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ABSTRACT: SCOPE: Cow's milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cow's milk β-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age. METHODS AND RESULTS: Oral sensitization to cow's milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to αS1- and κ-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice. CONCLUSION: Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.
Molecular Nutrition & Food Research 10/2012; · 4.30 Impact Factor
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ABSTRACT: Despite a sequence homology of 90% between bovine and caprine β-caseins (CN), IgE antibodies from patients allergic to goat's milk (GM), but tolerant to cow's milk (CM), recognize caprine β-CN without cross-reacting with bovine β-CN. We investigated this lack of cross-reactivity by evaluating the IgE-reactivity toward peptides isolated from plasmin hydolysates of bovine and caprine β-CN.
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 β-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.
The restricted specificity of the IgE response toward the caprine β-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.
Molecular Nutrition & Food Research 09/2012; 56(10):1532-40. · 4.30 Impact Factor
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ABSTRACT: 2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated.
Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71) or at Cys(73) -Cys(115) rendered Arg(16/20) or Arg(114) susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58) or at Cys(59) -Cys(107) led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products.
Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation.
Molecular Nutrition & Food Research 04/2012; 56(4):548-57. · 4.30 Impact Factor
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ABSTRACT: In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.
Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.
1D (1)H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D (1)H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins.
PLoS ONE 01/2012; 7(7):e39785. · 4.09 Impact Factor
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Fany Blanc,
Yvonne M Vissers,
Karine Adel-Patient,
Neil M Rigby,
Alan R Mackie,
A Patrick Gunning,
Nikolaus K Wellner,
Per S Skov,
Laetitia Przybylski-Nicaise,
Barbara Ballmer-Weber,
Laurian Zuidmeer-Jongejan,
Zsolt Szépfalusi,
Janneke Ruinemans-Koerts,
Ad P H Jansen,
Hervé Bernard, Jean-Michel Wal,
Huub F J Savelkoul,
Harry J Wichers,
E N Clare Mills
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ABSTRACT: Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1.
Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein.
Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.
Molecular Nutrition & Food Research 11/2011; 55(12):1887-94. · 4.30 Impact Factor
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ABSTRACT: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ-free (GF) mice exhibit Th2-polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant.
β-lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ- and αS1-caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG-reactivation, IL-4, IL-5, IL-13 and IFN-γ productions by splenocytes were higher in GF mice than in CV mice. Heat-treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat-treatment increased protein immunogenicity and led to the production of anti-BLG and anti-κ-casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL-4, IL-5 and IL-13 in BLG-reactivated splenocytes from CV mice.
Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.
Molecular Nutrition & Food Research 11/2011; 55(11):1700-7. · 4.30 Impact Factor
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Karine Adel-Patient,
Valeria D Guimaraes,
Alain Paris,
Marie-Françoise Drumare,
Sandrine Ah-Leung,
Patricia Lamourette,
Marie-Claire Nevers,
Cécile Canlet,
Jérôme Molina,
Hervé Bernard,
Christophe Créminon, Jean-Michel Wal
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ABSTRACT: 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.
PLoS ONE 01/2011; 6(1):e16346. · 4.09 Impact Factor
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ABSTRACT: Cow's milk (CM) allergy affects about 2% of infants. The allergenicity of dietary proteins, including those from CM, has been related to their digestibility although the generality of the link and its causality remains to be demonstrated. In this study we use an in vitro digestion system, to investigate the digestibility of β-lactoglobulin (blg) during gastrointestinal transit and to assess the impact of this process on blg allergenic reactivity in CM allergic children.
Blg digesta were prepared using an in vitro digestion protocol simulating either gastric digestion alone or followed by duodenal digestion with or without phosphatidylcholine (PC). Biochemical analysis of blg digesta was performed by SDS-PAGE and their concentration was measured by a sandwich ELISA. Assessment of their allergenic reactivity was done in vitro by EAST inhibition, specific basophil activation (basotest) and lymphocyte proliferation (PCNA-flow cytometry) assays using sera and cells from patients allergic to blg and in vivo by skin prick testing (SPT) of these patients.
Blg was only broken down to smaller peptides after gastro-duodenal digestion although a sizeable amount of intact protein still remained. Digestion did not modify the IgE binding capacity of blg except for gastro-duodenal digestion performed in the absence of PC. These results are consistent with the quantity of intact blg remaining in the digesta. Overall both gastric and gastroduodenal digestion enhanced activation of sensitized basophils and proliferation of sensitized lymphocytes by blg. However, there was a tendency towards reduction in mean diameter of SPT following digestion, the PC alone during phase 1 digestion causing a significant increase in mean diameter.
