Allergic Donors Research Articles

Andolast Acts at Different Cellular Levels to Inhibit Immunoglobulin E Synthesis

The anti-asthmatic agent andolast is thought to inhibit the release of allergic mediators, but its mechanism of action is not fully understood. We investigated whether the compound inhibits immunoglobulin E (IgE) synthesis and tested the hypothesis that andolast affects immunoglobulin class switching. Interleukin (IL)-4 and the interaction of CD40 expressed on B cells with its ligand on T cells are necessary for IgE synthesis. Thus, peripheral blood mononuclear cells (PBMCs) from 40 asthmatic, 16 non-asthmatic allergic, and 9 normal donors were stimulated with IL-4 and/or anti-CD40 antibody. T cells from 9 additional allergic donors were activated with anti-CD3/CD28 antibodies to express IL-4 mRNA. After incubation in the absence or presence of test compounds, immunoglobulin concentrations were measured by enzyme immunoassay, and mRNA levels were analyzed by RT-PCR. Andolast significantly inhibited IgE synthesis by stimulated PBMCs from both asthma patients and combined allergic/normal donors. In mechanistic studies, andolast was found to act at different cellular levels. Firstly, it reduced by about 45 percent (p<0.05) the levels of IL-4 mRNA in T cells stimulated with anti-CD3/CD28. Secondly, andolast reduced by about 36 percent (p<0.05) the expression of epsilon germline transcripts in PBMCs stimulated with IL-4/anti-CD40. Thirdly, the effect of andolast on immunoglobulin synthesis was selective in that the production of IgG4 antibodies was not significantly inhibited. Our findings, while supporting the evidence that andolast is effective for the treatment of asthma, provide new insights into its mechanism of action.

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

It has been suggested that allergic diseases are caused by defective suppression of allergen-specific Th2 cells by CD4(+)CD25(+) regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25-expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127(lo), allowing discrimination between these distinct T cell subpopulations. The aim of this study was to study whether the putative regulatory subset defined as CD4(+)CD25(+)CD127(lo) was involved in grass pollen-reactive T cell responses. Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non-atopic controls out of season. Grass pollen-induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4(+)CD25(+), CD4(+)CD25(+)CD127(hi) or CD4(+)CD25(+)CD127(lo) T cells. Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non-atopic controls. Depletion of all CD25(+) T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4(+)CD25(+)CD127(lo) cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non-atopic group. Our study showed that T cells from grass pollen-allergic patients and non-atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.

Molecular profiles: A new tool to substantiate serum banks for evaluation of potential allergenicity of GMO

Assessment of the allergenicity of GMOs involves performing a test with a panel of sera obtained from allergic donors. However, there is no clear indication of how to characterize the above-mentioned panel. The patient selection criteria should take into account the geographical location of patients, the intensity and nature of the environmental allergens in the area and the potential cross-reactivity among allergenic molecules. Sera for serum banks, obtained from patients with demonstrated food allergy, should be subjected to a further characterization by screening with a panel of relevant allergenic molecules. A representative panel of these sera should be used in the allergenicity assessment. Finally, the “in vitro” methodologies should have the adequate specificity and sensitivity, and the integrity of the molecules tested should be guaranteed.

Allergy as an organ and a systemic disease

Allergic disorders are viewed generally as organ diseases and thus referred to organ specialists, such as the ear, nose and throat specialist for rhinitis, the pulmonologist for asthma, the dermatologist for dermatitis, and so on. Indeed, the systemic nature of allergy is made evident by the fact that the same individual may develop during the life different manifestations to a given allergen. This is true for example in sensitisation to house dust mites, which may start in childhood as atopic dermatitis and later express as asthma or rhinitis. The major player in driving the immune response is the T lymphocyte, and the T helper subpopulations--Th1 and Th2--as well as the T regulatory cells, are involved in orienting tolerance or reactivity to allergens. Interesting observations on the systemic or organ-specific actions of T cells were obtained by transplantations from allergic donors to non-allergic recipients. Bone marrow is able to transfer all allergic manifestations, while lung transplantation transfers only asthma. A number of factors are involved in the expression of allergy as a systemic or organ disease and deserve deeper investigations. They include the antigen presenting cells, the homing of T cells, the cytokine and chemokine pattern, and the adhesion molecules.

IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK Cells

IL-33 is an IL-1 family member recently identified as the ligand for T1/ST2 (ST2), a member of the IL-1 receptor family. ST2 is stably expressed on mast cells and T(h)2 effector T cells and its function has been studied in the context of T(h)2-associated inflammation. Indeed, IL-33 induces T(h)2 cytokines from mast cells and polarized mouse T cells and leads to pulmonary and mucosal T(h)2 inflammation when administered in vivo. To better understand how this pathway modulates inflammatory responses, we examined the activity of IL-33 on a variety of human immune cells. Human blood-derived basophils expressed high levels of ST2 receptor and responded to IL-33 by producing several pro-inflammatory cytokines including IL-1 beta, IL-4, IL-5, IL-6, IL-8, IL-13 and granulocyte macrophage colony-stimulating factor. Next, utilizing a human T(h)2-polarized T cell culture system derived from allergic donor blood cells, we found that IL-33 was able to enhance antigen-dependent and -independent T cell responses, including IL-5, IL-13 and IFN-gamma production. IL-33 activity was also tested on V alpha 24-positive human invariant NKT (iNKT) cells. In the presence of alpha-galactosylceramide antigen presentation, IL-33 dose dependently enhanced iNKT production of several cytokines, including both IL-4 and IFN-gamma. IL-33 also directly induced IFN-gamma production from both iNKT and human NK cells via cooperation with IL-12. Taken together, these results indicate that in addition to its activity on human mast cells, IL-33 is capable of activating human basophils, polarized T cells, iNKT and NK cells. Moreover, the nature of the responses elicited by IL-33 suggests that this axis may amplify both T(h)1- and T(h)2-oriented immune responses.

Citrus/Cydonia Comp. Can Restore the Immunological Balance in Seasonal Allergic Rhinitis‐Related Immunological Parameters In Vitro

In two in vitro studies, we examined the immunological (pathways of the) effects of Citrus/Cydonia comp. from, respectively, a healthy and an allergic donor; peripheral blood mononuclear cells (PBMCs) were isolated out of peripheral blood and analyzed in vitro after polyclonal stimulation of T-cells. The differentiation capacity and the influence with regard to Th1 (IFN-γ) and Th2 (IL-5) cells were examined. Citrus/Cydonia comp. has a selective effect on the differentiation of T-cells by producing relatively more IL-10 than IL-12. By that, it also seems to have an effect on the induction of regulatory (IL-10 producing) T-cell subsets. It is in vitro capable of neutralizing (to some extent) the changes, characteristic to allergic rhinitis, with regard to the maturation, differentiation, and activity of the immune system. Thus, Citrus/Cydonia comp. can potentially restore the disturbed immune state of rhinitis patients, which essentially could be sufficient to make allergic symptoms disappear permanently.

Galectin-3 Functions as an Adhesion Molecule to Support Eosinophil Rolling and Adhesion under Conditions of Flow

Allergic inflammation involves the mobilization and trafficking of eosinophils to sites of inflammation. Galectin-3 (Gal-3) has been shown to play a critical role in eosinophil recruitment and airway allergic inflammation in vivo. The role played by Gal-3 in human eosinophil trafficking was investigated. Eosinophils from allergic donors expressed elevated levels of Gal-3 and demonstrated significantly increased rolling and firm adhesion on immobilized VCAM-1 and, more surprisingly, on Gal-3 under conditions of flow. Inhibition studies with specific mAbs as well as lactose demonstrated that: 1) eosinophil-expressed Gal-3 mediates rolling and adhesion on VCAM-1; 2) alpha(4) integrin mediates eosinophil rolling on immobilized Gal-3; and 3) eosinophil-expressed Gal-3 interacts with immobilized Gal-3 through the carbohydrate recognition domain of Gal-3 during eosinophil trafficking. These findings were further confirmed using inflamed endothelial cells. Interestingly, Gal-3 was found to bind to alpha(4) integrin by ELISA, and the two molecules exhibited colocalized expression on the cell surface of eosinophils from allergic donors. These findings suggest that Gal-3 functions as a cell surface adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.

