Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen's egg allergy in adults.

BACKGROUND: Although hen's egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffer from a persistent or newly onset hen's egg allergy, making accurate diagnosis in adults necessary. However, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children. METHODS: Hen's egg allergic (n = 16) and tolerant (n = 19) adults were selected by sensitization towards recombinant components rGal d 1 and/or 3. Sensitization profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterization of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. RESULTS: Overall, sIgE titres against hen's egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitization to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen's egg allergic adults with objective symptoms.

[EXTRACORPOREAL BLOOD CORRECTION METHODS AND THEIR IMPACT ON PRODUCTS OF MICROBIAL METABOLISM IN PATIENTS WITH SEPSIS].

Sepsis is a disease with a high death-rate and is accompanied by profound metabolic disturbances. Interference of microbe metabolic products with biochemical processes in human organism is present in case of severe infection. But there is little information about integration of microbe and human metabolism in septic patients. We evaluated an indol level in healthy individuals and septic patients. It was revealed that septic patients have higher indol levels. Hemoperfusion through "Ovosorb" sorbing agent allows to decrease indol concentration to normal levels. Application of hemosorbtion in combination with magnetic blood processing allows achieving faster and more effective indol removal.

Oral immunotherapy induces local protective mechanisms in the gastrointestinal mucosa.

BACKGROUND: Oral immunotherapy (OIT) is a promising treatment for food allergy. Studies are needed to elucidate mechanisms of clinical protection and to identify safer and potentially more efficacious methods for desensitizing patients to food allergens. OBJECTIVE: We established a mouse model of OIT to determine how the dose or form of antigen may affect desensitization and to identify mechanisms of desensitization. METHODS: Increasing doses of egg white or ovomucoid as OIT were administered orally to sensitized mice. The impact of OIT on anaphylaxis elicited by oral allergen challenge was determined. Allergen-specific antibody and cytokine responses and mast cell and basophil activation in response to OIT were measured. Gene expression in the small intestine was studied by microarray and real-time PCR. RESULTS: OIT resulted in desensitization but not tolerance of mice to the allergen. OIT did not result in desensitization of systemic effector cells, and protection was localized to the gastrointestinal tract. OIT was associated with significant changes in gene expression in the jejunum, including genes expressed by intestinal epithelial cells. Extensively heated ovomucoid that does not trigger anaphylaxis when given orally to sensitized mice was as efficacious as native ovomucoid in desensitizing mice. CONCLUSIONS: OIT results in clinical protection against food-induced anaphylaxis through a novel mechanism that is localized to the intestinal mucosa and is associated with significant changes in small intestinal gene expression. Extensively heating egg allergen decreases allergenicity and increases safety while still retaining the ability to induce effective desensitization.

Oral immunotherapy with immunodominant T-cell epitope peptides alleviates allergic reactions in a Balb/c mouse model of egg allergy.

BACKGROUND: Allergen-specific T-cell epitopes are obvious targets for immunotherapeutic interventions in allergic disease. T-cell epitope peptides given orally may provide a practical way of inducing tolerance and preventing allergy. OBJECTIVE: This study investigates oral immunotherapy (OIT) with T-cell epitope peptides of the dominant egg-white allergen ovomucoid (Ovm) in a Balb/c mouse model of egg allergy. METHODS: Groups of mice were orally sensitized to Ovm and subsequently administered Ovm T-cell epitopes [single peptide 157-171 (SP) or multiple peptide (157-171)(3) (MP)], followed by oral challenge with Ovm. Outcomes post oral challenge were measured as clinical signs, serum histamine, antibody activity (IgG, IgE, IgG1, IgG2, IgA), cytokines (IL-4, IFN-γ, IL-12p70, IL-10, TGF-ß, and IL-17), and T regulatory cells (Tregs). RESULTS: Clinical signs were less frequent in both SP and MP groups (P ≤ 0.05). Specific IgE was less and IgA was more in both groups; however, SP-treated mice had less histamine and IgG1 and more IgG2-related antibodies indicating a bias toward the type-1 response (P ≤ 0.05). Concentration of type-2 cytokine interleukin-4 (IL-4) was significantly less in both groups and IL-12p70 and IL-10 were more in SP-treated mice (P ≤ 0.001). Interferon-γ, IL-17, and TGF-ß did not differ significantly. There was significant increase in the percentage of CD4+FOXP3+ and CD4+CD25+ cells in the SP group, indicating the significant role of Tregs in immune regulation. CONCLUSION: In summary, we demonstrated that OIT with SP and MP comprising the immunodominant regions of Ovm was safe and significantly reduced subsequent frequency of allergy to Ovm, and validated potential use of Ovm T-cell epitope as an immunoregulator.

Intake of Diet Including 1% Ovomucoid for 4 Weeks Induces Oral Desensitization in Ovomucoid-Specific Allergic Mouse Model.

