Maternal Synbiotic Supplementation with B. breve M-16V and scGOS/lcFOS Shape Offspring Immune Development and Gut Microbiota at the End of Suckling.

Immune system development during gestation and suckling is significantly modulated by maternal environmental and dietary factors. Breastfeeding is widely recognized as the optimal source of nutrition for infant growth and immune maturation, and its composition can be modulated by the maternal diet. In the present work, we investigated whether oral supplementation with Bifidobacterium breve M-16V and short-chain galacto-oligosaccharide (scGOS) and long-chain fructo-oligosaccharide (lcFOS) to rat dams during gestation and lactation has an impact on the immune system and microbiota composition of the offspring at day 21 of life. On that day, blood, adipose tissue, small intestine (SI), mesenteric lymph nodes (MLN), salivary gland (SG), cecum, and spleen were collected. Synbiotic supplementation did not affect the overall body or organ growth of the pups. The gene expression of Tlr9, Muc2, IgA, and Blimp1 were upregulated in the SI, and the increase in IgA gene expression was further confirmed at the protein level in the gut wash. Synbiotic supplementation also positively impacted the microbiota composition in both the small and large intestines, resulting in higher proportions of Bifidobacterium genus, among others. In addition, there was an increase in butanoic, isobutanoic, and acetic acid concentrations in the cecum but a reduction in the small intestine. At the systemic level, synbiotic supplementation resulted in higher levels of immunoglobulin IgG2c in plasma, SG, and MLN, but it did not modify the main lymphocyte subsets in the spleen and MLN. Overall, synbiotic maternal supplementation is able to positively influence the immune system development and microbiota of the suckling offspring, particularly at the gastrointestinal level.

Salivary concentrations of secretory leukocyte protease inhibitor and matrix metallopeptidase-9 following a single bout of exercise are associated with intensity and hydration status.

AIM: To investigate the effects of exercise on salivary concentrations of inflammatory markers by analyzing a panel of 25 inflammatory markers in subjects who had participated in bicycle ergometer tests varying in workload and hydration status. METHODS: Fifteen healthy young men (20-35 years) had performed 4 different exercise protocols of 1 hour duration in a randomly assigned cross-over design, preceded by a rest protocol. Individual workloads depended on participant's pre-assessed individual maximum workload (Wmax): rest (protocol 1), 70% Wmax in hydrated (protocol 2) and dehydrated (protocol 3) state, 50% Wmax (protocol 4) and intermittent 85%/55% Wmax in 2 min blocks (protocol 5). Saliva samples were collected before (T0) and immediately after exercise (T1), and at several time points after exercise (2 hours (T3), 3 hours (T4), 6 hours (T5) and 24 hours (T6)). Secretory Leukocyte Protease Inhibitor (SLPI), Matrix Metallopeptidase-9 (MMP-9) and lactoferrin was analyzed using a commercial ELISA kit, a panel of 22 cytokines and chemokines were analyzed using a commercial multiplex immunoassay. Data was analyzed using a multilevel mixed linear model, with multiple test correction. RESULTS: Among a panel of 25 inflammatory markers, SLPI concentrations were significantly elevated immediately after exercise in all protocols compared to rest and higher concentrations reflected the intensity of exercise and hydration status. MMP-9 showed a significant increase in the 70% Wmax dehydrated, 50% Wmax and intermittent protocols. CONCLUSIONS: Salivary concentrations of SLPI and MMP-9 seem associated with exercise intensity and hydration status and may offer non-invasive biomarkers to study (local) inflammatory responses to different exercise intensities in human studies.

An evening of alcohol consumption negatively impacts next-day immune fitness in both hangover-sensitive drinkers and hangover-resistant drinkers.

