Biomarkers of severity and threshold of allergic reactions during oral peanut challenges.

BACKGROUND: Oral food challenge (OFC) is the criterion standard to assess peanut allergy (PA), but it involves a risk of allergic reactions of unpredictable severity. OBJECTIVE: Our aim was to identify biomarkers for risk of severe reactions or low dose threshold during OFC to peanut. METHODS: We assessed Learning Early about Peanut Allergy study, Persistance of Oral Tolerance to Peanut study, and Peanut Allergy Sensitization study participants by administering the basophil activation test (BAT) and the skin prick test (SPT) and measuring the levels of peanut-specific IgE, Arachis hypogaea 2-specific IgE, and peanut-specific IgG4, and we analyzed the utility of the different biomarkers in relation to PA status, severity, and threshold dose of allergic reactions to peanut during OFC. RESULTS: When a previously defined optimal cutoff was used, the BAT diagnosed PA with 98% specificity and 75% sensitivity. The BAT identified severe reactions with 97% specificity and 100% sensitivity. The SPT, level of Arachis hypogaea 2-specific IgE, level of peanut-specific IgE, and IgG4/IgE ratio also had 100% sensitivity but slightly lower specificity (92%, 93%, 90%, and 88%, respectively) to predict severity. Participants with lower thresholds of reactivity had higher basophil activation to peanut in vitro. The SPT and the BAT were the best individual predictors of threshold. Multivariate models were superior to individual biomarkers and were used to generate nomograms to calculate the probability of serious adverse events during OFC for individual patients. CONCLUSIONS: The BAT diagnosed PA with high specificity and identified severe reactors and low threshold with high specificity and high sensitivity. The BAT was the best biomarker for severity, surpassed only by the SPT in predicting threshold. Nomograms can help estimate the likelihood of severe reactions and reactions to a low dose of allergen in individual patients with PA.

Acid Stripping of Surface IgE Antibodies Bound to FcεRI is Unsuitable for the Functional Assays that Require Long-Term Culture of Basophils and Entire Removal of Surface IgE.

Basophils are rare granulocytes and dysregulated functions of these cells are associated with several atopic and non-atopic allergic diseases of skin, respiratory system and gastrointestinal tract. Both cytokines and immunoglobulin E (IgE) are implicated in mediating the basophil activation and pathogenesis of these disorders. Several reports have shown that healthy individuals, and patients with allergic disorders display IgG autoantibodies to IgE and hence functional characterization of these anti-IgE IgG autoantibodies is critical. In general, anti-IgE IgG autoantibodies modulate basophil activation irrespective of allergen specificity by interacting with constant domains of IgE. Therefore, an ideal solution to prove the functions of such anti-IgE IgG autoantibodies would be to completely eliminate type I high affinity immunoglobulin E receptor (FcɛRI)-bound IgE from the surface of basophils and to demonstrate in an unequivocal manner the role of anti-IgE IgG autoantibodies. In line with previous reports, our data show that FcɛRI on peripheral blood basophils are almost saturated with IgE. Further, acetic acid buffer (pH 4) efficiently removes these FcɛRI-bound IgE. Although immediately following acetic acid-elution of IgE had no repercussion on the viability of basophils, following 24 hours culture with interleukin-3 (IL-3), the viability and yield of basophils were drastically reduced in acid-treated cells and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3-induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, our results show that acid buffers could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens. However, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE to validate the role of anti-IgE IgG autoantibodies that interact with FcɛRI-bound IgE irrespective of allergen specificity.

Basophil CD63 expression in chronic spontaneous urticaria: correlation with allergic sensitization, serum autoreactivity and basophil reactivity.

