In vitro determination of the allergenic potential of technologically altered hen's egg.

Hen's egg allergy represents one of the most common and severe IgE-mediated reactions to food in infants and young children. It persists, however, in many cases also lifelong. Therefore, the aim of this study was the detailed analysis of a technological process used to reduce the allergenic potential of hen's egg. The investigation focused on the pasteurized egg as starting material, intermediate, and final products of a nine-step manufacturing process performed for use of eggs in convenience products appropriate for allergic individuals. The steps consisted of a combination of various heat treatments and enzymatic hydrolyses. The alterations were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, enzyme allergosorbent test (EAST) inhibition, and mass spectrometry. Thereby it could be demonstrated that the allergenic potential of the raw material was reduced from step to step, and despite the known stability against heat and proteolysis of certain egg proteins, the total allergenic potential was finally below 1/100 that of the starting material without a significant change in texture and flavor as evaluated in various products.

Food products and allergy development, prevention and treatment.

In westernized countries allergic diseases have reached epidemic proportions. Food is frequently a perpetrator of allergy but, in turn, modified food and selected food ingredients can become valuable intervention tools in the fight against allergy. There are two basic approaches towards mitigation of food allergy through nutrition: to reduce the allergenicity of raw food materials by physical, chemical or genetic methods or to influence host immunity towards a non-allergic state using various food ingredients. Dietary intervention for the prevention and therapy of allergy is an emerging field where initial findings from animal studies are now being validated in human trials. Nevertheless, to consolidate the utility of such interventions, more pre-clinical and clinical studies remain necessary.

Oral Immunotherapy With Partially Hydrolyzed Wheat-Based Cereals: A Pilot Study.

To date, only few studies have assessed oral immunotherapy (OIT) for wheat allergy and often describe severe adverse reactions during therapy. We developed partially hydrolyzed wheat-based cereals (pHC), which were used in a multicenter, open-label, OIT pilot study, in immunoglobulin E-mediated wheat allergy children (NCT01332084). The primary objective of the study was to test whether wheat allergic patients tolerate pHC and primary end point was the presence or not of immediate adverse reactions to pHC during the 1-day initial escalation phase (stepwise increased doses of pHC), with evaluation of the maximum dose tolerated. Of the 9 patients enrolled in the trial, 4 discontinued OIT because of mild to severe reactions at the initial escalation phase. The 5 patients who passed the escalation phase consumed pHC daily for 1 to 6 months. One of these patients withdrew due to noncompliance, whereas the 4 others completed the study and successfully passed the wheat challenge test at the end of the study. About 60% of the adverse events were unrelated to the study product. Our study provides preliminary evidence that pHC is tolerated by a subset of wheat allergic patients. Further studies are warranted to test its efficacy as a potential therapeutic option for wheat allergic patients.

Animal models in food allergy: assessment of allergenicity and preventive activity of infant formulas.

Food allergies occur in about 5-10% of the overall infant and small-child population. Cow's milk protein allergy (CMPA) is the most common in young infants, with a 2-4% incidence. When breastfeeding is not possible, hypoallergenic (HA) cow's milk based formulas are usually given during the first months of life for prevention of CMPA. Depending on primary (sensitization) or secondary (triggering) prevention, the requested quality of HA formulas may be different. Besides in vitro methods, in vivo and ex vivo animal models are helpful in assessing residual allergenicity and the preventive effect of HA formulas. The sensitizing capacity of a formula can be examined by either the parenteral rat (IgE), the guinea pig (IgG1a mediated) or the oral mouse (IgE) models. The triggering IgE mediated allergenicity is tested by a parenteral rat model with oral gavage for intestinal mast cell protease (RMCPII) release. These animal models are also used for testing the oral tolerance inducing capacities of formulas. Together with cellular in vitro assays, animal models are very helpful in predicting allergenicity and the tolerogenic potential of HA infant formulas.

Immunomodulatory potential of partially hydrolyzed ß-lactoglobulin and large synthetic peptides.

