PURPOSE OF REVIEW: Immunoglobulin A (IgA) mediates immune exclusion of antigens in the gut. Notably, IgA plays also a role in the prevention of IgE-mediated allergies and induction of immune tolerance. The present review addresses the role of IgA in the manifestation of IgE-mediated allergies, including allergen-specific immunotherapy (AIT), the regulation of IgA production, and the mechanism of IgA in immune cell activation. RECENT FINDINGS: The majority of studies report an association of IgA with the induction of immune tolerance in IgE-mediated allergies. However, reports on the involvement of humoral and mucosal IgA, IgA subtypes, monomeric and polymeric IgA, and the mechanism of IgA-mediated immune cell activation are confounding. Effects by IgA are likely mediated by alteration of microbiota, IgE-blocking capacity, or activation of inhibitory signaling pathways. However, the precise mechanism of IgA-regulation, the contribution of serum and/or mucosal IgA, and IgA1/2 subtypes, on the manifestation of IgE-mediated allergies, and the underlying immune modulatory mechanism are still elusive.

Turmeric (Curcuma longa) contains various compounds that potentially improve health. Bisacurone is a turmeric-derived compound but has been less studied compared to other compounds, such as curcumin. In this study, we aimed to evaluate the anti-inflammatory and lipid-lowering effects of bisacurone in high-fat diet (HFD)-fed mice. Mice were fed HFD to induce lipidemia and orally administered bisacurone daily for two weeks. Bisacurone reduced liver weight, serum cholesterol and triglyceride levels, and blood viscosity in mice. Splenocytes from bisacurone-treated mice produced lower levels of the pro-inflammatory cytokines IL-6 and TNF-α upon stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS), and TLR1/2 ligand, Pam3CSK4, than those from untreated mice. Bisacurone also inhibited LPS-induced IL-6 and TNF-α production in the murine macrophage cell line, RAW264.7. Western blot analysis revealed that bisacurone inhibited the phosphorylation of IKKα/β and NF-κB p65 subunit, but not of the mitogen-activated protein kinases, p38 kinase and p42/44 kinases, and c-Jun N-terminal kinase in the cells. Collectively, these results suggest that bisacurone has the potential to reduce serum lipid levels and blood viscosity in mice with high-fat diet-induced lipidemia and modulate inflammation via inhibition of NF-κB-mediated pathways.

Cholesterol and its oxidized forms, oxysterols, are ingested from foods and are synthesized de novo. Cholesterol and oxysterols influence molecular and cellular events and subsequent biological responses of immune cells. The amount of dietary cholesterol influence on the levels of LDL cholesterol and blood oxysterols plays a significant role in the induction of pro-inflammatory state in immune cells, leading to inflammatory disorders, including cardiovascular disease. Cholesterol and oxysterols synthesized de novo in immune cells and stroma cells are involved in immune homeostasis, which may also be influenced by an excess intake of dietary cholesterol. Dietary compounds such as β-glucan, plant sterols/stanols, omega-3 lipids, polyphenols, and soy proteins, could lower blood cholesterol levels by interfering with cholesterol absorption and metabolism. Such dietary compounds also have potential to exert immune modulation through diverse mechanisms. This review addresses current knowledge about the impact of dietary-derived and de novo synthesized cholesterol and oxysterols on the immune system. Possible immunomodulatory mechanisms elicited by cholesterol-lowering dietary compounds are also discussed.

The concept of food and aging is of great concern to humans. So far, more than 300 theories of aging have been suggested, and approaches based on these principles have been investigated. It has been reported that antioxidants in foods might play a role in human aging. To clarify the current recognition and positioning of the relationship between these food antioxidants and aging, this review is presented in the following order: (1) aging theories, (2) food and aging, and (3) individual food antioxidants and aging. Clarifying the significance of food antioxidants in the field of aging will lead to the development of strategies to achieve healthy human aging.

In the late 2010s, artificial intelligence (AI) technologies became complementary to the research areas of food science and nutrition. This review aims to summarize these technological advances by systematically describing the following: the use of AI in other fields (eg, engineering, pharmacy, and medicine); the history of AI in relation to food science and nutrition; the AI technologies currently used in the agricultural and food industries; and some of the important applications of AI in areas such as immunity-boosting foods, dietary assessment, gut microbiome profile analysis, and toxicity prediction of food ingredients. These applications are likely to be in great demand in the near future. This review can provide a starting point for brainstorming and for generating new AI applications in food science and nutrition that have yet to be imagined.

Hen's eggs are one of the most common causes of food allergy. Although hen's eggs are known to cause more gastrointestinal symptoms than other foods, it is not known whether there is a difference in organ-specific symptoms between egg yolk (EY) and egg white (EW). The present study aimed to determine whether there are organ-specific differences in the immediate symptoms of EY and EW in patients with hen's egg allergies. We retrospectively investigated the immediate symptoms and treatment contents of those who had a positive result in an oral food challenge (OFC) of boiled whole EY or 10 g of boiled EW in our hospital from January 2013 to July 2019. We compared 80 patients in the EY-OFC-positive group with 106 patients in the EW-OFC-positive group. The EY-OFC-positive group had significantly fewer respiratory symptoms and significantly more gastrointestinal symptoms than the EW-OFC-positive group and had significantly more gastrointestinal symptoms only. In terms of treatment, significantly fewer patients in the EY-OFC-positive group required beta 2-agonist inhalation, and a significantly higher proportion of patients did not require treatment. Compared to EW, EY is more likely to cause gastrointestinal symptoms and less likely to cause respiratory symptoms. It may be necessary to discriminate between EY and EW allergy during diagnosis.

PURPOSE OF REVIEW: The incidence of allergies is increasing and has been associated with several environmental factors including westernized diets. Changes in environment and nutrition can result in dysbiosis of the skin, gut, and lung microbiota altering the production of microbial metabolites, which may in turn generate epigenetic modifications. The present review addresses studies on pectin-mediated effects on allergies, including the immune modulating mechanisms by bacterial metabolites. RECENT FINDINGS: Recently, microbiota have gained attention as target for allergy intervention, especially with prebiotics, that are able to stimulate the growth and activity of certain microorganisms. Dietary fibers, which cannot be digested in the gastrointestinal tract, can alter the gut microbiota and lead to increased local and systemic concentrations of gut microbiota-derived short chain fatty acids (SCFAs). These can promote the generation of peripheral regulatory T cells (Treg) by epigenetic modulation and suppress the inflammatory function of dendritic cells (DCs) by transcriptional modulation. The dietary fiber pectin (a plant-derived polysaccharide commonly used as gelling agent and dietary supplement) can alter the ratio of Firmicutes to Bacteroidetes in gut and lung microbiota, increasing the concentrations of SCFAs in feces and sera, and reducing the development of airway inflammation by suppressing DC function. Pectin has shown immunomodulatory effects on allergies, although the underlying mechanisms still need to be elucidated. It has been suggested that the different types of pectin may exert direct and/or indirect immunomodulatory effects through different mechanisms. However, little is known about the relation of certain pectin structures to allergies.