BACKGROUND: Survey research found poorer baseline immune fitness for self-reported hangover-sensitive drinkers compared to hangover-resistant drinkers. However, up to now a limited number of clinical studies revealed mixed results regarding the relationship between the concentrations of biomarkers of systemic inflammation in blood or saliva with hangover severity, and could not differentiate between hangover-sensitive drinkers and hangover-resistant drinkers. The aim of this study was to assess immune fitness and saliva biomarkers of systemic inflammation at multiple timepoints following an alcohol day and alcohol-free control day. METHODS: The study had a semi-naturalistic design. In the evening before the test days, participants were not supervised. They could drink ad libitum drinking on the alcohol test day and refrained from drinking alcohol on the control day. Activities and behaviors on the alcohol and control day were reported the follow morning. On both test days, from 09:30 to 15:30, hourly assessments of immune fitness (single-item scale) and overall hangover severity (single-item scale) were made and saliva samples were collected for biomarker assessments. RESULTS: N = 14 hangover-resistant drinkers and n = 15 hangover-sensitive drinkers participated in the study. The amount of alcohol consumed on the alcohol day did not significantly differ between the hangover-resistant group (mean (SD) of 13.5 (7.9) alcoholic drinks) and the hangover-sensitive group (mean (SD) of 12.4 (4.4) alcoholic drinks). All hangover-sensitive drinkers reported having a hangover following the alcohol day (overall hangover severity score 6.1 (on a 0-10 scale) at 09:30, gradually decreasing to 3.3 at 15:30), whereas the hangover-resistant drinkers reported no hangover. On the control day, immune fitness of the hangover-sensitive group was significantly poorer than the hangover-resistant group. On the alcohol day, both groups showed a significant reduction in immune fitness. The effect was evident throughout the day, but significantly more pronounced in the hangover-sensitive group than the hangover-resistant group. No significant differences between the groups were found at any time point on the two test days for saliva concentrations of Interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α. CONCLUSIONS: Whereas hangover-sensitive drinkers reported a hangover following an alcohol day and hangover-resistant drinkers did not, both groups reported significantly reduced immune fitness throughout the day. However, the reduction in immune fitness among hangover-sensitive drinkers was significantly more pronounced in comparison to the hangover-resistant group.

Effect of Rotavirus Infection and 2'-Fucosyllactose Administration on Rat Intestinal Gene Expression.

Viral infections are described as modifying host gene expression; however, there is limited insight regarding rotavirus (RV) infections. This study aimed to assess the changes in intestinal gene expression after RV infection in a preclinical model, and the effect of 2-fucosyllactose (2'-FL) on this process. From days 2 to 8 of life, rats were supplemented with the dietary oligosaccharide 2'-FL or vehicle. In addition, an RV was inoculated on day 5 to nonsupplemented animals (RV group) and to 2'-FL-fed animals (RV+2'-FL group). Incidence and severity of diarrhea were established. A portion from the middle part of the small intestine was excised for gene expression analysis by microarray kit and qPCR. In nonsupplemented animals, RV-induced diarrhea upregulated host antiviral genes (e.g., Oas1a, Irf7, Ifi44, Isg15) and downregulated several genes involved in absorptive processes and intestinal maturation (e.g., Onecut2, and Ccl19). The 2'-FL-supplemented and infected animals had less diarrhea; however, their gene expression was affected in a similar way as the control-infected animals, with the exception of some immunity/maturation markers that were differentially expressed (e.g., Ccl12 and Afp). Overall, assessing the expression of these key genes may be useful in the evaluation of the efficacy of nutritional interventions or treatments for RV infection.

Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

An In Vitro and In Vivo Translational Research Approach for the Assessment of Sensitization Capacity and Residual Allergenicity of an Extensive Whey Hydrolysate for Cow's Milk-Allergic Infants.

INTRODUCTION: Hypoallergenic formulas prepared from hydrolyzed cow's milk proteins are often used for the management of cow's milk allergy (CMA) in infants. In this study, both in vitro assays and an in vivo mouse model for CMA were used to assess the sensitizing and allergenic potential of a newly developed, extensive whey hydrolysate (eWH). METHODS: Gel permeation chromatography was used to characterize the molecular weight distribution of the peptides. Residual antigenicity was measured using a beta-lactoglobulin ELISA as well as with immunoblotting using anti-beta-lactoglobulin (BLG) and anti-alpha-lactalbumin antibodies. In vitro residual allergenicity was assessed using huFcεRIα-RBL-2H3 cells sensitized with anti-bovine BLG human IgE. In vivo sensitizing and allergenic potential was assessed in a CMA mouse model by measuring the acute allergic skin response, anaphylactic shock score, body temperature, serum mMCP-1, whey-specific IgE, and cytokines. RESULTS: There was no in vitro residual antigenicity and allergenicity observed of the eWH. Mice sensitized with eWH showed no acute allergic skin reaction after challenge with whey, confirmed by an absence of whey-specific IgE and anaphylactic symptoms and decrease in body temperature and mMCP-1 levels. CONCLUSIONS: Results from our in vitro and in vivo translational approach to assess sensitization capacity and residual allergenicity indicate that the newly developed eWH is safe for use in CMA infants. This was subsequently confirmed in a clinical study in which this eWH was tolerated by more than 90% (with 95% confidence) of infants or children with confirmed CMA.

