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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 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.

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 β-defensin-3 (AvβD3) 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 AvβD3 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 (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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2010 S. Karger AG, Basel.)

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.

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.

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.

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.

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.

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.

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.

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.

This paper presents a kinetic study of the intrasplenic circulation of three formulations of the protein antigen conalbumin including the soluble form and two liposomal formulations, one encapsulated in the internal aqueous milieu and one surface-linked to the liposomal vehicle. These formulations differ not only in their physical status but also in their immunostimulating properties and were chosen in an attempt to correlate the movements of antigen in lymphoid tissues with the immune response elicited. The presence of conalbumin was followed over a period of 21 days using, as a detection system, an antibody that we developed and which allows for the visualization of antigenic peptides such as those presented at the surface of antigen-presenting cells (APC). The results demonstrate that the amount of antigen accessing to the spleen, its time of residency and the pathway it follows are all profoundly influenced by the form under which it penetrates the immune system. The results also indicate that the marked initial preferences of an antigen for either B cells, marginal zone macrophages (MZM) or metallophilic macrophages (MM) are of fundamental importance in determining the fate of this antigen in the spleen. It is concluded that the exact formulation of an antigen is as crucial to the regulation of the immune response as is the nature of this antigen. It is further concluded that liposomes can be used efficiently to modify the formulation of an antigen and can contribute as such to the induction of specific immune functions by driving the antigen towards some privileged immune cell populations.

Inflammation is homeostatic process associated with a variety of cellular injuries resulting from infections, toxicosis, and physical trauma. The studies on inflammation in avian species are limited. To understand the inflammation-induced changes, 4-wk-old male broiler chickens were subjected to experimental inflammation by a subcutaneous injection of croton oil (inflammatory) with changes in serum measured over time and were compared with birds treated similarly with olive oil (injected control). Croton oil treatment significantly elevated serum interleukin (IL)-6 concentrations and heterophil counts by 6 and 16 h postinjection, respectively, which returned to the basal levels of controls at 16 and 24 h, respectively. Croton oil treatment affected the serum protein profiles of chickens as assessed by SDS-PAGE and densitometric analyses. Compared with olive oil-injected or noninjected chicken sera, there were increases in the density of protein bands corresponding to molecular weights (MW) of 42, 65, 200, and 219 kDa and decreases in bands corresponding to 49 kDa (serum albumin) and a 56-kDa protein in chickens treated with croton oil. Most of these changes were evident at 24 h and lasted through 48 h. The protein band corresponding to 65 kDa was further characterized using two-dimensional gel electrophoresis and N-terminal sequence analyses. A sequence similarity search in the Genbank database using the first 22 amino acids yielded a complete homology with chicken ovotransferrin. Western blot analysis using antichicken serum transferrin or antichicken ovotransferrin antibodies also confirmed the 65-kDa protein band to be ovotransferrin. Under nonreducing conditions, the ovotransferrin standard also showed an apparent MW corresponding to 65 kDa, like the serum transferrin. The serum ovotransferrin was found to be glycosylated using a glycoprotein stain. Although the significance of ovotransferrin in avian inflammation is not clear, these results show that it is a major acute phase protein (APP) in chickens.

Liposomal antigens are potent adjuvants of humoral and cell-mediated immunity. Although this property requires as an essential condition a physical association between the antigen and the phospholipid vehicle, the nature of the association, i.e., encapsulation or surface linkage, markedly influences the outcome of the elicited response. Available evidence suggests that macrophages are involved in this fine tuning of the immune response in a manner that is not yet clearly established. It is postulated that this might be related to their capacity to interact differently with surface-linked and encapsulated formulations. Using conalbumin as a model antigen, we address the question by analyzing the movements of encapsulated and surface-linked antigen as well as those of MHC-II molecules in macrophages in a pulse-chase immunoelectron microscopic study carried out over a 24-hr period. The antigen was followed using a polyclonal serum specifically raised against fragmented conalbumin (fCA) that allows the detection of processed antigen and of some MHC-peptide complexes. The results indicate that, in macrophages, the two liposomal formulations affect macrophage morphology in distinct ways and circulate through the various subcellular compartments with different kinetics. On the basis of the overall results, we conclude that surface-linked antigen gains access less readily to the endogenous presentation pathway than encapsulated antigen but can favor a more sustained activation of the immune system through its production of exosome-like structures and its more thorough utilization of the MHC-II pathway.

