sRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion.

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

Dietary Intervention with Whey Protein Concentrate Does Not Affect Toll-like Receptor Responses and Gene Expression Patterns in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants.

Pollen Sensitization Can Increase the Allergic Reaction to Non-Cross-Reactive Allergens in a Soy-Allergic Patient.

During and after the pollen season, an increase in food-triggered allergic symptoms has been observed in pollen-food syndrome patients, possibly due to seasonal boosting of pollen-IgE levels. It has been suggested that consumption of birch-pollen-related foods plays a role in seasonal allergenic inflammation. However, whether this increased pollen sensitization during the pollen season can also affect the allergenicity of allergens that are non-cross-reactive with birch pollen remains in question. This study presents the case of a patient with soy allergy and pollinosis, who experiences worsening of gastrointestinal (GI) symptoms during the birch pollen season even though the eliciting food factor does not cross-react with birch pollen allergens and their homologs (e.g., Bet v 1 and Gly m 4). The results showed a notable increase in sIgE for Gly m 4 (3.3 fold) and Bet v 1 (2.6 fold) during the birch pollen season compared to outside the birch pollen season, while Gly m 5 and Gly m 6 showed only a slight increase (1.5 fold). The basophil activation test (BAT) showed that in this patient Gly m 5 and Gly m 6 are clinically relevant soy allergens, which correlates with the reported clinical symptoms to processed soy. Moreover, the BAT against raw soy shows an increase in basophil activation during the birch pollen season and a negative basophil activation result outside the birch pollen season. Thus, the worsening of GI symptoms could possibly be due to an increase in IgE receptors, an over-reactive immune system, and/or significant intestinal allergic inflammation. This case highlights the importance of including allergens that do not cross-react with birch pollen and using a functional assay such as the BAT to evaluate clinical relevance when assessing birch pollen seasonal influence on soy allergenicity.

Soy Gly m 8 sIgE Has Limited Value in the Diagnosis of Soy Allergy in Peanut Ara h 2-Sensitized Adults.

INTRODUCTION: Recently, specific IgE (sIgE) sensitization against Gly m 8 (soy 2S albumin) has been described as a good diagnostic marker for soy allergy (SA). The aim of this study was to evaluate the diagnostic value of Gly m 8 by determining the sensitization profiles based on the homologues soy allergens Bet v 1, Ara h 1, Ara h 2, and Ara h 3. METHODS: Thirty soy-allergic adults were included; sIgE to total soy extract, Gly m 8, Gly m 4, Gly m 5, Gly m 6, Bet v 1, Ara h 1, Ara h 2, and Ara h 3 were determined. Sensitization patterns were analyzed and determined. The clinical relevance of sIgE of Gly m 8 sensitization was measured by assessing its capacity to degranulate basophils in Gly m8-sensitized patients by an indirect basophil activation test (iBAT). RESULTS: Based on the sIgE patterns of sensitization, two groups of SA patients were identified: (i) peanut-associated SA group (all patients were sensitized to one or more of the peanut compounds) and (ii) non-peanut/PR-10-associated SA group (22 patients were sensitized to Gly m 4 and Bet v 1 but not to any of the peanut compounds). A high and significant correlation between total soy extract and Gly m 6 (R2 = 0.97), Gly m 5 (R2 = 0.85), and Gly m 8 (R2 = 0.78) was observed. A nonsignificant correlation was observed between the levels of sIgE of Gly m 8 versus Ara h2. The iBAT results showed that Gly m 8 did not induce basophil degranulation in any of the peanut-associated patients, indicating that the Gly m8 sensitizations were not clinically relevant. CONCLUSIONS: Gly m 8 was not a major allergen in the selected soy-allergic population. The iBAT results indicated that Gly m 8 was not able to induce basophil degranulation in sIgE Gly m 8-sensitized soy-allergic patients. Thus, Gly m 8 would have no added value in the diagnosis of SA in the present study population.

Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge.

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses.

Direct Binding of Bovine IgG-Containing Immune Complexes to Human Monocytes and Their Putative Role in Innate Immune Training.