BACKGROUND: The underlying causes and factors contributing to the disease severity of chronic spontaneous urticaria (CSU) are unknown. OBJECTIVE: Given the important role of basophils in the pathogenesis of urticaria and that CD63 serves as a useful marker for basophil activation and detecting, CD63 expression of basophils is a reliable tool for diagnosing allergy and hypersensitivity reactions to different allergens; the objective of this study was to investigate whether the level of basophil CD63 expression is correlated with allergen sensitization, serum autoreactivity and basophil reactivity in patients with CSU. METHODS: Basophil-enriched leucocytes were separated from the blood of 64 patients with chronic urticaria (54 CSU patients and 10 symptomatic dermographism patients), 18 healthy control subjects and seven atopic donors without urticaria. Flow cytometry was then used to detect CD63 expression on the cell membrane of basophils from all samples. Analysis was also preformed on basophils incubated with sera from CSU patients with positive or negative autologous serum skin test (ASST). RESULTS: CD63 expression was significantly higher in the basophils from patients with CSU than in those from patients with symptomatic dermographism and the healthy control group. The levels of CD63 expression in CSU patients with ASST+ and/or allergen sensitization were higher than those with ASST- and/or no allergen sensitization patients. Incubation with ASST+ serum resulted in an increased expression of CD63 in the basophils of ASST+ CSU patients, whereas no such response was observed in healthy controls or ASST- CSU patients. CONCLUSION: The increased CD63 expression in basophils from CSU patients may correlate with allergen sensitization, autoreactivity of serum and basophil reactivity. Our results suggest that CD63 may contribute new insight into the pathogenesis of CSU.

Single-step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient.

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.

Expression and function of Angiopoietins and their tie receptors in human basophils and mast cells.

The Angiopoietin/Tie system is a key regulator of vascular remodeling, maturation, angiogenesis and lymphangiogenesis. In humans there are three angiopoietins: Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2), and Angiopoietin-4 (Ang4). Ang1 and Ang2 are the best characterized angiopoietins. The angiopoietin receptor system consists of two type I tyrosine kinase receptors (Tie1 and Tie2). Tie2 binds all known angiopoietins. We sought to characterize Ang1, Ang2, Tie1 and Tie2 expression and functions in human basophils and mast cells. Basophils, LAD-2 cells and Human Lung Mast Cells (HLMCs) constitutively express Ang1 and Ang2 mRNA. Intracellular staining for Ang1 and Ang2 was stronger in basophils than in mast cells. Immunoelectron microscopy demonstrated Ang1 in cytoplasmic vesicles of basophils. The protein kinase C activators phorbol diester (PMA) and bryostatin 1 (Bryo1) stimulated basophils to rapidly release a large amount of Ang1. PMA-induced Ang1 release was inhibited by brefeldin A. Tie1 and Tie2 mRNAs were expressed in basophils, LAD-2 and HLMCs. Basophils, LAD-2 and HLMCs expressed Tie1 on the cell surface. HLMCs and LAD-2 expressed Tie2 on the cell surface, whereas basophils did not. Ang1, but not Ang2, induced migration of mast cells through the engagement of Tie2. Neither Ang1 nor Ang2 induced basophil chemotaxis. We have identified a novel mechanism of cross-talk between human basophils and mast cells mediated by the Ang1/Tie2 system that might be relevant in the orchestration of inflammatory and neoplastic angiogenesis.

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two-photon fluorescence microscopy.

Two-photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two-photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two-photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto-second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 microW 800 nm fs have a relatively poor resolution, whereas the 50 microW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.

Quantity and Quality of Basophil RNA Depend on the RNA Extraction Technique.

Basophils have been suggested to express low quantities of RNA, challenging the study of gene expression within these cells. However, the purification technique employed might have an impact on the quantity and quality of RNA purified from basophils. This chapter describes a method which gives an optimal RNA output using a TRIzol-based method in contrast to a commercial kit.

Detection of Mast Cells and Basophils by Immunohistochemistry.

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

Identification of Murine Basophils by Flow Cytometry and Histology.

Here, we describe how murine basophils can be detected in vivo by flow cytometry and immunofluorescence staining. Basophils constitute a homogeneous population of CD4-CD19-CD49b+IgE+ cells in flow cytometric analysis. When IgE levels are low, one can also use anti-FcεRI or anti-CD200R3 antibodies instead of anti-IgE. For immunofluorescence staining, we use an anti-Mcpt8 antibody since Mcpt8 is a specific marker for murine basophils. We describe how to prepare the tissue to cut cryo-sections and how to perform the staining using a tyramide-based amplification kit.

