Major Cow's Milk Research Articles

Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture

Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4.A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value.These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples.This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, “epitopes-ALL,” and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.

Regulation of the expression of casein alpha S1 and S2 genes in the bovine mammary epithelial cells by STAT5A

Cow milk is rich in protein. Major cow milk proteins include casein α S1 (CSN1S1), casein α S2 (CSN1S2), casein β (CSN2), casein kappa (CSN3), lactalbumin α (LALBA), and β-lactoglobulin (LGB). These milk proteins are produced through gene expression in the mammary epithelial cells. Little is known about the molecular mechanism that mediates the expression of milk protein genes in cows. In this study, we tested the hypothesis that the expression of milk protein genes in cows is mediated by STAT5A, a transcription factor that is induced to bind and activate the transcription of target genes by extracellular signals such as prolactin. To circumvent the need of prolactin-responsive bovine mammary epithelial cells, we generated a plasmid that expresses a constitutively active bovine STAT5A variant, bSTAT5ACA. Transfection of the bovine mammary epithelial cell line MAC-T cells with the bSTAT5ACA expression plasmid caused a more than 100,000-fold and 600-fold increase in the expression of CSN1S1 and CSN1S2 mRNAs, respectively, compared with transfection of the wild-type bovine STAT5A (bSTAT5A) expression plasmid. Transfection of bSTAT5ACA, however, had no significant effect on the expression of CSN2, CSN3, LALBA, or LGB mRNA in MAC-T cells. Transfection of bSTAT5ACA caused a more than 260-fold and 120-fold increase in the expression of a luciferase reporter gene linked to the bovine CSN1S1 and CSN1S2 promoters in MAC-T cells, respectively, compared with that of bSTAT5A. The bovine CSN1S1 and CSN1S2 promoters each contain a putative STAT5 binding site, and gel-shift and super-shift assays confirmed bSTAT5ACA binding to both sites. These results together suggest that STAT5A plays a major role in regulating the expression of CSN1S1 and CSN1S2 genes in the bovine mammary epithelial cells and that STAT5A regulates the expression of these genes at least in part by binding to the STAT5 binding sites in their promoter regions. These results also suggest that STAT5A does not play a major role in regulating the expression of other major milk protein genes.

Real-Life Use of Component-Specific IgE in IgE-Mediated Cow's Milk Protein Allergy in a Spanish Paediatric Allergy Centre.

In Spain, IgE-mediated cow's milk protein allergy (CMPA) affects approximately 0.69% of infants. Molecular diagnosis may be useful for monitoring natural spontaneous tolerance development in CMPA. The aim of this study was to retrospectively analyse a cohort of paediatric patients with IgE-mediated CMPA who were avoiding milk products awaiting natural tolerance and determine the relationship between disease persistence and major cow's milk allergens. A retrospective chart review of 200 patients diagnosed with IgE-mediated CMPA between 2011 and 2020 was conducted. Patients strictly avoided milk products until an oral food challenge was performed. The main outcome was the introduction of liquid milk following a negative oral food challenge and its correlation with IgE and SPT measurements of milk components at diagnosis. Secondary outcomes included the rate of allergic reactions and anaphylaxis during the treatment period and its correlation with IgE and SPT measurements. Of the 200 charts analysed, 122 patients had a negative oral food challenge to milk (61.0%) (95% confidence interval (CI): 54.1-67.5) following a period of strict avoidance of milk. Higher levels of component-specific IgE, especially casein, were associated with failure in the oral food challenge (p = 0.02). Allergic reactions were experienced by 106 children (53%), of which 34 (17%; 95% CI: 12.4-22.8) had anaphylactic reactions. The risk of anaphylaxis was not predicted by raised IgE levels. While a large proportion of children acquired natural tolerance to cow's milk following a period of strict avoidance, IgE-mediated CMPA persisted in many children. Casein IgE levels at diagnosis were raised in those who failed to achieve natural tolerance. Allergic reactions to milk, including anaphylaxis, occurred commonly, but this was not predicted by raised IgE levels or SPT measurements.

Immunoglobulin G4 in eosinophilic esophagitis: Immune complex formation and correlation with disease activity.