Interaction of suplatast tosilate (IPD) with chloride channels in human blood eosinophils: a potential mechanism underlying its anti‐allergic and anti‐asthmatic effects

Alterations in chloride ion channels have been implicated in the induction of changes in cell shape and volume. Because blood and tissue eosinophilia are hallmarks of bronchial asthma, in this study we examined the role of chloride channels in the underlying effects of suplatast tosilate (IPD), an anti-allergic drug, in human blood eosinophils. Eosinophils were isolated and purified from the blood of allergic asthmatic donors. Chloride ion currents were recorded using the whole-cell patch-clamp technique in freshly isolated eosinophils. The current-voltage relationship of whole-cell currents in human blood eosinophils was calculated and recorded. The effect of chloride channel blockers was examined on superoxide release, eosinophil chemotaxis as measured by the Boyden chamber, and eosinophil adhesion to endothelial cells. Radioligand binding studies with [3H]IPD and competition curves with chloride channel blockers were performed. IPD increased both inward and outward chloride currents in human blood eosinophils. IPD in 1 ng/mL did not have significant effect on chloride current. However, at 5 ng/mL IPD activated both outward and inward currents in human blood eosinophils. Chloride channel blockers inhibited IPD-induced respiratory burst in eosinophils, eosinophil chemotaxis, and eosinophil adhesion to endothelial cells. All these effects of IPD on chloride current and the resultant functional responses in human blood eosinophils were not due to its basic salt, p-toluenesulphonic acid monohydrate. Human blood eosinophils contained specific binding sites for [3H]IPD with K(D) and B(max) values of 187.7+/-105.8 nm and 58.7+/-18.7 fmol/10(6) cells, respectively. Both NPPB and DIDS competed, in a dose-dependent manner, for the specific binding of [3H]IPD in human blood eosinophils. These data suggest that the anti-allergic and anti-asthmatic effects of IPD could be due to its interaction with chloride channels in human blood eosinophils.

Differential activation of dendritic cells by nerve growth factor and brain‐derived neurotrophic factor

Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well. The aim of this study was to investigate the influence of neurotrophins on DC function. Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta. Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found. These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.

Differences in the antibody response to a mucosal bacterial antigen between allergic and non-allergic subjectsSmoke-free legislation reduces exposure in children

Background: The immune response to bacterial antigens on mucosal surfaces may be modified in individuals allergic to aeroallergens due to a maturational or genetic difference or from the interaction between...

Human dendritic cells transfected with allergen‐DNA stimulate specific immunoglobulin G4 but not specific immunoglobulin E production of autologous B cells from atopic individuals in vitro

Atopic/allergic diseases are characterized by T helper 2 (Th2)-dominated immune responses resulting in immunoglobulin E (IgE) production. DNA-based immunotherapies have been shown to shift the immune response towards Th1 in animal models. In further studies we showed that human dendritic cells (DC) transfected with allergen-DNA are able to stimulate autologous CD4(+) T cells from atopic individuals to produce Th1 instead of Th2 cytokines and to activate interferon-gamma (IFN-gamma)-producing CD8(+) T cells. The aim of this study was to analyse whether DC transfected with allergen-DNA are also able to influence immunoglobulin production of B cells from atopic donors. For this purpose, human monocyte-derived DC from grass-pollen allergic donors were transfected with an adenovirus encoding the allergen Phleum pratense 1 and cocultured with B cells, autologous CD4(+) T cells, and CD40 ligand-transfected L-cells. B cells receiving help from CD4(+) T cells stimulated with allergen-transfected dendritic cells produced more allergen-specific IgG4 compared to stimulation with allergen protein pulsed DC or medium, while total IgG4 production was not affected. In contrast, specific IgE production was not enhanced by stimulation with allergen-DNA transfected DC compared to medium and inhibited compared to allergen protein-pulsed DC with similar effects on total IgE production in vitro. Allergen-DNA transfected dendritic cells are able to direct the human allergic immune response from Th2-dominance towards Th1 and Tc1 also resulting in decreased IgE and increased IgG4 production.