We propose a new oral immunotherapy (OIT) method that includes a small amount of a food allergen in the diet. However, it is not clear whether this method will induce oral desensitization and immune tolerance. Therefore, we investigated the therapeutic effectiveness using a 1% food allergen diet in an allergic mouse model. C3H/HeJ mice were sensitized to ovomucoid (OM) in alum four times at 12-d intervals. Sensitized mice were divided into two groups: the OIT group (19% casein diet with 1% OM) and the non-treated group (20% casein diet without OM). The non-sensitized mice served as the non-allergy group. The OIT treatment was performed for 4 wk. To assess desensitization and immune tolerance, we performed oral and intraperitoneal OM challenges, assessed vascular permeability of the dorsal skin, and measured allergic biomarkers. The OIT group exhibited significantly lower oral symptom scores and vascular permeability than the non-treated group, but the two groups did not differ in intraperitoneal allergy symptom scores. Furthermore, the OIT group had significantly higher OM-specific IgA levels in their plasma than the non-treated group. However, the plasma levels of OM-specific IgE, IgG1, and IgG2a were not significantly different between the OIT and the non-treated groups. These results suggest that the proposed OIT using an OM-supplemented diet may induce desensitization, but not immune tolerance, in an OM allergic mouse model.

Identification and Characterization of Glycopeptides from Egg Protein Ovomucin with Anti-Agglutinating Activity against Porcine K88 Enterotoxigenic Escherichia coli Strains.

Ovomucin is a glycoprotein from egg white with potential to act as an anti-adhesive agent against infectious diseases. This study aimed to determine whether ovomucin or ovomucin hydrolysates could prevent adhesion of two porcine K88 enterotoxigenic Escherichia coli (ETEC) strains. Adhesion was assessed in vitro using a hemagglutination assay. Ovomucin hydrolysates, but not intact ovomucin, prevented adhesion of ETEC to porcine erythrocytes. The ovomucin hydrolysate prepared by acid protease II exhibited the best anti-agglutinating activity against both strains; this hydrolysate was fractionated by cation exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC). The most active fractions, F3(9) and F7(1), with minimal inhibitory concentrations of 0.03 and 0.25 g/L against strains ECL13795 and ECL13998, respectively, were subjected to structural characterization. Six identified glycopeptides were all derived from α-ovomucin, composed of a pentasaccharide core of two N-acetylglucosamine and three mannose residues (GlcNAc2Man3) and a bisecting N-acetylglucosamine (GlcNAc). The terminal ß-linked galactose from these glycopeptides could be one of the binding sites for K88ac fimbriae.

Nitration of ß-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice.

BACKGROUND: We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. OBJECTIVE: The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. DESIGN: BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen ß-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. RESULTS: A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. CONCLUSION: Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

[Inhibitors of proteolytic enzymes in the treatment of diabetes mellitus]. / Ingibitory proteoliticheskikh fermentov v terapii sakharnogo diabeta.

A new approach to overcome the degradation of insulin by proteolytic enzymes and its targeting to the blood through the digestive apparatus was developed. The approach is based on the immobilization of insulin into the polymeric hydrogel which is modified by ovomucoid--glycoprotein, inhibitor of proteolytic enzymes. Oral administration of this system to rabbits and rats, (in contrast to the hydrogels modified by proteolytic enzymes inhibitors without polysaccharide part), statistically significantly lowered blood glucose level.

[Effect of soybean trypsin inhibitor and chicken ovomucoid on the immunogenic and sensitizing properties of various dietary proteins]. / Vliianie soevogo ingibitora tripsina i kurinogo ovomukoida na immunogennye i sensibiliziruiushchie svoistva nekotorykh pishchevykh belkov.

Adult male guinea pigs were sensitized by intragastric administration of bovine serum albumin (BSA) and chick ovalbumin (OA) and their mixtures with soybean Kunitz trypsin inhibitor (SBTI) and chick ovomucoid (OM). Sensitization of the animals was evaluated by the anaphylactic shock reaction and also by the levels of serum specific IgG antibodies against BSA and OA as measured in the solid phase radioimmunoassay. The experiment revealed pronounced desensitizing properties of SBTI combined both with OA and BSA. OM produced no effect on the animal sensitization caused by OA and enhanced the BSA-induced sensitization. The results obtained demonstrate the necessity of differential approach to the evaluation of the action of varying trypsin inhibitors on food sensitization.

Ablation of ovomucoid-induced allergic response by desensitization with recombinant ovomucoid third domain in a murine model.

