Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Heating affects protein digestion of skimmed goat milk under simulated infant conditions.

The objective of this research was to analyse the effects of heating on digestion of skimmed goat milk proteins. Most previous goat milk digestion studies evaluated the digestion only based on the supernatant. In this study, digestion of skimmed goat milk was studied in both supernatant and gastric clot. The results indicated that, compared to mild temperature heated samples (≤75 °C), samples heated at ≥80 °C showed more extensive gastric clot formation with a higher protein digestion rate, but also resulted in a larger amount of undigested whey proteins due to its severe aggregation. For the peptidome, ß-casein was the major source of bioactive peptides. The samples heated at 65 °C showed higher bioactive peptide abundances, whereas at temperatures higher than 75 °C, it was reduced due to cleavage into smaller peptides. Overall, this study showed that different heating temperatures induced different whey protein denaturation degrees, which affected their digestion in skimmed goat milk..

Clostridioides difficile toxin A-mediated Caco-2 cell barrier damage was attenuated by insect-derived fractions and corresponded to increased gene transcription of cell junctional and proliferation proteins.

Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.

Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture.

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

Enhanced Uptake of Processed Bovine ß-Lactoglobulin by Antigen Presenting Cells: Identification of Receptors and Implications for Allergenicity.

SCOPE: ß-lactoglobulin (BLG) is a major cow milk allergen encountered by the immune system of infants fed with milk-based formulas. To determine the effect of processing on immunogenicity of BLG, this article characterized how heated and glycated BLG are recognized and internalized by APCs. Also, the effect of heat-induced structural changes as well as gastrointestinal digestion on immunogenicity of BLG is evaluated. METHODS AND RESULTS: The binding and uptake of BLG from raw cow milk and heated either alone (BLG-H) or with lactose/glucose (BLG-Lac and BLG-Glu) to the receptors present on APCs are analyzed by ELISA and cell-binding assays. Heated and glycated BLG is internalized via galectin-3 (Gal-3)and scavenger receptors (CD36 and SR-AI) while binding to the receptor for advanced glycation end products (R AGE) does not cause internalization. Receptor affinity of BLG is dependent on increased hydrophobicity, ß-sheet exposure and aggregation. Digested glycated BLG maintained binding to sRAGE and Gal-3 but not to CD36 and SR-AI, and is detected on the surface of APCs. This suggests a mechanism via which digested glycated BLG may trigger innate (via RAGE) and adaptive immunity (via Gal-3). CONCLUSIONS: This study defines structural characteristics of heated and glycated BLG determining its interaction with APCs via specific receptors thus revealing enhanced immunogenicity of glycated versus heated BLG.

Curdlan, zymosan and a yeast-derived ß-glucan reshape tumor-associated macrophages into producers of inflammatory chemo-attractants.

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible ß-glucans (oatßG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived ß-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived ß-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses.

Current Understanding of the Structure and Function of Fungal Immunomodulatory Proteins.

Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi, which are extensively studied for their immunomodulatory activity. Currently, more than 38 types of FIPs have been described. Based on their conserved structure and protein identity, FIPs can be classified into five subgroups: Fve-type FIPs (Pfam PF09259), Cerato-type FIPs (Pfam PF07249), PCP-like FIPs, TFP-like FIPs, and unclassified FIPs. Among the five subgroups, Fve-type FIPs are the most studied for their hemagglutinating, immunomodulating, and anti-cancer properties. In general, these small proteins consist of 110-125 amino acids, with a molecular weight of ~13 kDa. The other four subgroups are relatively less studied, but also show a noticeable influence on immune cells. In this review, we summarized the protein modifications, 3-dimensional structures and bioactivities of all types of FIPs. Moreover, structure-function relationship of FIPs has been discussed, including relationship between carbohydrate binding module and hemagglutination, correlation of oligomerization and cytokine induction, relevance of glycosylation and lymphocyte activation. This summary and discussion may help gain comprehensive understanding of FIPs' working mechanisms and scope future studies.

Hydrophobicity drives receptor-mediated uptake of heat-processed proteins by THP-1 macrophages and dendritic cells, but not cytokine responses.

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to ß-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

Peptide Release after Simulated Infant In Vitro Digestion of Dry Heated Cow's Milk Protein and Transport of Potentially Immunoreactive Peptides across the Caco-2 Cell Monolayer.

Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

Wheat-derived arabinoxylans reduced M2-macrophage functional activity, but enhanced monocyte-recruitment capacity.

