MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells.

BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.

Shellfish allergens: tropomyosin and beyond.

IgE-mediated shellfish allergy constitutes an important cause of food-related adverse reactions. Shellfish are classified into mollusks and crustaceans, the latter belonging to the class of arthropoda. Among crustaceans, shrimps are the most predominant cause of allergic reactions and thus more extensively studied. Several major and minor allergens have been identified and cloned. Among them, invertebrate tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, and hemocyanin are the most relevant. This review summarizes our current knowledge about these allergens.

Cannabis sativa allergy: looking through the fog.

IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross-allergies, mostly for plant foods. This clinical entity, designated as the 'cannabis-fruit/vegetable syndrome', might also imply cross-reactivity with tobacco, natural latex and plant-food-derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between nonspecific lipid transfer proteins or thaumatin-like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63-/CD203c-based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.

New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?

BACKGROUND: Allergy to fruit and vegetables exhibit geographic variation regarding the severity of symptoms and depending on the sensitization profile of the patient. These sensitization profiles and routes remain incompletely understood. Cannabis is a very popular drug and derived from Cannabis sativa, a plant containing lipid transfer proteins (LTP) also known as important allergens in plant and fruit allergies. In this study we sought to elucidate a potential connection between C. sativa allergy and plant food allergies. METHODS: A case-control study involving 21 patients consulting for plant food allergies. Twelve patients were cannabis allergic and 9 had a pollen or latex allergy without cannabis allergy. Testing for cannabis IgE implied measurement of specific IgE, skin testing and basophil activation tests. Allergen component analysis was performed with a microarray technique. RESULTS: Plant food allergy in patients with documented cannabis allergy had more severe reactions than patients without cannabis allergy and frequently implied fruits and vegetables that are not observed in a (birch) pollen-related food syndrome. With the exception of 1 patient with cannabis allergy, all were sensitized to nonspecific (ns)-LTP. CONCLUSION: Our data suggest that illicit cannabis abuse can result in cannabis allergy with sensitization to ns-LTP. This sensitization might result in various plant-food allergies. Additional collaborative studies in different geographical areas are needed to further elucidate on this hypothesis.

Age-dependent sensitization to the 7S-vicilin-like protein Cor a 11 from hazelnut (Corylus avellana) in a birch-endemic region.

BACKGROUND: Hazelnut (Corylus avellana) allergy exhibits age and geographically distinct sensitization patterns that have not yet been fully resolved. OBJECTIVE: To study sensitization to Cor a 11 in different age groups of hazelnut-allergic patients and infants with atopic dermatitis (AD) sensitized to hazelnut in a birch-endemic region. METHODS: Sera from 80 hazelnut-allergic patients, 33 infants under 1 year of age with AD (24 sensitized and 9 not sensitized to hazelnut), 32 healthy control individuals, and 29 birch pollen-allergic but hazelnut-tolerant individuals were tested for immunoglobulin (Ig) E reactivity to Cor a 11 by ImmunoCAP. IgE reactivity to Cor a 1.01, Cor a 1.04, Cor a 8, and Cor a 9 was studied by ISAC microarray. RESULTS: Forty patients (22 preschool children, 10 schoolchildren, and 8 adults) with systemic reactions on consumption of hazelnut were sensitized to Cor a 11 (respective rates of 36%, 40%, and 12.5%). Forty patients (6 preschool children, 10 schoolchildren, and 24 adults) reported oral allergy syndrome but only 2 of them (of preschool age) were sensitized to Cor a 11. Two (8%) of the AD infants sensitized to hazelnut showed IgE reactivity to Cor a 11. This reactivity was not observed in any of the AD infants without sensitization to hazelnut, in any of the birch-pollen allergic patients without hazelnut allergy, or in any of the healthy control individuals. CONCLUSION: Sensitization to Cor a 11 in a birch-endemic region is predominantly found in children with severe hazelnut allergy, a finding that is consistent with observations concerning sensitization to Cor a 9.

