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Cross-linking of IgE-receptor complexes by rigid bivalent antigens greater than 200 A in length triggers cellular degranulation.

We have examined the effect of cross-linking IgE-receptor complexes with variable receptor-receptor distances on the transmembrane signaling that leads to degranulation of rat basophilic leukemia cells. Linear polymers of the biotin-binding protein avidin were generated with bis biotin-1,12-diamidododecane, and a dinitrophenyl-biotin conjugate was bound at each end of the polymers to form a series of rigid bivalent haptens of well-defined length. The polymers were fractionated by size with nondenaturing PAGE, electro-eluted, and tested for their ability to stimulate degranulation of rat basophilic leukemia cells sensitized with anti-DNP IgE. We found that hexamers of avidin (of length greater than or equal to 240 A) were as effective in triggering degranulation as dimers (of length approximately 80 A), while the monomeric avidin antigen (of length approximately 40 A) elicited a poorer degranulation response from the cells. The mechanism by which aggregation of cell surface receptors can initiate signal transduction is discussed in light of these results.

Animals↗

The autologous serum skin test in a cohort of chronic idiopathic urticaria patients compared to respiratory allergy patients and healthy individuals.

BACKGROUND: A positive autologous serum skin test (ASST) is considered to reflect the presence of anti-FceRI and/or anti-IgE autoantibodies that are capable of activating mast and basophil cell degranulation. The ASST is regarded as a reliable in vivo test in chronic idiopathic urticaria (CIU) patients, with diagnostic, therapeutic and prognostic implications. However, positive ASST results have also occasionally been demonstrated in patients with other diseases and in healthy subjects. OBJECTIVE: To evaluate the specificity and sensitivity of the ASST in a cohort of CIU patients compared to a cohort of respiratory-allergic patients and a group of normal individuals. METHODS: ASST was performed in a cohort of 116 subjects, 47 subjects with seasonal allergic rhinitis, 32 respiratory-symptom-free CIU patients, and 37 healthy individuals. RESULTS: The results were compared statistically to those of the CIU patients. The intradermal injection of autologous serum induced a weal and flare reaction in 17/32 (53.1%) CIU patients; 14/47 (29.8%) patients with seasonal allergic rhinitis (P=0.06) and in 15/37 (40.5%) of the healthy controls (P=0.34). The sensitivity and specificity of the ASST in the CIU patients and the seasonal allergic rhinitis patients was 53 and 28%, respectively. When comparing the CIU patients with the healthy controls the sensitivity and specificity was 55 and 31%, respectively. The positive and negative predictive values of the ASST when comparing CIU patients with healthy controls were 53 and 59.5%, respectively. The positive and negative predictive values of the ASST in the CIU patients compared to seasonal allergic rhinitis patients were 53 and 70%, respectively. CONCLUSION: The relatively low sensitivity and specificity of the ASST in the CIU patients compared to the seasonal allergic rhinitis patients and healthy controls warrants a more critical interpretation of the ASST in chronic idiopathic urticaria.

Adolescent↗

Blocking antibodies to inhalant allergens and asthma.

The mechanisms of specific immunotherapy are not still established. Among the lot of immunological changes, induced by immunotherapy, the increase of specific IgG 1, and then of IgG 4 antibodies, during the first months is well demonstrated. The skin-tests, the histamine-release and the human basophil degranulation are significantly decreased after incubation of allergen with the serum of desensitized patients. This antigen neutralizing capacity (blocking antibodies) disappeared when IgG 4 were suppressed from the serum by the mean of activated columns linked either with allergen, or protein A or antihuman IgG4 antibodies. Conversely, the total amount of serum blocking activity was found using pure IgG 4. However, the role of IgG 4 antibodies remains a subject of controversy. Since a clear correlation has yet to be established with symptom scores.

Allergens↗

[Value of cellular immunity tests (lymphocyte transformation test and leukocyte migration test) in the detection of sensitivity to drugs].