Interleukin-3 Promotes the Expression of E-NPP3/CD203C on Human Blood Basophils in Healthy Subjects and in Patients with Birch Pollen Allergy

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Design, engineering and in vitro evaluation of MHC class‐II targeting allergy vaccines

The worldwide increasing incidence of allergic diseases requires the development of new, efficient vaccination strategies, the only curative treatment with a long-lasting effect. Current allergen-specific immunotherapy protocols suffer from limited efficacy and a long treatment time. We engineered modular antigen translocating (MAT) molecules for intracellular targeting of allergens to the major histocompatibility class-II (MHC-II) presentation pathway to enhance antigen presentation. MAT-fusions were evaluated for their ability to localize intracellularly, to induce proliferation, and for their influence on cytokine patterns in peripheral blood mononuclear cells (PBMCs) cultures. We show that MAT-allergen fusions are able to rapidly translocate into the cytoplasm of PBMCs, whereas naked recombinant allergens are only marginally taken up. MAT vaccines accumulate intracellularly and induce strong proliferation of PBMC cultures at concentrations 10-100 times lower than the corresponding naked allergens, indicating an enhanced presentation through the MHC-II presentation pathway. In PBMC cultures of allergic donors, MAT vaccines induce a cytokine shift from a T(H)2 to a T(H)1 profile, resulting in a stronger and earlier secretion of INF-gamma and Interleukin (IL)-10, and a decreased secretion of IL-4, IL-5 and IL-2, compared with those induced by the corresponding recombinant allergens. Modular antigen translocation vaccines induce strong proliferation responses in PBMC cultures at low concentration and induce a T(H)1/T(H)2 shift in the cytokine profile, reflecting those reported to occur in successfully desensitized allergic patients. Therefore, MAT molecules represent promising lead compounds for the development of potent allergy vaccines.

Differential Immunomodulating Effects of Inactivated Probiotic Bacteria on the Allergic Immune Response

Bacterial stimulation plays an important role in modulating the allergic immune response. The aim of this study was to investigate the effects of inactivated probiotic Lactobacillus acidophilus and non-pathogenic Escherichia coli strain Nissle on the phenotype and function of T- and B-cells. Peripheral blood mononuclear cells from patients with grass-pollen allergy (n=10) and non-allergic patients (n=19) were co-stimulated with inactivated bacteria and grass-pollen allergen. Expression of CD23, CD80, CD86 and CD69 were analysed, and the intracellular production of interleukin-4 and interferon-gamma was measured by direct ex vivo flow cytometry after stimulation. Both bacteria induced a significant up-regulation of CD69 expression on T-lymphocytes (p=0.001). CD23 expression was significantly increased following stimulation with allergen (p=0.008), but reduced after stimulation with Lactobacillus and significantly reduced with E. coli plus allergen (p=0.029). CD80 expression was reduced after stimulation with Lactobacillus in the allergic group only (p=0.021). By contrast, CD86 expression was significantly increased after stimulation with Lactobacillus (p=0.049) and distinctly increased with E. coli in both groups (p=0.001). The cytokine patterns of CD69-positive T-lymphocytes from allergic patients showed a TH2-dominated response after allergen stimulation (interferon-gamma/interleukin-4-ratio 0.2), directed into a T-helper1-like response by stimulation with both types of bacteria (interferon-gamma/interleukin-4-ratios 1.5-2.0 in both groups). These data show that both types of bacteria modulate the allergic immune response by the alteration of CD23 and co-stimulatory molecule expression. Regarding cytokine production, the data suggest a differential response to both bacteria depending on the atopic state, but a clear promotion of T-helper1-dominated response in allergic donors.