Digestion did not reduce the allergenic reactivity of blg to a clinically insignificant extent, PC inhibiting digestion and thereby protecting blg allergenic reactivity. SPT reactivity was reduced compared to blg immunoreactivity in in vitro tests.
Clinical and translational allergy. 01/2011; 1(1):6.
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Yvonne M Vissers,
Fany Blanc,
Per Stahl Skov,
Phil E Johnson,
Neil M Rigby,
Laetitia Przybylski-Nicaise,
Hervé Bernard, Jean-Michel Wal,
Barbara Ballmer-Weber,
Laurian Zuidmeer-Jongejan,
Zsolt Szépfalusi,
Janneke Ruinemans-Koerts,
Ad P H Jansen,
Huub F J Savelkoul,
Harry J Wichers,
Alan R Mackie,
Clare E N Mills,
Karine Adel-Patient
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ABSTRACT: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut.
Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6.
Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.
PLoS ONE 01/2011; 6(8):e23998. · 4.09 Impact Factor
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Valeria Guimaraes,
Marie-Françoise Drumare,
Didier Lereclus,
Michel Gohar,
Patricia Lamourette,
Marie-Claire Nevers,
Marie-Lisa Vaisanen-Tunkelrott,
Hervé Bernard,
Blanche Guillon,
Christophe Créminon, Jean-Michel Wal,
Karine Adel-Patient
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ABSTRACT: A pepsin resistance test performed at pH 1.2 and with high pepsin to protein ratio is one of the steps of the weight-of-evidence approach used for assessment of allergenicity of new proteins. However, the use of other in vitro digestibility tests, performed in more physiologically relevant conditions and in combination with immunological assays so as to increase the value of the information gained from the studies of stability of a novel protein to digestion for the overall allergenicity assessment, has been proposed. This study then aimed to investigate the stability to digestion of Cry1Ab protoxin and toxin, insecticidal proteins expressed in genetically modified crops, using simulated gastric fluid (SGF) at different pH values and pepsin-to-substrate ratios, in the presence or absence of physiological surfactant phosphatidylcholine (PC). Electrophoresis and immunoblot patterns and residual immunoreactivity of digesta were analyzed. Although Cry1Ab protoxin is extensively degraded at pH 1.2 with high pepsin-to-protein ratio, it is only slightly degraded at pH 2.0 and conserved its immunoreactivity. Furthermore, Cry1Ab proteins were demonstrated to be stable in a more physiologically relevant in vitro digestibility test (pH 2.5, pepsin-to-substrate ratio 1:20 (w/w) with PC). Factors such as pH, SGF composition, and pepsin-to-substrate ratio then greatly influence the digestion of Cry1Ab proteins, confirming that new and more physiologically relevant in vitro digestibility tests should be also considered to study the relationship between the resistance of a protein to digestion and its allergenicity.
Journal of Agricultural and Food Chemistry 02/2010; 58(5):3222-31. · 2.82 Impact Factor
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ABSTRACT: 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.
Clinical and vaccine immunology: CVI 11/2009; 17(1):36-42. · 2.37 Impact Factor
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ABSTRACT: Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.
Analytical and Bioanalytical Chemistry 06/2009; 395(1):139-46. · 3.78 Impact Factor
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ABSTRACT: 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.
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.
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.
These preliminary results demonstrate the efficiency of the intranasal administration of LL-BLG for specific therapy against cows' milk-related allergy.
International Archives of Allergy and Immunology 05/2009; 150(1):25-31. · 2.40 Impact Factor
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ABSTRACT: Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.
Molecular Nutrition & Food Research 06/2008; 52 Suppl 2:S166-75. · 4.30 Impact Factor
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ABSTRACT: 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.
Clinical and Vaccine Immunology 04/2007; 14(3):226-33. · 2.55 Impact Factor
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ABSTRACT: This review article deals with the assessment of cytotoxic and allergenic potential of bioactive proteins and peptides. It is evident that `novel' foods or nutraceuticals containing bioactive proteins and peptides must fulfill their proposed “health claim”. Furthermore, there is a need to assess their potential to exert adverse effects before they can be made widely available to consumers. A brief overview of compounds (i.e. proteins and peptides of animal and plant origin) and mechanisms involved in cytotoxic and allergenic (adverse) reactions is given along with some recent results obtained from ongoing studies. There are numerous proteins and peptides of plant and animal origin that are known to exhibit cytotoxic effects. There is evidence that many cytotoxic compounds described in the literature exclusively affect malignant cells leading to the assumption that a cancer protective effect could exist for such bioactive proteins and peptides. All the constituents that are responsible for the allergenicity of foods (as well as of pollens) are proteinaceous in nature. Some protein breakdown products, i.e. peptide fragments, may conserve part of the allergenicity of the native protein and thus can also be considered as allergens. The molecular basis of IgE recognition underlying cow's milk protein allergy is described. Some results from studies on volunteers fed caseinophosphopeptides or potentially hypotensive milk protein hydrolysates illustrate the major difference between allergenicity and immunogenicity. The data presented on the relationship between the structure of food proteins and peptides and their allergenicity shows the difficulty in trying to assess the “non-allergenicity” of products derived from an allergenic source, even if the process used involved extensive hydrolysis of the native protein(s). A `weight of evidence approach' for assessing the potential allergenicity of a novel protein with no history of prior allergenicity is also presented with regard to the current EU Regulations.