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-gamma concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.

Overexpressing LFA-1 (CD11a/CD18) on antigen specific CD4+ T cells makes the cells proliferate to normally subthreshold stimuli, including self-Ia molecules without specific antigen. The mechanisms by which this occurs are unknown, but potentially include transmission of an increased costimulatory signal, overstabilization of normally low affinity TCR-Ia interactions, or both. A role for increased costimulatory signaling was tested by culturing control and CD18-transfected antigen-specific T cells clones with anti-CD3 and anti-CD11a. Minimal calcium fluxes were detected, but increased protein tyrosine phosphorylation was observed in the transfectants. However, the proliferative response to graded amounts of these antibodies were identical in the transfectants and controls, suggesting that increased signaling alone was insufficient to cause the increased responsiveness. To test for overstabilization, transfected and control clones were cultured with syngeneic Mø with or without antigen. The transfected but not control cells downregulated TCR expression in response to Mø alone, thus demonstrating successful TCR signaling to a low affinity interaction. These results indicate that LFA-1 overexpression permits TCR signal transmission to a normally subthreshold stimulus presented by Mø, consistent with overstabilization. LFA-1 overexpression also causes increased tyrosine phosphorylation, but this alone is not sufficient to cause a proliferative response to low level stimuli.

Background: It has been shown that antigen presentation by resting B cells can induce tolerance to intravenously administered protein antigens, but the role of resting B cells in the induction of oral tolerance is unclear., Methods: Mice continuously treated since birth with rabbit anti-mouse IgM serum for 5 weeks were depleted of B cells. When 4 weeks old, B cell-depleted mice drank 10% chicken egg white (EW) for 5 days. Ten weeks later, they were immunized with 10 microgram of ovalbumin in alum and their T helper 2 (Th2) immune responses were tested., Results: Th2 cell-mediated IgE and IgG1 antibody responses and spleen cell production of IL-4 and IL-5 were suppressed by prior EW feeding during anti-IgM treatment. When anti-IgM-treated spleen cells collected 1 week after EW ingestion were transferred to naïve recipients, active suppression of Th2 immune responses was also demonstrated., Conclusions: Although resting small B cells aggregate in the mantle zone of follicles of intestinal Peyer's patches, the present data suggest that they are not antigen-presenting cells in the induction of oral tolerance of Th2 immune responses to oral antigens., (Copyright 2000 S. Karger AG, Basel.)

Background: We occasionally see egg-allergic children who develop contact urticaria to hen's egg despite the absence of the overt symptoms on ingestion. The mechanisms remain to be elucidated., Methods: Twenty-one subjects with positive reactions to 20-min patch tests for egg-white antigens were divided into subgroups with positive (n = 10) and negative (n = 11) results to oral challenge tests by the same antigens. We measured IgE antibody for egg white and its components, and IgE-binding activities to digestive enzyme-treated ovomucoid by RAST inhibition., Results: There were no significant differences in IgE antibody titers to egg white (positive vs negative: 30.3% vs 15.3%, P=0.130), ovomucoid (21.5% vs 10.2%, P= 0.078), ovotransferrin (9.9% vs 3.7%, P = 0.105), and lysozyme (3.4% vs 2.9%, P=0.944), except ovalbumin (16.8% vs 5.6%, P=0.024), between the positive and negative subjects in the provocation tests. In contrast, the concentration (1.93 microg/ml) of pepsin-treated ovomucoid needed for 50% RAST inhibition in the challenge-positive subjects was significantly (P=0.0003) lower than that (114.9 microg/ml) of negative subjects. Similar but less significant differences were obtained when ovomucoid fragments treated with chymotrypsin (0.91 microg/ml vs 6.86 microg/ml, P=0.014) and trypsin (0.75 microg/ml vs 4.67 microg/ml, P= 0.041) were used as inhibitors., Conclusions: We suggest that IgE antibodies from subjects showing contact urticaria despite the absence of reactions to the ingestion of egg white recognize the epitope(s) unstable to digestive enzymes.

