Glycation of Whey Proteins Increases the Ex Vivo Immune Response of Lymphocytes Sensitized to ß-Lactoglobulin.

Glycation is a spontaneous reaction accompanying the thermal processing and storage of food. It can lead to changes in the allergenic and immunogenic potential of protein. This study aimed to evaluate the effect of the glycation of α-lactalbumin and ß-lactoglobulin (ß-lg) on the ex vivo response of ß-lg sensitized lymphocytes. C57BL/6 mice were immunized intragastrically (i-g) or intraperitoneally (i-p) with ß-lg. The humoral response of the groups differed only with respect to the IgE level of the i-p group. Cellular response was studied after stimulation with antigen variants. The lymphocytes from the i-g/group mesenteric lymph nodes, stimulated with ß-lg before and after glycation, presented a higher percentage of CD4 and CD8 T cells compared to the i-p/group. The cytokine profile of the i-p/group splenocytes stimulated with antigens showed elevated levels of pro-inflammatory IL-17A regardless of protein modification. In conclusion, the ex vivo model proved that the glycation process does not reduce protein immunogenicity.

OVA-Experienced CD4+ T Cell Transfer and Chicken Protein Challenge Affect the Immune Response to OVA in a Murine Model.

Chicken meat is often a major component of a modern diet. Allergy to chicken meat is relatively rare and occurs independently or in subjects allergic to ovalbumin (OVA). We examined the effect of adoptive transfer of OVA-CD4+ T cells on the immune response to OVA in mice fed chicken meat. Donor mice were injected intraperitoneally with 100 µg of OVA with Freund's adjuvant two times over a week, and CD4+ T cells were isolated from them and transferred to naïve mice (CD4+/OVA/ChM group), which were then provoked with OVA with FA and fed freeze-dried chicken meat for 14 days. The mice injected with OVA and fed chicken meat (OVA/ChM group), and sensitized (OVA group) and healthy (PBS group) mice served as controls. Humoral and cellular response to OVA was monitored over the study. The CD4+/OVA/ChM group had lowered levels of anti-OVA IgG and IgA, and total IgE. There were significant differences in CD4+, CD4+CD25+, and CD4+CD25+Foxp3+ T cells between groups. OVA stimulation decreased the splenocyte proliferation index and IFN-γ secretion in the CD4+/OVA/ChM group compared to the OVA group. IL-4 was increased in the OVA/ChM mice, which confirms allergenic potential of the egg-meat protein combination. Transfer of OVA-experienced CD4+ T cells ameliorated the negative immune response to OVA.

Differences in Regulatory Mechanisms Induced by ß-Lactoglobulin and κ-Casein in Cow's Milk Allergy Mouse Model-In Vivo and Ex Vivo Studies.

The presence of various proteins, including modified ones, in food which exhibit diverse immunogenic and sensitizing properties increases the difficulty of predicting host immune responses. Still, there is a lack of sufficiently reliable and comparable data and research models describing allergens in dietary matrices. The aim of the study was to estimate the immunomodulatory effects of ß-lactoglobulin (ß-lg) in comparison to those elicited by κ-casein (κ-CN), in vivo and ex vivo, using naïve splenocytes and a mouse sensitization model. Our results revealed that the humoral and cellular responses triggered by ß-lg and κ-CN were of diverse magnitudes and showed different dynamics in the induction of control mechanisms. ß-Lg turned out to be more immunogenic and induced a more dominant Th1 response than κ-CN, which triggered a significantly higher IgE response. For both proteins, CD4+ lymphocyte profiles correlated with CD4+CD25+ and CD4+CD25+Foxp3+ T cells induction and interleukin 10 secretion, but ß-lg induced more CD4+CD25+Foxp3- Tregs. Moreover, ex vivo studies showed the risk of interaction of immune responses to different milk proteins, which may exacerbate allergy, especially the one caused by ß-lg. In conclusion, the applied model of in vivo and ex vivo exposure to ß-lg and κ-CN showed significant differences in immunoreactivity of the tested proteins (κ-CN demonstrated stronger allergenic potential than ß-lg), and may be useful for the estimation of allergenic potential of various food proteins, including those modified in technological processes.

Effect of Low-Immunogenic Yogurt Drinks and Probiotic Bacteria on Immunoreactivity of Cow's Milk Proteins and Tolerance Induction-In Vitro and In Vivo Studies.

