Human Basophil Degranulation Research Articles

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation

BackgroundSiglec-3 (CD33) is a major Siglec expressed on human mast cells and basophils and engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ObjectiveWe sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). MethodsDirect and indirect basophil activation tests (BAT) were used to assess both antigen-specific (peanut) and antigen non-specific (polyclonal anti-IgE) stimulation. Whole blood from allergic donors was used for direct BAT, whereas non-food allergic donor blood was passively sensitized with peanut-allergic plasma in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for one hour or overnight, then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine (fMLP) for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. ResultsIncubation for one hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to fMLP, providing evidence that this inhibition is IgE-pathway specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. ConclusionsPre-treating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L prior to antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Sequential engagement of adhesion molecules and cytokine receptors impacts both piecemeal and anaphylactic degranulation of human basophils.

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L-, CD49d- or CD11b crosslinking and IL-3 or IL-33 stimulation in different orders followed by anti-IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL-3 induced PMD in basophils and combined with CD62L- or CD11b crosslinking, IL-3 potentiated the degranulation regardless of sequential order. IL-3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti-IgE. CD62L- and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL-3 increased CD63 expression following anti-IgE. IL-3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL-33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE-dependent nor IgE-independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine-tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied.

Sputum basophils from allergic asthmatic patients do not express IL‐7Rα that is essential for TSLP signalling

AbstractBasophils are crucial in regulating allergic reactions via immediate secretion of multiple mediators upon IgE‐induced degranulation. IL‐3 regulates the development and activation of human basophils while epithelial cytokine thymic stromal lymphopoietin (TSLP) is another key regulator for murine basophils. Despite association of increased TSLP with exaggerated basophil responses in oesophageal biopsies, the effects of TSLP in regulating human basophil degranulation and activation are under debate. In this study, we aimed to examine whether human basophils responded to TSLP by co‐expression of TSLP receptors, TSLPR and IL‐7Rα (CD127), upon in vitro activation and in sputum of allergic asthmatic patients. Flow cytometric analysis of fresh basophils from healthy controls revealed no detectable TSLPR and CD127. Further flow cytometric analysis of basophils from healthy controls in vitro stimulated by multiple established basophil modulators for 24 hours showed induction of TSLPR but not CD127 by IL‐3 (10 ng/ml), anti‐IgE (10 μg/ml) and C5a (50 ng/ml). One‐hour stimulation of basophils from allergic asthmatic patients and healthy controls by TSLP (50 ng/ml) with or without IL‐3 (10 ng/ml) and anti‐IgE (10 μg/ml) had no effect on induction of CD63+ degranulated basophils. Similarly, TSLP (50 ng/ml) with or without IL‐3 (10 ng/ml) and anti‐IgE (10 μg/ml) did not induce IL‐4 and IL‐13 production by basophils from healthy controls. Further ex vivo analysis revealed that sputum basophils from allergic asthmatic patients did not express CD127. We conclude that human basophils from healthy controls and allergic asthmatic patients do not respond to TSLP as lacking CD127.

Targeting Inhibitory Siglec-3 In Vitro to Suppress Peanut-Induced Human Basophil Degranulation

Siglec 3 (CD33) is the major Siglec expressed on human basophils, and engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We sought to inhibit in vitro basophil degranulation to peanut by simultaneously targeting both CD33 and the FcεRI receptor complex by pre-treating with monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L).

The Use of Bruton's Tyrosine Kinase Inhibitors to Treat Allergic Disorders.

Purpose of reviewStudies show that inhibitors of Bruton’s tyrosine kinase (BTKis), currently FDA-approved for the treatment of B cell malignancies, can prevent IgE-mediated reactions through broad inhibition of the FcεRI signaling pathway in human mast cells and basophils. This review will summarize recent data supporting the use of these drugs as novel therapies in various allergic disorders.Recent findingsRecent studies have shown that BTKis can prevent IgE-mediated degranulation and cytokine production in primary human mast cells and basophils. Two oral doses of the second-generation BTKi acalabrutinib can completely prevent moderate passive systemic anaphylaxis in humanized mice and even protect against death during severe anaphylaxis. Furthermore, two doses of ibrutinib can reduce or eliminate skin prick test responses to foods and aeroallergens in allergic subjects. BTKis in development also show efficacy in clinical trials for chronic urticaria. Unlike other therapies targeting IgE, such as omalizumab, BTKis appear to have rapid onset and transient effects, making them ideal candidates for intermittent use to prevent acute reactions such as IgE-mediated anaphylaxis.SummaryThese studies suggest that BTKis may be capable of preventing IgE-mediated anaphylaxis, paving the way for future trials in food allergy and urticaria.