Hypoallergenicity assessment of an extensively hydrolyzed whey-protein formula in cow's milk allergic infants.

BACKGROUND: Extensively hydrolyzed formulas are recommended for the dietary management of infants with cow's milk allergy (CMA). OBJECTIVES: Hypoallergenicity, growth, and gastrointestinal (GI) tolerability of a new extensively hydrolyzed whey-protein formula (eHWF) in CMA children were assessed. METHODS: In this prospective, randomized, international, multi-center study (Trial NL3889), 34 children with confirmed CMA (74% IgE-mediated) underwent a double-blind, placebo-controlled food challenge (DBPCFC) with an eHWF developed with non-porcine enzymes, supplemented with prebiotic short-chain galacto- and long-chain fructo-oligosaccharides (0.8 g/L, ratio 9:1), arachidonic acid (0.35/100 g), and docosahexaenoic acid (0.35/100 g). If tolerant to the eHWF, children participated in a 7-day open food challenge with this eHWF. Anthropometrics and GI tolerability were assessed in an optional 16-weeks follow-up. RESULTS: Of the 34 children who started the DBPCFC with the eHWF, 25 subjects (19 boys, mean age: 61 weeks, 18 with IgE-mediated CMA) completed the DBPCFC and 7-day open challenge without major protocol deviations and tested negative at both challenges. One child experienced a late moderate eczematous allergic reaction in the optional follow-up period, indicating the need for close monitoring of subjects starting new formula. Weight and length gain followed the World Health Organization growth curves. Changes in frequency and consistency of stools upon test formula intake were transient. CONCLUSIONS: The newly developed eHWF is a suitable option in CMA treatment as all subjects tolerated the product. This result is in line with the international criteria for hypoallergenicity (American Academy of Pediatrics) that state that more than 90% of CMA children must tolerate the formula. Use of the formula is also associated with normal growth curves and GI tolerability. TRIAL REGISTRATION: Trial NL3889, https://www.trialregister.nl/trial/3889.

Preventive Effect of a Postbiotic and Prebiotic Mixture in a Rat Model of Early Life Rotavirus Induced-Diarrhea.

Rotavirus (RV) is the main cause of gastroenteritis in children. Prebiotics and, more recently, postbiotics are used for preventing and treating gastrointestinal infections. The aim of this study was to analyze the effects of a LactofidusTM, short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) mixture, and their combination on RV infection, in a rat model, for early life diarrhea. Fifteen litters of suckling rats were intragastrically administered daily with the vehicle, the prebiotic mixture, the postbiotic or the combination. The RV was inoculated on day 5 and then fecal samples were clinically evaluated daily. Viral shedding, intestinal permeability assay, in vitro blocking assay, immunoglobulin profiles, and anti-RV response were assessed at day 8 and 16 of life. Cecal microbiota composition, intestinal gene expression, and short chain fatty acids (SCFAs) were analyzed at day 16. The incidence and severity of diarrhea were significantly reduced by all the supplementations. Moreover, they showed blocking activity, changes in the immunoglobulin profiles, in gut microbiota, and in the intestinal gene expression. The prebiotic mixture reduced gut permeability and changed the SCFA profile, whereas the postbiotic enhanced the expression of Toll-like receptors (TLRs). The combination preserved most of the individual observed effects, and furthermore, complementary effects, such as an increase in white blood cells and lymphocytes recruitment, as well as upregulation of TLR7 and TLR9 gene expression.

