Cow's Milk Allergen Research Articles

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.

Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

BackgroundLactic 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 β-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23.ResultsUsing 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 μg/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.ConclusionBLG 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.

The challenge of cow milk protein allergy

Hypersensitivity to cow milk proteins is one of the main food allergies and affects mostly but not exclusively infants, while it may also persist through adulthood and can be very severe. Different clinical symptoms of milk allergy have been established. The diagnosis of milk allergy differs widely due to the multiplicity and degrees of symptoms, and can be achieved by skin or blood tests. Cow milk contains more than 20 proteins (allergens), that can cause allergic reactions. Casein fractions and β-lactoglobulin are the most common cow milk allergens. Human milk is free of β-lg, similar to camel milk. On the contrary, β-lg is a major whey protein in cow, buffalo, sheep, goat, mare and donkey milk. Caseins in milk of the different species differ in fraction number, amino acid composition, and their peptide mappings. β-Casein is the major fraction in goat casein, which is similar to human casein and different from cow casein. The peptide mappings of goat α-la and β-lg are completely different from those of cow milk. Different procedures can reduce the allergenicity of cow milk proteins by heat or enzymatic treatment to some degree. Allergies to milk proteins of non-bovine mammals have also been documented due to cross-reactivity between cow milk proteins and their counterpart in other species, and even between goat and sheep caseins. Genetic polymorphisms of milk proteins play an important role in eliciting different degrees of allergic reactions. Goat milk lacking α-s1-casein, which is the main casein in cow milk, is less allergenic than goat milk with α-s2-casein, which is more typical for many goat breeds. Several studies have reported real and dramatic benefits from using goat, camel, mare or even soy milk as alternatives in cases of cow milk allergy and they can be considered hypoallergenic. However, therapeutic benefits vary with the degree of severity of the allergy and may be only around 60% of all cases, since other studies revealed allergenicity to occur also for any of those other milks.

Constitutive Delivery of Bovine β-Lactoglobulin to the Digestive Tracts of Gnotobiotic Mice by EngineeredLactobacillus casei

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

Changes in humoral responses to β‐lactoglobulin in tolerant patients suggest a particular role for IgG4 in delayed, non‐IgE‐mediated cow's milk allergy

The major cow's milk allergen beta-lactoglobulin (beta-LG) is relatively resistant to enzymatic degradation and may therefore be involved in non-immunoglobulin (Ig)E-mediated cow's milk allergy (CMA) with delayed gastrointestinal symptoms. Serum levels of beta-LG-specific IgG(1), IgG(4), IgE, and IgA were compared in clinically reactive and tolerized IgE-mediated and non-IgE-mediated CMA with delayed gastrointestinal symptoms (n = 29) and controls (n = 10). Tolerance was associated with decreased beta-LG-specific IgE, IgG(1), and IgG(4) levels in both patient groups. However, the significantly increased beta-LG-specific IgG(4) levels in clinically reactive non-IgE-mediated CMA patients, and its median 36-fold reduction in tolerant patients, suggested a possible immunopathological role for IgG(4) in delayed CMA. Similarly, the significantly increased beta-LG-specific IgE levels in IgE-mediated CMA patients were decreased 44-fold in tolerant patients. The tolerant patients had apparently shifted the humoral immune response from a beta-LG-specific IgE- and/or IgG(4)-dominated immune response to an IgA-dominated immune response as the IgA/IgE or IgA/IgG(4) ratios increased 90- and 15-fold in tolerant IgE-mediated-, and non-IgE-mediated CMA patients, respectively. Thus, the marked difference in beta-LG-specific Ig ratios suggested a tolerance-induced inhibition of a Th(2)-type of immune response with significantly increased IgA dominance in both CMA patient groups.

