Component resolved diagnosis of egg yolk is an indispensable part of egg allergy

Introduction and objectives It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk. Materials and methods Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis. Results Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994. Conclusions Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably (AU)

Component resolved diagnosis of egg yolk is an indispensable part of egg allergy.

INTRODUCTION AND OBJECTIVES: It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk. MATERIALS AND METHODS: Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis. RESULTS: Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994. CONCLUSIONS: Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably.

Geographic variation in baseline innate immune function does not follow variation in aridity along a tropical environmental gradient.

Geographic variation in aridity determines environmental productivity patterns, including large-scale variability in pathogens, vectors and associated diseases. If disease risk decreases with increasing aridity and is matched by immune defense, we predict a decrease in innate immune function along a gradient of increasing aridity from the cool-wet forest to the hot-dry Sahel, from south to north in Nigeria. We sampled blood and measured five innate immune indices from 286 Common Bulbuls Pycnonotus barbatus between 6 and 13°N. We sampled in the dry season; we resampled the first location (Jos) also as the last sample location to test temporal change in immune function. Immune indices did not decrease with aridity. One immune index, nitric oxide concentration showed a weak quadratic pattern. In Jos, ovotransferrin concentration, haemagglutination and haemolysis titres increased 12 weeks into the dry season, contrary to expectations that immune indices should decrease with increased dryness. In this tropical system, innate immune function does not decrease with increasing aridity but temporal factors within a location may influence immune function more strongly than spatial variation in aridity, suggesting that immune variation does not follow a simple environmental productivity pattern. Consequently, caution should probably be exercised in predicting effects of climate variability on immune function or disease risk.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Ovotransferrin enhances intestinal immune response in cyclophosphamide-induced immunosuppressed mice.

Ovotransferrin (OVT), a glycoprotein from avian egg, which has a variety of biological activities and immunomodulatory effects. The purpose of this research was to demonstrate the effect of OVT on intestinal immunomodulatory function which used a mouse model of cyclophosphamide (CP) induced intestinal immunosuppression and injury by intraperitoneal injection of 80 mg/kg. Effects of OVT on intestinal immunomodulatory function in CP-induced immunosuppression mice were detected by flow cytometry, real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot. Results showed that OVT effectively increased the major histocompatibility complex class II (MHC-II) and cluster of differentiation 83 (CD83) levels to enhance intestinal dendritic cells (DCs) maturation and promoted the expression of cytokines and gene of tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4) and interleukin-10 (IL-10). Furthermore, the imbalance ratio of the Th1 and Th2 in the intestine was regulated to produce an immune response and the expression of immunoglobulin A (IgA) and secretory immunoglobulin A (sIgA) were increased to promote humoral immunity by OVT-treated. Meanwhile, cyclophosphamide treatment induces activation of p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) to causes intestinal damage and blockage of p38 MAPK, JNK and ERK activation contributed to the effect of OVT on the repair of intestinal damage. These results indicated that OVT may have immunomodulatory function and could be potential functional factor to regulate body intestinal immunity.

Iron-induced chelation alleviates the potential allergenicity of ovotransferrin in a BALB/c mouse model.

Ovotransferrin (OVT) is one of the main egg allergens with 2 iron-binding sites. Several studies have demonstrated that iron-chelation decreased the allergenicity of milk allergen and birch pollen allergens. Therefore, we hypothesized that iron-chelation could also reduce the allergenicity of OVT. Apo-OVT (iron-free OVT, the natural state in egg white) and Holo-OVT (iron-chelated OVT) were prepared, and the allergenicity of them were assessed and compared using a BALB/c mouse model as well as dendritic cells (DCs) based on antigen uptake. Mice were orally sensitized with Apo-OVT or Holo-OVT using cholera toxin as adjuvant. Clinical signs of allergy, morphological structure of jejunum, specific antibody levels, mast cell protease-1 (mMCP-1) concentrations, cytokines and antigen uptake by DCs were determined after the mice were challenged with Apo-OVT or Holo-OVT. Results showed that both Apo-OVT and Holo-OVT induced intestinal allergy, but no systematic allergic symbols were observed. Serum levels of mouse mast cell protease-1 (mMCP-1) and specific IgE in Apo-OVT group were lower than in control group, and no significant difference between Apo-OVT group and Holo-OVT group (P>.05). The levels of OVT-specific IgG and IgG1, as well as the Th-1 cytokine interferon gamma and Th2-type cytokine interleukin-13 in Holo-OVT sensitized mice were significantly decreased compared to Apo-OVT group (P<.05), while no significant difference with control group (P>.05). However, DCs took in less Apo-OVT than Holo-OVT. Overall, iron-induced chelation could alleviate the potential allergenicity of OVT in vivo.

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli.

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients' sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid.

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Special consideration is required for the component-resolved diagnosis of egg allergy in infants.

