammation-related mechanisms may increase the production of reactive oxygen species. Therefore, both oxidative and inflammatory mechanisms are interconnected and underlie the molecular basis of many diseases. The present chapter aims to describe the main molecular mechanisms of oxidative stress and inflammation.

Inflammatory bowel disease (IBD), which includes Crohn’s 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.

We investigated whether the administration of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 modulate the expression of genes in the intestinal mucosa of obese Zucker rats. Forty-eight Zucker-Leprfa/fa and 16 Zucker lean Lepr+/fa rats were used. Eight Zucker lean Lepr+/fa and 8 Zucker-Leprfa/fa rats were euthanized as a reference. The remaining 40 Zucker-Leprfa/fa rats were then assigned to receive 1010 colony forming units (CFU) of one of the three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo by oral administration for 30 days. An additional group of 8 Zucker lean Lepr+/fa rats received the placebo for 30 days. Over 27,000 rat genes were studied using a DNA array. Four animals per group were used. Total RNA was extracted from intestinal mucosa and cDNA was synthesized, fragmented and labeled. Labeled cDNA was hybridized using GeneChip kits, and the latter were scanned. Intensity values of each probe were processed and normalized to obtain an individual value for each set of probes.

Obesity and asthma are two chronic conditions that affect millions of people. Genetic and lifestyle factors such as diet, physical activity, and early exposure to micro-organisms are important factors that may contribute to the escalating prevalence of both conditions. The prevalence of asthma is higher in obese individuals. Recently, two major phenotypes of asthma with obesity have been described: one phenotype of early-onset asthma that is aggravated by obesity, and a second phenotype of later-onset asthma that predominantly affects women. Systemic inflammation and mechanical effect, both due to the expansion of the adipose tissue, have been proposed as the main reasons for the association between obesity and asthma. However, the mechanisms involved are not yet fully understood. Moreover, it has also been suggested that insulin resistance syndrome can have a role in the association between these conditions. The intestinal microbiota is an important factor in the development of the immune system, and can be considered a link between obesity and asthma. In the obese state, higher lipopolysaccharide (LPS) serum levels as a consequence of a microbiota dysbiosis have been found. In addition, changes in microbiota composition result in a modification of carbohydrate fermentation capacity, therefore modifying short chain fatty acid (SCFA) levels. The main objective of this review is to summarize the principal findings that link obesity and asthma.

The following are available online at https://www.mdpi.com/2072-6643/11/8/1761/s1: Figure S1, Rarefaction curves; Figure S2, Alpha diversity measured by means of the Shannon index (H); Figure S3, Bacterial beta diversity; Table S1, Gastrointestinal microbiome normalized data, This paper will be part of Carmen Tenorio Jimenez’s doctorate, which is being completed as part of Programa de Doctorado en Seguridad de los Alimentos at the University of Jaén, Spain. We also wish to thank all the participants who generously contributed to the study., Previous studies have reported that probiotics may improve clinical and inflammatory parameters in patients with obesity and metabolic syndrome (MetS). Lactobacillus (L.) reuteri V3401 has shown promising results on the components of MetS in animal studies. We aimed to evaluate the effects of L. reuteri V3401 together with healthy lifestyle recommendations on adult patients with MetS. Methods: We carried out a randomized, crossover, placebo-controlled, single-center trial in which we included 53 adult patients newly diagnosed with MetS. Patients were block randomly allocated by body mass index (BMI) and sex to receive a capsule containing either the probiotic L. reuteri V3401 (5 × 109 colony-forming units) or a placebo once daily for 12 weeks. Anthropometric variables, biochemical and inflammatory biomarkers, as well as the gastrointestinal microbiome composition were determined. Results: There were no differences between groups in the clinical characteristics of MetS. However, we found that interleukin-6 (IL-6) and soluble vascular cell adhesion molecule 1 (sVCAM-1) diminished by effect of the treatment with L. reuteri V3401. Analysis of the gastrointestinal microbiome revealed a rise in the proportion of Verrucomicrobia. Conclusions: Consumption of L. reuteri V3401 improved selected inflammatory parameters and modified the gastrointestinal microbiome. Further studies are needed to ascertain additional beneficial effects of other probiotic strains in MetS as well as the mechanisms by which such effects are exerted., This study is part of the grant entitled “Guía para la sustanciación de declaraciones de salud en alimentos: funciones inmune, cognitiva y síndrome metabólico”, funded by the company Biosearch life (Granada, Spain), reference 3006, managed by Fundación General Universidad de Granada, Spain.