We studied degranulation of human basophils (HBDT), lymphoblast transformation and leucocyte migration in 8 cases of patients who were sensitive to penicillin (type 1 hypersensitization of the Gell and Coombes classification). The HBDT was negative in 7 of the cases, whilst the lymphoblast transformation and leucocyte migration tests were positive in 6 cases and negative in 2. These latter tests are more efficient than HBDT in the detection of human sensitization to penicillin.

Cell Migration Inhibition↗

Sensitive flow cytometric method to test basophil activation influenced by homeopathic histamine dilutions.

OBJECTIVE: In an experimental setting, human basophil degranulation was triggered by anti-IgE to measure the effects from homeopathic solutions in an in-vitro cell system. A 3-color flow cytometric method with enhanced accuracy was established. As an example we looked at the influence of histamine on anti-IgE activation of basophils. METHODS: Basophils were identified in the flow cytometer by their physical properties in the forward and side scatter light depiction and by gating on CD2(-), CD14(-), CD16(-), CD19(-), HLA-DR(-) negative and CD123-positive cells. CD63 expression on the cell surface of the anti-IgE-activated basophils served as an activation marker. RESULTS: With this method we were able to study basophil function of the 0.6-3.9% basophils out of the mononuclear blood cell fraction and to document their activation status upon anti-IgE activation. Optimal activation occurs at 0.6 microg/ml final anti-IgE concentration; not less than 10,000 basophils have to be counted per batch to reduce the variation of the measurement. The fixation method was able to stabilize activation for two days. After investigation and reduction of the source of measurement variability, an unequivocally inhibited basophil activation was documented in a partly optimized system with homeopathic dilutions of histamine (10(-22)M, 10(-23)M, 10(-24)M, and 10(-25)M histamine). Dilutions greater than 10(-20)M histamine (Avogadro's number 6.02 x 10(23)) account for less than 1.36 molecules of histamine in the test sample, indicating a true homeopathic effect. CONCLUSIONS: This test system is adequate for studying the effects of highly diluted mediators on basophil activation by anti-IgE. The systematic application of this experimental arrangement is recommended to study the effects of homeopathic dilutions on basophils.

Antibodies, Anti-Idiotypic↗

Effects of malathion metabolites on degranulation of and mediator release by human and rat basophilic cells.

In the present study, the effects of malathion and malathion derivatives on histamine and beta-hexosaminidase release by RBL-1 cells, rat peritoneal mast cells (RPMC), and human peripheral blood basophils (HPBB) and cutaneous mast calls were examined. One hour of incubation of RBL-1 cells with all organophosphate compounds tested, except for malathion and malathion monoacid, led to an increase in histamine release. beta-Hexosaminidase, an enzyme released by basophilic cells and a biochemical marker of degranulation, was not released from RBL-1 cells after 1 h of exposure to organophosphate compounds. Within 4 h, all compounds tested increased the release of histamine and beta-hexosaminidase. Longer exposures led to a decrease in the concentration of the compound that was required to cause mediator release. Exposure of RPMC to organophosphate compounds, with the exception of malathion monoacid and malathion (30 min) or malathion monoacid (1 h), led to the release of histamine, but not beta-hexosaminidase. Incubation of HPBB with malaoxon (51.4 +/- 2.8% total histamine released), malathion diacid (25.7 +/- 2.9%), beta-malathion monoacid (31.4 +/- 2.8%), and isomalathion (57.1 +/- 17.1%) for 1 h led to the release of histamine. Only malaoxon and isomalathion caused beta-hexosaminidase release from HPBB after a 1-h incubation. Incubation of cutaneous mast cells with malaoxon and beta-monoacid for 4 h led to increased release of histamine and beta-hexosaminidase at levels comparable to compound 48/80. These data suggest that malathion metabolites can cause rapid release of histamine from basophilic cells from a variety of origins and species. With prolonged incubation, malathion itself caused the release of mast-cell mediators, suggesting that the cells may be capable of metabolizing malathion. These data also indicate a disparity between the release kinetics of two different mast-cell mediators contained in granules by organophosphates, and that there are different mechanisms of mediator release.

