South East Asian Nutrition Surveys (SEANUTS) II - a multi-country evaluation of nutrition and lifestyle indicators in children aged 12 years and below: Rationale and Design.

OBJECTIVE: To assess the nutritional status, growth parameters and lifestyle behaviours of children between 0.5-12 years in nationally representative samples in Malaysia, Indonesia, Thailand, and Vietnam. DESIGN: A cross-sectional study was conducted in the four countries, between May 2019 and April 2021. Data collected can be categorized into four categories: (1) Growth - anthropometry, body composition, development disorder, (2) Nutrient intake and dietary habits - 24-hour dietary recall, child food habits, breast feeding and complementary feeding, (3) Socio-economic status - food insecurity and child health status/environmental, and (4) Lifestyle behaviours - physical activity patterns, fitness, sunlight exposure, sleep patterns, body image and behavioural problems. Blood samples were also collected for biochemical and metabolomic analyses. With the pandemic emerging during the study, a COVID-19 questionnaire was developed and implemented. SETTING: Both rural and urban areas in Malaysia, Indonesia, Thailand, and Vietnam. PARTICIPANTS: Children who were well, with no physical disability or serious infections/injuries and between the age of 0.5-12 years old were recruited. RESULTS: The South East Asian Nutrition Surveys II recruited 13,933 children. Depending on the country, data collection from children were conducted in schools and commune health centres, or temples, or sub-district administrative organizations. CONCLUSIONS: The results will provide up-to-date insights into nutritional status and lifestyle behaviours of children in the four countries. Subsequently, these data will facilitate exploration of potential gaps in dietary intake among Southeast Asian children and enable local authorities to plan future nutrition and lifestyle intervention strategies.

Babies, Bugs, and Barriers: Dietary Modulation of Intestinal Barrier Function in Early Life.

The intestinal barrier is essential in early life to prevent infection, inflammation, and food allergies. It consists of microbiota, a mucus layer, an epithelial layer, and the immune system. Microbial metabolites, the mucus, antimicrobial peptides, and secretory immunoglobulin A (sIgA) protect the intestinal mucosa against infection. The complex interplay between these functionalities of the intestinal barrier is crucial in early life by supporting homeostasis, development of the intestinal immune system, and long-term gut health. Exclusive breastfeeding is highly recommended during the first 6 months. When breastfeeding is not possible, milk-based infant formulas are a safe alternative. Breast milk contains many bioactive components that help to establish the intestinal microbiota and influence the development of the intestinal epithelium and the immune system. Importantly, breastfeeding lowers the risk for intestinal and respiratory tract infections. Here we review all aspects of intestinal barrier function and the nutritional components that impact its functionality in early life, such asmicronutrients, bioactive milk proteins, milk lipids, and human milk oligosaccharides. These components are present in breast milk and can be added to milk-based infant formulas to support gut health and immunity.

Effect of milk fat-based infant formulae on stool fatty acid soaps and calcium excretion in healthy term infants: two double-blind randomised cross-over trials.

BACKGROUND: Palmitic acid (PA) is predominantly esterified at the SN-2 position of triacylglycerols in human milk. PA at the SN-2 position is more efficiently absorbed and results in reduced formation of PA soaps, as well as reduced fatty acid (FA) and calcium malabsorption. Bovine milk fat (MF), a natural source of SN-2-palmitate, was used in the fat blend of infant formulae (IF) in the current study to investigate its effect on stool fatty acid soaps, calcium excretion and stool characteristics. METHODS: Two double-blind, randomised cross-over trials (CS1, CS2) were conducted in parallel with healthy term, formula-fed infants aged 9-14 weeks. After a two-week run-in period, infants in CS1 (n = 17) were randomly allocated to receive either a 50% MF-based formula (50MF) or a 100% vegetable fat (VF) formula; in CS2 (n = 18), infants received either a 20% MF-based formula (20MF) or the VF formula, in a 2 × 2-week cross-over design. At the end of each two-week intervention period, stool samples were collected for FA, FA soaps and calcium excretion analysis and stool consistency was assessed according to the Amsterdam Infant Stool Scale (AISS). RESULTS: MF-based groups showed no significant difference in PA in stools compared to VF group, although reduced stool PA soaps (CS1: 111.28 ± 18.33 vs. 220.25 ± 29.35 mg/g dry weight, p < 0.0001; CS2: 216.24 ± 25.16 vs. 233.94 ± 35.12 mg/g dry weight, p = 0.0023), total FA soaps and calcium excretion (CS1: 46.40 ± 5.27 vs. 49.88 ± 4.77 mg/g dry weight, p = 0.0041; CS2: 46.20 ± 4.26 vs. 50.47 ± 6.71 mg/g dry weight, p = 0.0067) were observed. Furthermore, the 50MF group showed a favourable lower mean stool consistency score compared to the VF group (1.64 ± 0.49 vs. 2.03 ± 0.19, p = 0.0008). CONCLUSIONS: While the use of bovine MF in IF did not affect PA concentrations in stool, lower excretion of palmitate soaps, total FA soaps and calcium was seen in healthy term infants. 50MF formula also showed improved stool consistency. The use of MF in IF could be an interesting approach to improve gut comfort and stool characteristics in infants, warranting further research. TRIAL REGISTRATION: Netherlands Trial Registry Identifier: NTR6702. Date registered: December 01, 2017.