Impact of Lactic Acid Bacteria on Dendritic Cells from Allergic Patients in an Experimental Model of Intestinal Epithelium

Lactic acid bacteria (LAB) are Gram positive nonpathogenic commensal organisms present in human gastrointestinal tract. In vivo, LAB are separated from antigen-presenting cells such as dendritic cells (DC) by the intestinal epithelial barrier. In this study, the impact of one LAB strain (Lactobacillus casei ATCC393) on human monocyte-derived DC from allergic and healthy donors was assessed by using a polarized epithelium model. Confocal and flow cytometer analyses showed that immature DC efficiently captured FITC-labelled L. casei through the epithelial layer. After interaction with L. casei, DC acquired a partial maturation status (i.e., CD86 and CD54 increase) and increased their interleukin (IL)-10 and IL-12 production. Interestingly, after activation by L. casei in the presence of experimental epithelium, DC from allergic patients instructed autologous naïve CD4+ T cells to produce more interferon-γ than without the epithelium. Thus by modulating human DC reactivity, LAB and intestinal epithelium might modify T cell immune response and regulate the development of allergic reaction.

Environmental Polycyclic Aromatic Hydrocarbons (PAHs) Enhance Allergic Inflammation by Acting on Human Basophils

Diesel exhaust particles (DEPs) have been implicated in the worldwide increased incidence of allergic airway diseases over the past century. There is growing evidence that DEP-associated polycyclic aromatic hydrocarbons (PAHs) participate in the development and maintenance of immunoglobulin (Ig) E-mediated allergic diseases. To address this issue we investigated the impact of U.S. Environmental Protection Agency (EPA) priority PAHs as well as of PAH-containing airborne extracts on antigen-induced CD63 upregulation and mediator release from human basophils. Whole blood samples from birch pollen allergic and control subjects were incubated in the presence of organic extracts of urban aerosol (AERex) or EPA-PAH standard with or without rBet v 1. Basophils were analyzed for CD63 expression as a measure of basophil activation by using multiparameter flow cytometry. In addition, purified basophils from birch pollen allergic donors were incubated for 2 h in the presence of 1 μM benzo[a]pyrene (BaP) or phenanthrene (Phe) and then stimulated with rBet v 1 for 45 min. Supernatants were assayed for histamine, interleukin (IL)-4, and IL-8 by means of enzyme-linked immunosorbent assay (ELISA). Basophils exposed in vitro simultaneously to AERex or EPA-PAH standard and rBet v 1 expressed CD63 significantly more than with antigen alone. PAHs synergized with rBet v 1 dose dependently, but did not activate basophils from nonallergic donors. BaP and Phe significantly enhanced cytokine secretion (IL-4, IL-8) and histamine release from purified basophils without antigen added, and secretion was not further enhanced by rBet v 1 stimulation. In conclusion, PAHs from roadside emissions can directly activate sensitized basophils to cytokine secretion and drive proallergic processes through enhanced Fcε RI-coupled mediator release from human basophils.

Differential Inhibition of Primary versus Preactivated T Cells by Pimecrolimus but Not by Tacrolimus in vitro

Background: Recent studies in murine models of allergic contact dermatitis have shown that systemic treatment with pimecrolimus in contrast to tacrolimus did not inhibit the sensitization phase, whereas both compounds equivalently suppressed the inflammatory response in sensitized animals. This finding indicated a differential sensitivity of antigen-naïve and primed T cells towards pimecrolimus and tacrolimus. Methods: T cells obtained from healthy and allergic donors were subjected to primary and secondary stimulation by allogeneic or staphylococcal superantigen-presenting dendritic cells (DC). Human skin-derived, allergen-specific T cell clones from an atopic dermatitis patient were activated by anti-CD3 antibodies or by specific allergen-presenting DC. The inhibition of T cell proliferation and cytokine release by graded doses of calcineurin inhibitors was evaluated. Results: Primary stimulation of T cells was inhibited by pimecrolimus with an approximately 8-fold lower potency as compared with tacrolimus. In contrast, the secondary response of ex vivo expanded T cells activated by allogeneic or staphylococcal superantigen-presenting DC was inhibited by both compounds with equivalent potency. Likewise, both drugs showed very similar potency to inhibit the proliferation and cytokine synthesis from antigen- stimulated T cell clones and the induction of cytokines in Jurkat T cells. Conclusion: These data indicate that pimecrolimus has a selectivity for antigen-primed memory T cells not seen with tacrolimus.