Tolerance development in cow's milk-allergic infants receiving amino acid-based formula: A randomized controlled trial.

BACKGROUND: Tolerance development is an important clinical outcome for infants with cow's milk allergy. OBJECTIVE: This multicenter, prospective, randomized, double-blind, controlled clinical study (NTR3725) evaluated tolerance development to cow's milk (CM) and safety of an amino acid-based formula (AAF) including synbiotics (AAF-S) comprising prebiotic oligosaccharides (oligofructose, inulin) and probiotic Bifidobacterium breve M-16V in infants with confirmed IgE-mediated CM allergy. METHODS: Subjects aged ≤13 months with IgE-mediated CM allergy were randomized to receive AAF-S (n = 80) or AAF (n = 89) for 12 months. Stratification was based on CM skin prick test wheal size and study site. After 12 and 24 months, CM tolerance was evaluated by double-blind, placebo-controlled food challenge. A logistic regression model used the all-subjects randomized data set. RESULTS: At baseline, mean ± SD age was 9.36 ± 2.53 months. At 12 and 24 months, respectively, 49% and 62% of subjects were CM tolerant (AAF-S 45% and 64%; AAF 52% and 59%), and not differ significantly between groups. During the 12-month intervention, the number of subjects reporting at least 1 adverse event did not significantly differ between groups; however, fewer subjects required hospitalization due to serious adverse events categorized as infections in the AAF-S versus AAF group (9% vs 20%; P = .036). CONCLUSIONS: After 12 and 24 months, CM tolerance was not different between groups and was in line with natural outgrowth. Results suggest that during the intervention, fewer subjects receiving AAF-S required hospitalization due to infections.

Effects of a Postbiotic and Prebiotic Mixture on Suckling Rats' Microbiota and Immunity.

Human milk serves as a model for infant formula providing nutritional solutions for infants not able to receive enough mother's milk. Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as prebiotics, probiotics and postbiotics. The aim of this study was to examine the effects of the supplementation with a postbiotic (LactofidusTM) and its combination with the prebiotics short-chain galactooligosaccharides (scGOS) and long-chain fructooligosaccharides (lcFOS) in a preclinical model of healthy suckling rats. Pups were supplemented daily with LactofidusTM (POST group) and/or scGOS/lcFOS (P+P and PRE groups, respectively). Body weight and fecal consistency were analyzed. At the end of the study, immunoglobulin (Ig) profile, intestinal gene expression, microbiota composition and short chain fatty acid (SCFA) proportion were quantified. The supplementation with all nutritional interventions modulated the Ig profile, but the prebiotic mixture and the postbiotic induced differential effects: whereas scGOS/lcFOS induced softer feces and modulated microbiota composition and SCFA profile, Lactofidus™ upregulated Toll-like receptors gene expression. The use of the combination of scGOS/lcFOS and Lactofidus™ showed the effects observed for the oligosaccharides separately, as well as showing a synergistic impact on animal growth. Thus, the combined use of both products seems to be a good strategy to modulate immune and microbial features in early life.

Limited Lactosylation of Beta-Lactoglobulin from Cow's Milk Exerts Strong Influence on Antigenicity and Degranulation of Mast Cells.

BACKGROUND: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity. PRIMARY OBJECTIVE: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS). SECONDARY OBJECTIVE: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells. METHODS: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used. RESULTS: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding (p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells (p < 0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.

Optimization of a human milk-directed quantitative sIgA ELISA method substantiated by mass spectrometry.

Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk-tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)-based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk-specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk-derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk-specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits.

Immune Responses after Heavy Alcohol Consumption: Cytokine Concentrations in Hangover-Sensitive and Hangover-Resistant Drinkers.