A network-based approach for identifying suitable biomarkers for oral immunotherapy of food allergy.

BACKGROUND: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, concerns with regards to safety and long-term efficacy of OIT remain. There is a need to identify biomarkers that predict, monitor and/or evaluate the effects of OIT. Here we present a method to select candidate biomarkers for efficacy and safety assessment of OIT using the computational approaches Bayesian networks (BN) and Topological Data Analysis (TDA). RESULTS: Data were used from fructo-oligosaccharide diet-supported OIT experiments performed in 3 independent cow's milk allergy (CMA) and 2 independent peanut allergy (PNA) experiments in mice. Bioinformatical approaches were used to understand the data structure. The BN predicted the efficacy of OIT in the CMA with 86% and indicated a clear effect of scFOS/lcFOS on allergy parameters. For the PNA model, this BN (trained on CMA data) predicted an efficacy of OIT with 76% accuracy and shows similar effects of the allergen, treatment and diet as compared to the CMA model. The TDA identified clusters of biomarkers closely linked to biologically relevant clinical symptoms and also unrelated and redundant parameters within the network. CONCLUSIONS: Here we provide a promising application of computational approaches to a) compare mechanistic features of two different food allergies during OIT b) determine the biological relevance of candidate biomarkers c) generate new hypotheses to explain why CMA has a different disease pattern than PNA and d) select relevant biomarkers for future studies.

Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.

BACKGROUND: Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.

Butyrate Enhances Desensitization Induced by Oral Immunotherapy in Cow's Milk Allergic Mice.

BACKGROUND: In previous studies, we showed that a fructo-oligosaccharide- (FOS-) supplemented diet enhanced oral immunotherapy (OIT) efficacy in a mouse model for cow's milk allergy. Fermentation of FOS by intestinal bacteria leads to production of short-chain fatty acids (SCFA) including butyrate. AIM: To investigate the contribution of butyrate in the enhanced efficacy of OIT + FOS. METHODS: C3H/HeOuJ mice were sensitized and received OIT with or without FOS or butyrate supplementation. After treatment, whole blood was collected to conduct a basophil activation test (BAT) and allergen challenges were performed to measure acute allergic symptoms. CD4 + CD25 + regulatory T cells (Tregs) were isolated from treated mice or differentiated in vitro and used in a bone marrow-derived mast cell (BMMC) suppression assay. Cecum content was collected to analyze SCFA concentrations. RESULTS: Allergen-induced basophil activation was reduced in OIT + butyrate samples compared to OIT. Accordingly, the acute allergic skin response and mast cell degranulation upon challenge were reduced in OIT + butyrate and OIT + FOS mice compared to sensitized controls. Butyrate was increased in the cecum content of OIT + FOS mice compared to OIT mice and sensitized controls. Treg-mediated BMMC suppression was enhanced after in vivo butyrate and FOS exposure in combination with OIT but with a more pronounced effect for butyrate. CONCLUSION: Butyrate supplementation enhanced OIT-induced desensitization of basophils and mast cells and Treg functionality. Only OIT + FOS treatment induced potential microbial alterations, shown by increased butyrate levels in cecum content. Both butyrate and FOS are promising candidates to improve OIT efficacy in human studies to treat food allergies.

Dietary Supplementation with Nondigestible Oligosaccharides Reduces Allergic Symptoms and Supports Low Dose Oral Immunotherapy in a Peanut Allergy Mouse Model.

SCOPE: A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side effects. Non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) reduce allergy development in murine models. Therefore, it is hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model. METHODS AND RESULTS: After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice are fed a 1% scFOS/lcFOS or control diet and receive OIT (1.5 or 15 mg PE). Hereafter, mice are exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE-specific antibody production, and numbers of various immune cells. scFOS/lcFOS increases short-chain fatty acid levels in the caecum and reduce the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induce protection against anaphylaxis, whereas 1.5 mg OIT alone does not. OIT, with or without scFOS/lcFOS, induces PE-specific immunoglobulin (Ig) IgG and IgA levels and increases CD103+ dendritic cells in the mesenteric lymph nodes. CONCLUSIONS: scFOS/lcFOS and scFOS/lcFOS combined with low dose OIT are able to protect against a peanut-allergic anaphylactic response.