Bovine milk IgG (bIgG) was shown to bind to and neutralize the human respiratory synovial virus (RSV). In animal models, adding bIgG prevented experimental RSV infection and increased the number of activated T cells. This enhanced activation of RSV-specific T cells may be explained by receptor-mediated uptake and antigen presentation after binding of bIgG-RSV immune complexes (ICs) with FcγRs (primarily CD32) on human immune cells. This indirect effect of bIgG ICs on activation of RSV-specific T cells was confirmed previously in human T cell cultures. However, the direct binding of ICs to antigen-presenting cells has not been addressed. As bovine IgG can induce innate immune training, we hypothesized that this effect could be caused more efficiently by ICs. Therefore, we characterized the expression of CD16, CD32, and CD64 on (peripheral blood mononuclear cells (PBMCs), determined the optimal conditions to form ICs of bIgG with the RSV preF protein, and demonstrated the direct binding of these ICs to human CD14+ monocytes. Similarly, bIgG complexed with a murine anti-bIgG mAb also bound efficiently to the monocytes. To evaluate whether the ICs could induce innate immune training more efficiently than bIgG itself, the resulted ICs, as well as bIgG, were used in an in vitro innate immune training model. Training with the ICs containing bIgG and RSV preF protein-but not the bIgG alone-induced significantly higher TNF-α production upon LPS and R848 stimulation. However, the preF protein itself nonsignificantly increased cytokine production as well. This may be explained by its tropism to the insulin-like growth factor receptor 1 (IGFR1), as IGF has been reported to induce innate immune training. Even so, these data suggest a role for IgG-containing ICs in inducing innate immune training after re-exposure to pathogens. However, as ICs of bIgG with a mouse anti-bIgG mAb did not induce this effect, further research is needed to confirm the putative role of bIgG ICs in enhancing innate immune responses in vivo.

Receptor Mediated Effects of Advanced Glycation End Products (AGEs) on Innate and Adaptative Immunity: Relevance for Food Allergy.

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.

The Basophil Activation Test for Clinical Management of Food Allergies: Recent Advances and Future Directions.

The basophil activation test (BAT) is an ex vivo functional assay that measures by flow cytometry the degree of basophil degranulation after stimulation with an allergen. In recent years, there has been an increased interest in the diagnostic value of the BAT as it has the potential to mimic the clinical phenotype of sIgE sensitized patients, in contrast to allergen-specific IgE levels. This diagnostic potential would be of particular interest for food allergies present early in life such as peanut, cow's milk and eggs, which require an expensive, time-consuming and patient unfriendly oral food challenge (OFC) for diagnosis. However, routine applications of the BAT for clinical use are not yet feasible due to the lack of standardized protocols and large clinical validation studies. This review will summarize the current data regarding the application of the BAT in food allergy (FA) for cow's milk, egg and peanut, being the most common causes of FA in children. Additionally, it will discuss the hurdles for widespread clinical use of the BAT and possible future directions for this diagnostic procedure.

No Glycation Required: Interference of Casein in AGE Receptor Binding Tests.

For the determination of the binding of heated cow's milk whey proteins such as ß-lactoglobulin to the receptors expressed on immune cells, inhibition ELISA with the soluble form of the receptor for advanced glycation end products (sRAGE) and scavenger receptor class B (CD36) has been successfully used in the past. However, binding to heated and glycated caseins in this read-out system has not been tested. In this study, inhibition ELISA was applied to measure the binding of cow's milk casein alone, as well as all milk proteins together, which underwent differential heat treatment, to sRAGE and CD36, and we compared those results to a dot blot read out. Moreover, binding to sRAGE and CD36 of differentially heated milk protein was measured before and after in vitro digestion. Casein showed binding to sRAGE and CD36, independent from the heat treatment, in ELISA, while the dot blot showed only binding to high-temperature-heated milk protein, indicating that the binding is not related to processing but to the physicochemical characteristics of the casein. This binding decreased after passage of casein through the intestinal phase.

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.

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.

Binding of CML-Modified as Well as Heat-Glycated ß-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity.

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, ß-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to ß-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of ß-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 ß-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.

Differential Effects of Dry vs. Wet Heating of ß-Lactoglobulin on Formation of sRAGE Binding Ligands and sIgE Epitope Recognition.

The effect of glycation and aggregation of thermally processed ß-lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity < 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (<73 °C) and high temperatures (>90 °C) in the presence or absence of the milk sugar lactose. Nε-(carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow's milk BLG.

Toll-Like Receptor-Dependent Immunomodulatory Activity of Pycnogenol®.

BACKGROUND: Pycnogenol® (PYC), an extract of French maritime pine bark, is widely used as a dietary supplement. PYC has been shown to exert anti-inflammatory actions via inhibiting the Toll-like receptor 4 (TLR4) pathway. However, the role of the other receptors from the TLR family in the immunomodulatory activity of PYC has not been described so far. AIM: The aim of this study was to investigate whether PYC might exert its immunomodulatory properties through cell membrane TLRs (TLR1/2, TLR5, and TLR2/6) other than TLR4. Moreover, the effect of gastrointestinal metabolism on the immunomodulatory effects of PYC was investigated. FINDINGS: We showed that intact non-metabolized PYC dose-dependently acts as an agonist of TLR1/2 and TLR2/6 and as a partial agonist of TLR5. PYC on its own does not agonize or antagonize TLR4. However, after the formation of complexes with lipopolysaccharides (LPS), it is a potent activator of TLR4 signaling. Gastrointestinal metabolism of PYC revealed the immunosuppressive potential of the retentate fraction against TLR1/2 and TLR2/6 when compared to the control fraction containing microbiota and enzymes only. The dialyzed fraction containing PYC metabolites revealed the capacity to induce anti-inflammatory IL-10 secretion. Finally, microbially metabolized PYC affected the colonic microbiota composition during in vitro gastrointestinal digestion. CONCLUSIONS: This study showed that gastrointestinal metabolism of PYC reveals its biological activity as a potential inhibitor of TLRs signaling. The results suggest that metabolized PYC acts as a partial agonist of TLR1/2 and TLR2/6 in the presence of the microbiota-derived TLR agonists (retentate fraction) and that it possesses anti-inflammatory potential reflected by the induction of IL-10 from THP-1 macrophages (dialysate fraction).