This study investigated immunological changes during an alcohol hangover, and the possible difference between hangover-resistant and hangover-sensitive drinkers in terms of immune reactivity. Using a semi-naturalistic design, N = 36 healthy social drinkers (18 to 30 years old) provided saliva samples on a control day (after drinking no alcohol) and on a post-alcohol day. Hangover severity was rated directly after saliva collection. Cytokine concentrations, interleukin (IL)-1ß, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α, and hangover severity were compared between both test days and between hangover-sensitive and -resistant drinkers. Data from N = 35 drinkers (17 hangover-sensitive and 18 hangover-resistant) were included in the statistical analyses. Relative to the control day, there were significant increases in saliva IL-6 and IL-10 concentrations on the post-alcohol day. No significant differences in cytokine concentrations were found between hangover-sensitive and hangover-resistant drinkers, nor did any change in cytokine concentration correlate significantly with hangover severity. In line with previous controlled studies assessing cytokines in blood, the current naturalistic study using saliva samples also demonstrated that the immune system responds to high-level alcohol intake. However, further research is warranted, as, in contrast to previous findings in blood samples, changes in saliva cytokine concentrations did not differ significantly between hangover-sensitive and hangover-resistant drinkers, nor did they correlate significantly with hangover severity.

Immunological Outcomes of Allergen-Specific Immunotherapy in Food Allergy.

IgE-mediated food allergies are caused by adverse immunologic responses to food proteins. Allergic reactions may present locally in different tissues such as skin, gastrointestinal and respiratory tract and may result is systemic life-threatening reactions. During the last decades, the prevalence of food allergies has significantly increased throughout the world, and considerable efforts have been made to develop curative therapies. Food allergen immunotherapy is a promising therapeutic approach for food allergies that is based on the administration of increasing doses of culprit food extracts, or purified, and sometime modified food allergens. Different routes of administration for food allergen immunotherapy including oral, sublingual, epicutaneous and subcutaneous regimens are being evaluated. Although a wealth of data from clinical food allergen immunotherapy trials has been obtained, a lack of consistency in assessed clinical and immunological outcome measures presents a major hurdle for evaluating these new treatments. Coordinated efforts are needed to establish standardized outcome measures to be applied in food allergy immunotherapy studies, allowing for better harmonization of data and setting the standards for the future research. Several immunological parameters have been measured in food allergen immunotherapy, including allergen-specific immunoglobulin levels, basophil activation, cytokines, and other soluble biomarkers, T cell and B cell responses and skin prick tests. In this review we discuss different immunological parameters and assess their applicability as potential outcome measures for food allergen immunotherapy that may be included in such a standardized set of outcome measures.

eNose breath prints as a surrogate biomarker for classifying patients with asthma by atopy.

BACKGROUND: Electronic noses (eNoses) are emerging point-of-care tools that may help in the subphenotyping of chronic respiratory diseases such as asthma. OBJECTIVE: We aimed to investigate whether eNoses can classify atopy in pediatric and adult patients with asthma. METHODS: Participants with asthma and/or wheezing from 4 independent cohorts were included; BreathCloud participants (n = 429), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes adults (n = 96), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes pediatric participants (n = 100), and Pharmacogenetics of Asthma Medication in Children: Medication with Anti-Inflammatory Effects 2 participants (n = 30). Atopy was defined as a positive skin prick test result (≥3 mm) and/or a positive specific IgE level (≥0.35 kU/L) for common allergens. Exhaled breath profiles were measured by using either an integrated eNose platform or the SpiroNose. Data were divided into 2 training and 2 validation sets according to the technology used. Supervised data analysis involved the use of 3 different machine learning algorithms to classify patients with atopic versus nonatopic asthma with reporting of areas under the receiver operating characteristic curves as a measure of model performance. In addition, an unsupervised approach was performed by using a bayesian network to reveal data-driven relationships between eNose volatile organic compound profiles and asthma characteristics. RESULTS: Breath profiles of 655 participants (n = 601 adults and school-aged children with asthma and 54 preschool children with wheezing [68.2% of whom were atopic]) were included in this study. Machine learning models utilizing volatile organic compound profiles discriminated between atopic and nonatopic participants with areas under the receiver operating characteristic curves of at least 0.84 and 0.72 in the training and validation sets, respectively. The unsupervised approach revealed that breath profiles classifying atopy are not confounded by other patient characteristics. CONCLUSION: eNoses accurately detect atopy in individuals with asthma and wheezing in cohorts with different age groups and could be used in asthma phenotyping.