Recent studies have shown deposition of immunoglobulin G4 (IgG4) and food proteins in the esophageal mucosa of eosinophilic esophagitis (EoE) patients. Our aims were to assess whether co-localization of IgG4 and major cow's milk proteins (CMPs) was associated with EoE disease activity and to investigate the proteins enriched in proximity to IgG4 deposits. This study included adult subjects with EoE (n = 13) and non-EoE controls (n = 5). Esophageal biopsies were immunofluorescence stained for IgG4 and CMPs. Co-localization in paired samples from active disease and remission was assessed and compared to controls. The proteome surrounding IgG4 deposits was evaluated by the novel technique, AutoSTOMP. IgG4-food protein interactions were confirmed with co-immunoprecipitation and mass spectrometry. IgG4-CMP co-localization was higher in the active EoE group compared to paired remission samples (Bos d 4, p = .02; Bos d 5, p = .002; Bos d 8, p = .002). Co-localization was also significantly higher in the active EoE group compared to non-EoE controls (Bos d 4, p = .0013; Bos d 5, p = .0007; Bos d 8, p = .0013). AutoSTOMP identified eosinophil-derived proteins (PRG 2 and 3, EPX, RNASE3) and calpain-14 in IgG4-enriched areas. Co-immunoprecipitation and mass spectrometry confirmed IgG4 binding to multiple food allergens. These findings further contribute to the understanding of the interaction of IgG4 with food antigens as it relates to EoE disease activity. These data strongly suggest the immune complex formation of IgG4 and major cow's milk proteins. These immune complexes may have a potential role in the pathophysiology of EoE by contributing to eosinophil activation and disease progression.

Insight into the molecular-level details of αs1 casein interactions with IgG: Combining with LC-MS/MS and molecular modelling techniques

αs1-Casein (αs1-CN) is a major cow milk allergen, while the tertiary structure of αs1-CN and conformational epitopes of αs1-CN have not been clarified. Here, a reasonable three-dimensional structure of αs1-CN was established using ab initio methods, and hot-spot residues and epitopes were investigated by combining molecular dynamics simulation, peptides synthesis, and ELISA. Obtained results demonstrated that the binding mechanism between αs1-CN and IgG was located on three main regions: a helical structure zone (E77-Q97), the flexible loop zone (Y154-T174), and a flexible C-terminal (N190-L198), mainly connecting via hydrogen bond and ionic bonds. The hydrolysates produced by papain with lowest antigenicity (12.43%), which could considerably destroy the essential epitopes of αs1-CN confirmed by epitope synthesis, and LC-MS/MS. The results reported herein would provide novel insights into the interface interactions between αs1-CN and IgG, and prove valuable for developing hypoallergenic infant-formula and peptide vaccines for allergen-specific immunotherapy.

Pressure assisted thermal pasteurization (PATP) of hypoallergenic and low protein ready-to-feed (RTF) infant formula fortified with bioactives: A novel approach

Infant Milk Formula (IMF) is an instant alternative to breastfeeding which is being investigated continuously to mimic the composition of human milk. Hypoallergenic IMF is recommended for infants who cannot tolerate cow milk due to allergy. Paediatric obesity resulted in burgeoning demand for low protein infant formula, especially in Asia. Beta-lactoglobulin (β-Lg), a major cow milk allergen to infants, is present in cow milk as 3–4 g/L, whereas, interestingly, it is absent in human milk. Besides, human milk contains Fe3+ binding lactoferrin (LF) and Ca2+ binding alpha-lactalbumin (α-Lac) about 10 and 3 times, respectively, higher than cow milk. Therefore, a mechanism to formulate a hypoallergenic IMF with low protein content could be the addition of LF and α-Lac and reducing β-Lg at the same time using an appropriate processing method. Hence, as a pragmatic approach, this study considered a pasteurized form of ready-to-feed (RTF) by applying Pressure Assisted Thermal Pasteurization (PATP) achieved from HPP of 600 MPa for 15 s applied at 57.1 °C to attain 72.3 °C after pressurization. In parallel, conventional HTST was also applied at 72 °C for both 15 and 30 s to compare PATP's effect. HTST and PATP retained >90% and > 75% LF respectively whereas PATP induced higher retention of α-Lac compared to HTST. However, HTST retained >60% β-Lg while PATP induced a high denaturation of β-Lg with 28% retention only. Moreover, both PATP and HTST induced around 5 log reduction of E. coli (ATCC 8739) in IMF. Therefore, the current investigation potentially indicates a route to develop a PATP-processed hypoallergenic and low protein RTF formula, a niche product.