Coagulation factors induce human skin mast cell and basophil degranulation via activation of complement 5 and the C5a receptor

Coagulation factors induce human skin mast cell and basophil degranulation via activation of complement 5 and the C5a receptor

Piperidinyl-embeded chalcones possessing anti PI3Kδ inhibitory properties exhibit anti-atopic properties in preclinical models

Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes involved in membrane signalization pathways. Attempts to administer inhibitors with broad activity against different isoforms have failed due to toxicity. Conversely the PI3Kδ isoform is much more selectively expressed, enabling therapeutic targeting of this isoform. Of particular interest PI3Kδ is expressed in human basophils and its inhibition has been shown to reduce anti-IgE induced basophil degranulation, suggesting that PI3Kδ inhibitors could be useful as anti-allergy drugs. Herein, we report for the first time the activity of compounds derived from chalcone scaffolds as inhibitors of normal human basophil degranulation and identified the most active compound with anti-PI3Kδ properties that was investigated in preclinical models. Compound 18, namely 1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)phenyl]-3-(2,4,6-trimethoxyphenyl)-prop-2-en-1-one, was found to inhibit normal human basophil degranulation in a dose-dependent manner. In a murine model of ovalbumin-induced asthma, compound 18 was shown to reduce expiratory pressure while its impact on the inflammatory infiltrate in alveolar lavage and total lung was dependent on the route of administration. In a DNFB-induced model of atopic dermatitis compound 18 administered systemically proved to be as potent as topical betamethasone. These results support the anti-atopic and allergic properties of the title compound and warrant further clinical development.

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

BackgroundP2Y purinergic receptors (P2YR) are G protein-coupled receptors that are stimulated by extracellular nucleotides. They mediate cellular effects by regulating cAMP production, protein kinase C activation, inositol trisphosphate generation, and Ca2+ release from intracellular stores. The P2Y6 receptor of this family is selectively stimulated by UDP, and selectively inhibited by MRS2578. In the present study, we examined the effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in human basophils. MethodsBasophils were purified from human peripheral blood. The mRNA expression of genes encoding P2YR and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) was measured by RT-PCR. Intracellular Ca2+ influx via UDP/P2Y6 receptor signaling in basophils was detected using a calcium probe. The effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in basophils was confirmed by measuring CD63 expression by flow cytometry. Autocrine secretion of nucleotides was detected by HPLC analysis. ResultsWe showed that purified basophils express P2Y6 mRNA and that UDP increased intracellular Ca2+, which was reduced by MRS2578 treatment. UDP promoted IgE-dependent degranulation. Furthermore, MRS2578 inhibited IgE-dependent degranulation in basophils. HPLC analysis indicated that basophils spontaneously secrete UTP. In addition, basophils expressed the extracellular nucleotide hydrolases ENTPDase2, ENTPDase3, and ENTPDase8. ConclusionsThis study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

Nanoparticle-allergen interactions mediate human allergic responses: protein corona characterization and cellular responses.