Seeking Windows of Opportunity to Shape Lifelong Immune Health: A Network-Based Strategy to Predict and Prioritize Markers of Early Life Immune Modulation.

A healthy immune status is strongly conditioned during early life stages. Insights into the molecular drivers of early life immune development and function are prerequisite to identify strategies to enhance immune health. Even though several starting points for targeted immune modulation have been identified and are being developed into prophylactic or therapeutic approaches, there is no regulatory guidance on how to assess the risk and benefit balance of such interventions. Six early life immune causal networks, each compromising a different time period in early life (the 1st, 2nd, 3rd trimester of gestations, birth, newborn, and infant period), were generated. Thereto information was extracted and structured from early life literature using the automated text mining and machine learning tool: Integrated Network and Dynamical Reasoning Assembler (INDRA). The tool identified relevant entities (e.g., genes/proteins/metabolites/processes/diseases), extracted causal relationships among these entities, and assembled them into early life-immune causal networks. These causal early life immune networks were denoised using GeneMania, enriched with data from the gene-disease association database DisGeNET and Gene Ontology resource tools (GO/GO-SLIM), inferred missing relationships and added expert knowledge to generate information-dense early life immune networks. Analysis of the six early life immune networks by PageRank, not only confirmed the central role of the "commonly used immune markers" (e.g., chemokines, interleukins, IFN, TNF, TGFB, and other immune activation regulators (e.g., CD55, FOXP3, GATA3, CD79A, C4BPA), but also identified less obvious candidates (e.g., CYP1A2, FOXK2, NELFCD, RENBP). Comparison of the different early life periods resulted in the prediction of 11 key early life genes overlapping all early life periods (TNF, IL6, IL10, CD4, FOXP3, IL4, NELFCD, CD79A, IL5, RENBP, and IFNG), and also genes that were only described in certain early life period(s). Concluding, here we describe a network-based approach that provides a science-based and systematical method to explore the functional development of the early life immune system through time. This systems approach aids the generation of a testing strategy for the safety and efficacy of early life immune modulation by predicting the key candidate markers during different phases of early life immune development.

Strain-Specific Probiotic Properties of Bifidobacteria and Lactobacilli for the Prevention of Diarrhea Caused by Rotavirus in a Preclinical Model.

Probiotic supplementation with different lactobacilli and bifidobacterial strains has demonstrated beneficial effects in infectious diarrhea caused by rotavirus (RV) in young children. Preclinical models of RV infection might be a good strategy to screen for the efficacy of new probiotic strains or to test their comparative efficacy. Neonatal Lewis rats were supplemented with Bifidobacterium breve M-16V, Lactobacillus acidophilus NCFM, Lactobacillus helveticus R0052, or Lactobacillus salivarius PS2 from days 2-14 of life. On day five, animals received RV SA-11 orally. Fecal samples were collected daily, weighed, and scored for the calculation of severity and incidence of diarrhea. In addition, fecal pH and fecal viral shedding were measured. Animals were sacrificed at the end of the study and their blood was obtained for the quantification of RV-specific immunoglobulins. RV infection was induced in ~90% of the animals. All probiotics caused a reduction of several clinical variables of severity and incidence of diarrhea, except L. salivarius PS2. L. acidophilus NCFM, B. breve M-16V, and L. helveticus R0052 seemed to be very effective probiotic strains. In addition, all Lactobacillus strains reduced the viral elimination one day post-inoculation. No differences were detected in the specific anti-RV humoral response. The present study highlights the strain-specific effects of probiotics and identifies promising probiotics for use in ameliorating and preventing RV-induced diarrhea in children, for example by including them in infant formulas.

Network-Based Selection of Candidate Markers and Assays to Assess the Impact of Oral Immune Interventions on Gut Functions.