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

Scopeβ‐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 resultsThe 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, β‐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).ConclusionsThis 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.

A molecular docking and molecular dynamics simulation study on the interaction between cyanidin-3-O-glucoside and major proteins in cow's milk.

The objective of this study was to investigate the molecular interaction and complex stability of four major cow's milk (CM) proteins (α-LA, β-LG, αs1 -CA, and β-CA) with cyanidin-3-O-glucoside (C3G) using computational methods. The results of molecular docking analysis revealed that hydrogen bond and hydrophobic interaction were the main binding forces to maintain the stability of the C3G-CM protein complexes. Molecular dynamics simulation results showed that all complexes except for C3G-αs1 -CA were found to reach equilibrium within 50ns of simulation. αs1 -CA and β-CA switched to a more compact conformation after binding with C3G. Additionally, the radius of gyration, number of hydrogen bond, radial distribution function, and interaction energy showed that β-CA is the best C3G carrier protein among the four CM proteins. This study can provide valuable information for CM proteins to serve as C3G delivery carriers. PRACTICAL APPLICATIONS: Anthocyanins (ACNs) are flavonoid-based pigments that play an important functional role in regulating human's health. Cow's milk (CM) proteins are the most representative protein-based carriers that can improve the short-term bioavailability and stability of ACNs. Thus, it is important to study the interactions between ACNs and CM proteins at the molecular level for the development of effective ACNs delivery carriers. Our study showed that caseins (αs1 -CA and β-CA) had more hydrophobic and hydrogen-bonding sites with cyanidin-3-O-glucoside (C3G) than whey proteins using computational methods. Among the four CM proteins, β-CA was the best C3G carrier protein showing the best interaction stability with C3G. Thus, it is helpful for us to screen effective ACNs carriers from multiple protein sources by computational methods.

Prevention of Allergy to a Major Cow's Milk Allergen by Breastfeeding in Mice Depends on Maternal Immune Status and Oral Exposure During Lactation.

Background: The high incidence of food allergy in childhood points to the need of elucidating early life factors dictating allergy susceptibility. Here, we aim to address in a mouse model how the exposure to a major cow's milk allergen through breastmilk of mothers with different immune status influences food allergy outcome in offspring.Methods: BALB/cJ future dams were either kept naïve, or sensitized through the oral route using cholera toxin (“orally sensitized”) or through the i.p. route using alum (“i.p. sensitized”), or rendered fully tolerant (oral gavage without any adjuvant) to bovine β-lactoglobulin (BLG). After mating with naïve males and delivery, mothers were orally exposed or not to BLG during the whole lactation. Then, eight groups of lactating mothers were considered: naïve, i.p. sensitized, orally sensitized, or tolerant, each exposed or not during lactation. In order to specifically address breastmilk effects on their allergy susceptibility, pups from naïve-synchronized mothers were cross-fostered by the different groups of treated dams and lactating mothers at delivery. In some experiments, mothers kept their own pups to address a possible in utero effect. BLG antigen, BLG-specific antibodies, and BLG-immune complexes were measured in breastmilk from the different lactating mother groups. Allergic sensitization was monitored in 5-weeks old female offspring (n = 7–8/group of lactating mothers) by determining BLG-specific antibodies in plasma and splenocytes cytokine secretion after i.p. injections of BLG/alum. Allergic reaction to oral BLG challenge was evaluated by measuring mMCP1 in plasma.Results: Offspring was protected from one allergic i.p. sensitization when nursed by i.p. sensitized mothers, independently of BLG exposure during lactation. Orally sensitized dams conferred protection in offspring solely when exposed to BLG during lactation, while naïve mothers did not provide any protection upon BLG exposure. The levels of protection correlated with the levels of BLG-specific antibodies and BLG-immune complex in breastmilk. There was a trend for decreased sensitization in offspring breastfed by tolerant and exposed mothers, which was not associated with transfer of specific antibodies through breastmilk. Protection provided by nursing by treated/exposed mothers was not persistent after a boost i.p. injection of the progeny and then did not protect them from an allergic reaction induced at this time point. No additional in utero effects were evidenced.Conclusion: Our study demonstrates the strong potential of breastmilk to modulate immune response to a major cow's milk allergen in the progeny. It highlights the importance of maternal immune status and of her consumption of the allergen during lactation in dictating the outcomes in offspring. This opens perspectives where modulating maternal immune status might increase the chance of cow's milk allergy prevention in breastfed children.