Humoral and cellular responses to cow milk proteins in patients with milk‐induced IgE‐mediated and non‐IgE‐mediated disorders

Cow milk allergy (CMA) is one of the most common food allergies in childhood. Patients with CMA present with a wide range of immunoglobulin (Ig)E- and non-IgE-mediated clinical syndromes. Limited information is known about the specific humoral and cellular responses to cow milk proteins in these various forms of CMA. The aim of the study was to determine IgE, IgA, IgG1 and IgG4 antibody levels and lymphocyte proliferative responses to the major cow milk allergens in patients with IgE- and non-IgE-mediated CMA. One hundred and forty cow milk allergic patients, 6 months to 22 years of age, were included in the study. One hundred and thirteen patients had IgE-mediated CMA, 11 had milk protein-induced enterocolitis syndrome and 16 had allergic eosinophilic gastroenteritis. Twenty-one patients without food allergy, 8 months to 18 years of age, served as controls. Serum IgE, IgA, IgG1 and IgG4 antibodies to alpha-, beta-, and kappa-casein, alpha-lactalbumin and beta-lactoglobulin were measured using enzyme-linked immunosorbent assays. For a subset of these patients, we performed lymphocyte proliferation assays to the various milk allergens. Patients with IgE-mediated CMA had higher specific IgE concentrations to casein compared with whey proteins (P < 0.001). In this group of patients, there was a positive correlation between IgE levels and levels of the other isotypes for all four milk proteins (P < 0.001). In general, the caseins were the more allergenic and antigenic proteins in all groups of patients. Patients with enterocolitis syndrome produced less milk protein-specific IgG4 (P < 0.05) and had a trend for higher IgA antibody levels when compared to the control group. Lymphocyte proliferative responses in all groups with CMA were significantly higher than controls (P < 0.05), although this response was similar in patients with IgE- and non-IgE-mediated CMA. There is a distinct pattern of humoral antibody response in the different forms of CMA. Patients with IgE-mediated CMA have an elevated polyisotypic response to cow milk protein. The relative lack of specific IgG4 production in patients with enterocolitis syndrome may be involved in the pathogenesis of the disease. In general, caseins appear to be the predominant allergen in patients with CMA.

Oral administration of recombinant Lactococcus lactis expressing bovine β‐lactoglobulin partially prevents mice from sensitization

The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

Cow's milk allergens identification by two‐dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.

Ménière’s disease and allergy: allergens and cytokines

The aim of this article is to evaluate the role of allergy in the pathogenesis of Meniere's disease by means of cytokine profiles, allergic parameters and lymphocyte subgroups. A total of 46 patients aged between 26-68 years diagnosed with Meniere's disease between 1993-2002 were recruited to this study. The control group consisted of 46 healthy volunteers who were from the same age group, living in the same region and possessing similar socioeconomic indicators. Lymphocyte subgroups were measured from the peripheral blood by employing Becton Dickinson (BD) monoclonal CD4, CD8, CD23 antibodies. IFN-gamma, IL4, total IgE levels, and specific IgE levels pertaining to tree, fungus, fruit, egg-white, cow's milk, wheat flour, corn flour, beef, and rice allergens, in all seasons, were measured and compared in the patient and control groups. In patient serum samples there were positive correlations between CD23 and IgE, CD8 and IgE, CD4/CD8 and IgE, and CD23 and CD8 (p <0.01). There were negative correlations between IL-4 and IFN-gamma, IFN-gamma and IgE, and a positive correlation between IL-4 and IgE. Total IgE levels were above the normal values in 19/46 (41.3 per cent) of the patient group, but the ratio was nine out of 46 (19.5 per cent) in the control group. A history of allergy was found in 31/46 (67.3 per cent) when the patients were questioned. The ratio of a history of allergy was 16/46 (34.7 per cent) in the control group. When specific IgE levels were evaluated the ratio of patients with all the panels negative was eight out of 46 (17.9 per cent), but it was 31/46 (67.3 per cent) in the control group. This study found that the prevalence of allergy was higher in patients with Meniere's disease than in the control group. Thus the authors suggest that allergy should be taken into account when patients with this disease are treated.

Modification of IgE binding during heat processing of the cow's milk allergen beta-lactoglobulin.

The effect of heat treatment on the IgE binding ability of beta-lactoglobulin, as pure protein or in whole milk, was studied by inhibition of IgE antibody binding using FEIA-CAP inhibition. A slight but significant decreased IgE binding was seen between unheated and heat-treated beta-lactoglobulin solution at 74 degrees C (IC(50) = 2.03 and 3.59 microg/mL, respectively, p = 0.032). A more pronounced decrease was found at 90 degrees C with an IC(50) of 8.45 microg/mL (p = 0.014). The inhibition of IgE binding of milk after heat treatment at 90 degrees C was also significantly decreased (p = 0.007). However, at all heat treatments, a similar total amount of IgE antibodies could be inhibited at a sufficiently high concentration of beta-lactoglobulin. The inhibiting ability of beta-lactoglobulin was significantly impaired in some fermented acidified milk products such as yogurt as compared to that in nonfermented milk (p < 0.001). There was only a small difference of IgE binding between the native forms of genetic variants A and B.