Improved Efficacy of Oral Immunotherapy Using Non-Digestible Oligosaccharides in a Murine Cow's Milk Allergy Model: A Potential Role for Foxp3+ Regulatory T Cells.

BACKGROUND: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, there are some concerns regarding its safety and long-term efficacy. The use of non-digestible oligosaccharides might improve OIT efficacy since they are known to directly modulate intestinal epithelial and immune cells in addition to acting as prebiotics. AIM: To investigate whether a diet supplemented with plant-derived fructo-oligosaccharides (FOS) supports the efficacy of OIT in a murine cow's milk allergy model and to elucidate the potential mechanisms involved. METHODS: After oral sensitization to the cow's milk protein whey, female C3H/HeOuJ mice were fed either a control diet or a diet supplemented with FOS (1% w/w) and received OIT (10 mg whey) 5 days a week for 3 weeks by gavage. Intradermal (i.d.) and intragastric (i.g.) challenges were performed to measure acute allergic symptoms and mast cell degranulation. Blood and organs were collected to measure antibody levels and T cell and dendritic cell populations. Spleen-derived T cell fractions (whole spleen- and CD25-depleted) were transferred to naïve recipient mice to confirm the involvement of regulatory T cells (Tregs) in allergy protection induced by OIT + FOS. RESULTS: OIT + FOS decreased acute allergic symptoms and mast cell degranulation upon challenge and prevented the challenge-induced increase in whey-specific IgE as observed in sensitized mice. Early induction of Tregs in the mesenteric lymph nodes (MLN) of OIT + FOS mice coincided with reduced T cell responsiveness in splenocyte cultures. CD25 depletion in OIT + FOS-derived splenocyte suspensions prior to transfer abolished protection against signs of anaphylaxis in recipients. OIT + FOS increased serum galectin-9 levels. No differences in short-chain fatty acid (SCFA) levels in the cecum were observed between the treatment groups. Concisely, FOS supplementation significantly improved OIT in the acute allergic skin response, %Foxp3+ Tregs and %LAP+ Th3 cells in MLN, and serum galectin-9 levels. CONCLUSION: FOS supplementation improved the efficacy of OIT in cow's milk allergic mice. Increased levels of Tregs in the MLN and abolished protection against signs of anaphylaxis upon transfer of CD25-depleted cell fractions, suggest a role for Foxp3+ Tregs in the protective effect of OIT + FOS.

The efficacy of oral and subcutaneous antigen-specific immunotherapy in murine cow's milk- and peanut allergy models.

BACKGROUND: Antigen-specific immunotherapy (AIT) is a promising therapeutic approach for both cow's milk allergy (CMA) and peanut allergy (PNA), but needs optimization in terms of efficacy and safety. AIM: Compare oral immunotherapy (OIT) and subcutaneous immunotherapy (SCIT) in murine models for CMA and PNA and determine the dose of allergen needed to effectively modify parameters of allergy. METHODS: Female C3H/HeOuJ mice were sensitized intragastrically (i.g.) to whey or peanut extract with cholera toxin. Mice were treated orally (5 times/week) or subcutaneously (3 times/week) for three consecutive weeks. Hereafter, the acute allergic skin response, anaphylactic shock symptoms and body temperature were measured upon intradermal (i.d.) and intraperitoneal (i.p.) challenge, and mast cell degranulation was measured upon i.g. challenge. Allergen-specific IgE, IgG1 and IgG2a were measured in serum at different time points. Single cell suspensions derived from lymph organs were stimulated with allergen to induce cytokine production and T cell phenotypes were assessed using flow cytometry. RESULTS: Both OIT and SCIT decreased clinically related signs upon challenge in the CMA and PNA model. Interestingly, a rise in allergen-specific IgE was observed during immunotherapy, hereafter, treated mice were protected against the increase in IgE caused by allergen challenge. Allergen-specific IgG1 and IgG2a increased due to both types of AIT. In the CMA model, SCIT and OIT reduced the percentage of activated Th2 cells and increased the percentage of activated Th1 cells in the spleen. OIT increased the percentage of regulatory T cells (Tregs) and activated Th2 cells in the MLN. Th2 cytokines IL-5, IL-13 and IL-10 were reduced after OIT, but not after SCIT. In the PNA model, no differences were observed in percentages of T cell subsets. SCIT induced Th2 cytokines IL-5 and IL-10, whereas OIT had no effect. CONCLUSION: We have shown clinical protection against allergic manifestations after OIT and SCIT in a CMA and PNA model. Although similar allergen-specific antibody patterns were observed, differences in T cell and cytokine responses were shown. Whether these findings are related to a different mechanism of AIT in CMA and PNA needs to be elucidated.