Some β-mannans, including those in coffee bean and soy, contain a mannose backbone with β-(1→4) bonds. Such mannooligosaccharides could have immunological functions involving direct interaction with immune cells, in addition to acting as prebiotics. This study aimed at assessing the immunological function of mannooligosaccharides with β-(1→4) bond, and elucidating their mechanism of action using bone marrow-derived murine dendritic cells (BMDCs). When BMDCs were stimulated with the mannooligosaccharides, only β-Man-(1→4)-Man significantly induced production of cytokines that included IL-6, IL-10, TNF-α, and IFN-β, and enhanced CD4+ T-cell stimulatory capacity. Use of putative receptor inhibitors revealed the binding of β-Man-(1→4)-Man to TLR4/MD2 complex and involvement with the complement C3a receptor (C3aR) for BMDC activation. Interestingly, β-Man-(1→4)-Man prolonged the production of pro-inflammatory cytokines (IL-6 and TNF-α), but not of the IL-10 anti-inflammatory cytokine during extended culture of BMDCs, associated with high glucose consumption. The results suggest that β-Man-(1→4)-Man is an immunostimulatory molecule, and that the promotion of glycolysis could be involved in the production of pro-inflammatory cytokine in β-Man-(1→4)-Man-stimulated BMDCs. This study could contribute to development of immune-boosting functional foods and a novel vaccine adjuvant.

Evidence has suggested that major peanut allergen Ara h 1 activates dendritic cells (DCs) via interaction with DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), a C-type lectin receptor, and contributes to development of peanut allergy. Since macrophages, as well as DCs, play a crucial role in innate immunity, we investigated whether natural Ara h 1 (nAra h 1) activates two different subsets of macrophages, human monocyte derived macrophage type 1 (hMDM1: pro-inflammatory model) and type 2 (hMDM2: anti-inflammatory model). hMDM1 and hMDM2 predominantly produced pro-inflammatory cytokines (IL-6 and TNF-α) and an anti-inflammatory cytokine (IL-10) in response to nAra h 1, respectively. hMDM2 took up nAra h 1 and expressed DC-SIGN at higher levels than hMDM1. However, small interfering RNA knockdown of DC-SIGN did not suppress nAra h 1 uptake and nAra h 1-mediated cytokine production in hMDM2. Inhibitors of scavenger receptor class A type I (SR-AI) suppressed the response of hMDM2, but not of hMDM1, suggesting that SR-AI is a major receptor in hMDM2 for nAra h 1 recognition and internalization. nAra h 1 appears to exert stimulatory capacity on DC and macrophages via different receptors. This study advances our understanding how a major peanut allergen interacts with innate immunity.

Macadamia nuts, the seeds of Macadamia tetraphylla or Macadamia integrifolia of family Proteaceae, are a familiar food item. Previous case reports of macadamia nut allergy demonstrated the possibility of false negative IgE antibody test results and cross-reactivity of macadamia nut with hazelnut.1-5 However, the clinical features of childhood macadamia nut allergy and the cross-reactivity of macadamia nut with foods other than hazelnut remain understudied.

SCOPE: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. METHODS AND RESULTS: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. CONCLUSION: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA: Artv1) or a hypoallergenic variant (rFlaA: Artv1(hyp)) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins' immune stimulatory capacity. Both rFlaA: Artv1 and rFlaA: Artv1(hyp) induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA: Artv1 (but not rFlaA: Artv1(hyp)) was paralleled by a strong secretion of IFN-gamma. In summary, we provided evidence that incorporating hypoallergens into flagellin: allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Vaccine platforms that can be flexibly loaded with antigens can contribute to decrease response time to emerging infections. For many pathogens and chronic infections, induction of a robust cytotoxic T lymphocytes-mediated response is desirable to control infection. Antigen delivery into the cytoplasm of antigen presenting cells favors induction of cytotoxic T cells. By fusion of the cell-permeable translocation motif (TLM)-peptide to the capsid-forming core protein of hepatitis B virus, and by insertion of the strep-tag in the spike tip (a domain that protrudes from the surface of the capsid), cell-permeable carrier capsids were generated that can be flexibly loaded with various antigens. Loading with antigens was demonstrated by electron microscopy, density gradient centrifugation and surface plasmon resonance spectroscopy. Confocal immunofluorescence microscopy showed that cell-permeable carrier capsids mediate transfer of cargo antigen into the cytoplasm. Using cell-permeable carrier capsids loaded with ovalbumin as model antigen, activation of antigen presenting cells and ovalbumin-specific CD8(+) T-cells, which correlates with enhanced specific killing activity, was found. This demonstrates the capacity of TLM-carrier-capsids to serve as universal antigen carrier to deliver antigens into the cytoplasm of antigen presenting cells, which leads to enhanced MHC class I-mediated presentation and induction of antigen-specific cytotoxic T lymphocytes response.

Background. TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim. To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods. Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam(2)CysK(4) (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naive mice and in a mouse model of OVA-induced intestinal allergy. Results. CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4(+) TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC: TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-alpha production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions. Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.

Background: Due to reduced allergic potency, hypoallergenic variants have been suggested as safer and potentially more efficacious alternative to the corresponding wild-type allergens in allergen-specific immunotherapy. Here, we aimed at investigating the efficacy of recombinant Bet v 1B2, a hypoallergenic folding variant of Bet v 1, in epicutaneous immunotherapy to suppress asthmatic features using a murine model of birch pollen allergy. Methods and Results: Before, or after sensitization with rBet v 1 plus ALUMW and intranasal challenges with birch pollen extract, BALB/c mice received epicutaneous immunization (EPI) with rBet v 1, or rBet v 1B2 on their depilated back. Prophylactic EPI with rBet v 1B2, but not with rBet v 1, suppressed serum levels of Bet v 1-specific IgE antibodies and reduced the number of eosinophils and the concentrations of Th2 cytokines in bronchoalveolar lavage. In an established allergic condition, serum levels of Bet v 1-specific IgE antibodies were similar between PBS-treated control mice and EPI-treated mice. However, therapeutic EPI with rBet v 1B2, but not with rBet v 1, significantly suppressed the development of airway inflammation and lung function impairment. Conclusion: This study is the first to show the effect of therapeutic EPI with a recombinant form of a hypoallergenic folding variant on the suppression of asthmatic features. Our results suggest that rBet v 1B2 along with its reduced IgE-binding capacity could be a preferred therapeutic allergen than wild-type rBet v 1 in epicutaneous immunotherapy of birch pollen-induced allergic asthma, in particular due to a lower risk of allergic side effect.