Monoclonal sheep antibodies have great potential for biomedical, veterinary and agricultural purpose. Although conventional sheep monoclonal antibodies can be generated by a modified hybridoma technology, the procedures are not routine. Here, we describe a method to generate recombinant sheep antibody fragments from immunised animals using a modified phage display system. Total RNA from pooled spleens of sheep immunised with the model antigens human serum albumin and conalbumin were used to amplify immunoglobulin V gene repertoires and an efficient two-step cloning method was employed to rapidly construct a phage display single-chain Fv (scFv) library. A total of 14 different scFvs were isolated and characterised. Sequence analysis indicated typical ovine immunoglobulin characteristics. Thirteen Vlambda and 11 VH genes were identified that could be grouped into the sheep Vlambda families I, II, VI and a single VH family. Soluble monomeric scFvs, produced in the periplasm of Escherichia coli, were subjected to affinity measurement via surface plasmon resonance analysis and affinities typical of the secondary immune response were observed. The method described here should be of value for the study of sheep immunology as well as for biorecognition in general.

The crystal structure of a complex involving the D10 T cell receptor (TCR), 16-residue foreign peptide antigen, and the I-Ak self major histocompatibility complex (MHC) class II molecule is reported at 3.2 angstrom resolution. The D10 TCR is oriented in an orthogonal mode relative to its peptide-MHC (pMHC) ligand, necessitated by the amino-terminal extension of peptide residues projecting from the MHC class II antigen-binding groove as part of a mini beta sheet. Consequently, the disposition of D10 complementarity-determining region loops is altered relative to that of most pMHCI-specific TCRs; the latter TCRs assume a diagonal orientation, although with substantial variability. Peptide recognition, which involves P-1 to P8 residues, is dominated by the Valpha domain, which also binds to the class II MHC beta1 helix. That docking is limited to one segment of MHC-bound peptide offers an explanation for epitope recognition and altered peptide ligand effects, suggests a structural basis for alloreactivity, and illustrates how bacterial superantigens can span the TCR-pMHCII surface.

We show that macrophages of X-linked immunodeficient mice with a mutant nonfunctional Bruton's tyrosine kinase produce less NO than wild-type macrophages in response to a variety of stimuli. Induction of the inducible NO synthase (iNOS) protein, the transcription factor IFN regulatory factor-1 involved in iNOS expression, and the transcription factor STAT-1 involved in regulating IFN regulatory factor-1 induction are all poorer in X-linked immunodeficient than in wild-type macrophages. On the other hand, induction of IL-12 is higher in X-linked immunodeficient than in wild-type macrophages. Macrophage IL-12 induction is enhanced by iNOS inhibitors such as aminoguanidine and thiocitrulline and is inhibited by NO generation via sodium nitroprusside. There is relative enhancement of IFN-gamma production by immune T cells from mice immunized under aminoguanidine cover. Our data thus suggest that Bruton's tyrosine kinase participates in signaling for iNOS induction via IFN regulatory factor-1 in macrophages and that NO is an inhibitor of IL-12 induction.

Human foods are usually prepared by cooking. Boiling of chicken egg-white (EW) led to decreased allergenicity, and abrogated intestinal uptake of immunoreactive ovalbumin (OVA) when fed to mice. Therefore, the effects of oral administration of boiled EW were examined further in BALB/c mice. Specific IgE, IgG1 and IgG antibody responses were suppressed by raw EW, but not by EW boiled for 5 or 60 min, fed prior to sensitization with 10 microg OVA or 1 microg DNP-OVA in alum. Similar results were obtained when mice were sensitized with 10 microg conalbumin, ovomucoid or lysozyme in alum. BALB/c spleen cell proliferation and secretion of Th2 cytokines IL-4 and IL-5 during in vitro stimulation with OVA were also suppressed by feeding raw EW, but not by boiled EW. Although heat denaturation of proteins can minimize allergenicity, the present results suggest that over-cooking of proteins may affect their intestinal antigen processing and thus prevent the induction of oral tolerance.