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.

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytesin vitro.The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity withLactobacillus paracasei,Lactobacillus rhamnosusandBifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S–23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and namedL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibitedin vitrothe meningitis aetiological agentListeria monocytogenesand human rotavirus infections.B. breveCNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

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.

The present study was undertaken to investigate the effects of C-atrial natriuretic peptide (C-ANP4-23) in human adipose-derived stem cells differentiated into adipocytes over 10 days (1 μM for 4 h). The intracellular cAMP, cGMP and protein kinase A levels were determined by ELISA and gene and protein expression were determined by qRT-PCR and Western blot, respectively, in the presence or absence of C-ANP4-23. The levels of lipolysis and glucose uptake were also determined. C-ANP4-23 treatment significantly increased the intracellular cAMP levels and the gene expression of glucose transporter type 4 (GLUT4) and protein kinase, AMP-activated, alpha 1 catalytic subunit (AMPK). Western blot showed a significant increase in GLUT4 and phosphor-AMPKα levels. Importantly, the adenylate cyclase inhibitor SQ22536 abolished these effects. Additionally, C-ANP4-23 increased glucose uptake by 2-fold. Our results show that C-ANP4-23 enhances glucose metabolism and might contribute to the development of new peptide-based therapies for metabolic diseases.

Obesity is characterized by an excessive accumulation of fat in adipose tissue, which is associated with oxidative stress and chronic inflammation. Excessive H2O2 levels are degraded by catalase (CAT), the activity of which is decreased in obesity. We investigated the effects of inhibition of catalase activity on metabolism and inflammation by incubating human differentiated adipocytes with 10 mM 3-amino-1,2,4-triazole (3-AT) for 24 h. As expected, the treatment decreased CAT activity and increased intracellular H2O2 levels significantly. Glutathione peroxidase (GPX) activity was also reduced, and the gene expression levels of the antioxidant enzymes GPX4 and peroxiredoxins (1, 3 and 5) were inhibited. Interestingly, this occurred along with lower mRNA levels of the transcription factors nuclear factor (erythroid 2-like 2) and forkhead box O, which are involved in redox homeostasis. However, superoxide dismutase activity and expression were increased. Moreover, 3-AT led to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and increased tumor necrosis alpha and interleukin 6 protein and gene expression levels, while lowering peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein levels. These alterations were accompanied by an altered glucose and lipid metabolism. Indeed, adipocytes treated with 3-AT showed reduced basal glucose uptake, reduced glucose transporter type 4 gene and protein expression, reduced lipolysis, reduced AMP-activated protein kinase activation and reduced gene expression of lipases. Our results indicate that increased H2O2 levels caused by 3-AT treatment impair the antioxidant defense system, lower PPARγ expression and initiate inflammation, thus affecting glucose and lipid metabolism in human differentiated adipocytes.