Age-related sensitization profiles for hazelnut (Corylus avellana) in a birch-endemic region.

BACKGROUND: Symptoms of hazelnut allergy seem related to geographic and possibly age variations in allergen recognition. OBJECTIVE: To investigate sensitization profiles of hazelnut allergy in different age groups in a birch-endemic region using component resolved diagnosis (CRD) by microarray. METHODS: Sixty-five patients with hazelnut allergy, 27 healthy control individuals tolerant to hazelnut, and 34 birch pollen allergic but hazelnut tolerant individuals were included. All blood samples were analyzed using ISAC microarray. RESULTS: Twenty-nine patients with hazelnut allergy suffered from a systemic reaction (17 preschool children with a median age of 2 years, six school children, and six adults), whereas 36 patients reported an oral allergy syndrome (OAS; three preschool and nine school children and 24 adults). In the hazelnut allergic preschool children with systemic reactions, 65% were sensitized to Cor a 9, 12% to Cor a 8, 18% to Cor a 1.04, 6% to Cor a 1.0101, and 29% to Bet v 1. Of the school-aged systemic reactors, 50% were sensitized to Cor a 9, 17% to Cor a 8, 50% to Cor a 1.04 and Cor a 1.0101, and 67% to Bet v 1. In adults with hazelnut allergy, 3.3% were sensitized to Cor a 9, 6.7% to Cor a 8, 90% to Cor a 1.04 and Bet v 1, and 87% to Cor a 1.0101. In regard to systemic reactors in this group, 17% were sensitized to Cor a 9, 33% to Cor a 8 and Cor a 1.0101, and 50% to Cor a 1.04 and Bet v 1. In the patients with OAS, irrespective the age group, all were sensitized to Bet v 1 and over 97% to Cor a 1.04 and Cor a 1.0101. No sensitization to Cor a 9 or Cor a 8 was found in patients with only an OAS. Of the patients with birch pollen allergy, tolerant to hazelnut, none were sensitized to Cor a 9 or Cor a 8, 56% to Cor a 1.0101, 82% to Cor a 1.04, and 92% to Bet v 1. In healthy controls, no sensitization to components of hazelnut, hazel pollen or birch pollen was demonstrable. CONCLUSION: Hazelnut allergy in a birch-endemic region exhibits age-related sensitization profiles with distinct clinical outcomes that can be identified using CRD. The majority of hazelnut allergic preschool and school children in a birch-endemic region show systemic reactions on consumption of processed hazelnut, mostly being sensitized to the hazelnut legumin-like allergen Cor a 9 but unrelated to birch pollen allergy. In contrast, adults generally suffer from an OAS apparently as a result of cross-reactivity between Cor a 1.04 from hazelnut and Bet v 1 from birch pollen.

Human basophils: a unique biological instrument to detect the allergenicity of food.

BACKGROUND: Labeling of major food allergens is mandatory for the safety of allergic consumers. Although enzyme-linked immunosorbent assay, polymerase chain reaction, and mass spectrometry are sensitive and specific instruments to detect trace amounts of food proteins, they cannot measure the ability of food constituents to trigger activation of mast cells or basophils. AIM: We evaluated the basophil activation test as an instrument to determine the allergenic potential of trace amounts of food allergens in complex matrices. Peanut (Arachis hypogaea) allergy was selected as a proof-of-concept model. METHODS: The study population comprised 5 severely peanut-allergic patients (3 males/2 females; median age, 12 years) all sensitized to 3 major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) and 5 peanut-tolerant individuals (2 males/3 females; median age, 8 years). Basophils from patients and controls were stimulated with pure peanut extract and blank and peanut-spiked (0.1, 0.01, and 0.001 ppm) biscuits (baking time 11, 16, 21, 26 minutes) and chocolate extracts. RESULTS: Blank biscuits and chocolate did not induce cell activation in patients or controls. A comparison between patients and controls showed significantly higher activation of basophils after stimulation with 0.1 and 0.01 ppm of peanut-spiked biscuit at all baking times and peanut-spiked chocolate (P < .05). CONCLUSIONS: The basophil activation test is a highly sensitive and specific tool to detect traces of functionally active food allergens. For biscuits, its accuracy seems independent of baking time. Furthermore, it allows even the most sensitive patients to be included in study protocols.

