Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin(OVA)is the major allergenic protein that can induce T helper 2(Th2)-allergic reactions,for which current treatment options are inadequate.In this study,we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid(Lac/CA)-catalyzed crosslinking in conjunction with galactomannan(Man).The formation of high molecular weight crosslinked polymers and the Ig G-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity.Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF).Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines(interleukin(IL)-4,IL-5,and IL-13),and upregulated IFN-γand IL-10 expression.Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers(CD80 and CD86)and MHC class II molecules,and suppressed the expression levels of proinflammatory cytokines.The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Boeravinone B ameliorates allergic nasal inflammation by modulating the GATA-3/T-bet signaling pathway in a mouse model of allergic rhinitis

Objective:To evaluate the anti-allergic effect of boeravinone B against ovalbumin-induced allergic rhinitis in mice and explore its possible mechanism.Methods:For the induction of allergic rhinitis,mice were intraperitoneally sensitized and intranasally challenged with ovalbumin,as well as orally received various concentrations of boeravinone B.Nasal mucosal inflammation,and the levels of nitric oxide,β-hexosaminidase,IFN-γ,LTC-4,myeloperoxidase,Nrf2,HO-1,GATA-3,ROR-γ,T-bet,antioxidant parameters,and allergen-specific cytokines were assessed.Results:Boeravinone B markedly reduced ovalbumin-induced increase in the number of episodes of nasal sneezing,rubbing,and discharge,as well as the levels of IgE,IgG1,andβ-hexosaminidase(P<0.05).It also significantly reduced differential cell count,myeloperoxidase,oxide-nitrosative stress,and the levels of IL-1β,IL-4,IL-5,IL-6,IL-13,IL-17,tumor necrosis factor-α,GATA-3,and ROR-γwhile enhancing the level of T-bet.Conclusions:Boeravinone B is a potential therapeutic agent for allergic rhinitis by modulating various inflammatory mediators and immune responses.

Investigation into IgG/IgE binding capacity and gut microbiota of digestion products derived from glycated ovalbumin

Gut microbiota plays an important role in food allergy.The immunoglobulin G(IgG)/immunoglobulin E(IgE)binding capacity and human gut microbiota changes of digestion products derived from glycated ovalbumin(OVA)were investigated.Gastrointestinal digestion effectively destroyed the primary structure of glycated OVA,resulting in a significantly higher digestibility than gastric digestion,and more abundant peptides<3 kDa.Moreover,gastric and gastrointestinal digestion products have different fluorescence quenching and red shift of fluorescence peaks,and possess different conformational structures.These changes resulted in a decrease in 28.7%of the IgE binding capacity of gastrointestinal digestion products beyond that of pepsin.Moreover,gastrointestinal digestion products of glycated OVA increased significantly the proportion of Subdoligranulum,Collinsella,and Bifidobacterium.Therefore,gastrointestinal digestion products of glycated OVA altered human intestinal microbiota,reducing the risk of potential allergy.

Rosmarinic acid improves tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats

Objective:To evaluate the effect of rosmarinic acid on tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats.Methods:Rats were randomly divided into six groups:the control group,the asthmatic group,and the asthmatic groups treated with dexamethasone(1 mg/kg;oral gavage)or three doses of rosmarinic acid(0.5,1,and 2 mg/kg;oral gavage).For induction of asthma,rats received intraperitoneal injections and inhalation of ovalbumin.After 21 days,bronchoalveolar lavage fluid and lung samples were collected for histopathological analyses.Moreover,total and differential white blood cell counts were determined.Results:The rosmarinic acid-treated group had significantly lower tracheal smooth muscle responses to methacholine than the asthmatic group.In addition,rosmarinic acid reduced white blood cell count and the percentages of eosinophils,monocytes,and neutrophils while increasing the percentage of lymphocytes.Ovalbumin-induced lung pathological changes were significantly improved by treatment with rosmarinic acid.Conclusions:Rosmarinic acid improves tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats.

Oral administration of Bacillus coagulans TQ-35 alleviates allergic responses in OVA-sensitive BALB/c mice

Bacillus coagulans has been extensively studied so far,but there has been a lack of research on its usage in allergy.In this study,we designed to assess the effect of different concentrations of B.coagulans on food allergy in a BALB/c mouse model of ovalbumin(OVA)-induced food allergy and its effect on gut microbes.The assessment of symptoms,specific immunoglobulin E(IgE),T-cell differentiation,and related gene expression levels in sensitized mice by assay indicated that high doses of oral B.coagulans could alleviate allergic symptoms.Treatment with B.coagulans,in the high-dose group,significantly reduced IgE and IgG1 levels and modulated the balance of T helper type 1 cell(Th1)and Th2 and the expression of relevant genes in the spleen.16S rRNA analysis showed that probiotics improved the structure of the microbiota,in particular by boosting the percentage of Clostridia,Bacteroides vulgatus and Enterococcus faecium,and by increasing the abundance of microbial species,thereby modulating the immune system.Therefore,this study can provide insights into the practical application of B.coagulans doses to alleviate OVA allergy.