Immunomodulation by Processed Animal Feed: The Role of Maillard Reaction Products and Advanced Glycation End-Products (AGEs).

The immune system provides host protection to infection with pathogenic organisms, while at the same time providing tolerance upon exposure to harmless antigens. Thus, an impaired immune function is associated with increased susceptibility to infections with increased disease severity and thereby necessitating the therapeutic use of antibiotics. Livestock performance and feed efficiency, in addition to their health status, are dependent on the microbial load of their gut, the barrier function of the intestinal epithelium and the activity of the mucosal immune system, all of which can be modulated by dietary components. The majority of feeds that are consumed in pets and livestock have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars called Maillard reaction (MR). Maillard reaction products (MRPs) and advanced Maillard reaction products (AGEs) determine taste, smell, and color of many food products therefore the MR is highly relevant for the feed industry. MRPs interact with different types of immune receptors, including the receptor for advanced glycation end products (RAGE) and immunomodulatory potential of feed proteins can be modified by Maillard reaction. This MR has become an important concern since MRPs/AGEs have been shown to contribute to increasing prevalence of diet-related chronic inflammatory states in the gut with negative health consequences and performance. The immunomodulatory effects of dietary MRPs and AGEs in livestock and pet animals are far less well-described, but widely considered to be similar to the relevant concepts and mechanisms obtained in the human field. This review will highlight immunological mechanisms underlying initiation of the innate and adaptive immune responses by MRPs/AGEs present in animal feeds, which are currently not completely understood. Bridging this knowledge gap, and taking advantage of progress in the human field, will significantly improve nutritional quality of feed and increase the prevention of diet-mediated inflammation in animals.

Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism.

BACKGROUND: Autism spectrum disorder (ASD) is a group of heterogeneous, behaviorally defined disorders whereby currently no biological markers are common to all affected individuals. A deregulated immune response may be contributing to the etiology of ASD. The active metabolite of vitamin D3 has an immunoregulatory role mediated by binding to the vitamin D receptor (VDR) in monocyte, macrophages, and lymphocytes. The effects of vitamin D and interaction with the VDR may be influenced by polymorphism in the VDR gene. METHODS: Genetic association of four different VDR polymorphisms (Apa-I, Bsm-I, Taq-I, Fok-I) associated with susceptibility to the development of autism in children was investigated. RESULTS: We uniquely found an association between the presence of the T allele at position Taq-I and presence of the a allele at position Apa-I of the VDR gene with decreased ASD incidence. There was also an association between female gender and the presence of the T allele. We found no statistical significant correlation between VDR single nucleotide polymorphisms (SNPs) and vitamin D3 concentration in serum of ASD children. CONCLUSION: Genetic polymorphism in two SNP in VDR may be correlated with development of ASD symptoms by influencing functionality of vitamin D3 metabolism, while vitamin D3 levels were not significantly different between ASD and non-ASD children.

Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins.

The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers' choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins.

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.

SCOPE: Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for ß-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. METHODS AND RESULTS: Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. CONCLUSION: The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

An alternative inhibition method for determining cross-reactive allergens.

BACKGROUND: Inhibition assays are an useful tool to identify the allergen of primary sensitization of cross-reactive allergens. Classical ELISA-based inhibition assays are limited by both the availability of commercial standardized allergen extracts and the experience and knowledge needed for making home-made extracts. Moreover the direct comparison of the inhibition ELISAs outcomes between different laboratories is difficult because of different sources of used allergen extracts and a number of methodological variations. Therefore, we propose a novel ImmunoCap (Phadia, Thermofisher Scientific) based immunoinhibition method with the use of commercially available Caps as the allergen source. METHODS: The novel ImmunoCap based immunoinhibition method was developed and tested with sera from patients with a well-known cross-reactive sensitization for fig (Ficus carica) and ficus (Ficus benjamina). Results were compared with a classically applied inhibition method, i.e. addition of homemade allergen extract to patient serum. RESULTS: The amount of allergens (fig and ficus extracts) needed to reach a similar degree of inhibition was comparable for both inhibition methods. CONCLUSIONS: The ImmunoCap based inhibition assay, in addition to classical inhibition methods, is a valuable tool as the ImmunoCap analyzer and commercial allergens (Caps) are more widely available which makes the outcomes of inhibition tests comparable between different laboratories. Furthermore, in the ImmunoCap inhibition method the same protein source is used for both the inhibition of sIgE and sIgE measurement, which might be even more relevant when multiple cross-reactive allergens are tested.

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.