Allergic diseases are an immune disorder reacting to certain type of allergen(s). Remarkably only a small number of proteins of the plant and animal proteome act as allergens. Therefore, allergens have been clustered according to their common structural, biochemical and functional features. Evidence has accumulated that some allergens possess intrinsic adjuvant properties to stimulate the innate immunity. The adjuvant properties appear to contribute to the allergenicity of the respective proteins, namely the ability to cause allergic sensitization in susceptible subjects or allergic reactions in sensitized individuals. Here, we discuss how allergens interact with the innate immune cells, in particular dendritic cells and epithelial cells, via binding to pattern recognition receptors, exhibiting proteolytic activities and/or inducting type2 innate lymphoid cells (ILC2), thereby contributing to the sensitization and development of allergic diseases.

Background and Objective: To improve the efficacy and safety of tolerance induction for food allergies, identifying the tissues responsible for inducing intestinal inflammation and subsequent oral tolerance is important. We used OVA23-3 mice, which express an ovalbumin-specific T-cell receptor, to elucidate the roles of local and systemic immune tissues in intestinal inflammation. Methods and Results: OVA23-3 mice developed marked enteropathy after consuming a diet containing egg white (EW diet) for 10 days but overcame the enteropathy (despite continued moderate inflammation) after receiving EW diet for a total of 28 days. Injecting mice with anti-IL-4 antibody or cyclosporine A confirmed the involvement of Th2 cells in the development of the enteropathy. To assess the individual contributions of Peyer's patches (PPs), mesenteric lymph nodes (MLNs), and the spleen to the generation of effector CD4(+) T-cells, we analyzed the IL-4 production, proliferation in response to ovalbumin, and CD4(+) T-cell numbers of these tissues. EW feeding for 10 days induced significant IL-4 production in PPs, the infiltration of numerous CD4(+) T-cells into MLNs, and a decrease in CD4(+) T-cell numbers in spleen. On day 28, CD4(+) T-cells from all tissues had attenuated responses to ovalbumin, suggesting tolerance acquisition, although MLN CD4(+) T-cells still maintained IL-4 production with proliferation. In addition, removal of MLNs but not the spleen decreased the severity of enteropathy and PP-disrupted mice showed delayed onset of EW-induced inflammatory responses. Disruption of peripheral lymphoid tissues or of both PPs and MLNs almost completely prevented the enteropathy. Conclusions: PPs and MLNs coordinately promote enteropathy by generating effector T-cells during the initial and exacerbated phases, respectively; the spleen is dispensable for enteropathy and shows tolerogenic responses throughout EW-feeding. The regulation of PPs may suppress the initiation of intestinal inflammation, subsequently restricting MLNs and inhibiting the progression of food-allergic enteropathy.

Background: Maillard reaction (MR) modifies food allergens with various glycation structures during thermal processing of foods. Results: We show that pyrraline, a glycation structure, enhances potential allergenicity of ovalbumin, a model allergen, by promoting scavenger receptor class A-mediated allergen uptake by dendritic cells. Conclusion: Pyrraline could act as a pathogenesis-related component in food allergies. Significance: We reveal how MR links to food allergies. The Maillard reaction (also referred to as glycation) takes place between reducing sugars and compounds with free amino groups during thermal processing of foods. In the final stage of the complex reaction cascade, the so-called advanced glycation end products (AGEs) are formed, including proteins with various glycation structures. It has been suggested that some AGEs could have immunostimulatory effects. Here, we aimed to identify specific glycation structure(s) that could influence the T-cell immunogenicity and potential allergenicity of food allergens, using ovalbumin (OVA, an egg white allergen) as a model allergen. OVA was specifically modified with representative glycation structures: N-E-carboxymethyl lysine (CM-OVA), N-E-carboxyethyl lysine (CE-OVA), pyrraline (Pyr-OVA), or methylglyoxal-derived arginine derivatives (MGO-OVA). As well as AGE-OVA, a crude glycation product in thermal incubation of OVA with glucose, only Pyr-OVA, and not other modified OVAs, was efficiently taken up by bone marrow-derived murine dendritic cells (BMDCs). The uptake of Pyr-OVA was reduced in scavenger receptor class A (SR-A)-deficient BMDCs, but not in cells treated with inhibitors of scavenger receptor class B, galectin-3, or blocking antibodies against CD36, suggesting that pyrraline binds to SR-A. Compared with other modified OVAs, Pyr-OVA induced higher activation of OVA-specific CD4(+) T-cells in co-culture with BMDCs. Furthermore, compared with native OVA, AGE-OVA and Pyr-OVA induced higher IgE production in mice. Pyrraline could induce better allergen uptake by DCs via association with SR-A and subsequently enhance CD4(+) T-cell activation and IgE production. Our findings help us to understand how Maillard reaction enhances the potential allergenicity of food allergens.

Scope: Heated foods often present low allergenicity, and have recently been used in specific oral immunotherapy for food allergies. However, the influence of heating on tolerogenicity of food allergens is not well elucidated. Here, we investigated biochemical, allergenic, and tolerogenic properties of heated egg white (EW) using a murine model of food allergy. Methods and results: Raw EWs were treated at 80 degrees C for 15 min (80EW, mild heating condition), 100 degrees C for 5 min (100EW, cooking condition), or 121 degrees C for 40 min (121EW, retort pouch condition), and freeze-dried. A transgenic OVA23-3 mice model expressing T-cell receptor specific for ovalbumin (OVA, a major EW allergen) induced Th2 cells and IgE production, and presented intestinal inflammation when fed untreated EW diet. 80EW-fedmice presented only moderate inflammation but high Th2 responses. 100EW-fedmice did not present inflammation but induced tolerance as seen in reduced T-cell responses and IgE levels. 100EW demonstrated higher digestive stability and slower absorption in intestine, compared with untreated EW and 80EW. 121EW was strongly aggregated, was not absorbed well, and developed Th1 responses without tolerance induction. Conclusion: OVA in EW treated only under a particular heat condition (e.g. 100 degrees C for 5 min) lost allergenicity, but possessed tolerogenicity.