There is no effective therapy for milk allergy. The role of lactic acid bacteria (LAB) and probiotics in protection against allergy-related outcomes is still under investigation. The aim of the study was to evaluate the immunomodulative and therapeutic potential of yogurt drinks in cow's milk allergy (CMA) management. We compared immunoreactivity of α-casein (α-CN), ß-casein (ß-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) in 27 yogurt drinks fermented with different basic yogurt cultures, or yogurt cultures enriched with Lactobacillus plantarum and/or Bifidobacterium lactis strains, by competitive ELISA assay. Drinks with the lowest antigenic potential were used as allergoids for CMA therapy. BALB/c mice were sensitized via intraperitoneal injection of α-CN + ß-LG mixture with aluminum adjuvant, and gavaged with increasing doses of selected low-immunogenic drinks (YM-basic, or YM-LB-enriched with L. plantarum and B. lactis) to induce tolerance. Milk- or phosphate-buffered saline (PBS)-dosed mice served as controls. Compared to milk, the immunoreactivity of proteins in drinks increased or decreased, depending on the bacterial sets applied for fermentation. Only a few sets acted synergistically in reducing immunoreactivity. The selected low-immunogenic drinks stimulated allergic mice for profiling Th2 to Th1 response and acquire tolerance, and the effect was greater with YM-LB drink, which during long-lasting interventional feeding strongly increased the secretion of regulatory cytokines, i.e., IL-10 and TGF-ß, and IgA and decreased IL-4, IgE, and anti-(α-CN + ß-LG) IgG1. The studies revealed variations in the potency of yogurt bacteria to change allergenicity of milk proteins and the need for their strict selection to obtain a safe product for allergy sufferers. The YM-LB drink with reduced antigenic potential may be a source of allergoids used in the immunotherapy of IgE mediated CMA, but further clinical or volunteer studies are required.

Milk and Meat Allergens from Bos taurus ß-Lactoglobulin, α-Casein, and Bovine Serum Albumin: An In-Vivo Study of the Immune Response in Mice.

The mechanism of food allergy may vary. This study aimed to compare the effects of milk, yogurt, or beef meat supplementation on humoral and cellular immune responses in a mice model. Mice were divided into four groups: The "Milk group" was sensitized with a ß-lactoglobulin (ß-lg)/α-casein (α-CN) mixture and supplemented cow milk; the "Yogurt group" was sensitized with ß-lg/α-CN and supplemented yogurt; the "Beef group" was immunized with bovine serum albumin (BSA) and supplemented beef meat; and the "PBS group" received PBS in all procedures. ELISA was used to measure humoral response, including: Total IgE, specific IgG, and IgA. Cellular response was determined by phenotyping lymphocyte from lymphoid tissue and measuring the Th1/Th2 cytokine concentration with flow cytometry. The qPCR method was used for quantification of the fecal microbiota. The results obtained revealed a lower IgE level for the Yogurt group than for the Milk one. In the Yogurt group, the contribution of regulatory T cells to MLN and PP was higher compared to the other groups. We confirmed that diet supplementation with yogurt modulates the immune response to the prime allergen, and changes the activity of serum antibodies to milk proteins and BSA. Based on a specific antibodies level, we cannot exclude the possibility of CMA mice reaction against BSA.

Immunoreactive properties of α-casein and κ-casein: Ex vivo and in vivo studies.

The aim of this study was to evaluate the ex vivo and in vivo studies immune potential of α- and κ-casein. Ex vivo, naïve mouse splenocytes were stimulated with α- or κ-casein. After 120 h of culture, the proliferation index (PI), determined by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, did not vary for either antigen, suggesting similar ex vivo immunogenic potential of both casein fractions. In vivo, BALB/ccmdb mice were sensitized with α- or κ-casein and then gavaged with primary antigen. Mice immunized with α-casein had higher levels of IgG (216.33) and IgA (210.22) in serum at the end of the experiment compared with mice immunized with κ-casein (215 and 29.3 for IgG and IgA, respectively). The use of α-casein for mouse immunization and ex vivo lymphocyte stimulation resulted in higher concentrations of secreted cytokines (IL-4, IL-10) compared with κ-casein stimulation. This is consistent with increasing regulatory T cell (Treg) lymphocyte populations, independent of the antigen used for stimulation. In summary, the immunogenic potential of α- and κ-casein was similar. Humoral and cellular immune responses confirmed their strong, independent potential to induce B and T cells. We propose that the lymphocyte proliferation index be used as an initial screening for protein immunogenicity.