Attempts to modulate the allergenic response by hypoallergens aimed at eliminating IgE-binding epitopes have been established recently for allergen immunotherapy. Desensitization offers an alternative approach to mounting a protective immune response. We have shown previously that mutation of the decisive amino acids in the B cell epitope of the ovomucoid third domain suppresses IgE binding reactivity against human patient sera and we hypothesize that this hypoallergenic variant could be a potential candidate molecule for specific immunotherapy against an ovomucoid-induced IgE reaction. The aim of this study was to investigate whether hyposensitization with the ovomucoid-modified isoform could desensitize ovomucoid-sensitized mice. We mapped the immunodominant B cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid mutant isoform, having ablated allergenicity against patient sera, was used to desensitize ovomucoid-sensitized Balb/c mice by intraperitoneal injection. Female Balb/c mice were sensitized with intact ovomucoid molecule (Fovm) and desensitized with the modified isoform of the third domain of ovomucoid (GMFA). Intact ovomucoid-sensitized mice desensitized with phosphate-buffered saline (PBS) served as positive controls to maintain hypersensitivity. To gain insight into the efficacy of the modified ovomucoid variant in desensitization, effects on hypersensitivity reactions and histamine levels, followed by its association with antibody levels and cytokine profiles, were measured. Abrogation of the allergic response with complete suppression of anaphylactic symptoms and lower serum histamine levels was observed in the desensitized group by GMFA, accompanied by significantly reduced ovomucoid-specific IgE and IgG1 levels and enhanced specific IgG and IgG2a levels. The sensitized group showed severe anaphylactic symptoms, enhanced serum histamine concentrations and increased levels of specific IgE and IgG1. The level of interleukin (IL)-4 was decreased dramatically in the desensitized group and higher levels of interferon (IFN)-gamma were found, whereas mice sensitized with intact ovomucoid exhibited significantly higher levels of IL-4 favouring a Th2 skewed pathway. We demonstrate clearly that GMFA is able to ablate ovomucoid-induced allergic reactions in sensitized mice. This occurs via a suppression of specific IgE accompanied by an increase in suppressor T cell activity. This approach offers some promise for the development of treatment against ovomucoid-induced allergic response.

Water-soluble polymers with low critical solution temperature (LCST) as carriers for protein drug delivery.

The copolymers of N,N-diethylacrylamide and ovomucoid from duck egg white (an inhibitor of proteolytic enzymes) with low critical solution temperature (LCST) have been synthesized. The behaviour of these copolymers at the point of phase transition was investigated. It was shown that the relation between LCST and physiological activity for these copolymers is a function of their composition. The increase of ovomucoid content leads to the increase of LCST. When the content of ovomucoid rises above 0.2 mol%, LCST disappears. At the same time the physiological activity of obtained copolymers decrease with increasing of N,N-diethylacrylamide content.

Inhibition of specific immune responses by feeding protein antigens.

A profound and long-lasting state of specific immune unresponsiveness may be induced in adult inbred mice given a single dose of protein immunogens--such as ovalbumin or hemocyanin--by the digestive route. The degree of unresponsiveness induced by intragastric exposure to ovalbumin could not be achieved by intravenous injection of deaggregated ovalbumin solutions across a wide range of doses. Unresponsiveness induced by intragastric exposure to hapten-protein conjugates is specific to the carrier protein.

Comparison between the protective effects of mycobacterial 65-kD heat shock protein and ovomucoid in pristane-induced arthritis: relationship with agalactosyl IgG.

The IgG of patients with rheumatoid arthritis and mice with pristane induced arthritis (PIA) tends to lack the terminal galactose normally on the conserved N-acetylglucosamine linked beta 1-2 to mannose in IgG. The terminal N-acetylglucosamine (GlcNAc) residues of oligosaccharides on agalactosyl IgG may be an important component of the action of these glycoforms. Here, administration of ovomucoid, a glycoprotein rich in terminal GlcNAc, before pristane injection was found to reduce the incidence of PIA. This observation is the second report of an intraperitoneally administered antigen that reduces the incidence of PIA, mycobacterial 65-kD heat shock protein (hsp65) being the first. The suppressive effect of ovomucoid was not transferred from protected to naive recipients by spleen cells at the dose tested. By contrast, transfer of spleen cells from hsp65-protected mice to naive recipients conferred protection and this protection may be antibody-mediated. It is considered that ovomucoid and hsp65 protect against the development of PIA by different mechanisms.

Modulation of ovomucoid-specific oral tolerance in mice fed plant extracts containing lectins.

We investigated the effect of feeding extracts of four different legumes (red kidney bean (Phaseolus vulgaris), peanut (Arachis hypogaea), soyabean (Glycine max) and pea (Pisum sativum) on the specific immune response against a food protein. Mice were fed ovomucoid and the specific immune response was evaluated. Ovomucoid fed alone resulted in oral tolerance induction measured as both a reduced ovomucoid-specific spleen cell proliferation and antibody response. Feeding kidney-bean extract prevented induction of oral tolerance to ovomucoid measured as spleen cell proliferation in vitro. Pure kidney-bean lectin also prevented oral tolerance induction, suggesting that lectin in the kidney-bean extract caused inhibition of oral tolerance. Parenteral administration (intravenous and intraperitoneal) of pure kidney-bean lectin had no significant influence on oral tolerance induction. Soyabean extract also influenced the immune response against ovomucoid; however, this was not as pronounced as for kidney bean and was only significant (P<0.001) for the antibody response. No effect was observed when pea extract was fed and peanut extract had a non-significant effect on induction of oral tolerance and on the general immune response. Plasma antibodies against kidney-bean lectin, but not against the three other legume lectins, were detected. Our current findings show that other dietary components can influence the specific immune response against food proteins. Various dietary components may thus contribute to the onset of adverse immunological responses.