The immunomodulatory potential of fragments derived from the cow's milk allergen bovine ß-lactoglobulin (BLG) was assessed in a mouse model of oral tolerance (OT) [Adel-Patient, K.; Wavrin, S.; Bernard, H.; Meziti, N.; Ah-Leung, S.; Wal, J. M. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine ß-lactoglobulin. Allergy 2011, 66 (10), 1312-1321]. Native BLG (nBLG) and chemically denatured BLG (lacking S-S bridges, dBLG), products resulting from their hydrolysis using cyanogen bromide (CNBr) and some synthetic peptides, were produced and precisely characterized. CNBr hydrolysates correspond to pools of peptides of various sizes that are still associated by S-S bridges when derived from nBLG. nBLG, dBLG, and CNBr hydrolysate of nBLG efficiently prevented further sensitization. CNBr hydrolysate of dBLG was less efficient, suggesting that the association by S-S bridges of peptides increased their immunomodulatory potential. Conversely, synthetic peptides were inefficient even if covering 50% of the BLG sequence, demonstrating that the immunomodulatory potential requires the presence of all derived fragments of BLG and further supporting the use of partially hydrolyzed milk proteins to favor OT induction in infants with a risk of atopy.

Utility of animal models for evaluating hypoallergenicity.

The goal of this article is to describe the use of in vitro methods as well as in vivo and ex vivo animal models to assess residual allergenicity of hypoallergenic (HA) foods, especially infant formulas. These animal models are also used for testing the oral tolerance-inducing capacities of infant formula; thus, they are very helpful in predicting allergenicity and the tolerogenic potential of HA infant formulas, and in helping to prevent adverse reactions in sensitive infants.

IgE-mediated rat mast cell triggering with tryptic and synthetic peptides of bovine beta-lactoglobulin.

BACKGROUND: Immunoglobulin E (IgE) epitopes of beta-lactoglubulin (betaLG) have been identified by ELISA inhibition methods using sera from allergic patients. However, the functional capacity of these epitopes to stimulate mast cells is unknown. It is the goal of the present study to identify bivalent IgE epitopes of betaLG able to trigger target mast cells. METHODS: Peptides were obtained either by purification from tryptic hydrolysates of betaLG or by synthesis. They were examined for their triggering activity in vitro on peritoneal 3H-serotonin-labeled rat mast cells passively sensitized with IgE anti-betaLG antibodies. In vivo, rats immunized with betaLG were administered peptides by gavage for intestinal rat mast cell protease II release. RESULTS: Compared with intact betaLG, purified or synthetic tryptic-like betaLG peptides have a sharply decreased allergenicity. Peptide 149-162 retains the highest bivalent IgE epitope-mediated triggering capacity. CONCLUSION: A functional bivalent IgE epitope was identified at the C terminal end of betaLG.

Lactobacillus bulgaricus proteinase expressed in Lactococcus lactis is a powerful carrier for cell wall-associated and secreted bovine beta-lactoglobulin fusion proteins.

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine beta-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB partial differential, PrtB partial differential-BLG, and PrtB partial differential-T6) or 807 (PrtBdelta) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB partial differential-BLG yielded up to 170 microg per 10(9) CFU in the culture supernatant and 9 microg per 10(9) CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.

Immunostimulatory potential of beta-lactoglobulin preparations: effects caused by endotoxin contamination.

BACKGROUND: The immunomodulating potential residing in cow's milk proteins is currently receiving increasing attention because of growing interest in functional foods and the complex problem of cow's milk allergy. One of the major cow's milk allergens, whey protein beta-lactoglobulin, has previously been shown to mediate cellular activation in both human and murine immune cells. OBJECTIVE: We examined the response to different beta-lactoglobulin preparations in naive immune cells. METHODS: Splenocytes and cells from mesenteric lymph nodes derived from BALB/c mice bred and maintained on a milk-free diet were cultured in vitro with different beta-lactoglobulin preparations. Cell proliferation, cytokine production, and increases in intracellular glutathione were used as cellular activation markers. Moreover, the effect of beta-lactoglobulin on cytokine production in murine bone-marrow-derived dendritic cells was examined. RESULTS: We observed that some commercial beta-lactoglobulin preparations induced pronounced proliferation of both spleen cells and cells from mesenteric lymph nodes; production of TNF-alpha, IL-6, IL-1beta, and IL-10; and an increased level of intracellular glutathione in spleen cell cultures. Furthermore, TNF-alpha, IL-6, IL-1beta, and IL-10 production was induced in murine bone-marrow-derived dendritic cells. Purification of beta-lactoglobulin from raw milk using nondenaturating conditions, however, revealed that the beta-lactoglobulin per se did not possess the immunomodulatory activity. Eventually, the immunostimulatory effect was found to be caused by endotoxin contamination. CONCLUSION: These results identify endotoxin as the main immunostimulatory component present in some commercial beta-lactoglobulin preparations. Moreover, the present study makes it evident that immunomodulatory effects attributed to beta-lactoglobulin need to be reassessed.