Myricetin alleviates ovalbumin-induced allergic rhinitis in mice by regulating Th1/Th2 balance

Objective:To evaluate the effect of myricetin on ovalbumin(OVA)-induced allergic rhinitis in mice.Methods:Mice were sensitized and challenged using OVA(5%,500 mL)intraperitoneally and intranasally,respectively,on an alternative day for 14 days,followed by administration of myricetin(50,100,and 200 mg/kg)till day 21.Nasal symptoms,biochemical parameters,protein expressions,and histopathology were observed.Results:OVA-induced increased nasal symptoms including rubbing,sneezing,and discharge were significantly reduced by myricetin(100and 200 mg/kg)(P<0.05).Myricetin also protected against histamine challenge and attenuated elevated serum immunoglobulin E(IgE;total and OVA-specific),total IgG1,andβ-hexosaminidase levels,as well as leukotriene C4 and interleukins levels in nasal lavage fluid(P<0.05).Western blot analysis showed that myricetin significantly upregulated the protein expression of T-box expressed in T cells,while downregulating the protein expression of GATA binding protein 3,NF-κB,and IκB-α(P<0.05).Additionally,OVA-induced histopathological abberations in the nasal mucosa was markedly ameliorated by myricetin treatment(P<0.05).Conclusions:Myricetin exerts anti-allergic effects against OVAinduced allergic rhinitis via regulating Th1/Th2 balance.

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Preliminary Study on the Separation of Lysozyme,Ovotransferrin and Ovalbumin from Egg White

[Objective] A study on separation process of lysozyme, ovotransferrin and ovalbumin from egg white. [Method] The proteins were separated by ammonium sul-fates and ion-exchange chromatography. Purity of the proteins was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. [Result] The results showed that the proteins were electrophoresis-pure. The specific activity of lysozyme was increased from 144.13 to 2 235 U/mg, and purification factor was 15-fold. Lysozyme recovery rate was estimated to be 15.76%. Bacteriostasis rate of ovotransferrin was 48.84%. [Conclusion] The procedure for separating lysozyme, ovotransferrin and ovalbumin from egg white was simple, fast, low-cost and suitable for industrilization.

Protective effect of Bifidobacterium infantis CGMCC313-2 on ovalbumin-induced airway asthma and b-lactoglobulininduced intestinal food allergy mouse models

AIM To determine whether oral administration of Bifidobacterium infantis CGMCC313-2(B. infantis CGMCC313-2) inhibits allergen-induced airway inflammation and food allergies in a mouse model.METHODS Ovalbumin(OVA)-induced allergic asthma and b-lactoglobulin-induced food allergy mouse models were used in this study. Following oral administration of B. infantis CGMCC313-2 during or after allergen sensitization, histopathologic changes in the lung and intestine were evaluated by hematoxylin and eosin(HE) staining. In the allergic asthma mouse model, we evaluated the proportion of lung-infiltrating inflammatory cells. OVAspecific IgE and IgG1 levels in serum and cytokine levels in bronchoalveolar lavage fluid(BALF) were also assessed. In the food allergy mouse model, the levels of total Ig E and cytokines in serum were measured.RESULTS Oral administration of B. infantis CGMCC313-2 during or after allergen sensitization suppressed allergic inflammation in lung and intestinal tissues, while the proportion of infiltrating inflammatory cells was significantly decreased in the BALF of allergic asthma mice. Moreover, B. infantis CGMCC313-2 decreased the serum levels of total Ig E in food allergy mice, and reductions in IgE and IgG1 were also observed in OVA-induced allergic asthma mice. The expression of interleukin-4(IL-4) and IL-13 in both serum and BALF was suppressed following the administration of B. infantis CGMCC313-2, while an effect on serum IL-10 levels was not observed.CONCLUSION B. infantis CGMCC313-2 inhibits the secretion of allergen-induced IgE, IL-4 and IL-13, and attenuates allergic inflammation.

The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant-free condition

Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when QVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens.

