Ageing is associated with a changing immune system, leading to inflammageing (increased levels of inflammation markers in serum) and immunosenescence (reduced immune cells and reduced responses ...towards pathogens). This results in reduced vaccination responses and increased infections in elderly. Much is known about the adaptive immune system upon ageing, but less is known about the innate immune system. Therefore, the aim of this study was to compare innate immune function of Toll like receptor (TLR)-mediated responses between elderly and young adult women. To this end, elderly and young adult women were compared to study the effect of ageing on the relative prevalence and reactivity to TLR-mediated responses of myeloid- and plasmacytoid dendritic cells (mDC, pDC). In addition, TLR expression and inflammatory markers in serum were investigated. Elderly women had reduced numbers of circulating pDCs. In addition, pDCs and mDCs of elderly women responded differently towards TLR stimulation, especially TLR7/8 mediated stimulation was reduced, compared to young adults. In serum, markers involved in inflammation were generally increased in elderly. In conclusion, this study confirms and extends the knowledge about immunosenescence and inflammageing on innate immunity in elderly women.
Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive ...molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers.
Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk.
EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression.
In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05).
Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk.
Background: Nutritional supplements, such as bovine lactoferrin (bLF), have been studied for their immunomodulatory properties, but little is known of their effect on the gut microbiota composition ...of the elderly when supplemented alone or combined with other nutritional supplements such as prebiotics and micronutrients. In the present study, fecal samples from a double-blind, placebo-controlled nutritional intervention study were analysed. At baseline (T1), 25 elderly women were distributed into two groups receiving dietary intervention (n = 12) or placebo treatment (n = 13) for 9 weeks. During the first 3 weeks of the study (T2), the intervention group consumed 1 g/day bLF, followed by 3 weeks (T3) of 1 g/day bLF and 2.64 g/day active galactooligosaccharides (GOS), and 3 weeks (T4) of 1 g/day bLF, 2.64 g/day GOS and 20 μg/day of vitamin D. The placebo group received maltodextrin, in dosages matching those of the intervention group. Fecal bacterial composition was profiled using partial 16S rRNA gene amplicon sequencing. Short-chain fatty acids (SCFA) were determined in fecal water as were levels of calprotectin, zonulin, and alpha-1-antitrypsin, as markers of gastrointestinal barrier and inflammation. Results: A significant increase was observed in the relative abundance of the genus Holdemanella (p < 0.01) in the intervention group compared to the placebo at T1. During T2, Bifidobacterium relative abundance increased significantly (p < 0.01) in the intervention group compared to the placebo, and remained significantly higher until the end of the study. No other effect was reported during T3. Furthermore, concentrations of SCFAs and calprotectin, zonulin and alpha-1-antitrypsin did not change during the intervention, although zonulin levels increased significantly within the placebo group by the end of the intervention. Conclusions: We conclude that supplementation of bLF enhanced the relative abundance of Holdemanella in the fecal microbiota of healthy elderly women, and further addition of GOS enhanced the relative abundance of Bifidobacterium.
As of late, evidence has been emerging that the Maillard reaction (MR, also referred to as glycation) affects the structure and function of food proteins. MR induces the conformational and chemical ...modification of food proteins, not only on the level of IgG/IgE recognition, but also by increasing the interaction and recognition of these modified proteins by antigen-presenting cells (APCs). This affects their biological properties, including digestibility, bioavailability, immunogenicity, and ultimately their allergenicity. APCs possess various receptors that recognize glycation structures, which include receptor for advanced glycation end products (RAGE), scavenger receptors (SRs), galectin-3 and CD36. Through these receptors, glycation structures may influence the recognition, uptake and antigen-processing of food allergens by dendritic cells (DCs) and monocytes. This may lead to enhanced cytokine production and maturation of DCs, and may also induce adaptive immune responses to the antigens/allergens as a result of antigen uptake, processing and presentation to T cells. Here, we aim to review the current literature on the immunogenicity of AGEs originating from food (exogenous or dietary AGEs) in relation to AGEs that are formed within the body (endogenous AGEs), their interactions with receptors present on immune cells, and their effects on the activation of the innate as well as the adaptive immune system. Finally, we review the clinical relevance of AGEs in food allergies.
Immunomodulating proteins and peptides are formed during the hydrolysis of cow's milk proteins. These proteins are potential ingredients in functional foods used for the management of a range of ...immune related problems, both in infants and adults. However, the mechanism behind these effects is unknown. We hypothesize that the interaction of peptides with Toll-like receptors (TLRs) can induce immune effects, since these receptors are known to sample many dietary molecules in the intestine in order to regulate immune effects. To investigate this, we compared the immune effects and TLR activation and inhibition by whey and casein hydrolysates with different hydrolysis levels. We first measured cytokine production in primary peripheral blood mononuclear cells stimulated with either whey, casein, or their hydrolysates. IL-10 and TNFα were induced by whey hydrolysates (decreasing with increasing hydrolysis level), but not by casein hydrolysates. Next, the activation of TLR 2, 3, 5 and 9 receptors were observed by intact and mildly hydrolysed whey proteins only and not by casein hydrolysates in TLR reporter cell lines. Many casein hydrolysates inhibited TLR signaling (mainly TLR 5 and 9). These results demonstrate that the effects of cow's milk proteins on the immune system are protein type and hydrolysis dependent. TLR signaling is suggested as a possible mechanism for differences in effect. This knowledge contributes to a better understanding of the immune effects of hydrolysates and the design of infant formula, and nutrition in general, with specific immunoregulatory effects.
Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed ...food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.
Food allergy incidence has increased worldwide over the last 20 years. For prevention of food allergy, current guidelines do not recommend delaying the introduction of allergenic foods. Several ...groundbreaking studies, such as the Learning Early About Peanut Allergy study, showed that the relatively early introduction of this allergenic food between 4–6 months of age reduces the risk of peanut allergy. However, less is known about the introduction of cow’s milk, as many children already receive cow’s-milk-based formula much earlier in life. This can be regular cow’s milk formula with intact milk proteins or hydrolyzed formulas. Several recent studies have investigated the effects of early introduction of cow’s-milk-based formulas with intact milk proteins on the development of cow’s milk allergy while breastfeeding. These studies suggest that depending on the time of introduction and the duration of administration of cow’s milk, the risk of cow’s milk allergy can be reduced (early introduction) or increased (very early introduction followed by discontinuation). The aim of this narrative review is to summarize these studies and to discuss the impact of early introduction of intact cow’s milk protein—as well as hydrolyzed milk protein formulas—and the development of tolerance versus allergy towards cow’s milk proteins.
Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. ...Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.
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.
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