Specific IgG4 antibodies to cow's milk proteins in pediatric patients with eosinophilic esophagitis

Allergen-specific IgG4 (sIgG4) antibodies are often associated with tolerance, but sIgG4 antibodies to causally relevant foods have been reported recently in adults with eosinophilic esophagitis (EoE). Prevalence and levels of food sIgG4 are not well established in the general pediatric population. We sought to investigate serum food sIgG4 with component diagnostics in children with EoE and children from an unselected birth cohort and to explore the effects of sex, age, and milk consumption on sIgG4 levels. Sera from 71 pediatric patients with EoE and 210 early adolescent children from an unselected birth cohort (Project Viva) were assayed for sIgG4 and specific IgE (sIgE) to major cow's milk (CM) proteins (α-lactalbumin, β-lactoglobulin, and caseins) and to wheat, soy, egg, and peanut proteins. In the EoE cohort high-titer sIgG4 (≥10μg/mL) to CM proteins was more common than in control sera and achieved odds ratios for EoE ranging from 5.5 to 8.4. sIgE levels to CM proteins were mostly 4 IU/mL or less in patients with EoE, such that sIgG4/sIgE ratios were often 10,000 or greater. When adjusted for age and milk consumption, high-titer sIgG4 to CM proteins was strongly associated with EoE, with an odds ratio of greater than 20 to all 3 CM proteins in boys. sIgG4 to CM proteins are common and high titer in children with EoE. Although it is not clear that this response is pathogenic, sIgG4 levels imply that these antibodies are an important feature of the local immune response that gives rise to EoE.

A rapid analytical method of major milk proteins byreversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with rapid and automated analysis, good separation, high resolution, high accuracy and reproducible results was successfully developed and used to separate and quantify the major cow milk proteins within 30min analytical time. Standard solutions of single purified cow milk proteins were used to develop calibration equations. The RP-HPLC method was validated with respect to intra-day repeatability, inter-day precision, linearity, accuracy and limit of detection (LOD). The recoveries of the RP-HPLC analyses of major milk proteins from cows ranged 71.0-114%, the inter-day precision was expressed as the relative standard deviation, and the ranged from 1.51 to 4.60% and the LOD ranged from 0.08g/L to 0.28g/L. Major proteins in cow were quantified according to the chromatographic profiles. Results showed that a rapid RP-HPLC method for quantifying the major cow milk proteins was developed, which could be used to determine milk protein contents in the dairy industry.

Immune status of the mother and early exposure through breastfeeding both influence the protection of the progeny from food allergy development to a major cow's milk allergen

Early tolerance induction is an attractive approach for primary prevention of food allergies. Using a mouse model, we aimed to assess the role of the immune status of the mother and the role of exposure during breastfeeding on the food allergy induced in the progeny. Females BALB/c mice were either orally tolerized or orally sensitized to bovine β-lactoglobulin (BLG), a major cow’s milk allergen. Naive mice were used as controls. Females were then mated with age matched naive males. At delivery, pups were bred by their mothers or pups from sensitized/tolerised mother were replaced by pups from naive mothers to focus on the breastfeeding effect. Mothers were then exposed or not to BLG by gavage every other day until weaning. BLG, specific Ig and BLG-IgG immune complexes were assayed in breast milk. When 5-week old, progeny was experimentally sensitized to BLG and sensitization levels were assessed by measurement of BLG-specific IgE and IgG1. The allergic reaction was assessed by mMCP1 measurement in sera obtained 1hr after an oral challenge with BLG. BLG-specific IgE, IgA and IgG1 and BLG-IgG1 immune complexes were detected only in milk from sensitized mothers. Exposition to BLG during breastfeeding increased these levels. Pups from and bred by naive mothers exposed to BLG during breastfeeding demonstrated the same sensitization and allergic reaction levels than control pups. Conversely, a significant protection of the progeny was observed in pups from naive or orally-sensitized mothers breastfed by orally sensitized mothers. The protective effect was enhanced if the mothers are exposed to BLG during breastfeeding, correlating with the level of IgG1-BLG complexes in breast milk. Protection of the progeny was less efficient when mother were orally tolerized and occurred only if there is exposure to the allergen during breastfeeding. Altogether, these results thus demonstrate that protection of the progeny from food allergy depends on the immune status of the mother, i.e. naive vs orally sensitized or tolerized. Importantly, exposition of the non-naive mother to the allergen by the oral route during breastfeeding allows enhancing this protective effect.