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA: Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10(-/-), TLR5(-/-) mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA: Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA: Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA: Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA: Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5(-/-) mDC the rflaA: Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10(-/-) mDC with DO11.10 T cells the lack of rflaA: Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA: Ova showed strongest preventive effect. Stimulation with rflaA: Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.

Food allergies are abnormal responses to a food triggered by the immune system. The majority of allergenic foods are often subjected to thermal processing before consumption. The Maillard reaction is a non-enzymatic reaction between reducing sugars and compounds with free amino groups such as amino acids and proteins, and takes place during thermal processing and storage of foods. Among many other effects the reaction leads to modification of proteins with various types of glycation structures such as N epsilon-(carboxymethyl-)lysine (CML), pentosidine, pyrraline and methylglyoxal-H1, which are collectively called advanced glycation end-products (AGEs). Notably, evidence has accumulated that some glycation structures of AGEs function as immune epitopes. Here we discuss the possible involvement of food allergen AGEs in the pathogenesis of food allergies.

The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR)2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly. BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. (C) 2012 Elsevier Ltd. All rights reserved.

Background: The Toll-like receptor (TLR) 5 agonist flagellin is associated with immunomodulatory functions. Objective: We sought to investigate whether Listeria monocytogenes-derived flagellin A (flaA) can modulate ovalbumin (OVA)-specific T-cell responses and prevent OVA-induced intestinal allergy. Methods: Bone marrow-derived myeloid dendritic cells from BALB/c, C57BL/6, or TLR signaling-deficient (MyD88(-/-)) mice were stimulated with rOVA, rflaA, rflaA plus rOVA, or a recombinant fusion protein consisting of rflaA and rOVA (rflaA: OVA). The immunomodulating properties of rflaA plus rOVA and rflaA: OVA were investigated by means of DC-T-cell coculture with CD4(+) T cells from OVA-T-cell receptor transgenic or OVA/alum-immunized mice. rflaA: OVA was applied as a prophylactic and therapeutic vaccine in a murine model of intestinal allergy. Results: rflaA: OVA induced upregulation of TLR5 and dose-dependent IL-6 and IL-10 secretion by myeloid dendritic cells. IL-10 contributed to repressing IL-4 and IFN-gamma secretion by OVA-T-cell receptor transgenic CD4(+) T cells. Moreover, rflaA: OVA suppressed CD4(+) T cells derived from T(H)2-biased mice on OVA/alum immunization. In a murine model of intestinal allergy, prophylactic vaccination with rflaA: OVA reduced T-cell activation. Protection from intestinal allergy included suppression of OVA-specific IgE while inducing OVA-specific IgG(2a). Equimolar amounts of rflaA or rOVA provided alone or as a mixture did not have comparable effects. Moreover, therapeutic vaccination was shown to reduce allergic symptoms and T-cell activation in the spleen. Conclusion: The rflaA: OVA fusion protein showed strong TLR-mediated immunomodulating capacities probably attributed by the proximity of adjuvant and allergen, leading to the prevention of intestinal allergy in a murine disease model. Therefore recombinant flaA: allergen fusion proteins are promising vaccine candidates for intervention in patients with IgE-mediated allergy. (J Allergy Clin Immunol 2011; 128: 1340-8.)

Background: Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. Objective: We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. Methods: A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. Results: Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. Conclusion: Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy. (J Allergy Clin Immunol 2011;128:1022-30.)

Scope: Oral immunotherapy (OIT) involving continuous oral administration of allergenic foods has gained attention as a therapy for food allergies. To study the influence of oral administration of allergenic foods on gastrointestinal symptoms including inflammation, we established a mouse model of food-induced intestinal allergy. Methods and results: BALB/c mice were fed an egg white (EW) diet containing ovalbumin (OVA, a major EW allergen) after intraperitoneal sensitisation with OVA and Alum. The mice on the EW diet for one wk presented gastrointestinal symptoms (i.e. weight loss and soft stools) and inflammation in the small intestines (i.e. duodenum, jejunum and ileum). Further continuous EW diet resolved the weight loss but not the soft stools. Splenic CD4(+) T-cells of EW diet-fed mice on the continuous diet showed less proliferation and cytokine production compared with those of control mice, suggesting tolerance induction by the diet. The continuous EW diet reduced levels of OVA-specific IgE antibodies, but significantly aggravated the inflammation in the jejunum. Conclusion: Our mouse model would be useful to investigate inflammatory and regulatory mechanisms in food-induced intestinal allergies. Our results suggest potential gastrointestinal inflammation in patients undergoing OIT as continuous administration of allergenic foods, even though the therapy may induce clinical tolerance.

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a typical type 1 allergy induced by CJ pollen and one of the most common allergic diseases in Japan. New immunotherapies have been developed for treatment of CJ pollinosis. We focus here on new immunotherapies for CJ pollinosis including sublingual immunotherapy with crude extract of CJ antigen, oral immunotherapy with transgenic rice expressing CJ allergens, a peptide vaccine using T cell epitopes of CJ allergens, DNA vaccines encoding either the CJ allergen gene or T cell epitope gene, and adjuvant-conjugated vaccines using CJ allergen conjugated with adjuvants such as CpG oligodeoxynucleotide or pullulan. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

We determined whether a major Japanese cedar pollen allergen (Cry j 1) conjugated with CpG oligodeoxynucleotide would enhance allergen-specific Th1 responses in mice. Cry j 1 conjugated with CpG (Cry j 1-CpG) induced IL-12 in the spleen cells of naive mice. Cry j 1-CpG immunization of BALB/c mice suppressed anti-Cry j 1 IgE response and enhanced anti-Cry j 1 IgG(2a) to subsequent Cry j 1 and alum adjuvant injection. CD4(+)T cells isolated from the spleens in mice immunized with Cry j 1-CpG produced higher IFN-gamma levels than did CD4(+)T cells obtained from mice as negative controls. Our results suggested that Cry j 1-CpG immunization can induce Cry j 1-specific Th1 immune responses, thereby inhibiting IgE response to the pollen allergen. (C) 2010 Elsevier Ltd. All rights reserved.