BackgroundEngineered nanomaterials (ENMs) interact with different biomolecules as soon as they are in contact, resulting in the formation of a biomolecule ‘corona’. Hence, the ‘corona’ defines the biological identity of the ENMs and could affect the response of the immune system to ENM exposure. With up to 40 % of the world population suffering from type I allergy, a possible modulation of allergen effects by binding to ENMs is highly relevant with respect to work place and consumer safety. Therefore, the aim of this present study was to gain an insight into the interactions of gold nanoparticles with different seasonally and perennially occurring outdoor and indoor allergens.MethodsGold nanoparticles (AuNPs) were conjugated with the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1). The AuNP-allergen conjugates were characterized by means of TEM negative staining, dynamic light scattering (DLS), z-potential measurements and hyperspectral imaging. Furthermore, 3D models were constructed, based on the characterization data, to visualize the interaction between the allergens and the AuNPs surface. Differences in the activation of human basophil cells derived from birch/grass pollen- and house dust mite-allergic patients in response to free allergen and AuNP-allergen conjugates were determined using the basophil activation assay (BAT). Potential allergen corona replacement during BAT was controlled for using Western blotting. The protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was assessed, by an enzymatic activity assay and a cellular assay pertaining to lung type II alveolar epithelial cell tight junction integrity.ResultsThe formation of a stable corona was found for all three allergens used. Our data suggest, that depending on the allergen, different effects are observed after binding to ENMs, including enhanced allergic responses against Der p 1 and also, for some patients, against Bet v 1. Moreover elevated protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was found.ConclusionIn summary, this study presents that conjugation of allergens to ENMs can modulate the human allergic response, and that protease activity can be increased. Graphical Cross-linking of IgE receptors and degranulation of human basophils due to epitope alignment of nanoparticle-coated allergens. Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0113-0) contains supplementary material, which is available to authorized users.

Role of basophils in the pathogenesis of minimal change nephrotic syndrome: A literature review.

A number of studies have verified that minimal change nephrotic syndrome (MCNS) may result from the dysfunction of T cells and B cells, although the precise mechanisms are yet to be elucidated. It is widely recognized that MCNS is a T helper (Th)2-dominant glomerular disease caused by an imbalanced Th1/Th2 immune response. Increased levels of the Th2 cytokines, interleukin (IL)-4 and IL-13, have been demonstrated to be closely associated with disease activity. In addition, basophils can affect the Th1/Th2 balance by enhancing the Th2 response and impairing the Th1 response, which are then involved in the development of numerous diseases. However, whether basophils are vital in the pathogenesis of MCNS remains unknown. Frequent positivity of the human basophil degranulation test in patients with MCNS has been observed. Thus, basophils should be analyzed in order to determine their role in the pathogenesis of MCNS.

Mechanism of phosphatidylserine inhibition of IgE/FcεRI-dependent anaphylactic human basophil degranulation via CD300a

The process that leads to histamine release from human basophils constitutes a continuum that starts with piecemeal degranulation and eventually converts to anaphylactic degranulation (AND).1,2 These degranulation events are accompanied by mobilization of distinct cellular compartments and expression of surface molecules that are not or barely expressed on resting basophils. Two different histamine-containing compartments, which show distinct early signaling requirements and kinetics, have been described, namely, the CD203c compartment and the CD63 compartment.

Immunomodulating and Anti-Allergic Effects of Negroamaro and Koshu Vitis vinifera Fermented Grape Marc (FGM)

Polyphenols contained in FGM from Negroamaro (N) and Koshu (K) Vitis vinifera have been shown to exhibit several immunomodulating activities. For instance, mice affected by experimental colitis when administered with K-FGM showed an attenuation of the inflammatory process. In murine asthma, K-FGM reduced IgE production and eosinophil number in bronchial alveolar lavage fluid. In vitro, both N- and K-FGM were able to induce T regulatory cells in terms of Foxp-3 molecule expression and release of interleukin-10. In another set of experiments both N- and K-FGM were able to balance rate of proliferation/apoptosis/necrosis of normal human peripheral lymphocytes, thus indicating the property of these compounds to maintain immune homeostatic mechanisms in the host. On the other hand, N- and K-FGM inhibited human basophil degranulation, thus, confirming our previous results obtained with rat basophilic leukemia cells. Finally, N- and K-FGM also decreased oxidative burst of human polymorphonuclear cells and monocytes.Taken together, these findings imply the potential clinical usefulness of FGM administration in inflammatory/allergic conditions, such as chronic asthma.