Gastro‐duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential

The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments M(r) L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P<0,05; Wilxocon's signed rank test). The Th-2 type cytokine polarization of T cells from peanut allergic donors (IFN-gamma/IL-13 ratio and IFN-gamma/IL-4 ratio of CFSE(low) CD4(+) T cells) remained unchanged regardless of the level of digestion. Histamine release of basophils from peanut allergic individuals was induced to the same extent by native Ara h 1 and its digestion products. Gastro-duodenal digestion fragments of Ara h 1 retain T cell stimulatory and IgE-binding and cross-linking properties of the intact protein.

Comparison of human tonsillar mast cell localization and ultrastructural observations between IgE-mediated allergic and nonallergic donors

Mast cells (MCs) are important effector cells in mucosal defense and allergic inflammation. Lymphoid tissues such as tonsils and adenoids also have modest numbers of MCs. However, the role of MCs in lymphoid tissues is not well known. In this study, we showed the local distribution of MCs in tonsils from IgE-mediated allergic or nonallergic donors, studied their ultrastructure, and specified their surrounding cell types. Tonsils were obtained from IgE-mediated allergic or nonallergic donors suffering from chronic tonsillitis or hyperplastic tonsils. The localization and distribution of MCs and IgE binding to MCs were determined by immunohistochemistry using antibodies against tryptase, c-kit, and IgE. In addition, mast cell structure was examined using electron microscopy. In both allergic and nonallergic donors, MCs were distributed mainly in the interfollicular area and the perivascular area of connective tissue. We found that the total number of MCs in tonsils was almost the same in both donor groups. However, the number of MCs in the interfollicular area was significantly higher in nonallergic versus allergic donors. Moreover, the electron microscopy revealed that MCs localized in perivascular areas of connective tissue have scroll-type granules, and MCs in the interfollicular areas contained both particle and scroll-type granules. In addition, the MCs that were surrounded by CD4+ lymphocytes in the interfollicular area showed empty granules, whereas MCs in the perivascular area, not surround by CD4+, were intact. This implies that these MCs were degranulated, and this probably was caused by CD4+ cells. Taken together, these findings suggest that tonsillar MCs distribute differently in allergic and nonallergic donors, and that MCs in the interfollicular area might be activated by direct contact with CD4+ T cells.

Evidence of in vivo basophil activation in chronic idiopathic urticaria

Approximately 40% of chronic idiopathic urticaria (CIU) subjects have autoantibodies to either FcepsilonRIalpha or IgE. The effect of such autoantibodies on circulating basophil activation status is unknown. The expression of cell surface activation markers on basophils from CIU, non-allergic, and allergic subjects were compared. Further, the relationship between marker expression and serum factors reported in CIU, such as histamine-releasing activity (HRA) and immunoreactivity to FcepsilonRIalpha were examined. Peripheral blood was obtained from CIU, allergic, and non-allergic donors and fractionated by density gradients. Enriched basophils (1-12%) were analysed by flow cytometry for expression of activation markers including CD63, CD69, and CD203c. Dilutions of serum (5-50%) were analysed for HRA on basophils from a normal donor. Serum was tested for immunoreactivity by western blotting to a standard cell lysate prepared from an RBL-SX38 cell line transfected with human FcepsilonRIalpha. CIU subjects (n=9) and allergic subjects (n=8) exhibited enhanced expression of CD63 and CD69, as compared with non-allergic subjects (n=7); however, no difference was seen among groups for CD203c expression. Five CIU and two non-allergic subjects had evidence of significant serum HRA (>20%), whereas two CIU, two allergic, and three non-allergic subjects had evidence of serum immunoreactivity to FcepsilonRIalpha. Serum HRA and serum immunoreactivity to FcepsilonRIalpha were not associated with enhanced surface marker expression. Basophil activation marker expression is increased in CIU subjects and is not associated with serum factors. In addition, serum HRA and FcepsilonRIalpha immunoreactivity are not unique to CIU, or related to enhanced circulating basophil marker expression.