BACKGROUND: There are few reports regarding differences in reactivity to the major egg allergens according to children's age, although component-resolved diagnosis is gradually being used. OBJECTIVE: To investigate differences in reactivity to major egg allergens among various age groups of children with egg allergy. METHODS: Twenty-seven patients diagnosed with egg allergy were included. Egg allergy was defined as a convincing history of reproducible symptoms within 2 hours of egg consumption and an egg white-specific IgE level of at least 0.35 kUA/L. Patients were divided into 3 age groups: younger than 12 months (group A, 7 subjects), 12 to 23 months (group B, 8 subjects), and at least 24 months (group C, 12 subjects). Immunoblotting and enzyme-linked immunosorbent assay investigated IgE reactivity toward ovalbumin, ovomucoid, and ovotransferrin in eggs. RESULTS: Immunoblotting analysis showed that all patients in group A reacted to ovalbumin, whereas reactions to other proteins were not detected. All patients in group B displayed a reaction to ovalbumin and ovomucoid. IgE binding to ovotransferrin was shown in 3 patients in group B. All patients in group C displayed reactivity to ovalbumin, 5 patients showed a reaction to ovomucoid, and 8 patients displayed a reaction to ovotransferrin. As a patient's age increased, specific IgE binding to ovalbumin and ovotransferrin increased (P = .011 and .004). CONCLUSION: IgE reactivity to egg allergens differs according to children's ages.

Affinity purification of egg-white allergens for improved component-resolved diagnostics.

BACKGROUND: Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD. METHODS: Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals. RESULTS: Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%). CONCLUSIONS: In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.

Primary structure of potential allergenic proteins in emu (Dromaius novaehollandiae) egg white.

The emu (Dromaius novaehollandiae) egg is considered promising as an alternative egg product. To obtain basic biochemical information on emu egg white, the major protein compositions in emu and chicken egg whites and the primary structures of potential allergenic proteins were compared. The dominant protein in emu egg white was ovotransferrin (OVT), followed by ovalbumin (OVA) and TENP protein. The OVA and ovomucoid (OVM) levels in emu egg white were estimated as significantly lower than those in chicken egg white by Western blotting and enzyme-linked immunosorbent assays using anti-chicken OVA or OVM antibodies. Lysozyme and its enzymatic activity were not detected in emu egg white. OVT, OVA, and OVM genes were also cloned, and their nucleotide and amino acid sequences were determined. The protein sequences of OVT, OVA, and OVM from emu showed lower similarities to those of chicken than other avian species, such as quail and turkey. These results emphasize the low allergenicity of emu egg white and its potential as an alternative to chicken egg white.

Allergy to quail's egg without allergy to chicken's egg. case report.

INTRODUCTION: We present a case of quail's egg allergy without allergy to chicken's egg. CASE: Girl of 10.5 years old who presents anaphylactic reaction after she ate an uncooked quail's egg. She had eaten boiled quail's egg before. She eats chicken's eggs without clinical symptoms. METHODS: We made a prick to chicken's egg and prick-by-prick to uncooked quail's and raw chicken's egg. We determined specific IgE to chicken's egg; electrophoresis and IgE by immunoblot to eggs from chicken, duck, goose, and quail. RESULTS: We obtained negative results to prick, prick-by-prick and specific IgE to chicken's egg. Prick-by-prick to quail's egg was positive. By immunoblot we recognised a protein in quail's egg white, which is ovotransferrin without any similar bands in other species' eggs. CONCLUSIONS: The protein that we recognised is a specific protein of quail's egg. These proteins did not cross-react with proteins of chicken's egg. Cooking may degrade such proteins.

The panel of egg allergens, Gal d 1-Gal d 5: Their improved purification and characterization.

Egg proteins represent one of the most important sources evoking food allergic reactions. In order to improve allergy diagnosis, purified and well-characterized proteins are needed. Although the egg white allergens Gal d 1, 2, 3 and 4 (ovomucoid, ovalbumin, ovotransferrin, and lysozyme) are commercially available, these preparations contain impurities, which affect exact in vitro diagnosis. The aim of the present study was to set up further purification protocols and to extend the characterization of the physicochemical and immunological properties of the final batches. The egg white allergens Gal d 1-4 were purified from commercial preparations, whereas Gal d 5 (alpha-livetin) was purified from egg yolk. The final batches of Gal d 1-5 consisted of a range of isoforms with defined tertiary structure. In addition, the IgE binding capacity of the purified egg allergens was tested using allergic patients' sera. The allergen batches will be further used to set up allergen specific diagnostic assays and to screen a larger collection of patients' sera.

Detection of four distinct groups of hen egg allergens binding IgE in the sera of children with egg allergy.