Background Thrombophilia is defined as an inherited or acquired abnormality of hemostasis predisposing to thrombosis. While the most common thrombophilia has a genetic origin and is manifested by elevated circulating antiphospholipid antibodies, about 40% of cases presenting with thrombosis are acquired. Factor V Leiden G1691A, prothrombin G20210A, MTHFR C677T, and Factor XII C46T mutations are associated with the risk of developing thrombophilia. Methods In this study, a method using single base extension assay coupled with fluorescent detection and capillary electrophoresis was applied to simultaneously detect G1691A, G20210A, C677T and C46T mutations in 1499 patients from Spain with suspicion of thrombotic disease. Results Out of these individuals, 5.4% were heterozygous for G20210A mutation, 9.21% were heterozygous and 0.20% homozygous for G1691A mutation, 46.36% were heterozygous and 20.71% homozygous for MTHFR mutation, and 30.41% were heterozygous and 3.4% homozygous for C46T mutation. Conclusion We applied an accurate, simple, semi-automatic, and cost-effective method to simultaneously detect the main thrombophilia-related mutations, allowing us to determine the frequency of these mutations in a Spanish population.

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.

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.

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.

Background Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defence against oxidizing agents and in reductive biosynthetic reactions. Many variants of G6PD have been described, mostly produced from missense mutations, with wide ranging levels of enzyme activity and associated clinical symptoms. Method A single base extension assay is used, yielding a single base difference of the extended products. Primers are designed to amplify products of different sizes with distinct fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. Results We present the first application of a multiplex multicolour assay to detect 15 of the most frequent G6PD-related mutations in Spain, which are studied in three multiplex reactions. Capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high-resolution discrimination between wild-type and mutant alleles, even though various mutations may be present in the multiplex analysis. Conclusion The analytical method described herein offers greater diagnostic power in Spanish and Mediterranean populations and would facilitate automated genotyping in routine molecular diagnostics and large-scale genetic studies (e.g., newborn screening programs).

To identify novel proteins required for receptor-mediated endocytosis, we have developed an RNAi-based screening method in Drosophila S2 cells, based on uptake of a scavenger receptor ligand. Some known endocytic proteins are essential for endocytosis in this assay, including clathrin and α-adaptin; however, other proteins important for synaptic vesicle endocytosis are not required. In a small screen for novel endocytic proteins, we identified the Drosophila homologue of Vps35, a component of the retromer complex, involved in endosome-to-Golgi trafficking. Loss of Vps35 inhibits scavenger receptor ligand endocytosis, and causes mislocalisation of a number of receptors and endocytic proteins. Vps35 has tumour suppressor properties because its loss leads to overproliferation of blood cells in larvae. Its loss also causes signalling defects at the neuromuscular junction, including upregulation of TGFβ/BMP signalling and excessive formation of synaptic terminals. Vps35 negatively regulates actin polymerisation, and genetic interactions suggest that some of the endocytic and signalling defects of vps35 mutants are due to this function.

Background The action of probiotics has been studied in vitro in cells isolated from both mice and humans, particularly enterocytes (IECs), dendritic cells (DCs) and co-cultures of peripheral DCs and IECs. Peripheral DCs and murine DCs differ from human gut DCs, and to date there are no data on the action of any probiotic on co-cultured human IECs and human intestinal DCs. To address this issue, a novel transwell model was used. Human IECs (Caco-2 cells) grown in the upper chamber of transwell filters were co-cultured with intestinal-like human DCs grown in the basolateral compartment of the transwells. The system was apically exposed for 4 h to live probiotic L. paracasei CNCM I-4034 obtained from the faeces of breastfed infants or to its cell-free culture supernatant (CFS) and challenged with Salmonella typhi. The secretion of pro- and anti-inflammatory cytokines in the basolateral compartment was determined by immunoassay, and the DC expression pattern of 20 TLR signaling pathway genes was analysed by PCR array. Results The presence of the live probiotic alone significantly increased IL-1β, IL-6, IL-8, TGF-β2, RANTES and IP-10 levels and decreased IL-12p40, IL-10, TGF- β1 and MIP-1α levels. This release was correlated with a significant increase in the expression of almost all TLR signaling genes. By contrast, incubation of the co-culture with CFS increased IL-1β, IL-6, TGF-β2 and IP-10 production only when Salmonella was present. This induction was correlated with an overall decrease in the expression of all TLR genes except TLR9, which was strongly up-regulated. Conclusions The data presented here clearly indicate that L. paracasei CNCM I-4034 significantly increases the release of pro-inflammatory cytokines, enhances TLR signaling pathway activation and stimulates rather than suppresses the innate immune system. Furthermore, our findings provide evidence that the effects of probiotics in the presence of IECs and DCs differ from the effects of probiotics on cultures of each cell type alone, as reported by us earlier. Thus, co-culture systems such as the one described here are needed to characterise the effects of probiotics in vitro, highlighting the potential utility of such co-cultures as a model system. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0408-6) contains supplementary material, which is available to authorized users.