Structural and functional properties of hydrolyzed/glycosylated ovalbumin under spray drying and microwave freeze drying

Ovalbumin(OVA),the main protein in egg white,affects most of the functional properties of egg white protein in food processing.The aim of this study was to investigate the effects of spray drying(SD)and microwave freeze drying(MFD)on the preparation of hydrolyzed/glycosylated ovalbumin(HGOVA)and provide useful information on the applications of egg protein powders in the food industry.Results demonstrated that the structure of HGOVA was considerably changed,and its functional properties were improved compared with those of native OVA.SD and MFD processing did not lead to dissociation of HGOVA subunits.SD-HGOVA exhibited higher protein solubility,emulsifying activity,foaming capacities,and gel hardness than MFD-HGOVA.However,MFD-HGOVA was better than the SD-HGOVA in terms of color,emulsion stability,foam stability,water/oil absorption capacity,and thermal stability.Selection of an appropriate drying method could enhance the potential applications of HGOVA in the food industry.

Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyle-neimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-Nl was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the β-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

Electrochemical study of pyrite-ovalbumin interaction in relation to flotation

Ovalbumin （OVA）, chicken egg albumin, is an environmentally friendly, non-toxic, cheap surface active agent. It has electrochemically active sulfhydryl residues in molecular structure. OVA adsorption on pyrite as an alternative depressant was investigated by cyclic voltammetry, FTIR spectroscopy and flotation. Tests were conducted in a wide potential range in alkaline pH to clarify the role of electrochemical condition on the adsorption mechanisms and the rate of depression. In the absence of OVA, the floatability reached its maxima around slightly oxidizing condition. Beyond the range, it decreased presumably due to the formation of Fe-oxyhydroxides together with the hydrophilic S-containing species. OVA-pyrite interaction occurred in the whole examined potential range. Depressing effect of OVA increased gradually from reducing to slightly oxidizing potential, open circuit potential （OCP） of pyrite, mainly due to weak conformational changes in OVA molecules together with the hydrophobic interaction around OCP. The rate of depression reached its maxima at moderately oxidizing potentials, which was referred to electrostatic attraction. This level was almost kept at higher potentials owing to OVA adsorption as metal-chelates.

Protein Adsorption onto Nanosized Hydroxyapatite Particles for Controlled Drug Release

Nanosized hydroxyapatite（nsHAp） was synthesized to examine its possibility as a controlled release cartier of protein. To achieve effective protein release from nanosized hydroxyapatite, the study of the adsorptive properties of protein on nsHAp and different influence parameters such as pH, calcium, and phosphate concentrations during the adsorption process is necessary. Ovalbumin（OVA） was selected as the model of growth factors. The results show that the amount of OVA adsorbed onto nsHAp in acetic buffer（pH = 3.6） is more than that in acetic buffer（pH =5.6） because of the electric interaction. The amount of OVA adsorption in phosphate buffer solution（PBS） is smaller than that in acetic buffer because of surface complexation and surface hydroxylation. The presence of Ca^2＋ dramatically increases the adsorbed amount of OVA in acetic buffer on maintaining the same pH. Meanwhile, the release kinetics of OVA adsorbed onto nsHAp（nsHAp-OVA） was also examined. The amount of released OVA in PBS（pH = 5.6） was significantly smaller than that released in solution of pH = 7.0. All the results suggest that nanosized hydroxyapatite particles could be successfully used as controlled released carrier of protein.

Peripheral tolerance of antigen-specific Th cells induced with polyethylene glycol-conjugate of protein antigen

It has long been known that protein antigen conjugated with polyethylene glycol (PEG), a nonimmunogenic artificial polymer, induces immune tolerance of antigen-specific Th cells. However, the mechanism of this tolerance induction remains unknown. In this study, the response and differentiation of ovalbumin (OVA)-specific CD4+ Th cells upon exposure to tolerogenic PEG conjugate of OVA (PEG-OVA) were studied. Na?ve OVA-specific Th cells from OT-II mice were labeled with carboxyfluorescein succinimidyl ester (CFSE), transferred into histocompatible C57BL/6 mice, and then subsequently stimulated with either tolerogenic PEG-OVA or with OVA. Upon stimulation with tolerogenic PEG-OVA in vivo, these cells showed a robust proliferative response comparable to that observed by stimulation with OVA. Nevertheless, upon prolonged exposure to PEG-OVA, OVA-specific Th cells became anergic, showing a markedly reduced capacity to respond, and to produce IL-2 and other cytokines when stimulated with antigenic OVA323-339 peptide in vitro. There was also a significant reduction of the frequency of clonotypic TCR Vα2+CD4+ T cells in the spleens of OT-II mice treated with PEG-OVA. These features of response of na?ve OVA-specific Th cells upon sustained exposure to PEG-OVA were quite analogous to those reported for the same cells transferred into mice with systemic expression of the transgenic OVA gene. The highly enhanced stability in the circulation that was observed for PEG-OVA was likely the basis of its tolerogenic capacity.