The development of pulmonary allergic responses was examined in mice following pulmonary transfer of Ag (conalbumin)-specific Th2 cells. The levels of serum-specific IgE, cellular infiltrates, airway mucus goblet cells, and airway responsiveness were analyzed and compared with those in Ag-sensitized and -challenged mice. Pulmonary transfer of the conalbumin-specific Th2 clone (D10) induced, in an Ag-specific manner, high levels of the Th2 cytokines IL-4 and IL-5 in the bronchoalveolar lavage fluids and mucosal eosinophils, concomitant with an increase in airway responsiveness. The D10 cell-induced responses were seen in the absence of serum specific IgE. In the presence of Ag, the transferred D10 cells not only remained in the lungs, but also increased in number 72 h post-cell transfer. Although significantly higher levels of IL-4 and IL-5 in the bronchoalveolar lavage fluids were found in D10-transferred mice, the levels of pulmonary eosinophilia, mucus goblet cells, and airway responsiveness were significantly lower than those in Ag-sensitized and -challenged mice. These results demonstrate that although Ag-specific activation of Th2 cells at mucosal sites is able to mediate the recruitment of eosinophils and the subsequent induction of airway hyper-responsiveness, the more severe pulmonary allergic responses were observed only in mice sensitized and challenged with Ag.

Maternal transfer of TCR clonotypic Ab protected young NOD mice against the adoptive transfer of diabetes by the BDC 2.5 T cell clone. The effect of maternal anti-TCR Vbeta-8 Ab on T cell development and function has now been investigated. SJL/J mice, which lack TCR Vbeta-8, were immunized with soluble, chimeric D10 TCR-IgG1 containing Vbeta-8.2. The (SJL/J x AKR/J) F1 offspring of immunized female SJL/J mice were severely depleted of peripheral T cells bearing Vbeta-8 until 11 to 17 wk of age. The loss of Vbeta-8 expression did not appear to be due to modulation of cell surface TCR. Since the Vbeta-8+ T cell population was unperturbed in the (AKR/J x SJL/J) F1 offspring of D10 TCR-IgG1-immunized AKR/J mothers making D10 clonotypic Ab, the effect was immunologically specific. The deletion of Vbeta-8+ T cells had functional consequences. In the in vitro response to the superantigen, staphylococcal enterotoxin B, the usually observed participation of Vbeta-8.2+ T cells was largely suppressed, whereas the recruitment of Vbeta-3+ T cells remained unaltered. In control mice, T cell responses to the 134- to 146-residue peptide of conalbumin (pCA(134-146)) were biased toward use of Valpha-2/Vbeta-8.2 TCR. In D10 TCR-IgG1 maternally immunized (SJL x AKR/J) F1 mice, the T cell responses to pCA(134-146) were suppressed, and T cell lines derived from these in vitro were devoid of Vbeta-8.2 expression. With an increased understanding of TCR V gene usage in autoimmune diseases, similar strategies for the depletion of autoreactive T cells may become feasible in humans.

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell-mediated antigen presentation. We have previously shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen-presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen-pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti-I-A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1-trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I-A, from MBP-pulsed IrrSPL but blockade of I-A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I-A, R1-trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self-MHC complex strongly promotes acquisition of these complexes from professional APC.

The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.