Detection of Bovine Alpha-S1-Casein in Term and Preterm Human Colostrum with Proteomic Techniques

Due to increased social awareness of allergens and population hyper-sensitization, the reported incidence of allergic reactions to food allergens has increased over the past two decades. Cow's milk proteins (CMPs) are among the most common food allergens. The aim of this study was to use proteomics techniques to investigate cow's milk allergens in both full-term human colostrum and in preterm newborns mothers where both groups showed no prior allergen detection -- in order to understand whether cows milk allergens could be a cause of sensitization established through lactation. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using techniques detailed in this paper and which allowed for direct protein identification, beta-lactoglobulin was not detected in any of the colostrum samples. According to our results, bovine alpha 1 casein is considered a major cow's milk allergen, is readily secreted in human milk, and so could be considered a possible cause of sensitization in exclusively breastfed infants.

Structural changes and allergenic properties of β‐lactoglobulin upon exposure to high‐intensity ultrasound

The aim of this work was to investigate the effects of high-intensity ultrasound (sonication), on the structure and allergenicity of the major cow's milk allergen, beta-lactoglobulin (BLG). Structural changes upon sonication of BLG were monitored by circular dichroism spectroscopy, tryptophan emission fluorescence, hydrophobic dye and retinol binding, as well as digestibility and phenol-oxidase cross-linking capacity. Allergenicity was monitored in individual patients' sera, basophil activation test, and skin prick testing in 41 cow's milk allergy patients. Uncontrolled local temperature changes induced modifications in BLG secondary structure accompanied by formation of dimers, trimers, and oligomers of BLG that were more digestible by pepsin and had reduced retinol binding. Controlled temperature conditions induced changes in secondary structure of BLG without causing formation of oligomers, or changing protein's capacity to bind retinol. Both sonicated forms of BLG had more exposed hydrophobic surfaces than native BLG and underwent facilitated cross-linking reaction with phenol-oxidase. Sonication had a minor effect on IgE-binding properties of BLG. Sonication-induced structural changes in major whey allergen were not clinically significant in cow's milk allergy patients. Ultrasound can be a safe procedure for dairy processing as it maintains the nutritional value and does not increase allergenic potential of BLG.

Production of Fibronectin Binding Protein A at the Surface of Lactococcus lactis Increases Plasmid Transfer In Vitro and In Vivo

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine β-lactoglobulin

Food allergy is considered as resulting from an impaired development or a breakdown of oral tolerance. We aimed to induce oral tolerance to the major cow's milk allergen bovine β-lactoglobulin (BLG) or corresponding trypsin hydrolysates (BLG-Try) and to investigate the mechanisms involved. Wild-type BALB/cJ mice were gavaged on days 1-3 and 8-10 with different doses of native BLG (nBLG) or with nBLG-Try and were then sensitized on day 14 by i.p. administration of BLG in alum. Sensitization was assessed by measurement of BLG-specific antibodies in sera and of cytokines secreted by BLG-reactivated splenocytes. Elicitation of the allergic reaction was assessed by measurement of cytokines and mMCP-1 in sera collected 35 min after an oral challenge. Cellular and biochemical markers of the allergic reaction were also analysed in bronchoalveolar lavage fluids (BAL) collected 24 h after intra-nasal challenge. Analysis of the CD4(+) CD25(+) Foxp3(+) cells in different organs obtained 3 days after gavage and in vivo depletion of CD25(+) cells before oral tolerance induction were then performed. Systemic sensitization and elicitation of the allergic reaction were totally inhibited in mice gavaged with 2 mg of nBLG whereas nBLG-Try was far less efficient. A high percentage of CD4(+) Foxp3(+) cells were observed in BAL from tolerant mice, and a negative correlation between the number of eosinophils and the percentage of Foxp3(+) cells was evidenced. Efficient induction of CD4(+) CD25(+) Foxp3(+) cells after nBLG gavage and impaired oral tolerance induction after in vivo depletion of CD25 cells were then demonstrated. For the first time, allergen-induced Treg cells that inhibited both the sensitization and the elicitation of the allergic reaction were evidenced in gavaged wild-type mice.