Animals↗

Histamine-releasing autoantibodies in chronic urticaria.

Circulating histamine-releasing factors have been identified in the serum and plasma of chronic-urticaria patients by in vivo skin testing and in vitro histamine release from heterologous mixed leukocytes. Quantitative mast cell studies of serum skin test biopsies and electron microscopy indicate that the serum factors release histamine by mast cell degranulation. Peripheral blood basophils and total cellular blood histamine are reduced in chronic-urticaria patients suggesting that the circulating serum factors cause sustained degranulation. Histamine-releasing activity has been identified by skin testing in ultrafiltered serum fractions less than 30 kDa and greater than 100 kDa. In vitro histamine-releasing activity was confined to ultrafiltered serum fractions greater than 100 kDa and was present in IgG purified from some chronic-urticaria sera by protein G affinity chromatography. The dose-response relationship and kinetics of histamine release in vitro were similar to those of anti-human IgE. 'Desensitisation' of basophils by prior incubation with anti-IgE in the absence of calcium and competitive inhibition studies with myeloma IgE serum indicated that histamine-releasing autoantibodies in chronic-urticaria sera and purified IgG have the properties of anti-IgE. Plasma exchange in 4 patients with active chronic urticaria refractory to antihistamine therapy showing in vivo and in vitro histamine-releasing activity was followed by temporary remission of disease activity in 2 of them. It is possible that chronic urticaria is an autoimmune disease.

Animals↗

Rabbit F(ab')2 antihuman IgE is a universal skin test reagent in the evaluation of skin mast cell degranulation in vivo.

Antihuman IgE is often used to study basophil- and mast cell-mediator release in vitro but is infrequently used in vivo. To evaluate in vivo skin reactivity to anti-IgE, an affinity-purified rabbit F(ab')2 fragment of antihuman IgE was injected intradermally in 22 nonallergic and 27 allergic subjects. All 49 subjects (including a subject with less than 1 ng/ml of total serum IgE) had positive immediate cutaneous reactions to anti-IgE. Although total serum IgE level was weakly correlated (r = -0.51; p less than 0.005) with in vivo skin reactivity to anti-IgE for the entire population, allergic subjects did not have significantly increased skin reactivity compared to nonallergic subjects (p = 0.18), despite having higher total serum IgE levels (p less than 0.002). A late-phase cutaneous response (LPR) to anti-IgE occurred in 60% of the allergic and in 50% of the nonallergic subjects. Subjects with an LPR required approximately tenfold higher concentrations of anti-IgE to produce an immediate wheal of 10 mm compared to subjects who did not develop an LPR (p = 0.02), suggesting that the concentration of the stimulus injected is more important for the development of a LPR than the size of the immediate cutaneous response. Skin reactivity to codeine phosphate (a non-IgE-dependent secretagogue) was correlated with skin reactivity to anti-IgE (r = 0.47; p less than 0.05), suggesting that in vivo skin mast cell degranulation is partially a function of mast cell releasability.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

[Methods for the detection of drug allergies].

The following in vitro methods are predominantly discussed: Specific IgE determination (RAST). The test can be used to detect allergies of the immediate type such as anaphylactic shock and urticaria. Antibodies can be detected to a limited number of drugs such as penicillin, ACTH, TSH, insulin, asparaginase and proteins of animal sources. Degranulation of basophil leucocytes and histaminliberation have been used for many years. The practical value of the test has been limited but improved methods for analysis have given the tests hopes for a come-back. Cellular tests like lymphocyteproliferation and macrophage inhibition test (MIF) do not yet give such information which make them helpful as practical tests to detect drug allergy.

Basophils↗

Cellular allergen stimulation test (CAST) 2003, a review.