Characterization of the genes expressed in adipose tissue (AT) is key to understanding the pathogenesis of obesity and to developing treatments for this condition. Our objective was to compare the gene expression in visceral AT (VAT) between obese and normal-weight prepubertal children. A total of fifteen obese and sixteen normal-weight children undergoing abdominal elective surgery were selected. RNA was extracted from VAT biopsies. Microarray experiments were independently performed for each sample (six obese and five normal-weight samples). Validation by quantitative PCR (qPCR) was performed on an additional 10 obese and 10 normal-weight VAT samples. Of 1276 differentially expressed genes (p &lt, 0.05), 245 were more than two-fold higher in obese children than in normal-weight children. As validated by qPCR, expression was upregulated in genes involved in lipid and amino acid metabolism (CES1, NPRR3 and BHMT2), oxidative stress and extracellular matrix regulation (TNMD and NQO1), adipogenesis (CRYAB and AFF1) and inflammation (ANXA1), by contrast, only CALCRL gene expression was confirmed to be downregulated. In conclusion, this study in prepubertal children demonstrates the up- and down-regulation of genes that encode molecules that were previously proposed to influence the pathogenesis of adulthood obesity, as well as previously unreported dysregulated genes that may be candidate genes in the aetiology of obesity.

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., This study utilized fecal samples from the clinical trial NCT01479543 that was supported by Hero Spain S. A. through contract #3582 with the Fundacion General Empresa Universidad de Granada. Carolina Gomez-Llorente is the recipient of a postdoctoral fellowship of the University of Granada.

Quantitative-fluorescent polymerase chain reaction (QF-PCR) is a reliable, rapid, and economic technique for prenatal diagnosis of the most common abnormalities. However, conventional karyotyping is expensive and requires a much longer time to yield results. It is currently under debate whether the replacement or restriction of karyotyping reduces the quality of prenatal test results. This study was undertaken to determine the percentage of clinically significant chromosomal abnormalities that would not be detected if QF-PCR was the main analysis method and karyotyping reserved for cases with increased nuchal translucency (NT) and/or abnormal ultrasound findings and to estimate the difference in cost between QF-PCR and full karyotyping.Nine hundred twenty-eight pregnant women underwent an invasive procedure at our center between May 2009 and December 2012, yielding 580 (62.5%) chorionic villous samples and 348 (37.5%) amniotic fluid samples. Samples were studied by both QF-PCR and full karyotyping. Karyotyping and detailed ultrasound findings were retrospectively analyzed.If QF-PCR was the main analytic method and full karyotyping reserved for cases with elevated NT (≥4.5) and/or abnormal ultrasound findings, 12.7% of the patients would have required full karyotyping, 99% of the clinically significant chromosomal abnormalities would have been detected, and the cost would have been 54% lower than a policy of full karyotyping for all.Detailed prenatal ultrasound scan can reduce the need for conventional karyotyping as a complement to QF-PCR in most prenatal samples, offering rapid results and reducing parental anxiety and healthcare costs.