Generation of heat-killed E. coli expressing cow milk proteins for immunotherapy regiment for the milk allergic mice

Rationale Cow milk (CM) is one of the major causes of food hypersensitivity in children. However, there are no definitive prevention or treatment approaches for the cow milk allergy (CMA). Recently, we have demonstrated that the heat-killed E. coli (HKE) expressing mutated major peanut (PN) protein allergens was effective in protecting PN allergic C3J/HeJ mice from PN induced adverse reactions. Therefore, we proposed to employ the heat-inactivated E. coli expressing four major cow milk allergens (α S1-, α S2-, and β-caseins, and β-lactoglobulin) in attempt to reestablish oral tolerance to CM in the CM allergic mice. Moreover, in order to identify the antigen presenting cells involved in the initial antigen uptake we make use of the heat-killed E.coli expressing a fusion protein of α S1-casein adjacent to the enhanced green fluorescent protein (EGFP-α S1). Methods and results The target protein cDNAs were subcloned into the pET24b(+) vector and the final constructs were introduced into E.coli BL21 strain. The Western immunoblot analysis showed that the specific recombinant protein content in both the live and in the heat-killed bacteria is at least equals to that in the previously described PN expressing bacteria. The antigen uptake by the mouse spleen macrophages in vivo demonstrated that the EGFP-α S1 containing HKE could be detected by fluorescent microscopy inside the cell up to 3 hours after initial antigen exposure. Conclusions Clones of E.coli BL21 expressing the major milk protein allergens have been generated and characterized. Properties of these clones appear to make them a good tool for future immunotherapy study.

The Role of Cow Milk Allergy in Increasing the Severity of Atopic Dermatitis

Previous studies have shown that up to 33% of children with atopic dermatitis have experienced food hypersensitivity and among different kinds of food allergens Cow Milk (CM) has almost always been one of the most common food allergens in children. The aim of this study is to evaluate the cow milk allergy (CMA) as an increasing factor of severity of atopic dermatitis. One hundred and nineteen children (between 1.5 months and 12 years of age) with atopic dermatitis in the sense of Hanifin and Rajka's criteria entered this study and the severity of atopic dermatitis was identified via the SCORAD index. In order to make the diagnosis of cow milk allergy, a careful history, and a familial history of allergy was taken and the results of skin prick test (SPT) with CM and 4 other food allergen extracts, Radioallergosorbent test (RAST) with CM allergens and a food challenge test with cow milk (fresh or dried) were used. Also a total serum IgE determination and an eosinophil count (with a stool exam) were accomplished. The clinical manifestations of atopic dermatitis in patients was started from their first day of life up to 10 years of age. The family history in 83% of the patients was positive. Positive skin prick test and RAST with CM allergens were positive in 37.9% and 29.3% of cases respectively and the response to challenge test with cow milk was positive in 35 out of 40 patients and in total 44.5% had CMA according to a positive history of cow milk allergy and a positive outcome of the IgE tests (SPT and/or RAST) or a positive challenge test with CM allergens. The results showed that the most common food allergens in patients with atopic dermatitis are certainly cow milk allergens (44.5%) whereas other food allergens are tomato (29.41%), egg (28.57%), nuts (9.24%) and wheat (3.36%) according to the skin prick test. The mean total serum IgE was 307.11 ± 6.56 IU/ml (range = 6–5000) in children with CMA and 81.04 ± 5.97 IU/ml (range = 1–5000) in children without CMA while the mean eosinophil count was 569.52 ± 3.02 count/ml (range = 67–8500) and 314.22 ± 2.94 count/ml (range = 5–5000) respectively. The mean severity of atopic dermatitis according to the SCORAD index was 60.76 in children with CMA and 44.29 in children without CMA. The severity of atopic dermatitis in patients with CMA was significantly higher than patients without CMA (p < 0.0001). Also the mean total serum IgE and mean eosionophil counts in children with CMA were significantly higher than in children without CMA (P < 0.01 and p < 0.0001, respectively). It shows the important role of CM allergen proteins in the induction and in increasing the severity of AD in children.