The immunomodulatory properties of non-digestible polysaccharides (NDPs) have been recognized in in vitro and in vivo studies. The latter mostly demonstrated altered frequencies and inflammatory status of immune cells as clinical parameters. Most of the NDP activity will be exerted in the intestine where they can directly interact with macrophages. The predominant macrophage phenotype in the intestine is M2-like, with M1-like macrophages arising during inflammation. Here, we investigated transcriptional and functional impact on these macrophage phenotypes by NDP-treatment (i.e. yeast-derived soluble ß-glucan (yeast-ßG), apple-derived RG-I (apple-RGI), shiitake-derived ß-glucan (shiitake-ßG) or wheat-derived arabinoxylan (wheat-AX)). Wheat-AX, and to a lesser extent shiitake-ßG and apple-RGI but not yeast-ßG, reduced endocytosis and antigen processing capacity of M1- and M2-like macrophages. Moreover, the NDPs, and most notably wheat-AX, strongly induced transcription and secretion of a unique set of cytokines and chemokines. Conditioned medium from wheat-AX-treated M2-like macrophages subsequently demonstrated strongly increased monocyte recruitment capacity. These findings are in line with clinically observed immunomodulatory aspects of NDPs making it tempting to speculate that clinical activity of some NDPs is mediated through enhanced chemoattraction and modifying activity of intestinal immune cells.

Specific Polyunsaturated Fatty Acids Can Modulate in vitro Human moDC2s and Subsequent Th2 Cytokine Release.

Allergy is becoming a rapidly increasing problem worldwide, and in vitro models are frequently used to study the mechanisms behind the different types of allergic response. The dendritic cell (DC)-T-cell model can be used to study sensitization. However, lipopolysaccharide (LPS) is often used to maturate the DCs, but it gives rise to a DC1 phenotype, whereas Th2-driven inflammatory diseases such as allergy are characterized by the involvement of the DC2 phenotype. Our aim was to create a DC2-T-cell human model (human moDC2s) to study in vitro sensitization and validate the model using polyunsaturated fatty acids (PUFAs) that were previously shown to have immunomodulatory properties. We found that the generated DC2s expressed OX40L and drove naive T-cells into IL-13 production of CD4+ effector T-cells. In line with in vivo findings, n-3 long-chain (LC)PUFA docosahexaenoic acid (DHA) effectively decreased the DC2's surface expression of OX40L, as well as the IL-12p40 and IL-23 cytokine production by DC2s and subsequently lowered IL-13 production by DC2-induced effector T-cells. Similar cytokine production effects were found with eicosapentaenoic acid (EPA) and arachidonic acid (AA), whereas linoleic acid (LA) increased OX40L surface expression and subsequent T-cell-derived IL-13/IFNγ ratios, suggesting an increased risk of allergy development. Altogether, these data show that human moDC2s are able to induce Th2-type IL-13 secretion by T-cell differentiated in the presence of these DC2s and that this model can be differentially modulated by PUFAs. These results are in line with previous in vivo studies using PUFAs, indicating that this model may be of use to predict in vivo outcomes.

Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Hydrophobicity and aggregation, but not glycation, are key determinants for uptake of thermally processed ß-lactoglobulin by THP-1 macrophages.

The aim of this study is to investigate the immunological relevance of modifications of food protein structure due to thermal processing. We investigated the uptake of ß-lactoglobulin, treated with 3 different processing methods, by THP-1 macrophages: wet heating (60 °C in solution) and high- or low-temperature (130 °C or 50 °C, respectively) dry heating, combined with either of 8 types of saccharides or without saccharide. The processing method that was applied significantly affected the uptake of BLG by THP-1 macrophages, while the type of saccharide only had an influence in high-temperature dry heated samples. A set of physicochemical parameters of processed samples was determined, to determine the samples' molecular weight, hydrophobicity, amyloid-like structure, surface charge and secondary structure. Analysis of protein structure alterations indicated the uptake to be linked to the wet heating processing method and percentage of α-helix structure, amyloid-like structures, polymers, and hydrophobicity. We hypothesize that both amyloid-like structures and molecular weight were related to the increased hydrophobicity and therefore postulate that the exposure of hydrophobic regions is the leading physicochemical characteristic for the observed uptake of wet heated BLG samples by THP-1 macrophages. This work demonstrates how differential thermal processing of foods, through protein modification, can have an impact on its interaction with the immune system.

Consumption of ß-glucans to spice up T cell treatment of tumors: a review.