Uracil nucleotides enhance the degranulation of human basophils induced by anti-IgE antibody via a purinergic receptor (P6016)

Abstract The P2Y receptors (P2YRs) are a family of G protein-coupled purinergic receptors that are stimulated by ATP, ADP, UTP, UDP, and UDP-G, and their interaction induces a wide range of biological effects in various cells through phospholipase C activation, inositol triphosphate generation, and Ca2+ release from intracellular stores. In our previous study, we demonstrated that in neutrophils, ATP regulates chemotaxis and phagocytosis via P2YR. In the present study, we examined the distribution and function of P2YR in another innate immune cell type, basophils, the degranulation of which is associated with type I allergy. To date, P2YR have been subdivided into 8 different subclasses: P2Y1, 2, 4, 6, 11, 12, 13, and 14. Of these 8 subclasses, our RT-PCR results showed the predominant expression of P2Y2, P2Y4, and particularly P2Y6, which are known as uracil nucleotide-sensitive P2YRs. Interestingly, HPLC analysis indicated that basophils spontaneously secrete UTP, the ligand for P2Y2 and 4, whereas UDP, the ligand for P2Y6, is probably generated from UTP in a metabolic pathway. Although the addition of UTP and UDP did not directly induce basophil degranulation, these nucleotides clearly enhanced the degranulation induced by anti-IgE cross-linking. These results suggest that uracil nucleotides act as potent inducers of histamine release in basophils.

Soybean Isoflavones Suppress Basophil Degranulation in Vitro in Subjects with Food Allergy

S U N D A Y 431 Soybean Isoflavones Suppress Basophil Degranulation in Vitro in Subjects with Food Allergy Mohanapriya Kamalakannan, MS, Matthew Stadler, Hugh A. Sampson, MD, FAAAAI, Madhan Masilamani, PhD; Mt. Sinai School of Medicine, New York, NY, Mount Sinai School of Medicine, New York, NY. RATIONALE: Soybean isoflavones, genistein and daidzein, are well known for their anti-inflammatory and immunomodulatory properties. We have recently shown that dietary isoflavones suppress anaphylactic reactions in murine models of peanut allergy. Here we tested the effect of isoflavones on human basophil degranulation in vitroin whole blood samples from subjects with food allergy. METHODS: Whole blood (250mL) from healthy controls (n53), and peanut allergic (n55) and milk allergic (n57) subjects were pre-incubated with 100mM each of genistein+daidzein for 30 min followed by culture with peanut or milk extracts respectively +/100mM genistein+daidzein for 30 min, as well as control stimulations such as anti-IgE, fMLP or medium alone. Degranulation was stopped by adding 20mM EDTA and stained for anti-CD63 and basophil-specific markers followed by flow cytometric analysis. Basophil degranulationwasmeasured by percent foldchange in CD63cells in the basophil gate. Phosphorylation of signal transduction molecules such as Erk, Syk and p38-MAP kinase in basophils was tested by flow cytometry. RESULTS: Isoflavones suppressed human basophil degranulation induced by anti-IgE cross-linking in a dose-dependent fashion. Basophil degranulation from peanut and milk allergic subjects was suppressed in vitro by allergen+isoflavone treatment when compared to allergen-treatment alone (mean % fold change5 217 vs 313 for peanut; 581 vs 949 for milk, respectively). Erk phosphorylation was also suppressed in isoflavone-treated basophils. CONCLUSIONS: Based on the in vitro data and our published results in peanut allergy in murine models, isoflavones may provide a novel treatment option for food allergy.

The Effect of Stimuli on Basophil-Mediated Atopic Responses During Asthmatic Lying-In Women and in Newborns

Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.

CTAB-coated gold nanorods elicit allergic response through degranulation and cell death in human basophils

The effect of CTAB (cetyltrimethylammonium bromide)- or PEG (polyethylene glycol)-coated gold-nanorods (Au-NRs) on the non-IgE mediated allergic response was studied. We found that the CTAB-Au-NRs released more allergic mediators such as histamine and β-hexosaminidase from human basophil KU812, a common model for studying allergy, after 20 min incubation. Also, the CTAB-Au-NRs induced more apoptosis than the PEG-Au-NRs in KU812 24 h after treatment. These short- and long-term effects were not solely due to the CTAB residues in the supernatant desorbed from the Au-NRs.

