Direct Inhibition of the Allergic Effector Response by Raw Cow's Milk-An Extensive In Vitro Assessment.

The mechanisms underlying the allergy-protective effects of raw cow's milk are poorly understood. The current focus is mainly on the modulation of T cell responses. In the present study, we investigated whether raw cow's milk can also directly inhibit mast cells, the key effector cells in IgE-mediated allergic responses. Primary murine bone marrow-derived mast cells (BMMC) and peritoneal mast cells (PMC), were incubated with raw milk, heated raw milk, or shop milk, prior to IgE-mediated activation. The effects on mast cell activation and underlying signaling events were assessed. Raw milk was furthermore fractionated based on molecular size and obtained fractions were tested for their capacity to reduce IgE-mediated mast cell activation. Coincubation of BMMC and PMC with raw milk prior to activation reduced ß-hexosaminidase release and IL-6 and IL-13 production, while heated raw milk or shop milk had no effect. The reduced mast cell activation coincided with a reduced intracellular calcium influx. In addition, SYK and ERK phosphorylation levels, both downstream signaling events of the FcεRI, were lower in raw milk-treated BMMC compared to control BMMC, although differences did not reach full significance. Raw milk-treated BMMC furthermore retained membrane-bound IgE expression after allergen stimulation. Raw milk fractionation showed that the heat-sensitive raw milk components responsible for the reduced mast cell activation are likely to have a molecular weight of > 37 kDa. The present study demonstrates that raw cow's milk can also directly affect mast cell activation. These results extend the current knowledge on mechanisms via which raw cow's milk prevents allergic diseases, which is crucial for the development of new, microbiologically safe, nutritional strategies to reduce allergic diseases.

[Construction and identification of stable hFcεRIαßγ/RBL-2H3 cells for food allergy assessment].

OBJECTIVE: To establish an rat basophil leukemia(RBL)-2H3 cell line stably expressing human high affinity receptor containing alpha, beta and gamma chain(hFcεRIαßγ), in order to provide experimental materials for evaluating allergenicity of food. METHODS: The lentivirus was transfected into RBL-2H3 cells, and the mRNA expression of hFcεRIαßγ in cells was detected by real-time PCR and the protein expression of hFcεRIα was detected by flow cytometry. RESULTS: Sequencing result showed that recombinant lentiviral vector GV367-hFcεRIαßγ was successfully constructed. According to the result of experiments, lentivirus could effectively infect RBL-2H3 cells. The mRNA of hFcεRIαßγ and protein levels of hFcεRIα in RBL-2H3 cells were successfully overexpressed. CONCLUSION: The hFcεRIαßγ/RBL-2H3 cells were preliminarily constructed, which could be binded with human IgE and further used in the evaluation system of food allergy, compared to RBL-2H3 cells.

An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations.

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.

Assessment of Nasal Immunoglobulin E Level in Atopic and Non-atopic Rhinitis Patients: A Tool for Diagnosis of Local Allergic Rhinitis.

Many cases of AR can be miss-diagnosed due to deficiency in the conventional laboratory tools. Detection of local Ig E immune response and allergy associated genes may aid in diagnosis of these cases. The local immune response and the allergy associated genes of these suspected cases must be evaluated as they may help in their characterization. This study was conducted on 129 patients with chronic rhinitis to determine the frequency of LAR, and analyze the association of IgE receptor (FcεR1ß) gene polymorphism with LAR. All participants were subjected to clinical questionnaire, skin prick test, specific IgE measurement in serum and nasal secretions and analysis of FcεR1ß gene polymorphism. LAR constituted 24.8 % of total rhinitis cases and 44.4% of non-allergic cases. Cockroach was the main sensitizing agent in local allergic rhinitis in comparison with allergic cases (OR =0.11; 95% CI= 0.04-0.34; P<0.001). In LAR, nasal specific Ig E was significantly lower than that in AR patients (P < 0.001). FcεR1ß genotype TT was more frequently expressed in LAR and AR than non-allergic rhinitis (NAR) (P< 0.001). It is concluded that LAR is an emerging allergic condition that could be diagnosed by nasal specific IgE, and that FcεR1ß polymorphism is one of the genetic factors associated with AR and LAR.

An overview of the effects of anti-IgE therapies.