Exogenous nucleotides (NT) have been reported to exert a reparative role in animal models of intestinal and hepatic damage. Thus, the administration of NT in the diet of rats with thioacetamide-induced liver cirrhosis normalized many of the histological and biochemical alterations produced by this hepatotoxin. We are currently studying the mechanism by which NT exert this effect using cell culture models. The aim of this work was to investigate whether exogenous nucleosides (NS) modulate the proliferation of hepatocytes. We used fetal rat hepatocytes, which, unlike adult hepatocytes, are proliferative cells. Fetal rat primary hepatocytes were incubated with mixtures of NS, and cell proliferation was studied. NS added to the medium of fetal hepatocytes were taken up in a selective fashion by the cells. Cell proliferation was enhanced, as demonstrated by the induction of c-myc and h-ras gene expression as well as by the higher percentage of cells in S phase, and exogenous NS increased the expression of alpha-fetoprotein. These results suggest that exogenous NS may in fact stimulate proliferation of hepatic cells and help preserve the undifferentiated state of fetal rat hepatocytes.

We present the first application of a multiplex multicolour assay for the simultaneous detection of three of the most frequent mutations related to hereditary haemochromatosis (C282Y, H63D and S65C), using fluorescent detection and capillary electrophoresis. We describe two methods: the first is based on a single base extension assay, resulting in a single base difference of the extended products; and the second is a competitive allele-specific polymerase chain reaction (PCR), based on competition between allele-specific primers. Specificity of the latter primers is enhanced with a mismatch at the antepenultimate nucleotide. Primers are designed to amplify products of different sizes and with different fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. An advantage of the present approach is that capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high resolution discrimination between wild-type and mutant alleles, although different mutations may be present in the multiplex analysis. This will facilitate automated genotyping for routine molecular diagnostics and large-scale genetic studies.

The potential for the positive manipulation of the gut microbiome through the introduction of beneficial microbes, as also known as probiotics, is currently an active area of investigation. The FAO/WHO define probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts. However, dead bacteria and bacterial molecular components may also exhibit probiotic properties. The results of clinical studies have demonstrated the clinical potential of probiotics in many pathologies, such as allergic diseases, diarrhea, inflammatory bowel disease and viral infection. Several mechanisms have been proposed to explain the beneficial effects of probiotics, most of which involve gene expression regulation in specific tissues, particularly the intestine and liver. Therefore, the modulation of gene expression mediated by probiotics is an important issue that warrants further investigation. In the present paper, we performed a systematic review of the probiotic-mediated modulation of gene expression that is associated with the immune system and inflammation. Between January 1990 to February 2014, PubMed was searched for articles that were published in English using the MeSH terms “probiotics" and "gene expression" combined with “intestines", "liver", "enterocytes", "antigen-presenting cells", "dendritic cells", "immune system", and "inflammation". Two hundred and five original articles matching these criteria were initially selected, although only those articles that included specific gene expression results (77) were later considered for this review and separated into three major topics: the regulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver. Particular strains of Bifidobacteria , Lactobacilli , Escherichia coli , Propionibacterium , Bacillus and Saccharomyces influence the gene expression of mucins, Toll-like receptors, caspases, nuclear factor-κB, and interleukins and lead mainly to an anti-inflammatory response in cultured enterocytes. In addition, the interaction of commensal bacteria and probiotics with the surface of antigenpresenting cells in vitro results in the downregulation of pro-inflammatory genes that are linked to inflammatory signaling pathways, whereas other anti-inflammatory genes are upregulated. The effects of probiotics have been extensively investigated in animal models ranging from fish to mice, rats and piglets. These bacteria induce a tolerogenic and hyporesponsive immune response in which many genes that are related to the immune system, in particular those genes expressing anti-inflammatory cytokines, are upregulated. By contrast, information related to gene expression in human intestinal cells mediated by the action of probiotics is scarce. There is a need for further clinical studies that evaluate the mechanism of action of probiotics both in healthy humans and in patients with chronic diseases. These types of clinical studies are necessary for addressing the influence of these microorganisms in gene expression for different pathways, particularly those that are associated with the immune response, and to better understand the role that probiotics might have in the prevention and treatment of disease.