Mechanobiology of airway smooth muscle cells and its regulations in pathogenesis of asthma

Introduction Excessive narrowing of airways is the most important pathological feature of asthma,but its mechanism remains puzzling.One certain thing is that the contraction of airway smooth muscle(ASM)ultimately causes airway narrowing,thus both structural and functional alterations of airway smooth muscle(ASM)are thought as common final pathway responsible for the bronchial hyperresponsiveness(BHR),the hall mark of asthma.Many chemical and physical factors such as air pollutants,inflammatory agents,mechanical and geometrical properties of the microenvironment could influence structure and/or function of ASM cells.In addition,some re-

The effect of inhibitor peptide for ADAM8 in attenuating ovalbumin-induced murine asthma

Targeted peptides have been identified as showing great promise for treatment of various diseases including asthma.Asthma is considered of difficuIt-to-treat due to its unclear etiology,thus usually requiring life-long treatment.Current strategies for asthma therapy are hampered with undesirable side effects,poor targeting and failure in modulating airway hyperresponsiveness,leading to pressing need of developing more targeted and effective therapeutic sites for asthma.Recently,a disintegrin and metalloproteinase 8(ADAM8)have been shown to over-

Molecular dynamics simulations of ovalbumin adsorption at squalene/water interface

The adsorption of protein molecules to oil/water(O/W)interface is of critical importance for the product design in a wide range of technologies and industries such as biotechnology,food industry and pharmaceutical industry.In this work,with ovalbumin(OVA)as the model protein,the adsorption conformations at the O/W interface and the adsorption stability have been systematically studied via multiple simulation methods,including all-atom molecular dynamic(AAMD)simulations,coarse-grained molecular dynamic(CGMD)simulations and enhanced sampling methods.The computational results of AAMD and CGMD show that the hydrophobic tail of OVA tends to be folded under long time relaxation in aqueous phase,and multiple adsorption conformations can exist at the interface due to heterogeneous interactions raising from oil and water respectively.To further study the adsorption sites of the protein,the adsorption kinetics of OVA at the O/W interface is simulated using metadynamics method combined with CGMD simulations,and the result suggests the existence of multiple adsorption conformations of OVA at interface with the head-on conformation as the most stable one.In all,this work focuses on the adsorption behaviors of OVA at squalene/water interface,and provides a theoretical basis for further functionalization of the proteins in emulsion-based products and engineering.

FAB MS of Permethylated L-β-Aspartamido-Carbohydrates

Native glycopeptides are suitable to FAB mass spectrometric analysis due to their high polarity, low volatility and large molecular size. The deficiency in the FAB spectra of glycopeptides is the limited number of peaks and the low abundances of fragments. In order to obtain the information of carbohydrate sequence the permethylation of carbohydrate, a well known method, is recommended here. The L β aspartamido carbohydrate from chicken egg albumin was chosen as the model compound of glycopeptides. Our experiments showed obvious carbohydrate sequence in the spectra of permethylated aspartamido carbohydrates. The key conditions to perform the method are the methylation reagent and purification process for the permethylated derivatives.

Cyclodextrin/chitosan nanoparticles for oral ovalbumin delivery: Preparation, characterization and intestinal mucosal immunity in mice

A novel oral protein delivery system with enhanced intestinal penetration and improved antigen stability based on chitosan(CS) nanoparticles and antigen-cyclodextrin(CD) inclusion complex was prepared by a precipitation/coacervation method. Ovalbumin(OVA) as a model antigen was firstly encapsulated by cyclodextrin, either β-cyclodextrin( β-CD) or carboxymethyl-hydroxypropyl-β-cyclodextrin(CM-HP-β-CD) and formed OVA-CD inclusion complexes, which were then loaded to chitosan nanoparticles to form OVA loaded β-CD/CS or CM-HP-β-CD/CS nanoparticles with uniform particle size(836.3 and 779.2 nm, respectively) and improved OVA loading efficiency(27.6% and 20.4%, respectively). In vitro drug release studies mimicking oral delivery condition of OVA loaded CD/CS nanoparticles showed low initial releases at p H 1.2 for 2 h less than 3.0% and a delayed release which was below to 30% at p H 6.8 for further 72 h. More importantly, after oral administration of OVA loaded β-CD/CS nanoparticles to Balb/c mice, OVA-specific sIgA levels in jejunum of OVA loaded β-CD/CS nanoparticles were 3.6-fold and 1.9-fold higher than that of OVA solution and OVA loaded chitosan nanoparticles, respectively. In vivo evaluation results showed that OVA loaded CD/CS nanoparticles could enhance its efficacy for inducing intestinal mucosal immune response. In conclusion, our data suggested that CD/CS nanoparticles could serve as a promising antigen-delivery system for oral vaccination.