Measuring Histamine and Cytokine Release from Basophils and Mast Cells.

Basophils and mast cells are known for their capability to release both preformed and newly synthesized inflammatory mediators. In this chapter, we describe how to stimulate and detect histamine released from basophils in whole blood, purified basophils, in vitro cultured mast cells, and in situ skin mast cells (the latter by microdialysis), using either a solid phase assay or flow cytometry. We also give an example of an activation protocol for basophil and mast cell cytokine release and discuss approaches for cytokine detection.

Gene Silencing Approaches in Mast Cells and Primary Human Basophils.

The ability to silence gene expression is an invaluable tool for elucidating the importance of intracellular signaling proteins which contribute to the effector functions of mast cells and basophils. However, primary mast cells and their terminally differentiated blood counterpart, basophils, pose a difficult challenge for gene silencing approaches given not only their state of maturation and difficulty to transfect but also because their functions are readily altered by cell handling conditions. Here, we describe a method using lipofection which has been successfully employed to silence gene expression using siRNA in human LAD2 mast cells as well as primary human basophils.

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy.

[Determination of a new cow's milk-based formula allergenic capacity in 7 infants]. / Determinación de la capacidad alergénica de una nueva fórmula infantil a base de leche de vaca en 7 lactantes.

BACKGROUND: Cow's milk protein allergy is the main allergic problem during the first year of life, possibly owing to immune and gastrointestinal systems poor maturation. To prevent allergic reactions, the content and type of proteins in infant formulas resemble those of breast milk. We believe that reactions are due rather to the amount than to the type of protein. OBJECTIVE: To design a new formula with cow's milk that provides the infant with the main nutrients at an affordable cost and with lower risk for the development of allergies. METHODS: Three-phase project: product design, industrial production and ex vivo assay to assess for anemia and type I allergic reaction by CD63 expression in basophils. RESULTS: For every 100 calories, the content of protein was 2.0 g, carbohydrates 7.2 g and fat 0.5 g, which is higher than the indicated maximum value (4.5 g). Microbiologically, it was an innocuous food. CD63 expression was low in 57.1% of the babies and high in 42.9%. CONCLUSION: The new formula did not trigger any allergenic responses and can therefore be supplied to non-atopic infants.

Antecedentes: La alergia a las proteínas de la leche de vaca es el principal problema alérgico durante el primer año de vida, debido a la poca maduración de los sistemas inmunológico y gastrointestinal. Para evitar reacciones alérgicas, el contenido y tipo de proteínas de las fórmulas infantiles se asemejan a los de la leche materna. Conforme un principio de alergología, probablemente las reacciones se deben a la cantidad de proteínas más que al tipo. Objetivo: Diseñar una nueva fórmula con leche de vaca que aporte al lactante los principales nutrientes, a un bajo costo y con menor riesgo de padecer alergias. Métodos: Proyecto realizado en 3 fases: diseño del producto, producción industrial y ensayo ex vivo para evaluar mediante la expresión del CD63 en basófilos la presencia de anemia y reacción alérgica tipo I en lactantes. Resultados: Por cada 100 calorías, el contenido de proteínas fue de 2 g, de carbohidratos de 7.2 g y de grasa de 0.5 g, mayor al valor máximo indicado (4.5 g). Microbiológicamente, la fórmula láctea propuesta se trató de un alimento inocuo. La expresión del CD63 fue baja en 57.1 % de los lactantes y alta en 42.9 %. Conclusión: La nueva fórmula no desencadenó respuesta alergénica, por lo tanto, puede suministrarse a lactantes no atópicos.

Morphologic and Cytochemical Characteristics of the Blood Cells of the Yellow-Bellied Slider (Trachemys scripta scripta).

The increasing prevalence of yellow-bellied sliders (Trachemys scripta scripta) as pets in the European Union and also its utilization as animal models for experimental purposes makes crucial an accurate classification of their blood cells. The aim of this work was to provide a morphologic classification based on the cytochemical characteristics of the blood cells of 15 yellow-bellied sliders. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff and toluidine blue. Nuclear and cellular dimensions were also measured based on quick Romanowsky-type stained smears. Besides erythrocytes and thrombocytes, five types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. The cytochemical patterns of heterophils, eosinophils and basophils were unique compared to those described for other chelonians. This paper provides a useful guideline for clinical settings and further haematological studies of this species.