INTRODUCTION: Adoptive T-cell treatments of solid cancers have evolved into a robust therapy with objective response rates surpassing those of standardized treatments. Unfortunately, only a limited fraction of patients shows durable responses, which is considered to be due to a T cell-suppressive tumor microenvironment (TME). Here we argue that naturally occurring ß-glucans can enable reversion of such T cell suppression by engaging innate immune cells and enhancing numbers and function of lymphocyte effectors. AREAS COVERED: This review summarizes timely reports with respect to absorption, trafficking and immune stimulatory effects of ß-glucans, particularly in relation to innate immune cells. Furthermore, we list effects toward well-being and immune functions in healthy subjects as well as cancer patients treated with orally administered ß-glucans, extended with effects of ß-glucan treatments in mouse cancer models. EXPERT OPINION: Beta-glucans, when present in food and following uptake in the proximal gut, stimulate immune cells present in gut-associated lymphoid tissue and initiate highly conserved pro-inflammatory pathways. When tested in mouse cancer models, ß-glucans result in better control of tumor growth and shift the TME toward a T cell-sensitive environment. Along these lines, we advocate that intake of ß-glucans provides an accessible and immune-potentiating adjuvant when combined with adoptive T-cell treatments of cancer.

The Effect of Tomatine on Gene Expression and Cell Monolayer Integrity in Caco-2.

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1ß, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Water-Soluble Polysaccharide Extracts from the Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) with HMGCR Inhibitory Activity.

Water extracts from Pleurotus ostreatus containing no statins showed 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitory activity (in vitro) that might be due to specific water-soluble polysaccharides (WSPs); when isolated and deproteinized, increasing concentrations of the WSP extract induced higher inhibition. The WSP extract contained mainly ß-glucans, mannogalactans, and glycogen (e.g., α-glucans), although derivatives or fragments with lower molecular weights (between 14 and 3.5 kDa) were present and were able to induce the inhibitory activity. The extract contained more ß-(1→3)-glucans than ß-(11→3),(11→6)-glucans, and they partially survived digestion and managed to pass through Caco2 cell monolayers to the lower compartment after in vitro digestion and transport experiments. The WSP might also modulate Caco2 membrane integrity.

Immunomodulatory activity of protein hydrolysates derived from Virgibacillus halodenitrificans SK1-3-7 proteinase.

Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1ß and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1ß, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.

Bioavailability of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from Virgibacillus halodenitrificans SK1-3-7 proteinases hydrolyzed tilapia muscle proteins.

The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24h. After 8h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC50 value of 0.54mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3-4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC50 value of 0.29µM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable.

Adaptation of exercise-induced stress in well-trained healthy young men.

NEW FINDINGS: What is the central question of this study? Exercise is known to induce stress-related physiological responses, such as changes in intestinal barrier function. Our aim was to determine the test-retest repeatability of these responses in well-trained individuals. What is the main finding and its importance? Responses to strenuous exercise, as indicated by stress-related markers such as intestinal integrity markers and myokines, showed high test-retest variation. Even in well-trained young men an adapted response is seen after a single repetition after 1 week. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise. Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protégé Study (ISRCTN14236739; www.isrctn.com), we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained men (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% of maximal workload the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose and rhamnose solution was ingested. Markers of energy metabolism, lactulose-to-rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated 1 week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (1 h plasma lactulose-to-rhamnose ratio) and the serum interleukin-6, interleukin-10, fibroblast growth factor-21 and muscle creatine kinase concentrations were significantly increased compared with rest only during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be attributable to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise.

Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation.

Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE receptor (sRAGE). Samples consisting of mixtures of whey protein and lactose were heated at 130 °C. An in vitro infant digestion model was used to study the influence of heat treatment on the digestibility of whey proteins. The amount of sRAGE-binding ligands before and after digestion was measured by an ELISA-based sRAGE-binding assay. Water activity did not significantly affect the extent of digestibility of whey proteins dry heated at pH 5 (ranging from 3.3 ± 0.2 to 3.6 ± 0.1% for gastric digestion and from 53.5 ± 1.5 to 64.7 ± 1.1% for duodenal digestion), but there were differences in cleavage patterns of peptides among the samples heated at different pH values. Formation of sRAGE-binding ligands depended on the formation of aggregates and was limited in the samples heated at pH 5. Moreover, the sRAGE-binding activity of digested sample was changed by protease degradation and correlated with the digestibility of samples. In conclusion, generation of sRAGE-binding ligands during extensive heat treatment of whey protein/lactose mixtures is limited in acidic heating condition and dependent on glycation and aggregation.