Background: No egg white products have been clearly proven to be hypoallergenic. The role of egg white proteins in allergic reactions to eggs is still debatable., Objective: This study was designed to determine the importance of ovomucoid, an egg white protein, in the development of allergies to egg white., Methods: We performed a double-blind, placebo-controlled food challenge in subjects with high levels of IgE antibodies for egg white to compare the allergenicities of heated and ovomucoid-depleted egg white, freeze-dried egg white, and heated egg white. Levels of IgE antibodies for egg white, ovomucoid, ovalbumin, ovotransferrin, and lysozyme were measured in serum by RAST., Results: Twenty-one of 38 subjects with positive challenge responses to freeze-dried egg white had negative challenge responses to heated egg white, whereas 16 of 17 subjects (94.1%) with positive responses to heated egg white did not respond to the heated and ovomucoid-depleted egg white challenge. The subjects with positive challenge responses to freeze-dried egg white tended to have higher IgE antibody values to ovomucoid than those with negative responses. IgE antibody levels to ovomucoid were significantly higher in subjects with positive responses to a challenge with heated egg white than in those with no response. There were no significant differences in the levels of IgE antibodies to the other proteins, except ovomucoid, in the negative-response and positive-response groups in challenge tests with freeze-dried and heated egg white., Conclusion: The heated and ovomucoid-depleted egg white preparation was less allergenic than heated or freeze-dried preparations. Ovomucoid has a more important role in the pathogenesis of allergic reactions to egg white than other proteins in egg white.

Engagement of a TCR by its peptide Ag bound by MHC class II molecules leads to T cell activation, resulting in proliferation and cytokine production. This agonist response can be antagonized by analogue peptides containing single amino acid substitutions. We used T cell clones isolated from a mouse transgenic for the rearranged TCR alpha- and beta-chains of the D10.G4.1 (D10) Th2 clone specific for hen egg conalbumin peptide 134-146 (CA 134-146) to characterize antagonist peptides for the D10.TCR. The D10.TCR CD4 T cell clones proliferated in a dose-dependent manner to CA 134-146, and this proliferation was accompanied by secretion of IL-4 and/or IFN-gamma with Th1, Th2, and Th0 patterns. Proliferation of the clones was inhibited completely by CA 134-146 analogue peptides containing a substitution of the glutamic acid at position 8 with alanine (E8A) or threonine (E8T). The E8A and E8T peptides also antagonized the production of mRNA and subsequent cytokine secretion of IFN-gamma and/or IL-4. Our results, showing that antagonist peptides can inhibit both T cell proliferation and cytokine production in Th1, Th2, and Th0 clones all bearing the same TCR, demonstrate that the TCR:peptide interaction determines the outcome regardless of the phenotype of the clone. Thus, antagonism by peptides acts through the TCR.

Objective: To determine whether redirecting intestinal blood flow away from the liver via a portacaval shunt would protect distal immune responses in the spleen after hemorrhage., Design: Type C3H/HeN male mice in which a portacaval shunt had been established 2 to 3 weeks before the experiment were bled, their blood pressure was maintained at 35 mm Hg for 1 hour, and then they were resuscitated. Twenty-four hours later, the mice were killed, and mixed and adherent splenocyte cultures were established. The proliferative capacity of splenocytes, the release of interleukin-2 and interleukin-4, and the ability of splenic macrophages to present the antigen, conalbumin, were then determined as indexes of immunocompetence., Results and Conclusions: The results indicate that splenocyte proliferation and splenocyte interleukin-2, but not interleukin-4, release were decreased and that splenic macrophage antigen presentation declined after hemorrhage. However, animals in which a portacaval shunt had been established before hemorrhage showed no such changes in their splenocyte or splenic macrophage functions. Thus, the decline of splenic immune responses after hemorrhage seems to be due to mediators released from the gut. Such mediators, in turn, stimulate Kupffer cells to secrete additional agents that produce immunosuppressive exocrine effects on splenic immune functions.

Th2 cytokines are associated with the airway inflammation and hyperreactivity in allergic asthma and are potential targets for developing novel therapies. The efficacy of a mucosal cytokine-gene transfer approach was examined in a mouse model for allergic asthma. We showed first that mucosal IFN-gamma gene transfer results in a significant expression of IFN-gamma in the pulmonary epithelium. Significantly, this approach inhibits both Ag- and Th2-cell-induced pulmonary eosinophilia and airway hyperreactivity. These findings suggest that mucosal IFN-gamma gene transfer is effective in modulating pulmonary allergic responses and provides a basis for developing a novel therapeutic approach.