The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance to the commensal microbiota and food antigens. Different strains of probiotics possess the ability to finely regulate the activation of dendritic cells (DC), polarising the subsequent activity of T-cells. Nevertheless, information about their underlying mechanisms of action is scarce. In the present study, we investigated the immunomodulatory effects of a potentially probiotic strain,Lactobacillus rhamnosusCNCM I-4036, and its cell-free culture supernatant (CFS) on human DC challenged withEscherichia coli. The results showed that the levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8 and IL-12p70 were higher in the cells treated with liveL. rhamnosusthan in the cells treated with the CFS. In the presence ofE. coli, the supernatant was more effective than the probiotic bacteria in reducing the secretion of pro-inflammatory cytokines. In addition, liveL. rhamnosuspotently induced the production of transforming growth factor (TGF)-β1 and TGF-β2, whereas the CFS increased the secretion of TGF-β1. However, in the presence ofE. coli, both treatments restored the levels of TGF-β. The probiotic strainL. rhamnosusCNCM I-4036 and its CFS were able to activate the Toll-like receptor signalling pathway, enhancing innate immunity. The two treatments induced gene transcription ofTLR-9. LiveL. rhamnosusactivated the expression ofTLR-2andTLR-4genes, whereas the CFS increased the expression ofTLR-1andTLR-5genes. In response to the stimulation with probiotic/CFS andE. coli, the expression of each gene tested was notably increased, with the exception ofTNF-αandNFKBIA. In conclusion, the CFS exhibited an extraordinary ability to suppress the production of pro-inflammatory cytokines by DC, and may be used as an effective and safer alternative to live bacteria.

Background: Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible., Methods: Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives., Results: Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied., Conclusions: MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling., CGLL has a postdoctoral fellowship from the Plan Propio of the University of Granada.

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., Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada.

We carried out a molecular analysis of 350 chromosomes from 55 families originating from the South of Spain (Andalucia) who were diagnosed with cystic fibrosis (CF). We used polymerase chain reaction, followed by an oligonucleotide ligation assay (OLA) and sequence-coded separation using capillary electrophoresis. A frequency of 43.5% for DF508 was found, making it the most common CF mutation in our sample. Seven more mutations (G542X, R334W, R1162X, 2789+5G !A, R117H, DI507 and W1282X) were detected and accounted for 24.7% of the total. The remaining mutations (31.8%) were undetectable with the methodology used in this study. D 2001 Elsevier

Numerousin vitroandin vivostudies conducted using different probiotic micro-organisms have demonstrated their ability to interfere with the growth and virulence of a variety of enteropathogens. The reported beneficial effects of the use of probiotics to complement antibiotic therapy or prevent diarrhoea or gastrointestinal infection in infants have increased in recent years. In the present study, we demonstrated the capacity of supernatants obtained from three novel probiotics (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) isolated from the faeces of breastfed infants to inhibit the growth of enterotoxigenic and enteropathogenic (EPEC) bacteria, such asEscherichia coli,SalmonellaandShigella. To assess their potential antimicrobial activity, the 17 and 24 h cell-free supernatants broth concentrates (10 × ) having 1, 2 or 4 % of the three probiotics were incubated with EPEC bacteria strains. After 17 h of co-culture, the supernatants were able to inhibit the growth ofE. coli,SalmonellaandShigellaup to 40, 55 and 81 %, respectively. However, the inhibitory capacity of some supernatants was maintained or completely lost when the supernatants (pH 3·0) were neutralised (pH 6·5). Overall, these results demonstrated thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 produce compounds that exhibited strain-specific inhibition of enterobacteria and have the potential to be used as probiotics in functional foods.

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., This study was supported by Hero Spain S. A. through a number 3143 contract signed with the Fundación General Universidad de Granada Empresa and co-sponsored by a CDTI project, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government.