Leptin Enhances Survival and Induces Migration, Degranulation, and Cytokine Synthesis of Human Basophils

Basophils are the rarest leukocytes in human blood, but they are now recognized as one of the most important immunomodulatory as well as effector cells in allergic inflammation. Leptin, a member of the IL-6 cytokine family, has metabolic effects as an adipokine, and it is also known to participate in the pathogenesis of inflammatory reactions. Because there is an epidemiologic relationship between obesity and allergy, we examined whether basophil functions are modified by leptin. We found that human basophils express leptin receptor (LepR) at both the mRNA and surface protein levels, which were upregulated by IL-33. Leptin exerted strong effects on multiple basophil functions. It induced a strong migratory response in human basophils, similar in potency to that of basophil-active chemokines. Also, leptin enhanced survival of human basophils, although its potency was less than that of IL-3. Additionally, CD63, a basophil activation marker expressed on the cell surface, was upregulated by leptin, an effect that was neutralized by blocking of LepR. Assessments of basophil degranulation and cytokine synthesis found that leptin showed a strong priming effect on human basophil degranulation in response to FcεRI aggregation and induced Th2, but not Th1, cytokine production by the cells. In summary, the present findings indicate that leptin may be a key molecule mediating the effects of adipocytes on inflammatory cells such as basophils by binding to LepR and activating the cellular functions, presumably exacerbating allergic inflammation.

The Effect of Peptidoglycan Stimulation on Basophil-Mediated Atopic Responses During Pregnancy and in Newborns

Background. Toll-like receptor (TLR) molecules play critical roles in directing the course of atopic diseases by recognizing specific microbial products that activate immune effector cell function. Objective. We determined if basophils harvested from neonates genetically predisposed to atopic disease had different levels of TLR2 expression and determined whether putative TLR2 ligands mediated cytokine secretion. Methods. Blood samples were collected from 10 asthmatic and 12 healthy women and their newborns. Basophil histamine was measured using the human basophil degranulation test and TLR2 expression was determined using nucleic acid hybridization in situ and flow cytometry. IL-4 levels were quantified by ELISA following allergen stimulation. Results. The basophil degranulation index (DI) in granulocytes harvested from peripheral blood of asthmatic women was assessed following stimulation with either peptidoglycan (PGN) or Dermatophagoides farinae (Df) extract. The DI was significantly higher in atopic women than in healthy controls. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared with basophils purified from children born to nonatopic controls. Finally, TLR2 expression at the protein and mRNA levels was upregulated in cord blood basophils from neonates born to mothers with asthma following stimulation with PGN but not Df. Conclusion. These data suggested that TLR2-mediated innate immune responses play a role in augmenting allergic reactions through the modulation of basophil cytokine secretion and histamine release. Microbial components may activate basophils through TLR2 (especially for genetically predisposed infants) to release cytokines associated with an increased incidence of allergic diseases.

Differential effect of storage on molecular and ultra-molecular dilutions of histamine

We have worked for many years on a widely debated topic, namely the biological activity of so-called high dilutions of histamine in the negative feedback of human basophil activation mediated by the histamine H2 receptor. This choice of model was based on the fact that we wanted to test a chemically pure substance for which a series of agonists and antagonists were readily available. Various methods are possible for analysing basophil activation. We first used the so called human basophil degranulation test, which is based on cell staining with heparin specific dyes. We then proceeded to use flow cytometry, after having published the first application of the measurement of the CD63 marker for allergy diagnosis. In contrast to histamine release, this method may lend itself to a variety of protocols, depending on the selection and activation antibodies used. We began these studies with an anti-IgE/anti-CD63 method and then changed to an anti-IgE/anti-CD203c protocol, CD203c [1] being an earlier marker of the basophil activation cascade and less linked to the IgE dependent reaction [2]. Therefore, we ultimately used a protocol based on basophil selection by anti-IgE and an analysis of fMLP induced basophil activation by antiCD203c. These protocols led to the confirmation that high dilutions of histamine (15C to 18C,C standing for the n dilution to the one-hundredth of a mother solution (10 M) of histamine) were capable of inhibiting anti-IgE or fMLP induced basophil activation, this inhibition being reversed by cimetidine and being reproduced by 4-methylhistamine but not by 1or 3-methylhistamine [3–8]. The effect of cimetidine and the differential activity of the N-methylhistamine derivatives supported the specificity of the observed effect. We present here the effect of storage on the activity of these histamine dilutions.

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects. The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients. MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-gamma and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1. MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.