In our earlier studies we showed that successful immunotherapy of EAE in SJL/J mice can be achieved either by the use of antibodies to MHC class II antigens or by vaccination with synthetic peptide analogs of the beta chain of MHC class II molecules. We proposed that inhibition of EAE following vaccination with synthetic peptides derived from the beta chain of mouse I-A, was in part due to the generation of auto-anti-MHC class II antibodies that interfered with T cell sensitization. In our present study we show that suppression of EAE following vaccination results in poor sensitization of MBP reactive T cells, and that the lack of immune response is allele-specific. In F1(SJL(I-AS) x Balb/cI-Ad) mice, in which susceptibility to EAE is linked closely to the I-AS allele, vaccination with peptides from beta chain of I-AS results in inhibition of proliferative response to MBP and prevents the development of EAE. Vaccination with peptide from the beta chain of I-Ad did not affect either the development of immune response to MBP or the induction of EAE, indicating allele-specific suppression. Since global immunosuppression is not induced by vaccination with I-A peptides, we propose that this strategy can be extended to human autoimmune diseases wherein a clear association between certain MHC class II alleles and autoimmune disease is evident.

The biodistribution of liposomal antigens either encapsulated in or surface-linked to liposomes of similar composition was studied over time following intravenous injection and the results analyzed in relation to adjuvanticity. The two formulations were shown to behave very differently in vivo. While encapsulated antigen was rapidly focused to liver and spleen as expected, surface-linked antigen exhibited a more disseminated distribution which parallels that of the free protein. In dual-labelling experiments, it was also shown that encapsulated antigen remains associated with its liposomal vehicle in contrast to surface-linked antigen which is rapidly dissociated. This dissociation was apparently neither due to an exchange with plasma lipoproteins nor to a direct action of blood constituents. Besides, it was found that surface-linked antigen was rapidly accumulated in the carcass. We propose that the retention of the surface-linked antigen in the carcass results from a pre-processing of the protein involving more probably mononuclear phagocytes. This pre-processing might in turn favor the dissociation of the protein from the liposomes in a form that allows its dissemination in the whole organism and its interaction with more efficient antigen presenting cells such as for example Langerhans or dendritic cells.

Two proteins, a recombinant malaria protein (R32NS1) and conalbumin, were encapsulated in separate liposomes. The mechanisms of presentation of unencapsulated and liposome-encapsulated R32NS1 and conalbumin to antigen-specific T-cell clones were investigated in in vitro antigen presentation assays using murine bone marrow-derived macrophages (BMs) as antigen-presenting cells. A much lower concentration of liposomal antigen than of unencapsulated antigen was required for T-cell proliferation. Liposome-encapsulated conalbumin required intracellular processing by BMs for antigen-specific T-cell proliferation, as determined by inhibition with chloroquine, NH4Cl, leupeptin, brefeldin A, monensin, antimycin A, NaF, and cycloheximide and by treatment of BMs with glutaraldehyde. Liposome-encapsulated conalbumin therefore follows the classical intracellular antigen processing pathway described for protein antigens. Similarly, unencapsulated conalbumin also required intracellular processing for presentation to antigen-specific T cells. In contrast, both unencapsulated R32NS1 and liposome-encapsulated R32NS1 were presented to T cells by BMs without undergoing internalization and intracellular processing. These results suggest that the antigen itself is the major element that determines whether a requirement exists for intracellular processing of liposomal antigens by macrophages.

The capacity of CD4+ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by gamma-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

The immunoadjuvanticity of liposomal antigens, namely encapsulated and surface-linked conalbumin, was studied at different levels of an immune response including immunoglobulin production, blastogenic response and lymphokine production in sensitization conditions compatible with vaccine designs. The results demonstrated that both liposomal formulations stimulate all properties analyzed with respect to free antigen but significantly differ in some of their inductive capabilities, suggesting that they follow different routes in the immune network. Thus, although both liposomal antigens are capable of inducing potent humoral responses characterized by increased production of IgM and IgG2a, covalently linked antigen stimulates a quasi-polyclonal blastogenic response accompanied by the simultaneous secretion of IL-2 and IFN gamma, while encapsulated antigen which is less blastogenic mainly induces IL-2 secretion. We can conclude that: first, both antigenic formulations induce a Th1 type of activation and might therefore potentiate cell-mediated immunity, but surface-linkage favors a more rapid maturation of the response and a much more intense help induction. Second, although a strong adjuvanticity can be observed whatever the route of sensitization, namely intraperitoneally, intravenously or subcutaneously, the intravenous injections induce the better potentiation. The in vitro data were all compared to those obtained with naïve mice, allowing dissociation of the contribution of the in vitro rechallenge from that of the in vivo immunization. These differences observed between the liposomal antigens might be taken advantage of while formulating vaccines specifically suited to meet required needs and suggest that covalently linked antigen might be particularly useful in situations where induction of cell-mediated immunity is of prime importance.

