Circulating Ara h 6 as a marker of peanut protein absorption in tolerant and allergic humans following ingestion of peanut-containing foods.

BACKGROUND: Bioaccessibility of food allergens may be a key determinant of allergic reactions. OBJECTIVE: To develop a protocol allowing the detection of the major peanut allergen, Ara h 6, in the bloodstream following ingestion of low amounts of peanut and to compare Ara h 6 bioaccessibility by food matrix. We further assessed for differences in absorption in healthy versus peanut-allergic volunteers. METHODS: A blood pretreatment combining acidic shock and thermal treatment was developed. This protocol was then applied to blood samples collected from human volunteers (n = 6, healthy controls; n = 14, peanut-allergic patients) at various time-points following ingestion of increasing levels of peanut incurred in different food matrices (cookies, peanut butter and chocolate dessert). Immunodetection was performed using an in-house immunoassay. RESULTS: An original pretreatment protocol was optimized, resulting in irreversible dissociation of human antibodies-Ara h 6 immune complex, thus rendering Ara h 6 accessible for its immunodetection. Ara h 6 was detected in samples from all volunteers following ingestion of 300-1000 mg peanut protein, although variations in the kinetics of passage were observed between individuals and matrices. Interestingly, in peanut-allergic subjects, Ara h 6 could be detected following ingestion of lower doses and at higher concentrations than in non-allergic volunteers. CONCLUSIONS AND CLINICAL RELEVANCE: The kinetics and intensity of Ara h 6 passage in bloodstream depend on both individual and food matrix. Peanut-allergic patients appear to demonstrate higher absorption rate, the clinical significance of which warrants further evaluation.

Deep analysis of immune response and metabolic signature in children with food protein induced enterocolitis to cow's milk.

BACKGROUND: Food Protein-Induced Enterocolitis Syndrome (FPIES) is considered to be a non-IgE mediated food allergy. However, its pathogenesis remains poorly understood and biomarkers are lacking. We aimed to perform in-depth characterization of humoral and cellular immune responses in children with cow's milk (CM)-FPIES and investigated whether there is a FPIES metabolomic signature. METHODS: Children with CM-FPIES and control subjects with an IgE-mediated CM allergy (IgE-CMA), both avoiding CM, were recruited on the day of an oral food challenge. Blood samples were collected before the challenge. Total and specific levels of IgE, IgG1-4, IgA, IgM and IgD to various whey and casein allergens and to their gastroduodenal digestion products were measured in plasma, using plasma from CM-tolerant peanut allergic patients (IgE-PA, not avoiding CM) as additional controls. Cytokine secretion and cellular proliferation were analyzed after stimulation of PBMC with different CM allergens. Metabolomic profiles were obtained for plasma samples using liquid chromatography coupled to high-resolution mass spectrometry. RESULTS: Nine children with CM-FPIES and 12 control subjects (6 IgE-CMA and 6 IgE-PA) were included. In children with CM-FPIES, total Ig concentrations were lower than in control subjects, specific Ig against CM components were weak to undetectable, and no specific IgE against CM digestion products were detected. Moreover, in CM-FPIES patients, we did not find any Th cell proliferation or associated cytokine secretion after allergen reactivation, whereas such responses were clearly found in children with IgE-CMA. Plasma metabolic profiles were different between CM allergic patients, with significantly lower concentrations of various fatty acids and higher concentrations of primary metabolites such as amino acids in CM-FPIES compared to IgE-CMA patients. CONCLUSIONS: In CM-FPIES, both humoral and cellular specific immune responses are weak or absent, and this is not related to CM avoidance. A metabolomic signature was identified in patients with CM-FPIES that may be useful for the diagnosis and management of this disease.

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.

Specificity of IgE antibodies from patients allergic to goat's milk and tolerant to cow's milk determined with plasmin-derived peptides of bovine and caprine ß-caseins.

SCOPE: Despite a sequence homology of 90% between bovine and caprine ß-caseins (CN), IgE antibodies from patients allergic to goat's milk (GM), but tolerant to cow's milk (CM), recognize caprine ß-CN without cross-reacting with bovine ß-CN. We investigated this lack of cross-reactivity by evaluating the IgE-reactivity toward peptides isolated from plasmin hydolysates of bovine and caprine ß-CN. METHODS AND RESULTS: The IgE-binding capacity of plasmin-derived peptides was evaluated with sera from 10 CM-allergic patients and 12 GM-allergic/CM-tolerant patients. In CM-allergic patients, IgE reactivity of caprine fragments (f29-107) and (f108-207), but not (f1-28), was similar to that of the bovine counterparts. In contrast, all bovine fragments were poorly recognized by IgE antibodies from GM-allergic/CM-tolerant patients. The peptide (f29-107) was generally the most immunoreactive fragment of caprine ß-CN. By using synthetic peptides, the immunodominant IgE-binding epitope recognized by most GM-allergic/CM-tolerant patients was located in the caprine domain 49-79. CONCLUSION: The restricted specificity of the IgE response toward the caprine ß-CN in GM-allergic/CM-tolerant patients is mainly directed against the domain 49-79, which differs from its bovine counterpart by only three amino acid substitutions.

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse.

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.

Block copolymers have differing adjuvant effects on the primary immune response elicited by genetic immunization and on further induced allergy.