Identification of basogranulin (BB1) as a novel immunohistochemical marker of basophils in normal bone marrow and patients with myeloproliferative disorders.

In myeloproliferative disorders (MPDs), basophils typically increase in number in the bone marrow (BM) and blood. In chronic myeloid leukemia (CML), basophilia is a diagnostic and prognostic variable. However, no reliable approach for routine detection and enumeration of basophils in BM sections is available. We applied the antibasogranulin antibody BB1 on paraffin-embedded BM sections in 21 control samples (normal BM), 45 patients with CML, 9 with chronic idiopathic myelofibrosis, 11 with polycythemia vera, 19 with essential thrombocythemia, and 7 with indolent systemic mastocytosis. As assessed by immunostaining of serial BM sections, BB1+ cells coexpressed myeloperoxidase, histidine decarboxylase, and leukosialin but did not express B- or T-cell-restricted antigens. BB1+ BM cells were found to be highly elevated in patients with CML compared with normal BM or other MPDs, with maximum counts found in accelerated phase CML (median, 160 cells/mm(2)). In summary, BB1 (basogranulin) is a new immunohistochemical basophil marker that should allow quantification of basophils in CML at diagnosis and during therapy.

The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers.

AIMS: Compact tryptase-positive round cell infiltrates of the bone marrow (TROCI-BM) are very rare histopathological findings and may pose challenging problems with regard to the cell type involved (either mast cells or basophilic granulocytes) and the exact diagnosis. METHODS: A selected panel of immunohistochemical markers against mast cell and basophil related antigens, including CD25, CD34, CD117/Kit, and the 2D7 antigen (which is found only in basophilic granulocytes) on a total of 410 routinely processed bone marrow biopsy specimens (including 88 cases of systemic mastocytosis (SM), 20 cases of chronic myeloid leukaemia (CML), 92 cases of myeloid neoplasms other than CML, and 210 controls with normal/reactive bone marrows). RESULTS: In total, 17 cases with TROCI-BM could be identified: 11 SM (including two cases of well-differentiated SM and two mast cell leukaemias; MCL), 2 myelomastocytic leukaemia (MML), 2 CML with excess of basophils (secondary basophilic leukaemia (CMLba)), and 2 tryptase positive acute myeloid leukaemia (AML). Regarding the cell types involved, TROCI-BM cells were found to express CD117/Kit in all cases of SM and MCL. In MML and tryptase postitive AML, TROCI-BM cells were found to coexpress CD34 and Kit. The basophil specific antigen 2D7 was only detected in CD34/Kit negative TROCI-BM cells in two patients with CMLba. The activating point mutation D816V was detected in 8/11 patients with SM but not in any of the other haematological malignancies. CONCLUSIONS: In summary, a total of six rare myeloid neoplasms may present with a novel immunohistochemical phenomenon tentatively termed TROCI-BM.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7.

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif.

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.

Voltage-dependent ion channels on human basophils: do they exist?

The presence of voltage-dependent ion channels (particularly Ca2+ channels) on the surface of 'non excitable' cells such as human basophils is a matter of debate. Indeed, in basophils, Ca2+ entry or mobilization is not sufficient by itself to trigger secretion, although enhanced cytosolic Ca2+ concentration increases it. In order to address this question, we used a two-signal model and we report here experiments which suggest the presence of voltage-dependent structures directly or indirectly linked to membrane Ca2+ pathways. Indeed, it is known that, in the presence of PMA at threshold concentration (1st signal), elevation of cytosolic Ca2+ (2nd signal) induces histamine release. We observed that a depolarizing external solution (high K+) induced a Ca(2+)-dependent release of histamine from PMA-treated human basophils. High K+ alone did not induce histamine release. Although the voltage-sensitive component and the physiological relevance of this mechanism remain to be defined, these results suggest that this voltage-dependent Ca2+ influx in the human basophil could contribute to the up-regulation of histamine release.