Toll-like receptor ligands are Immune-modulatory components linking Innate and adaptive Immune responses and are considered to be promising vaccine components Objective of this study was to investigate the adjuvant activity of Listeria monocytogenesis-derived TLR5-ligand flagellin A (flaA) genetically fused to ovalbumin (Ova major chicken white egg allergen) in a murine in vitro system Recombinant flaA rOva and a fusion protein of rflaA and rOva (rflaA Ova) were over-expressed in Escherchia coli and purified by FPLC LPS depletion was confirmed by LAL test TLR5 binding was evaluated by human and murine TLR5-transgenic HEK 293 cells The Immune-modulatory effect of rflaA Ova and rflaA Ova modified by reduction and alkylation on purified BALB/c bone marrow-derived myeloid (mDC) and plasmacytoid dendritic cells (pDC) was investigated by flow cytometry and intracellular cytokine staining (ICS) Dose-dependent IL-8 secretion from transgenic HEK 293 cells confirmed binding of rflaA and rflaA Ova molecules to human and murine TLR5 Recombinant flaA showed similar biological reactivity to TLR5-ligand fliC derived from Salmonella typhimurium applied as positive control Compared to rflaA both rflaA Ova preparations induced higher expression of maturation markers (CD40 CD69 CD80 and CD86) on mDC whereas only CD69 and CD40 were upregulated on pDC Moreover IL-6 and IL-10 production by mDC was enhanced upon stimulation with rflaA Ova constructs in comparison to an equimolar mixture of both proteins whereas pDC did not show secretion of the investigated cytokines Any immunological effects of LPS can be excluded by depletion of endotoxins and the lack of IL-10 production upon proteinase K digestion of rflaA Ova In summary the rflaA Ova fusion proteins showed an enhanced immune modulating capacity in comparison to rflaA or the mixture of rflaA and antigen Since the rflaA Ova fusion proteins induce strong IL-10 induction they are considered as potential vaccine candidates to Improve allergen-specific immunotherapy (C) 2010 Elsevier Ltd All rights reserved

P>Advanced glycation endproducts (AGEs) of food proteins resulting from the Maillard reaction after cooking or heating may have particular importance in food allergy. The underlying immunological mechanisms are only poorly understood. The aim of the study was to examine the effects of AGE derived from the model food allergen ovalbumin (AGE-OVA) on dendritic cells (DCs), their immunostimulatory capacity and the T-cell response compared with regular OVA. For this purpose, human immature DCs were exposed to fluorescein isothiocyanate (FITC)-labelled AGE-OVA and FITC-labelled regular OVA and uptake was analysed by flow cytometry and fluorescence microscopy. Furthermore, autologous CD4+ T-cell proliferation and cytokine production induced by mature DCs loaded with AGE-OVA were compared with those induced by mature DCs loaded with OVA. Finally, expression of the receptor for advanced glycation endproducts (RAGE) and activation of the transcription factor nuclear factor (NF)-kappa B by AGE were investigated. Internalization of FITC-AGE-OVA by immature DCs was significantly increased compared with FITC-OVA. Blocking the mannose receptor, macropinocytosis or the scavenger receptor strongly reduced uptake of both FITC-OVA and FITC-AGE-OVA. In a comparison of CD4+ T cells co-cultured with AGE-OVA-loaded mature DCs versus those co-cultured with OVA-loaded mature DCs, AGE-OVA DCs were found to produce more interleukin (IL)-6 and to induce a stronger T helper type 2 (Th2) and a weaker Th1 cytokine response, while there was no difference in proliferation of CD4+ T cells. The expression of RAGE was higher on immature DCs compared with mature DCs. AGE-OVA-exposed immature DCs showed a stronger expression of RAGE and activation of the transcription factor NF-kappa B compared with OVA-loaded immature DCs. Our data indicate that AGE-OVA may be more immunogenic/allergenic than regular OVA.

Background: The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGES are suggested to function as pathogenesis-related immune epitopes in food allergy. Objective: This study aimed at defining the T-cell immunogenicity of food AGES by using ovalbumin (OVA) as a model allergen. Methods: AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGES, as well as mDCs deficient for these receptors. Results: Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs. Conclusion: SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens. (J Allergy Clin Immunol 2010;125:175-83.)

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocyrochemistry and Western immunoblot analysis on marine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 367) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism. (C) 2007 Elsevier Inc. All rights reserved.

Dendritic cells (DCs) are a subset of antigen-presenting cells (APCs) that are involved in the initiation and control of the immune response to antigens present at the interface with the environment. A limited number of groups have studied DCs in human and animal conjunctiva but no data is available concerning the different DC subsets present in the conjunctival tissue. The aims of this study are to characterize the phenotypes and numbers of DCs present in the murine model of allergic conjunctivitis using the technique of immunohistochemistry so as to aid the understanding of the mechanisms involved in allergic eye disease. A double immunofluorescence method was used to analyze the phenotypic distribution and density of DC subsets in the mouse conjunctival tissues of the allergic model using a panel of antibodies: CD11c, as a general marker of DCs, coupled with another DC subset marker such as Langerin for Langerhans cells (LCs), CD11b for myeloid DCs (mDCs) and mPDCA-1 for plasmacytoid DCs (pDCs). In the naive conjunctiva, mDCs were consistently detected in the subepithelial layer and substantia propria. In the epithelium and the subepithelial layer, very few LCs and virtually no pDCs were observed. Following allergen challenge, there was a marked influx of mDCs and pDCs, but no LCs, into the subepithelial layer and throughout the substantia propria. These results indicate that conjunctival DC subsets may play an important role in the immune-regulatory processes involved in the inflammatory component of allergic conjunctivitis. (c) 2007 Elsevier Inc. All rights reserved.

Chemokines have a clearly defined role in mobilizing the recruitment of leukocytes to both healthy and inflamed tissues. This review details work from our and other laboratories, indicating that beta-chemokines may play important roles (i) in driving the terminal differentiation of mast cell precursors in mucosal tissues and (ii) in providing priming or costimulatory signals required for mast cell activation, leading to an antigen-driven inflammatory response. These data stem from in vivo, ex vivo, and in vitro studies. Data are also presented that suggest that Fc epsilon RI:chemokine receptor cross talk may involve spatiotemporal dynamics that may control the strength and nature of the complex activating signals controlling mast cell effector function.

Marked inflammatory infiltration by activated leukocytes is a characteristic feature of allergic diseases. Elucidation of the mechanisms of leukocyte trafficking in allergic diseases would identity targets to establish novel anti-inflammatory strategies for treatment of these diseases. Leukocyte trafficking is controlled by tissue-specific expression of chemokines and chemokine receptor expression on the leukocyte, surface. Here, we review the role of chemokines and their receptors in leukocyte trafficking to inflammatory sites in allergic diseases and discuss therapeutic strategies targeting chemokine networks for treatment of these diseases. © 2007 Future Drugs Ltd.