Specific diagnosis of immediate type allergies, such as rhinoconjunctivis, asthma, urticaria/angioedema and anaphylaxis, particularly when IgE-mediated, traditionally rests on prick and/or intradermal skin tests and, since about 30 years, on the determination of allergen specific IgEs. Some cellular tests, i.e. tests determining the reactivity of blood cells in vitro, particularly basophils, to allergens, have been available for many years. The determination of histamine release has been widely used in allergy pathophysiological research but its routine application in allergy diagnosis has been restricted to few groups. Basophil degranulation, as determined by microscopic examination, was promoted by some groups in the 1980's but has been largely abandoned since around 10 years ago; an alternative cellular test, based on the determination of sulfidoleukotrienes (LTC4, LTD4, LTE4) produced by IL-3 primed basophils stimulated by allergens in vitro, has been proposed. This test became available commercially in 1993 under the name of CAST (Bühlmann Laboratories, Allschwil, Switzerland). The CAST assay has been used in allergy diagnosis in a variety of indications, such as inhalation allergies, allergies to insect venoms, foods, occupational allergens and various drugs. A large number of reports on CAST diagnostic value, however, have been anecdotal. A meta-analysis of validated and well controlled studies encompasses 37 studies, 1614 patients and 1145 controls. This should definitely establish the value of this diagnostic test, particularly in instances where other in vitro or in vivo diagnostic tests are not reliable, such as food or drug allergies, as well as in non-IgE-mediated immediate hypersensitivity reactions. However, a number of questions about the CAST diagnostic assay are still open or have not been systematically explored. This may explain, in addition to the practical limitations inherent to all allergy cellular tests, why CAST has not yet become a very widely used assay worldwide, having gained broad acceptance in some countries but not in others.

Allergens↗

Flow cytochemical analysis of atopic reactions.

The technicon H-6000 has the unique ability among automated hematology systems to discriminate basophils from other hematopoietic cells on the basis of astra blue positivity of heparin-containing granules. We therefore examined the ability of this flow cytochemistry system to predict allergic diathesis in vitro by examining the number of basophils detected before and after incubation with a variety of allergens. One hundred subjects with documented atopic reactions to known allergens were examined and compared to 13 patients who had undergone successful desensitization and 12 normal subjects who had no history of atopy. One subject who had a previous anaphylactic response to penicillin but was skin-test negative to the major antigenic determinant demonstrated 55% degranulation after incubation with penicillin G. Finally, the basophil degranulation as assessed by the Technicon H-6000 was compared to a histamine release assay to determine the sensitivity of the automated system. The results suggest that flow cytochemistry may prove a valuable tool in predicting the success of desensitization therapy for common allergens and may have the potential to screen atopic reaction in the routine hematology laboratory.

Allergens↗

Effect of horse gram lectin (Dolichos biflorus agglutinin) on degranulation of mast cells and basophils of atopic subjects: identification as an allergen.

Horse gram (Dolichos biflorus) is widely consumed in the tropical south Asian countries including rural areas of India. Since D. biflorus agglutinin (DBA) is an important dietary lectin in horse gram, we have studied its effect on the degranulation of mast cells and basophils of atopic subjects. Allergy to horse gram lectin has not been reported so far. Skin prick test (SPT) was performed with 100 microg/mL of DBA. DBA-specific IgE was detected by dot-blot, and ELISA. Histamine release (HR) assay was carried out using leukocytes from non-atopic and atopic subjects, and rat peritoneal exudate cells. Among the atopic group, 10 of 48 subjects (21%) were found to be positive for DBA by SPT, and none were positive in the non-atopic group (n=20). Two subjects out of the ten who tested positive for DBA by SPT were found to be sensitized to DBA as revealed by the presence of specific IgE by ELISA and dot-blot. The HR was found to be 2- to 3-fold higher in DBA-allergic subjects than in non-atopic and atopic subjects. Basophil HR by DBA was found to be similar in both non-atopic and atopic subjects. However, DBA induces activation of mast cells in vivo in a sub-population (21%) of atopic subjects. Two subjects have been identified as having food allergy to horse gram based on the presence of DBA-specific IgE with a positive correlation to basophil HR. This is the first report of food allergy to horse gram, and DBA has been identified as an allergen.

