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Information on the comparative digestibility of food allergens and non-allergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. Preheating effect on in vitro digestibility has not been fully examined. In this study we investigated the preheating effect of in vitro digestibility of several proteins and their proteolytic fragments in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Five major food allergens, ovalbumin (OVA), ovomucoid (OVM), beta-lactoglobulin (BLG), bovine serum albumin (BSA), soybean trypsin inhibitor (STI), four proteins of unproven allergenicity, horseradish peroxidase (HRP), ribulose-1,5-bisphosphate carboxylase/oxidase (RBC), phosphinothricin acetyltransferase (PAT) and zein from corn, and plant lectin, concanavalin A (Con A) were preheated (at 100 degrees C for 5 min) or not preheated, and then digested in SGF or SIF. Food allergens were relatively stable in both SGF and SIF. Among the allergens, digestibility of OVA in both SGF and SIF was markedly decreased, and BLG and STI were relatively stable after preheating. Digestibility of ConA in SGF and SIF was markedly decreased by preheating. Digestibility of non-allergenic proteins in SGF was higher than the allergenic proteins. From these results, because of the marked increase of the digestibility in several proteins by preheating, systematic information concerning the effect of food treatment on protein digestion is necessary to assess the relationship between allergenic potential and the digestibility of food protein.

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The incidence of type I allergic disorders has been increasing worldwide, particularly, the hypersensitivity to food. We first showed that apple condensed tannin (ACT) intake would inhibit the development of the oral sensitization and that the inhibition could correlate with the rise in the population of TCRcd-T cells in the intestinal intraepithelial lymphocytes (IEL) using W/W V mice and B10A mice which were ovalbumin (OVA)-orally sensitized. Serum OVA-specific immunoglobulin E and immunoglobulin G1 titers in the OVA-orally sensitized W/W V and B10A mice ad libitium fed ACT were extremely inhibited compared to those of the control. The ACT intakes of OVA-sensitized W/W V and B10A mice inhibited the immediate reduction of the body temperature or the rise in serum histamine induced by active systemic anaphylaxis. The proportions of the TCRcd-T cells in the IEL of the OVA-orally sensitized W/W V and B10A mice ad libitium fed ACT were significantly greater than that in the controls. Furthermore, ACT feeding by itself could induce the rise in the percentage of the TCRcd-T cells among the IEL of the W/W V and B10A mice. This suggests that the ACT intake may prevent the development of food allergies and this effect could be correlated with the rise in the percentage of TCRcd-T cells among the IEL.

BackgroundFor the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible.MethodsThe nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human FcεRI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1 : 100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen.ResultsSensitization with 15 pg/ml IgE was sufficient to detect IgE crosslinking–induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1 fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (P = 0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well (R = 0.9127, Spearman's test).ConclusionThe EXiLE test using RS-ATL8 cells is a promising in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.

Background: Many drugs are known to induce allergic reactions in the skin. The metabolic activation of drugs resulting in the formation of protein adducts is thought to be a first step in the induction of these allergic reactions. We postulated that dermal tissue might be a site of drug activation by cytochrome P450 (CYP) isozymes. Methods: Messenger RNA was extracted from cultured Langerhans cells, keratinocytes, fibroblasts and melanocytes from 6 individuals, and CYP mRNA expression was analyzed by RT-PCR. Results: CYP1A1, 1B1 and 2E1 were found in all four cell types. CYP2A6, 2C, 2D6, 3A5, 3A7 and 4B1 mRNA was expressed in a cell-type- and/or individual-specific manner. CYP1A2, 2A7, 2B6 and 3A4 mRNA was not detectable. Conclusions: The mRNA for a variety of CYP isozymes was expressed in all four types of skin cells examined. These CYP enzymes may be involved in the pathogenesis of drug-induced allergic reactions in the skin.

During the last decade there has been an exponential increase in data illustrating that the nervous and immune systems are not disparate entities.1,2) The nerve-mast cell relationship has served as a prototypic association and has provided substantial evidence for bi-directional communication between nerves and immune cells.3) Early studies elegantly described the non-random spatial association of nerves and mast cells in a variety of tissues in which actual membrane-membrane contacts could be observed. 4,5) To understand these events we have studied recently direct neurite-mast cell (RBL) communication using an in vitro co-culture approach and a calcium imaging by confocal laser scanning microscopy (CLSM). Our results showed clearly that nerve-mast cell communication can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication.6) Our findings have implications for the neuroimmune signaling cascade that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature. [6][7][8] In addition we studied by atomic force microscopy (AFM) the morphological structure of the interaction between neurites and mast cells (RBL) which was involved in the communication.9) AFM images showed that association between the growth cone of neurites and the RBL cell occurred over ca. 7 mm and that direct neurite-mast cell cross-talk induced degranulation in RBL cells via the neuropeptide substance P.In the present paper we have focused on the molecular mechanism of the membrane proteins involved in the synapse-like structure between neurites and mast cells. We used an in vitro co-culture approach comprising superior cervical ganglia (SCG) and mouse bone marrow-derived mast cells (BMMC) besides RBL cells. Mast cells are classified into two types. One is mucosal and another is connective tissue type. Both RBL cells and BMMCs belong to mucosal type mast cells. However, RBL cells are much more adhesive than BMMCs and would like to non-specifically stick to the supported matrigel-coated plate of glass dishes. They also have a malignant feature. In addition, nerves most commonly associated with mast cells in vivo contain substance P.5) Substance P in isolated neurons from SCG maintained in culture showed a 25-fold increase within 48 h in vitro.10) Therefore, we studied an in vitro co-culture approach comprising SCG and BMMC to understand precisely adhesion proteins that play a role in the neurite-mast cell communication.Cadherins are a family of calcium-dependent and homophilic cell adhesion proteins that play in the formation of synaptic plasticity. E-and N-cadherins, which are classic cadherins, are found in synapses and they appear to straddle the presynaptic and postsynaptic membranes surrounding the active zone and the postsynaptic density.11) Cadherins mediate strong adhesion through intracellular interaction with b-catenin, which in turn associates with a-ca...

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca 2ϩ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP ϩ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca 2ϩ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 Ϯ 0.04 vs. 0.05 Ϯ 0.01 m/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types. neuroimmunity; substance P; neurokinin-1 receptor; CD63; granule tracking ANALYSES OF NERVE-MAST CELL INTERACTIONS have been of significant importance in unequivocally establishing the concept of bidirectional neuroimmune communication (13,25). In addition to the anatomic association of mast cells and nerve fibers in many tissues (3,20), numerous studies have shown that mast cell activation can be evoked by nerve stimulation or the application of neurotransmitters and that mast cell-derived mediators can influence neuronal activity (7,18,24). For instance, the neuropeptide substance P has been shown to cause mast cell degranulation when used at high doses, whereas exposure to picomolar concentrations of substance P primes the mast cell, lowering the degranulation stimulation threshold to a second stimulus (10). We have used an in vitro model of mast cell-nerve interaction, composed of cocultures of the mucosal mast cell-like rat basophilic leukemic (RBL) cells and neurite-sprouting murine superior cervical ganglia (5). We showed that nerve-mast cell communication did not require transduction by an intermediate cell and that the RBL cell activation response following neurite stimulation was mediated largely via substance P release and through neurokinin (NK)-1 receptors (21). Here, we sought to further examine neurite-RBL cell interactions, to determine whether neuroninduced mast cell activation is a generalized whole cell response or if there is an additional level of subtlety to the neurite-RBL cell interaction that occurs at the specific...