Omalizumab, a humanized mAb that binds to the CH3 domain near the binding site for the high-affinity type-I IgE Fc receptors of human IgE, can neutralize free IgE and inhibit the IgE allergic pathway without sensitizing mast cells and basophils. We found that omalizumab in patients with severe persistent asthma (SPA) was an effective therapy for asthma and the following co-morbid conditions: chronic urticaria (CU), bee venom allergy, latex allergy, atopic dermatitis, food allergy and Samter's syndrome. Information on the use of omalizumab in treatment of asthma and other allergic diseases has improved our understanding that treatment acts on many levels, including regulating levels of inflammatory proteins, including cytokines (copper-containing alpha- 2-glycoprotein, total antioxidant capacity, MDA, NO, H2O2, CXCL8, IL-10, TGF-ß, GMCSF, IL-17, IL-1ß), MPV, Hs-CRP, eosinophil cationic peptide, vitamin-D (25(OH)D), homocysteine (Hcy), OX-2, d- dimer, albumin, and sApo-2L. The decrease in Hcy concentrations and increase in 25(OH)D also support the existence of a vascular endothelial protection mechanism. Mediators and cells classically involved in pro-coagulant and anticoagulant pathways together play a role in SPA and CU pathophysiology and omalizumab effect. The mechanism of action of omalizumab in the treatment of asthma is believed to be multifactorial, and includes effects mediated through altered production of redox metabolites, extrinsic coagulation pathway, oxidative markers-related mi RNA, TRAIL-related mi RNA, and regulation of production of known inflammatory proteins.

Single-particle tracking of immunoglobulin E receptors (FcεRI) in micron-sized clusters and receptor patches.

When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded FcεRI receptors aggregate at contact points and trigger degranulation and the release of immune activators. We used two-color total internal reflection fluorescence microscopy and single-particle tracking to show that most fluorescently labeled receptor complexes diffuse freely within these micron-size clusters, with a diffusion coefficient comparable to free receptors in resting cells. At later times, when the small clusters coalesce to form larger patches, receptors diffuse even more rapidly. In all cases, Monte Carlo diffusion simulations ensured that the tracking results were free of bias, and distinguished biological from statistical variation. These results show the diversity in receptor mobility in mast cells, demonstrating at least three distinct states of receptor diffusivity.

Disparity in FcεRI-induced degranulation of primary human lung and skin mast cells exposed to adenosine.

Inhaled and intravenously administered adenosine induces mast cell-mediated (histamine-dependent) bronchospasm in asthmatics without causing urticaria. A differential response to adenosine by human lung and skin mast cells is shown: low concentrations potentiate FcεRI-induced degranulation of human lung mast cells but not that of skin mast cells. Human lung mast cells were found to express ∼ 3-fold more A3AR messenger RNA (mRNA) than skin mast cells, suggesting the involvement of the G(i)-linked A3AR. Indeed, the adenosine-induced potentiation was sensitive to inhibition by pertussis toxin and, furthermore, could be induced with an A3AR-specific agonist. This study reveals a previously unrecognized disparity in the response to adenosine by primary human mast cells from lung and skin that might explain why adenosine induces a pulmonary but not dermatologic allergy-like response in vivo. In addition, we identify the A3AR as a potentiating receptor of FcεRI-induced degranulation, thereby implicating it in the in vivo bronchoconstrictive response to adenosine in asthmatics.

Suitable transmembrane domain significantly increase the surface-expression level of Fc(epsilon)RIalpha in 293T cells.

Evidence showed that the extracellular part of Fc(epsilon)RIalpha (FCR) with its own transmembrane domain (TMF) cannot be expressed as a transmembrane form in CHO cell line. However, FCR could be displayed on cell surface with the transmembrane domain (TM) of human IL2Ralpha (TMI). Theoretical analysis of TMF and TMI using TM prediction methods showed that TMI possessed strong orientation tendency to form "outside to inside" transmembrane mode from N-terminal to C-terminal, while TMF was prone to form "inside to outside" mode. Based on the analyzing results, the TM of Her2 (TMH) was studied and showed similar transmembrane mode as that of TMI, which implied that TMH might be a novel TM to obtain the surface display of FCR. Then, DNA sequences encoding TMH and TMF were fused to 3'-end of FCR gene, respectively. Fluorescent microscope observation indicated that FCR_TMH seemed to be located mainly on cell surface, while FCR_TMF appeared in endochylema. Flow cytometry analysis and Western blot also showed that the surface expression of FCR was enhanced significantly by TMH, while FCR_TMF could not be surface displayed in 293T cell. The experimental results were consistent with the theoretical predictions and demonstrated that the orientation tendency of TM may be very important in subcellular location of proteins.

Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract.

Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcepsilonRI was developed. Our objective was to evaluate the degranulation of three cell-lines expressing either the alpha-(RBL-hEI(a)-2B12 and RBL-30/25cells) or alpha-, beta-, and gamma-subunits (RBL SX-38) of the human FcepsilonRI by beta-hexosaminidase release. Purified human IgE and serum-derived polyclonal IgE from peanut-allergic subjects following challenge with anti-IgE or peanut protein extract, respectively, were utilized. Robust degranulation was induced in all three: RBL-30/25 (84%), -hEI(a)-2B12 (54%), SX-38 (94%), respectively, using purified IgE+anti-human IgE. Good release (18%, 40-45%, and 65%, respectively) occurred for one peanut-allergic subject+peanut extract with all cell-lines. With serum from three other peanut-allergic subjects, no beta-hexosaminidase release occurred with RBL-hEI(a)-2B12 cells+peanut extract, while only serum from one subject induced good degranulation, 30% and 60%, respectively, with RBL-30/25 and RBL SX 38 cells. Consistent degranulation with a potent food allergen (peanuts) was not observed. The assay's utility in safety assessment, predictive value and reproducibility for evaluating the cross-reactivity of proteins with allergens needs further investigation with additional proteins and well-characterized sera.

Physiologic assessment of allergic rhinitis in mice: role of the high-affinity IgE receptor (FcepsilonRI).

BACKGROUND: There have been few reports using animal models to study the development of allergic rhinitis. Characterization of such a model in mice would be advantageous given the availability of reagents and gene-manipulated strains. OBJECTIVE: We sought to develop a murine model of allergic rhinitis in the absence of lower airway changes. METHODS: After sensitization and challenge, both wild-type and FcepsilonRI-deficient mice were studied for their ability to develop early- and late-phase nasal responses. In the invasive approach, direct measurements of nasal airway resistance (R(NA)) were obtained; in the noninvasive approach using whole-body plethysmography, respiratory frequency and expiratory and inspiratory times were monitored. In both approaches, nasal responses were determined either acutely after challenge (early phase) or 24 hours after challenge (late phase). RESULTS: After challenge of sensitized mice, R(NA) significantly increased. In parallel, respiratory frequency significantly decreased and was highly correlated with the increases in R(NA). Sensitized wild-type mice had an early-phase nasal response and persistent nasal blockage (late-phase response) after allergen challenge. In contrast, sensitized and challenged FcepsilonRI alpha-chain-deficient mice did not have an early-phase nasal reaction and exhibited reduced nasal blockage and lower IL-13 levels in nasal tissue homogenates. CONCLUSIONS: These data indicate that FcepsilonRI is essential to development of an early-phase nasal response and contributes to the development of the late-phase nasal response. These invasive and noninvasive approaches provide new opportunities to evaluate the mechanisms underlying the development of nasal responses to allergen and to assess various therapeutic interventions.

Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.

To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Binding of bivalent ligand to cell surface IgE: can one detect ring formation?

It is well established that aggregation of cell surface immunoglobulin is involved in signal transduction by cells of the immune system. It is less well understood what special properties of these cell surface aggregates are important in initiating the signal cascade. Several authors have proposed that cells respond to the size (Fewtrell and Metzger (1980) J. Immun. 125, 701-710) as well as the stereochemistry (Ortega et al. (1989) Eur. J. Immun. 19, 2251-2256) of receptor aggregates. One approach to arriving at data relevant to this question has been to construct simple bivalent ligands that can bind to surface immunoglobulin. Several authors have suggested that when these bivalent ligands interact with surface immunoglobulin the formation of small stable cyclic complexes is highly favored. In this paper we consider whether it is possible to completely determine the parameters that describe the binding of a bivalent ligand to a bivalent receptor with the available experimental technology. We show that with the appropriate analysis procedure, using a modified equivalent site model, these parameters can be reliably determined from only three experiments even when there is a large amount of ring formation.