Allergic inflammation manifests as one of a number of diseases, including asthma, dermatitis, food allergy, vernal keratoconjunctivitis, and systemic anaphylaxis. Together these diseases affect nearly 25% of the Western world and are a leading health-care problem. The diseases are often biphasic, with an early phase driven primarily by mast cell degranulation and a late phase characterized by leukocyte recruitment. While chemokines are well known to be critical for leukocyte recruitment, their importance in early-phase reactions is poorly defined. We show here that administration of a single oral dose of a high affinity and highly selective CCR3 antagonist ablates both the early and late phase reactions in a mouse model of allergic conjunctivitis. A direct analysis of mast cells in the conjunctiva demonstrates that antagonism of the CCR3 receptor stabilizes the mast cell in vivo, thereby leading to the impaired early phase reaction. The late phase reaction is also strongly inhibited as characterized by both reduced eosinophilia and neutrophilia. These results constitute the first direct evidence that antagonism of CCR3 has clear potential for the treatment of allergic diseases.

Age-related macular maculopathy (ARM) and age-related macular degeneration (AMD) are the leading causes of blindness in the Western world. Despite the magnitude of this clinical problem, very little is known about the pathogenesis of the disease. In this study, we analysed the sera (using indirect immunohistochemistry and Western blot analysis) from a very large cohort of such patients and normal age-matched controls to detect circulating anti-retinal antibodies. Patients with bilateral drusen (n = 64) and with chorioretinal neovascularization (CNV) (n = 51) were recruited in addition to age-matched control subjects (n = 39). The sera were analysed for anti-retinal immunoglobulins on retinal sections. The data were then correlated with the clinical features graded according to the International Classification and Grading System of ARM and AMD. The sera of patients with drusen (93.75%) and CNV (82.27%) were found to have a significantly (P = 0.02) higher titre of autoantibodies to the retina in comparison with controls (8.69%), indicating significant evidence of involvement of the immune process in early stages of AMD. Subsequent statistical analysis of the drusen group showed significant progressive staining (P = 0.0009) in the nuclei layers from early to late stages of ARM. Western blotting confirmed the presence of anti-retinal immunoglobulins to retinal antigens. As anti-retinal immunoglobulins are present in patients with bilateral drusen and exudative AMD, these antibodies could play a significant role in the pathogenesis of AMD. Whilst we do not have evidence that these antibodies precede disease onset, the possibility that their presence might contribute to disease progression needs to be investigated. Finally, the eventual identification of the target antigens detected by these antibodies may permit the future development of new diagnostic methods for ARM and AMD.

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1a also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha. with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.

Apart from the FcεRI-mediated mechanism, mast cells are activated by chemokines. Evidence has accumulated indicating that there is cross-talk between the FcεRI-mediated signalling pathway and CC chemokine receptor (CCR)-mediated signalling pathways in mast cells. We have found that costimulation with IgE/antigen and CC chemokine ligand 3 (CCL3) enhances degranulation but inhibits chemotaxis of rat basophilic leukaemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells). We hypothesize that this signalling cross-talk in mast cells may play important roles in the orchestration and focusing of the allergic response. In this study, we have sought information about global protein networks either enhanced or inhibited following cross-talk between the FcεRI-mediated and CCR-mediated signalling pathways in mast cells. We believe this information may be useful for providing an understanding of mast cell function and in the establishment of new anti-inflammatory drugs for allergic diseases. Proteomics is a promising tool for studying protein profiles within biological samples and facilitates an understanding of the complex responses of an organism to a stimulus. Here, we show comparative data of protein profiles derived from FcεRI-engaged and/or CCR1-engaged RBL-CCR1 cells using protein chip array technology, a proteomic technology. We also discuss our view of the role of CC chemokines and CCRs in regulating multiple aspects of mast cell biology. Copyright © 2005 John Wiley &amp Sons Ltd.

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.

Here, we report the cross-talk between CC chemokine receptor 1 (CCR1)-mediated signaling and FceRI-mediated signaling in mast cells. Costimulation with r-human CCL3, a ligand of CCR1, and IgE/Ag synergistically enhanced degranulation via phosphatidylinositol 3-kinase-mediated cascade in rat basophilic leukemia-2H3 cells expressing human CCRL Mitogen activated protein (Map) kinase selective inhibitor SB202190 did not decrease, but PD98059 partially decreased the degranulation, suggesting a minor contribution of p38 kinase and ERK in the synergistic activation. However, phosphorylation of ERK and of p38 kinase was increased by the costimulation with CCL3 and Ag. The results suggest that simultaneous engagement of FcepsilonRI and CCR involves many events in mast cells and thus contribute to allergic inflammation.

Eotaxin-1 (CCL 11) regulates chemotaxis and/or activation of several kinds of immune cells such as eosinophils, basophils, mast cells (MC), and T lymphocytes. Two lines of evidence indicate that CCL11 may play an important role in conjunctival MC activation: 1) CCR3 expression on conjunctival MC, and 2) our investigation of allergic conjunctivitis in CCL11 -/- mice suggested that CCL11 may positively regulate for MC degranulation in the conjunctiva. CAT-213, monoclonal anti-eotaxin-1 human IgG(4) (Cambridge Antibody Technology) could potentially neutralise CCL11 mediated MC degranulation. We examined this potential using an ex-vivo human allergic conjunctivitis model we have developed, in which MC are passively sensitized by IgE and stimulated with anti-IgE. CAT-213 inhibited MC degranulation in the conjunctiva up to 30 % of positive control. These data suggest that CAT-213 may have therapeutic value with respect to allergic conjunctivitis.

Purpose: The aims of this research were to: (1) generate a rapid protocol for the sensitization of rodents to a defined allergen without footpad injections yet leading to both acute- and late-phase hypersensitivity reactions in the ocular surface; and (2) define detailed criteria for the assessment of clinical symptoms in the acute-phase response. Methods: With the approved methods for the use of experimental animals in research and existing sensitization protocols as a starting point, we developed and tested a new protocol with respect to its ability to generate an acute- and late-phase response on ocular challenge. Clinical symptoms were assessed by a trained ophthalmologist under masked conditions, and late-phase responses determined by histologic analysis of conjunctival tissue sections. Results: A new protocol for the rapid sensitization of mice, avoiding footpad injections, yet yielding both acute- and late-phase allergic responses, was developed. Detailed criteria for the assessment of disease severity were established and tested. Conclusion: This protocol establishes a murine model of allergic conjunctivitis that will be useful for both the study of the molecular and cellular basis of allergic reactions in the ocular surface and the testing of new therapies for this disease.