Heavily pigmented eyes tend to experience greater inflammation than lightly pigmented eyes following trauma and surgery. The purpose of these studies was to test the possibilities that: (i) melanin augments T cell-responses by depleting or neutralizing anti-inflammatory substances that are normally present in the aqueous humor, and (ii) melanin augments extraocular T cell-mediated inflammatory responses. Two types of experiments were performed. First, the capacity of melanin-adsorbed and non-adsorbed rabbit aqueous humor to inhibit the proliferative response of the T cell line D10.G4.1 to IL-1 was tested. Non-adsorbed aqueous humor inhibited T cell proliferation to the background level in unstimulated cultures, whereas melanin-adsorbed aqueous humor enhanced the proliferation of stimulated, but not resting T cells. Next, mice were sensitized to the antigen conalbumin, and challenged in the ear pinna with conalbumin alone, conalbumin + melanin, or melanin alone and delayed-type hypersensitivity (DTH) was measured. Challenge with melanin alone caused some ear swelling, and melanin increased the DTH response to conalbumin. Our findings are consistent with the notion that melanin can augment intraocular inflammation by depleting or neutralizing the inhibitory components of normal aqueous humor, possibly exposing stimulatory components that are normally masked.

Anti-TCR antibodies can activate or block the activation of T cells. In the present experiments, we have shown that different monoclonal antibodies to the same TCR can have either agonist or antagonist activity, and we have examined the relationship between these functional effects and the avidity of the antibody for the TCR. We show here that it is not the avidity of an anti-TCR antibody that determines whether it acts as an agonist or an antagonist. Moreover, we show that monovalent Fab fragments of agonist antibodies produce detectable changes in T cell behavior. These data suggest that T cell activation may involve not just aggregation of the TCR but also some induced change in individual ligated receptors, and that agonists may produce this change while antagonists do not. We argue that similar effects may apply to peptide-MHC ligands as well.

IgE is considered to be involved in immediate hypersensitive reactions (IHR) following egg ingestion. IgE antibody levels to egg-white (EW) antigens in the IHR-positive group (n = 19, mean age +/- SD = 5.2 +/- 4.5 yr) were higher than those in the IHR-negative group (n = 13, mean of age +/- SD = 3.6 +/- 2.2 yr). However, even in the IHR-negative group, some patients showed high IgE to EW. RAST inhibition tests with heat-treated (100 degrees C, 5, 10, and 30 min) egg-white antigens were performed on 13 serum samples from subjects with IHR and 9 serum samples from subjects without IHR. Heat treatment decreased the IgE-binding activity of egg white and it was speculated that IgE from IHR-negative subjects bound to relatively heat-unstable sites of egg-white antigens. Furthermore, we selected IHR-negative subjects (n = 8, mean of age +/- SD = 3.0 +/- 1.7 yr) with higher IgE antibody levels than the lowest limit of IgE to EW of the IHR-positive group and compared IgE to ovomucoid (OM), ovalbumin (OA), conalbumin (CA), and lysozyme (Ly) between these IHR-negative and positive groups. IgE-binding activities to egg-white components, including OA, CA, and Ly but not OM, were significantly decreased with heat treatment. The IHR-negative group showed significantly lower IgE to OM (untreated, 5, 10, 30 min treatment) and 5 min treated OA alone than the IHR-positive group, while no difference was found in IgE to other components between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)