Block copolymers were recently used to promote gene delivery in various tissues. Using a plasmid encoding a food allergen, bovine beta-lactoglobulin (BLG), we studied the effects of block copolymers on gene expression levels and primary immune response and on further induced allergy. Block copolymers (i.e., Tetronic 304, 908, and 1107) and various quantities of DNA were injected into the tibialis muscles of BALB/c mice. The BLG levels in injected muscle and the BLG-specific induced immune response were analyzed after injection. DNA-immunized mice were further experimentally sensitized with BLG, and the effects of block copolymer and DNA doses on allergic sensitization and elicitation were compared. Tetronic 304 induced a 12-fold increase in BLG production, while Tetronic 1107 increased the duration of BLG expression. Different Th1 primary specific immune responses were observed, either strong humoral and cellular (304), only cellular (1107), or weak cellular and humoral (908) responses. After BLG sensitization, increased BLG-specific IgG2a production was observed in all groups of mice independently of the presence and nature of the block copolymer. Increased BLG-specific IgG1 production was also detected after sensitization, except with Tetronic 1107. Compared with naked DNA, Tetronic 304 was the only block polymer that decreased BLG-specific IgE concentrations. However, after allergen challenge, Tetronic 1107 was the only block copolymer to reduce eosinophils and Th2 cytokines in bronchoalveolar lavage (BAL) fluid. Tetronic 304 amplified local inflammation. Each block copolymer elicited a different immune response, although always Th1 specific, in BALB/c mice.

Allergy therapy by intranasal administration with recombinant Lactococcus lactis Producing bovine beta-lactoglobulin.

BACKGROUND: In the last years, the use of probiotics such as lactic acid bacteria (LAB) has been proposed as an attractive alternative for the management of allergic diseases. A partial prevention from sensitization to bovine beta-lactoglobulin (BLG), one of the major cows' milk allergens, could be achieved in mice after intranasal administration with a recombinant LAB strain, Lactococcus lactis, producing BLG (LL-BLG). This study aimed to evaluate the effects of the LL-BLG strain in a therapeutic protocol. METHODS: Three groups of mice were first orally sensitized to cows' milk and then intranasally administered with either the LL-BLG strain, BLG protein alone or saline solution. Serum samples were collected to analyze BLG-specific IgE, IgG1 and IgG2a, and mice were further intranasally challenged with BLG to elicit a specific allergic reaction. RESULTS: Treatment with LL-BLG, but not with BLG alone, contributed to diminish IgG1 production in serum and bronchoalveolar lavage fluids. This was associated with decreased IL-4 production and enhanced IFN-gamma production by BLG-reactivated splenocytes, suggesting a switch from Th2- to Th1-immune response. Furthermore, we observed that administration of LL-BLG or LL locally reduced the allergic reaction induced after intranasal challenge, as evidenced by decreased release of IL-4 in bronchoalveolar lavage fluids. CONCLUSION: These preliminary results demonstrate the efficiency of the intranasal administration of LL-BLG for specific therapy against cows' milk-related allergy.

Current prophylactic and therapeutic uses of a recombinant Lactococcus lactis strain secreting biologically active interleukin-12.

The noninvasive and food-grade Gram-positive bacterium Lactococcus lactis is well adapted to deliver medical proteins to the mucosal immune system. In the last decade, the potential of live recombinant lactococci to deliver such proteins to the mucosal immune system has been investigated. This approach offers several advantages over the traditional systemic injection, such as easy administration and the ability to elicit both systemic and mucosal immune responses. This paper reviews the current research and advances made with recombinant L. lactis as live vector for the in situ delivery of biologically active interleukin-12, a potent pleiotropic cytokine with adjuvant properties when co-delivered with vaccinal antigens, at mucosal surfaces. Three well-illustrated examples demonstrate the high potential of interleukin-12-secreting lactococci strains for future prophylactic and therapeutic uses.

Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice.

The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the induction of a protective Th1 response by the association of L. lactis BLG and IL-12-producing L. lactis which inhibits the elicitation of the allergic reaction to BLG in mice and (ii) the efficiency of intranasal administration of BLG for the induction of tolerance.

Oral sensitization to peanut is highly enhanced by application of peanut extracts to intact skin, but is prevented when CpG and cholera toxin are added.

BACKGROUND: CpG oligonucleotides might offer an alternative to conventional immunotherapy in preventing and potentially reversing Th2-biased immune deregulation which leads to allergy. However, non-invasive ways of administration, especially in peanut-allergic patients, should be explored. METHODS: One hundred micrograms of whole peanut protein extract (PE) alone, or mixed with cholera toxin (CT, 50 microg) plus CpG (100 microg) as adjuvant, was applied on intact skin of mice (40 min, twice). Initiation of an immune response was monitored by detection of specific antibodies in sera. The effect of this pretreatment on a further oral sensitization by PE was then evaluated by assaying antibodies and cytokines specific for PE and purified allergens. Cytokine production in liver 40 min after skin application was also assayed. RESULTS: Two brief skin applications of PE alone highly potentiated further oral sensitization, as demonstrated by very intense specific IgE, IL-4 and IL-5 productions. Conversely, skin pretreatment with PE and CT + CpG efficiently prevented further sensitization via gastro-intestinal exposure. In both cases, the specificity of the antibodies and cytokines was the same as in control mice. CT + CpG treatment allowed the rapid production of IL-12 and TGFbeta in liver and of specific IgG2a in sera, suggesting the activation of Th1 and/or regulatory T cells. CONCLUSIONS: Oral sensitization to peanut is highly enhanced by a previous short exposure of allergens to intact skin. Conversely, the use of CT + CpG adjuvant for skin application efficiently prevents further oral sensitization. The potential of such treatment in specific immunotherapy needs to be evaluated.

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.