To assess the safety and efficacy of oral immune interventions, it is important and required by regulation to assess the impact of those interventions not only on the immune system, but also on other organs such as the gut as the porte d'entrée. Despite clear indications that the immune system interacts with several physiological functions of the gut, it is still unknown which pathways and molecules are crucial to assessing the impact of nutritional immune interventions on gut functioning. Here we used a network-based systems biology approach to clarify the molecular relationships between immune system and gut functioning and to identify crucial biomarkers to assess effects on gut functions upon nutritional immune interventions. First, the different gut functionalities were categorized based on literature and EFSA guidance documents. Moreover, an overview of the current assays and methods to measure gut function was generated. Secondly, gut-function related biological processes and adverse events were selected and subsequently linked to the physiological functions of the GI tract. Thirdly, database terms and annotations from the Gene ontology database and the Comparative Toxicogenomics Database (CTD) related to the previously selected gut-function related processes were selected. Next, database terms and annotations were used to identify the pathways and genes involved in those gut functionalities. In parallel, information from CTD was used to identify immune disease related genes. The resulting lists of both gut and immune function genes showed an overlap of 753 genes out of 1,296 gut-function related genes indicating the close gut-immune relationship. Using bioinformatics enrichment tools DAVID and Panther, the identified gut-immune markers were predicted to be involved in motility, barrier function, the digestion and absorption of vitamins and fat, regulation of the digestive system and gastric acid, and protection from injurious or allergenic material. Concluding, here we provide a promising systems biology approach to identify genes that help to clarify the relationships between immune system and gut functioning, with the aim to identify candidate biomarkers to monitor nutritional immune intervention assays for safety and efficacy in the general population. This knowledge helps to optimize future study designs to predict effects of nutritional immune intervention on gut functionalities.

Oligosaccharides Modulate Rotavirus-Associated Dysbiosis and TLR Gene Expression in Neonatal Rats.

Colonization of the gut in early life can be altered through multiple environmental factors. The present study aimed to investigate the effects of 2'-fucosyllactose (2'-FL), a mixture of short-chain galactooligosaccharides/long-chain fructooligosaccharides (scGOS/lcFOS) 9:1 and their combination (scGOS/lcFOS/2'-FL) on dysbiosis induced during rotavirus (RV) diarrhea in neonatal rats, elucidating crosstalk between bacteria and the immune system. The dietary interventions were administered daily by oral gavage at days 2-8 of life in neonatal Lewis rats. On day 5, RV SA11 was intragastrically delivered to induce infection and diarrhea assessment, microbiota composition, and gene expression of Toll-like receptors (TLRs) in the small intestine were studied. All dietary interventions showed reduction in clinical variables of RV-induced diarrhea. RV infection increased TLR2 expression, whereas 2'-FL boosted TLR5 and TLR7 expressions and scGOS/lcFOS increased that of TLR9. RV-infected rats displayed an intestinal dysbiosis that was effectively prevented by the dietary interventions, and consequently, their microbiota was more similar to microbiota of the noninfected groups. The preventive effect of 2'-FL, scGOS/lcFOS, and their combination on dysbiosis associated to RV diarrhea in rats could be due to changes in the crosstalk between gut microbiota and the innate immune system.

Immunomodulatory and Prebiotic Effects of 2'-Fucosyllactose in Suckling Rats.

Human milk oligosaccharides are unconjugated complex glycans present in high concentration in human milk that serve as pre-biotics and immunomodulators. They are not primarily absorbed or metabolized by the infant and reach the lower part of the intestinal tract unaltered. One of the main oligosaccharides found in human milk is 2'-fucosyllactose (2'-FL). This study aimed to investigate the effects of daily oral administration of 2'-FL in healthy suckling rats. From days 2 to 16 of life, rats were daily given the oligosaccharide (2'-FL) or vehicle (REF), weighed and their stool characteristics were assessed. On days 8 and 16 of life the morphometry, intestinal architecture, and cytokine release, mesenteric lymph nodes cell composition, plasma immunoglobulin concentrations, fecal microbiota composition, cecal short-chain fatty acids content, and the urinary metabolic profile were assessed. Animals given 2'-FL showed higher plasma IgG and IgA and more T cell subsets in the mesenteric lymph nodes on day 16. Moreover, at intestinal level, villus heights, and areas were increased on day 8. Cecal samples displayed a higher Lactobacillus proportion and a different urinary metabolic profile was observed on day 8, and a higher proportion of butyrate on day 16. In conclusion, supplementation of 2'-FL in early life has a pre-biotic and intestinal trophic effect and promotes maturation of the immune system.