Chemokines, representing a large superfamily of 8- to 15-kd proteins, were originally discovered through their ability to recruit various cell types into sites of inflammation. It is now clear that these molecules play a much wider role in immune homeostasis, playing key roles in driving the maturation, homing, and activation of leukocytes. In this review we analyze the roles chemokines play in the development, recruitment, and activation of leukocytes. Because signaling from the receptors drives these processes, signal transduction from chemokine receptors will also be reviewed. Taken together, we highlight the various points at which chemokines contribute to allergic inflammation and at which their targeting might contribute to new therapies for type I hypersensitivity reactions.

Background Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata ) as well as in humans. Most human patients and monkeys with pollinosis have specific IgE for Cry j 2, a major allergen of CJ pollen. Objective The main purpose of this study was to identify IgE B cell epitopes of Cry j 2 using a synthetic peptide in humans, monkeys and mice. Methods We synthesized 38 overlapping peptides that span the entire length of Cry j 2. We examined the B cell epitopes of Cry j 2 that are recognized by IgE in the sera of human patients and monkeys with pollinosis and immunized mice using synthetic peptides of Cry j 2. We also examined the reaction of Cry j 2-specific mouse monoclonal IgG antibodies to the peptides. Furthermore, we conducted a histamine release assay with leucocytes from a pollinosis patient using human serum albumin (HSA) conjugated with the peptides as a B cell epitope. Results We found that 16 of the 20 pollinosis patients who had specific IgE to Cry j 2 also exhibited IgE reaction with some Cry j 2 peptides. Of these 16 patients, 10 exhibited IgE reaction with Cry j 2 peptide no. 13 ((121)GQCKWVNGREICNDRDRPTA(140)). Five of the seven monkeys with CJ pollinosis exhibited a reaction with peptide no. 13. Furthermore, IgE in mice immunized with Cry j 2 and two mouse monoclonal IgG antibodies reacted with peptide no. 13. Peptide no. 13-conjugated HSA showed the release of histamine from basophils. Furthermore, to determine the minimum epitope in peptide no. 13, we conducted an enzyme-linked immunosorbent assay inhibition test. The core of the epitope in humans, monkeys and mice was (124)KWVNGREI(131). Conclusion We found that (124)KWVNGREI(131) is an important B cell epitope recognized by IgE in humans, monkeys and mice.

The world-wide effort to identify susceptibility genes for allergic diseases is motivated by the conviction that the identification of disease genes may permit the design of new classes of anti-inflammatory compounds. Molecules concerned with the allergic reaction, such as cytokines, chemokines, their receptors, major histocompatibility complex molecules, and transcription factors, could provide the candidate genes of the allergic diseases. On the basis of genetic studies, multiple research groups have attempted to identify a susceptibility gene for allergy using the candidate gene approach and/or genome-wide screening. Both of these approaches suggest genetic heterogeneity of allergic diseases. Many variants of candidate genes are or are not associated with particular diseases in different ethnic groups and the function of variants is now being investigated. Based on the information accumulated thus far and the information on the human genome sequence, future advances in research on genetic factors for allergic diseases will be likely lead to the establishment of more effective prophylaxis and therapy for these diseases.

In our previous study [Immunology 91 (1997) 161] using monoclonal antibodies (mAbs) specific to Cry j 1, a major allergen in Japanese cedar (Cryptomeria japonica) pollen, we identified five independent epitopes (EP-1-EP-5) on the molecule and found that EP-1 and EP-5 are the predominant allergic epitopes for humans and monkeys, respectively. In this study, we analyzed the epitopes recognized by IgE in the sera of 10 dogs sensitive to C. japonica pollen allergen using an IgE-ELISA inhibition method with these mAbs. The IgE reaction patterns varied among dogs. In eight of the 10 dogs, IgE recognized EP-5 which is a predominant allergic epitope for monkeys with the pollenosis. In four dogs, IgE recognized EP-1 which is a predominant allergic epitope for human patients with the pollenosis. In three dogs, IgE recognized EP-4 which is a heat-stable epitope. EP-5 is a predominant allergic epitope for dogs and some, but not all, dogs have IgE reaction patterns to the epitopes similar to those of humans. (C) 2001 Elsevier Science B.V. All rights reserved.

Background: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides, The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. Methods: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. Results and Conclusion: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides, IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152). Copyright (C) 2000 S.Karger AG, Basel.

We have previously found that most occurrences of anaphylaxis to live virus vaccines are caused by gelatin present in the vaccines as a stabilizer. After we published the evidence for the role of gelatin in anaphylaxis, Vaccine manufacturers in Japan began to eliminate gelatin from live virus vaccines. In the present study, we tried to estimate its incidence before the gelatin elimination was started. Physicians and vaccine manufacturers submitted serum samples from children with anaphylaxis to measles, mumps, rubella or varicella vaccine to National Institute of Infectious Diseases (NIID) for 3 years from April 1994 to March 1997. Specific IgE to gelatin was assayed at NIID or two manufacturers by the CAP and ELISA methods. There were 44 children with life-threatening severe anaphylaxis (airway obstruction or anaphylactic shock) during the 3-year period, 41 of whom had anti-gelatin IgE. There were 64 children with mild anaphylaxis (without airway obstruction); 62 had anti-gelatin IgE. There were 100 children with only systemic cutaneous signs; 81 had anti-gelatin IgE. The estimates for the incidence of the severe anaphylaxis in 1994-1996 are: 6.84, 7.31, 4.36, and 10.3 cases per million doses of gelatin-containing measles, rubella, mumps, and varicella vaccines, respectively. (C) 2000 Elsevier Science Ltd. All rights reserved.

Background: Most children with anaphylaxis to measles, mumps, and rubella vaccines had shown sensitivity to bovine gelatin that was included in the vaccines. Recently, it was found that bovine type I collagen, which is the main content in the gelatin, is a major allergen in bovine gelatin allergy, Fish meat and skin also contain type I collagen. Objective: The present study was designed to investigate IgE antibody to fish gelatin in children with fish allergy. Methods: Serum samples were taken from patients in 3 groups: (1) 10 patients with fish allergy and specific IgE to fish meat; (2) two patients with allergies to both fish meat and bovine gelatin and specific IgE to fish meat and bovine gelatin; and (3) 15 patients with atopic dermatitis and specific IgE to fish meat. Various fish gelatins (type I collagen) were prepared from fish skin. IgE antibody to fish gelatin was analyzed by using ELISA and immunoblotting. Results: Of 10 patients with fish allergy, 3 had specific IgE to fish gelatin. Of two patients with fish allergy and bovine gelatin allergy, all had specific IgE to fish gelatin. Of 15 patients with atopic dermatitis and specific IgE to fish meat, 5 had specific IgE to fish gelatin. Furthermore, IgE from pooled serum of the patients reacted with both the alpha 1 and alpha 2 chains of fish type I collagen in immunoblots. There is cross-reactivity among gelatins from various fishes, but there is little crossreactivity between fish and bovine gelatins. Conclusion: Some fish-sensitive patients possessed IgE antibody to fish gelatin. Fish gelatin (type I collagen) might be an allergen in subjects with fish allergy.