Adolescent↗

Cutaneous basophil hypersensitivity in atopic dermatitis.

The infiltration by basophils into delayed hypersensitivity skin test sites was examined in patients with atopic dermatitis, contact dermatitis and in normal healthy persons. Atopic dermatitis patients, with large amounts of IgE, injected intradermally with staphylococcal antigens showed reactions that were more transient and erythematous than those of normal persons. On histological examination there were numerous, degranulating basophils among the perivascular mononuclear cells. Normal persons, with small amounts of IgE showed typical mononuclear cell infiltration and few basophils. Patients with contact dermatitis, one with much IgE, responded to patch tests to potassium dichromate or to nickel sulphate by delayed type reactions, but on histology, two of the four patients showed a significant infiltration by basophils. It is considered that the erythematous response to the antigen in atopic dermatitis patients is related to the basophil infiltration.

Adult↗

Evidence of granule discharge in the bone marrow reserve heterophils after acute anaphylactic shock in the guinea pig.

The heterophils in circulating blood and bone marrow were investigated in sensitized guinea pigs after acute anaphylactic shock (AAS) induced by inhalatory antigen challenge. Using histochemical, ultrastructural and in vitro methods evidence was obtained of a granule discharge from bone marrow reserve heterophils (BMRH). The mean number of primary (Biebrich scarlet positive) granules in BMRH decreased from 91 +/- 23 in the control to 74 +/- 23 after AAS. After AAS about 40% of BMRH and numerous circulating heterophils exhibited in the cytoplasm lucent areas which were surrounded by aggregates of granules. Only about 33% of BMRH were not affected by AAS. The rest of the cells showed different stages of granule aggregation and the lucent areas in the cytoplasm. Bone marrow basophils exhibited an early stage of degranulation. Almost 5% of BMRH ingested basophilic granules after AAS. Neither mortality rates in cytotoxicity tests nor antigen binding capacity were different in the experimental and control groups. It is suggested that the heterophils have a regulatory function during the anaphylactic reaction.

Anaphylaxis↗

Autoimmunity and complement in the pathogenesis of chronic urticaria.

Chronic urticaria is a debilitating skin disease that is believed to have an underlying autoimmune etiology in 35% to 50% of cases. Patients with autoimmune urticaria have functional antibodies in their sera that release histamine from basophils and mast cells. The C5a component of complement is required for mast cell degranulation in this process and at least augments basophil histamine release. In this article, the evidence that is key to our understanding of autoimmunity and complement in the pathogenesis of a subset of patients with chronic urticaria is outlined. Some of the issues in testing for and treating autoimmune urticaria are discussed.

Autoimmunity↗

Macrophage inflammatory protein-1alpha and RANTES are present in nasal secretions during ongoing upper respiratory tract infection.

Macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were measured by enzyme-linked immunosorbent assay (ELISA) from virus-infected respiratory cell culture supernatants and from 100 nasal wash samples obtained from patients aged 8 d to 10 yr. The results of the nasal wash samples were analyzed in relation to the etiology of the viral infection. In vitro, respiratory syncytial virus (RSV) induced the production of MIP-1alpha, while both RSV and adenovirus were associated with the production of RANTES. Both MIP-1alpha and RANTES were detected in nasopharyngeal secretions from pediatric patients with acute upper respiratory tract RSV, adenovirus, influenza, and parainfluenza virus infection (p<0.001 by Fisher's exact test). As both of these chemokines have potent effects on the recruitment and degranulation of eosinophils and basophils, further understanding of their role in upper respiratory tract infections may provide valuable insights into the immunopathogenesis of respiratory viral infections.

Adenoviridae↗