Basophil activation reveals divergent patient-specific responses to thermally processed peanuts.

INTRODUCTION: The impact of processing on the allergenicity of peanut (Arachis hypogaea) proteins has traditionally been studied using immunoglobulin (Ig) E binding assay. However, as this technique does not assess the potential of an allergen to trigger basophils and mast cells, studies based on it can hardly be considered complete. We evaluated the effect of processing on peanut allergenicity using flow-cytometric quantification of in vitro basophil activation (basophil activation test [BAT]). PATIENTS AND METHODS: Basophils from 10 patients with severe peanut allergy and 3 peanut-tolerant individuals were stimulated with extracts from 5 raw and thermally processed peanut varieties. Data were compared using protein staining (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and IgE immunoblotting. RESULTS: Stimulation with different extracts resulted in patient-dependent and variety-dependent effects on basophil activation. SDS-PAGE revealed a considerable loss of identifiable bands, especially for the South Africa Common Natal, Argentina Runner, and US Virginia varieties. The results of IgE immunoblotting in patients were similar, irrespective of the responses observed in the BAT. CONCLUSIONS: The impact of thermal processing on the capacity of peanuts to trigger basophils seems highly divergent between patients and cannot be predicted using SDS-PAGE or IgE binding. BAT can be considered a complementary tool for the evaluation of food allergenicity.

Component-resolved diagnosis from latex allergy by microarray.

BACKGROUND: A positive specific IgE (sIgE) result for latex does not always mirror the clinical situation and is frequently found in individuals without overt latex allergy. OBJECTIVE: We sought to investigate the potential of component-resolved diagnosis (CRD) of latex allergy by microarray and to assess whether the technique allows discriminating genuine allergy from asymptomatic sensitization. METHODS: Twenty-six healthy controls without a history of latex allergy with a negative latex sIgE and skin test, 22 latex-allergic patients with a compelling history of latex allergy with a positive latex sIgE and prick test and 20 latex-sensitized individuals with a frequent asymptomatic exposure to natural rubber latex-containing devices with a negative latex skin test but a positive sIgE were also included. CRD was performed with the ImmunoCAP ISAC microarray and traditional singleplexed ImmunoCAP. RESULTS: In all patients, the diagnosis of latex allergy could be established by the combination of recombinant latex components present on the microarray (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02). Over three-quarters of our patients were sensitized for Hev b 5 and/or Hev b 6.02. Some patients also displayed reactivity for Hev b 1 and/or Hev b 3. In contrast, none of the individuals sensitized to natural rubber latex or control individuals demonstrated IgE reactivity for rHev b 1, rHev b 3, rHev b 5 or rHev b 6.02. Three-quarters of the patients sensitized to latex displayed a positive microarray result for recombinant latex profilin (rHev b 8). In contrast to the results obtained by traditional ImmunoCAP for bromelain, almost no sensitization for cross-reactive carbohydrates was demonstrated by bromelain spotted on the microarray. CRD by traditional singleplexed ImmunoCAP showed highly comparable results. CONCLUSION: CRD by microarray is a reliable tool for diagnosing latex allergy. In addition, the technique allows discrimination between genuine allergy and sensitization. CRD by microarray can improve the diagnosis of IgE-mediated latex allergy by discriminating between genuine allergy and sensitization. CRD by microarray is a reliable tool to diagnose latex allergy. In addition, the technique allows discrimination between a genuine allergy and simple sensitization.

Sensitization profiles in birch pollen-allergic patients with and without oral allergy syndrome to apple: lessons from multiplexed component-resolved allergy diagnosis.