BACKGROUND: There appears to be a lack of agreement in the literature on the allergenicity of hen egg proteins. This may be partly due to the use of impure proteins in some cases. Egg yolk proteins have also been largely ignored in such studies. We therefore set out to determine, using especially purified proteins, their relative allergenicity, and to observe whether there were any relationships between their potency and the sensitivity of patients to them. METHODS AND RESULTS: The sera of 40 patients with clinically observed hen egg hypersensitivity were tested for specific IgE binding to purified egg white and egg yolk proteins using the radioallergosorbent test (RAST). Statistical treatment by correspondence analysis of the percent radioactive uptakes in the RAST to the 8 proteins demonstrated that there were four distinct groups of patients reacting in a similar way to four discrete sets of proteins. CONCLUSIONS: The first three sets of allergens consisted of egg white proteins as follows: firstly, lysozyme and ovalbumin; secondly, ovomucoid; and thirdly, ovomucin. The fourth set contained the egg white protein ovotransferrin and the egg yolk proteins apovitellenins I and VI and phosvitin. The existence of patient groups may explain why various workers have reported different allergens to be important in egg hypersensitivity. A sufficiently large number of patients must be examined so as to give a representative distribution across each group, otherwise the results may be biased towards one allergen.

A novel Syk kinase-selective inhibitor blocks antigen presentation of immune complexes in dendritic cells.

The initiation of antigen presentation by dendritic cells requires proper internalization of antigens through various mechanisms. Internalization of immune complexes via Fc receptors has been shown to be around 100 times more efficient than the internalization of non-complexed antigens. Spleen tyrosine kinase (Syk) plays an essential role in the signaling cascade initiated by immunoglobulin receptors. We used a selective Syk inhibitor, 7-(3,4-dimethoxyphenyl)-N-1H-indazol-6-ylimidazo[1,2-c]pyrimidin-5-amine dihydrochloride (compound-D), to evaluate the role of Syk in antigen presentation by mouse bone marrow-derived dendritic cells. In line with our expectation, compound-D concentration-dependently inhibited the internalization of immune complexes but not that of antigen itself. Furthermore, when dendritic cells were pretreated with compound-D, the ability of dendritic cells to present immune complex antigens to Th2 cells was attenuated, parallel by a reduced release of interleukin-4 production in Th2 cells. Therefore, Syk kinase activity is a critical component in the process of Fcgamma receptor-mediated internalization of immune complex antigens in dendritic cells, and Syk kinase inhibitors may be beneficial in selectively suppressing antibody-mediated antigen presentation in allergic diseases.

Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles.

Unmethylated CpG dinucleotides present within certain specific sequence contexts in bacterial and synthetic DNA stimulate innate immune responses and induce cytokine secretion. Recently, we showed that CpG DNAs containing two 5'-ends, immunomers, are more potent in both regards. In this study, we show that an immunomer containing a synthetic CpR motif (R = 2'-deoxy-7-deazaguanosine) is a potent immunostimulatory agent. However, the profile of cytokine induction is different from that with immunomers containing a natural CpG motif. In general, a CpR immunomer induced higher interleukin (IL)-12 and lower IL-6 secretion. Compared with conventional CpG DNAs, both types of immunomers showed a rapid and enhanced activation of the transcription factor NF-kappaB in J774 cells. NF-kappaB activation by CpG DNA corresponded to degradation of IkappaBalpha in J774 cells. All three immunostimulatory oligonucleotides activated the p38 mitogen-activated protein kinase pathway as expected. Immunomers containing CpG and CpR motifs showed potent reversal of the antigen-induced Th2 immune response towards a Th1 type in antigen-sensitized mouse spleen cell cultures. Immunomers containing a CpR motif showed significant antitumor activity in nude mice bearing MCF-7 human breast cancer and U87MG glioblastoma xenografts. These studies suggest the ability for a divergent synthetic nucleotide motif recognition pattern of the receptor involved in the immunostimulatory pathway and the possibility of using synthetic nucleotides to elicit different cytokine response patterns.

A pool of central memory-like CD4 T cells contains effector memory precursors.

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.

Effects of ovotransferrin on chicken macrophages and heterophil-granulocytes.

Ovotransferrin (OTF) is an acute phase protein in chickens, serum levels of which increase in inflammation and infections. To understand the significance of OTF in inflammation, we studied its in vitro effects on HD11 cells, a macrophage cell line, and heterophils isolated from blood using a panel of variables indicative of cellular activation. These included the production of interleukin-6 (IL-6), nitrite, matrix metalloproteinase (MMP), oxidation of dichlorofluorescein diacetate for respiratory burst and the degranulation of heterophils by the loss of fluorescein isothiocyanate positive cytoplasmic granules. The results show that ovotransferrin stimulates the production of IL-6, nitrite and MMP by HD11 cells and augments phorbol ester-induced respiratory burst. Ovotransferrin stimulated heterophils to produce IL-6, and MMP, but failed to produce nitrite, enhanced respiratory burst activity and degranulation. These results suggest that ovotransferrin can modulate macrophage and heterophil functions in chickens.

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor.

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.