The efficacy of TCR antagonist peptides in inhibition of antigen-specific antibody production and T cell responses in vivo was evaluated. Among amino acid-substituted analogs of a peptide corresponding to residues 119-133 of bovine beta-lactoglobulin (p119-133), pR124Q and pD129S, prepared by substitution of Gin and Ser for Arg(124) and Asp(129), respectively, have been shown to display TCR antagonist activity for three out of four distinct p119-133-specific T cell clones and for polyclonal T cells derived from p119-133-immunized C57BL/6 mice. Both pD129S and pR124Q inhibited in vivo priming and subsequent activation of T cells by p119-133 when co-injected with p119-133 into mice, as shown by the decreased proliferation of T cells in response to p119-133 in vitro, pD129S significantly inhibited production of anti-p119-113 antibodies of IgG1, IgG2b and IgE isotype in vivo when co-injected into mice together with p119-133 at the time of the first immunization. However, pR124Q was totally ineffective in inhibition of the antibody responses. Anti-p119-133 antibodies from p119-133-immunized mice could bind to pR124Q but not to pD129S, suggesting that the difference in cross-reactivity is responsible for the different effect of these two peptides on specific antibody production. Our findings demonstrate that a single TCR antagonist peptide can inhibit antigen-specific polyclonal antibody production when this antagonist peptide does not cross-react with the antibody elicited in response to an antigenic peptide.

To develop a new immunotherapy for Japanese cedar (Cryptomeria japonica; CJ) pollinosis, we evaluated the use of DNA immunization by inoculating mice with plasmid DNA encoding Cry j 1 as a CJ pollen major allergen (pCACJ1). Repeated intramuscular (i.m.) inoculation of BALB/c mice with pCACJ1 produced anti-Cry j 1 antibody responses, which were predominately of the immunoglobulin G2a (IgG2a) type. Furthermore, this inoculation suppressed immunoglobulin E (IgE) and IgG1 antibody responses to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from mice inoculated with pCACJ1 i.m. secreted interferon-gamma (IFN-gamma), but not interleukin (IL)-4, in vitro upon stimulation with Cry j 1 as well as with p277-288, a peptide corresponding to the T-cell epitope of Cry j 1. In contrast, inoculation of BALB/c mice with pCACJ1 by gene gun injection caused response predominantly of the IgG1 type, and enhanced production of anti-Cry j 1 IgE antibodies to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from pCACJ1-innoculated mice by gene gun injection secreted both IFN-gamma and IL-4 in vitro, upon stimulation with Cry j 1 as well as with p277-288. These findings suggest that i.m. inoculation with pCACJ1 effectively elicits Cry j 1-specific T helper 1 (Th1)-type immune responses, resulting in inhibition of the IgE response to Cry j 1.

Background: Anaphylactic reactions to measles, mumps, and rubella vaccines, including gelatin as a stabilizer, have been reported. It had been found that most of these reactions to live vaccines are caused by the bovine gelatin included in these vaccines. Gelatin mainly includes denatured type I collagen, which consists of alpha 1 and alpha 2 chains. Objective: The current study was designed to investigate the IgE reactivity to alpha 1 and alpha 2 chains of bovine type I collagen in gelatin-sensitive children. Methods: Serum samples were taken from 10 children who had anaphylaxis to the vaccines and high levels of specific IgE to bovine gelatin. Bovine type I collagen was isolated from bovine skin and then separated to alpha 1 and alpha 2 chains by column chromatography, IgE reactivity to denatured type I collagen and its alpha 1 and alpha 2 chains was analyzed by immunoblotting, ELISA, and histamine release from the mast cells passive sensitized with IgE antibodies in pooled serum of the children. Results: All children had specific IgE to bovine type I collagen. Furthermore, IgE antibodies in their sera reacted with the alpha 2 chain but not with the alpha 1 chain. Similarly, the mast cells sensitized with pooled sera in the children showed alpha 2 chain-specific histamine release but not a1 chain-specific histamine release. Conclusion: In gelatin allergy denatured bovine type I collagen is a major allergen and IgE-binding sites exist in the alpha 2 chain of type I collagen.

The efficacy of T-cell receptor antagonist peptides in inhibition of antibody responses in vivo was evaluated. Substitution analogs of peptide 119-133 (p119-133) of bovine beta-lactoglobulin, R124Q ((124) Arg-->Gln) and D129S ((129)Asp-->Ser), have been shown to display TCR antagonist activity for three of four p119-133-specific T-cell clones. Both D129S and R124Q, when co-administered with p119-133 to mice, showed an inhibitory effect in vivo on T-cell priming and/or activation by p119-133, as evidenced by the decreased proliferation of lymph node T cells in response to p119-133. Production of p119-133-specific antibody was inhibited by D129S when mice were injected with p119-133 plus D129S at the first immunization, while co-immunization with R124Q instead led to increase in production of p119-133-specific antibodies. Anti-p119-133 antibodies showed cross-reactivity to R124Q but not to D129S, suggesting that p119-133-specific cells are efficiently activated by R124Q and R124Q-specific T cells. The present study suggests the necessity of selecting TCR antagonist peptides which escape recognition by pathogenic antibodies against allergens or autoantigens, for the purpose of inhibiting an undesirable antibody response.

T-cell receptor (TCR) antagonist peptides, which were originally found to inhibit T-cell responses in vitro in an antigen-specific fashion, are expected to be an effective tool for the selective inhibition of immune responses in vivo. To investigate their in vivo activities, we first identified TCR antagonist peptides for a panel of murine helper T-cell clones specific for the immunodominant determinant of a 119-133 region (p119-133) of bovine beta-lactoglobulin. D129A, which is an analog of p119-133 having a single substitution of Ala for a TCR contact residue of (129)Asp, showed an inhibitory effect on the proliferative responses of three of the four T-cell clones, identifying D129A as a TCR antagonist peptide. D129A was also inhibitory for the immune responses to p119-133 in vivo, evidenced by the reduction of the proliferation of lymph-node T cells and the anti-p119-133 antibody production when D129A was co-administered to mice with p119-133. These results illustrated the feasibility of the antigen analog approach to selective immunointervention, and also the usefulness of the in vitro T-cell culture system to screen peptide analog candidates which can be used in vivo.