Current Pharmaceutical Design 02/2007; 13(9):897-920. · 3.87 Impact Factor
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ABSTRACT: This review article deals with the assessment of cytotoxic and allergenic potential of bioactive proteins and peptides. It is evident that 'novel' foods or nutraceuticals containing bioactive proteins and peptides must fulfill their proposed "health claim". Furthermore, there is a need to assess their potential to exert adverse effects before they can be made widely available to consumers. A brief overview of compounds (i.e. proteins and peptides of animal and plant origin) and mechanisms involved in cytotoxic and allergenic (adverse) reactions is given along with some recent results obtained from ongoing studies. There are numerous proteins and peptides of plant and animal origin that are known to exhibit cytotoxic effects. There is evidence that many cytotoxic compounds described in the literature exclusively affect malignant cells leading to the assumption that a cancer protective effect could exist for such bioactive proteins and peptides. All the constituents that are responsible for the allergenicity of foods (as well as of pollens) are proteinaceous in nature. Some protein breakdown products, i.e. peptide fragments, may conserve part of the allergenicity of the native protein and thus can also be considered as allergens. The molecular basis of IgE recognition underlying cow's milk protein allergy is described. Some results from studies on volunteers fed caseinophosphopeptides or potentially hypotensive milk protein hydrolysates illustrate the major difference between allergenicity and immunogenicity. The data presented on the relationship between the structure of food proteins and peptides and their allergenicity shows the difficulty in trying to assess the "non-allergenicity" of products derived from an allergenic source, even if the process used involved extensive hydrolysis of the native protein(s). A 'weight of evidence approach' for assessing the potential allergenicity of a novel protein with no history of prior allergenicity is also presented with regard to the current EU Regulations.
Current pharmaceutical design 02/2007; 13(9):897-920. · 4.41 Impact Factor
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ABSTRACT: Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23.
Using a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SPUsp45 fused to its N-terminus. The fusion of SPUsp45 failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 microg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production.
BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow's milk allergy model in mice.
Microbial Cell Factories 02/2007; 6:12. · 3.55 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
International Archives of Allergy and Immunology 02/2007; 143(1):10-20. · 2.40 Impact Factor
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Riccardo Asero,
Barbara K Ballmer-Weber,
Kirsten Beyer,
Amedeo Conti,
Ruta Dubakiene,
Montserrat Fernandez-Rivas,
Karin Hoffmann-Sommergruber,
Jonas Lidholm,
Tihomir Mustakov,
Joanne N G Oude Elberink, [......],
Per Stahl Skov,
Ronald van Ree,
Berber J Vlieg-Boerstra,
Reinhard Hiller,
Jonathan O Hourihane,
Marek Kowalski,
Nikos G Papadopoulos, Jean-Michel Wal,
E N Clare Mills,
Stefan Vieths
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ABSTRACT: In June 2005, the work of the EU Integrated Project EuroPrevall was started. EuroPrevall is the largest research project on food allergy ever performed in Europe. Major aims of the project are to generate for the first time reliable data on the prevalence of food allergies across Europe and on the natural course of food allergy development in infants. Improvement of in vitro diagnosis of food allergies is another important aim of the project. The present review summarizes current knowledge about the clinical presentation of food allergy and critically reviews available diagnostic tools at the beginning of the project period. A major problem in diagnosis is a relatively poor 'clinical specificity', i. e. both positive skin tests and in vitro tests for specific IgE are frequent in sensitized subjects without food allergy symptoms. So far, no in vitro test reliably predicts clinical food allergy. EuroPrevall aims at improving the predictive value of such tests by proceeding from diagnosis based on allergen extracts to purified allergen molecules, taking into account the affinity of the IgE-allergen interaction, and evaluating the potential of biological in vitro tests such as histamine release tests or basophil activation tests including assays performed with permanently growing cell lines.
Molecular Nutrition & Food Research 02/2007; 51(1):135-47. · 4.30 Impact Factor