A Laboratory Method for Purification of Major Cow's Milk Allergens

Food allergy is responsible for the most frequent allergic reactions in children under 1 year of age. Diagnostic tests such as skin test or specific IgE assay usually need highly purified preparations of allergenic proteins. The aim of the present study was to purify three main cow's milk proteins: casein, α‐lactalbumin, and β‐lactoglobulin as allergenic extracts for first time in Iran.

Immunostimulatory potential of β-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 β-lactoglobulin, has previously been shown to mediate cellular activation in both human and murine immune cells. Objective We examined the response to different β-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 β-lactoglobulin preparations. Cell proliferation, cytokine production, and increases in intracellular glutathione were used as cellular activation markers. Moreover, the effect of β-lactoglobulin on cytokine production in murine bone-marrow–derived dendritic cells was examined. Results We observed that some commercial β-lactoglobulin preparations induced pronounced proliferation of both spleen cells and cells from mesenteric lymph nodes; production of TNF-α, IL-6, IL-1β, and IL-10; and an increased level of intracellular glutathione in spleen cell cultures. Furthermore, TNF-α, IL-6, IL-1β, and IL-10 production was induced in murine bone-marrow–derived dendritic cells. Purification of β-lactoglobulin from raw milk using nondenaturating conditions, however, revealed that the β-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 β-lactoglobulin preparations. Moreover, the present study makes it evident that immunomodulatory effects attributed to β-lactoglobulin need to be reassessed.

Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice.

Bovine beta-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice. We constructed a recombinant strain of L. lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L. lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producing L. lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L. lactis strain.

Mutational analysis of major, sequential IgE-binding epitopes in α s1-casein, a major cow's milk allergen

Background: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising. Objective: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of α s1-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity. Methods: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera. Results: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition. Conclusion: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity. (J Allergy Clin Immunol 2003;112:433-7.)

Elicitation of the allergic reaction in beta-lactoglobulin-sensitized Balb/c mice: biochemical and clinical manifestations differ according to the structure of the allergen used for challenge.

Mouse models of allergy are used to study the mechanisms of induction and perpetuation of bronchopulmonary hyper-reactivity (BHR) as related to eosinophils and specific IgE. Our aim was to adapt the current model for the study of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, and to further analyse the mechanisms of the acute and late allergic reaction. Female Balb/c mice were sensitized intraperitoneally with BLG and the influence of the adjuvant and of the BLG dose on the IgE response was analysed, IgE and IgG1 epitopes being characterized. Once optimized, this model was applied to the study of the active phase of allergy in the respiratory tract after a single airway challenge using native or denatured BLG, which contains only linear epitopes. An immediate allergic reaction was characterized by the rapid release of histamine into the bronchoalveolar lavage fluids. Prostaglandin (PG)D2 was only present when the standard histamine-releasing agent compound 48/80 or denatured BLG were used as triggers, whereas native BLG induced leukotriene release. Twenty-four hours after challenge, BHR, eosinophil influx, IL-4 and IL-5 production, plasma exudation and mucus production were very much increased, differently depending on the allergen structure, and indicated the occurrence of the late allergic reaction. Our results show that the murine model can be used to study the mechanisms of allergy to clinically relevant antigens, such as those contained in cow's milk. The acute allergic reaction, which depends on the structural feature of the allergen, is composed of two distinct pathways characterized by peptido-leukotrienes or PGD2 production, which may result from distinct activation intensities of mast cells, leading to distinct late reactions. This study thus demonstrates a clear link between the structural feature of a protein, and the physiopathology of the experimental asthmatic reaction.

Influence of heating and digestion on the B-cell epitopes of cow's milk allergens

Influence of heating and digestion on the B-cell epitopes of cow's milk allergens

Mutational analysis of IgE-binding epitopes of the major cow's milk allergen β-casein showed a heterogenous pattern of critical amino acids between individual patients and pooled sera

Mutational analysis of IgE-binding epitopes of the major cow's milk allergen β-casein showed a heterogenous pattern of critical amino acids between individual patients and pooled sera

Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin.

The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.