Visualization of clustered IgE epitopes on α-lactalbumin

alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. We performed molecular, structural, and immunologic characterization of alpha-La. Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

Contribution of IgE and immunoglobulin free light chain in the allergic reaction to cow's milk proteins

Cow's milk allergy (CMA) affects 2.5% of young infants. In previous murine studies it was observed that allergic sensitization to the major cow's milk allergens casein and whey led, respectively, to IgE-independent and IgE-dependent clinical responses. In this study the involvement of immunoglobulin free light chains (Ig-fLCs) in the hypersensitivity response to cow's milk proteins was explored in mice, and Ig-fLC serum levels were determined in children affected by CMA or atopic dermatitis (AD). Mice were orally sham, casein, or whey sensitized. Acute allergen-specific skin responses were determined, and serum immunoglobulin and Ig-fLC concentrations were measured. Ig-fLC dependency was validated by using the Ig-fLC blocker F991 in actively and passively sensitized mice. Ig-fLC serum concentrations were measured in a cohort of infants with CMA and infants with AD. After sensitization, no specific IgE was detectable in sera of casein-sensitized mice, whereas specific IgE levels were enhanced in whey-sensitized mice. Instead, Ig-fLC levels were increased in sera from casein-sensitized mice. Furthermore, blocking Ig-fLCs strongly diminished the allergic skin responses not only in casein-sensitized mice but also in mice transferred with splenocyte supernatants of casein-sensitized mice. In both patients with CMA and patients with AD, serum Ig-fLC concentrations were significantly enhanced. This study indicates that sensitization with cow's milk proteins can lead to both IgE-dependent and Ig-fLC-dependent allergic hypersensitivity responses. Also, in children affected with CMA or AD, serum Ig-fLC concentrations were increased, implying the relevance of Ig-fLC measurements in the diagnoses of human allergic disease.

Influence of the route of administration on immunomodulatory properties of bovine β-lactoglobulin-producing Lactobacillus casei

Because of their intrinsic immunomodulatory properties, some lactic acid bacteria were reported to modulate allergic immune responses in mice and humans. We recently developed recombinant strains of Lactobacillus casei that produce β-lactoglobulin (BLG), a major cow's milk allergen. Here, we investigated immunomodulatory potency of intranasal and oral administrations of recombinant lactobacilli on a subsequent sensitization of mice to BLG. Intranasal administration of the BLG-producing Lb. casei stimulated serum BLG-specific IgG2a and IgG1 responses, and fecal IgA response as well, but did not inhibit BLG-specific IgE production. In contrast, oral administration led to a significant inhibition of BLG-specific IgE production while IgG1 and IgG2a responses were not stimulated. After both oral and intranasal administrations, production of IL-17 cytokine by BLG-reactivated splenocytes was similarly enhanced, thus confirming the adjuvant effect of the Lb. casei strain. However, a mixed Th1/Th2 cell response was evidenced in BLG-reactivated splenocytes from mice intranasally pretreated, with enhanced secretions of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-4 and IL-5) whereas only production of Th1 cytokines, but not Th2 cytokines, was enhanced in BLG-reactivated splenocytes from mice orally pretreated. Our results show that the mode of administration of live bacteria may be critical for their immunomodulatory effects.

Evaluation Of Specific Sequential IgE- and IgG4-binding Epitopes, Recognized in Cow's Milk Allergic Patients during Specific Oral Desensitization, using Peptide Microarray Immunoassay.

RATIONALE: Cow's milk allergy is the most common food allergy in childhood. Oral desensitization may represent a valid alternative to elimination diet. Sequential IgE-binding epitopes have been characterized for the major cow's milk allergens, some of which are recognized only by patients with persistent allergy. The aim of this study was to identify specific IgE and IgG4 epitope binding patterns in milk allergic patients and the immunological changes that occurred after oral desensitization, using the microarray (MIA) technology.