BACKGROUND: Component-resolved diagnosis (CRD) using microarray technology has recently been introduced into the field of clinical allergology. OBJECTIVE: To further validate the use of CRD by microarray technology in allergy diagnosis. METHODS: Thiry-seven patients allergic to birch pollen were included. The discriminative value of apple-specific IgE (sIgE), recombinant Mal d 1 (rMal d 1) sIgE, apple skin prick test and rMal d 1 on the microarray was assessed between patients with a birch-related oral allergy syndrome to apple (OAS(+), n=20) and healthy control individuals (HC, n=8) without a history of inhalant allergies or apple-induced OAS. An additional comparative analysis was carried out with individuals allergic to birch pollen allergy without OAS (OAS(-); n=17). RESULTS: rMal d 1 coupled to the microarray constitutes a discriminative marker between OAS(+) and HC with a sensitivity 95% and a specificity of 100%. However, in parallel with the traditional sIgE assay, 15 out of 17 OAS(-) individuals (88%) also displayed IgE reactivity to rMal d 1 coupled to the microarray. OAS(-) individuals are more frequently sensitized to mite (about three to four times), cat and dog dander (about two to three times) and grass pollen (about 1.5 times) as compared with OAS(+) patients. CONCLUSION: At first glance, CRD by microarray seems to be a reliable instrument in the diagnosis of apple-mediated OAS in birch pollen allergy. However, for discriminating between sensitization and a real allergy, micro-arrayed rMal d 1 offers no advantage over conventional quantification of rMal d 1 sIgE. Most interestingly, within a single run, birch pollen-allergic patients without OAS to apple were shown to display a broader sensitization to classical inhalant allergens than birch pollen-allergic patients with an apple-related OAS.

Pre- and post-natal exposure to antibiotics and the development of eczema, recurrent wheezing and atopic sensitization in children up to the age of 4 years.

BACKGROUND: Little data are available on the relationship between indirect antibiotic exposure of the child in utero or during lactation and allergic diseases. On the other hand, several studies have been conducted on the association with direct post-natal antibiotic exposure, but the results are conflicting. OBJECTIVE: The aim of this study was to investigate pre- and post-natal antibiotic exposure and the subsequent development of eczema, recurrent wheeze and atopic sensitization in children up to the age of 4 years. METHODS: We conducted an aetiologic study in 773 children based on a prospective birth cohort project in which environmental and health information were collected using questionnaires. Antibiotic exposure was assessed as maternal antibiotic intake during pregnancy and during lactation and as medication intake of the child. The chronology of exposures and outcomes was taken into account during the data processing. At the age of 1 and 4 years, a blood sample was taken for the quantification of specific IgE. RESULTS: Prenatal antibiotic exposure was significantly positively associated with eczema, whereas no association was found with recurrent wheeze and atopic sensitization. We found a positive, although statistically not significant, association between antibiotic exposure through breastfeeding and recurrent wheeze. Neither eczema nor atopic sensitization was significantly associated with antibiotic exposure through breastfeeding. Finally, we observed a negative association between the use of antibiotics in the first year of life and eczema and atopic sensitization, and also between antibiotic use after the first year of life and recurrent wheeze, eczema and atopic sensitization. CONCLUSION: Indirect exposure to antibiotics (in utero and during lactation) increases the risk for allergic symptoms in children, while direct exposure to antibiotics appears to be protective. The biological mechanisms underlying these findings still need to be elucidated.

Flow-assisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow-up of wasp venom immunotherapy.

BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established. METHODS: To evaluate diagnostic performances of the basophil activation test (BAT) in wasp venom allergy, 80 patients with a definite history of wasp venom anaphylaxis (systemic reactors) and 14 wasp-stung asymptomatic controls (stung controls) were enrolled. Venom-induced basophil activation was analyzed flow cytometrically by double-labeling with anti-IgE and anti-CD63. Results were compared to wasp IgE levels and results of a venom skin test (VST). To establish whether the BAT constitutes a candidate marker to monitor VIT, the BAT was repeated in 22 patients on the 5th day of a build-up course and after 6 months of maintenance VIT. Whether the BAT could contribute in the decision of discontinuing VIT was assessed in a cross-sectional analysis in 30 patients receiving treatment for 3 years. RESULTS: Comparison between systemic reactors and stung controls revealed a sensitivity of 86.4% and specificity of 100% for venom IgE, and sensitivity of 81.8% for VST, respectively. In contrast to stung controls, patients demonstrated dose-dependent venom-induced basophil activation. The BAT attained a sensitivity of 83.8% and specificity of 100%. At the end of the build-up course, no effect of VIT on the BAT was demonstrable. When the BAT was repeated after 6 months of treatment, submaximal stimulation of the cells demonstrated a significant decreased CD63 expression (P < 0.04). Patients having VIT for 3 years also demonstrated significantly lower venom-induced CD63 expression (P < 0.001). After 3 years, 60% of the patients had a negative BAT for submaximal stimulation of the cells whereas only 17.9% of the patients had negativation of wasp IgE. CONCLUSIONS: The BAT is a reliable instrument for the diagnosis of wasp venom anaphylaxis and might constitute an instrument to monitor wasp VIT.

Hymenoptera venom allergy: taking the sting out of difficult cases.

BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy. OBJECTIVE: To assess whether the basophil activation test (BAT) constitutes an additional diagnostic instrument in patients with equivocal or negative specific immunoglobulin (Ig) E or venom skin test (VST) results. METHODS: One hundred eighteen patients with a compelling history of IgE-mediated hymenoptera venom allergy were enrolled. Venom-specific IgE was quantified by ImmunoCAP and VST was performed in all patients. Basophil activation was analyzed by flow cytometry after labeling with anti-IgE and anti-CD63. RESULTS: In 64 out of 118 patients, diagnosis was considered as definite and the entomologic description was confirmed by unequivocal and concordant positive specific IgE and VST results. In 53 of those 64 patients, BAT confirmed diagnosis, whereas the remaining 11 patients were nonresponsive in the BAT analysis. Forty-seven patients (40%) had a tentative diagnosis of venom allergy, as they had divergent specific IgE or VST results. In 31 of those patients, BAT was positive only for the suspected venom and helped to establish diagnosis of wasp and honeybee venom allergy in 28 and 3 patients, respectively. BAT was diagnostic in 7 patients with complete negative results for specific IgE and VST, despite clear entomologic identification. CONCLUSIONS: In about half the patients with diagnosis of venom allergy, unequivocal specific IgE and VST results are obtained and additional tests are not needed. In the remainder, diagnosis is less straightforward due to discrepant or negative specific IgE orVST results. In these patients, BAT constitutes a helpful additional instrument to identify the culprit venom and start venom immunotherapy accordingly.

Phenotypic and functional characterization of in vitro cultured human mast cells.

BACKGROUND: Mast cell progenitor cells, derived from CD34+ hematopoietic stem cells, enter the circulation and subsequently mucosal or connective tissues where they mature to mast cells. Upon activation, mast cells increase the expression of activation markers, e.g. CD63, and release histamine amongst other mediators. Traditionally, release of these mediators is quantified using assays measuring their extracellular concentration in the supernatant of stimulated cells. METHODS: Human mast cells (HuMC) were cultured from peripheral blood, phenotypically characterized, passively sensitized with allogenic IgE antibodies and finally stimulated by anti-IgE that crosslinks IgE/FcεRI complexes. Alterations in the number of cells positive for CD63 and release of histamine were quantified simultaneously by flow cytometry. RESULTS: In culture, two distinct CD45+ cell populations were identified: CD117+ CD203c+hi and CD117- CD203c+low cells. Both populations showed positivity for FcεRI, tryptase and chymase, and contained histamine. Activation resulted in a significant increase of cells positive for CD63+ up to 21% (range: 11-39) for CD117+ CD203c+hi cells (P = 0.005), and 27% (18-55) CD63+ for CD117- CD203c+low cells (P = 0.02). Baseline histamine content was higher for CD117+ CD203c+hi cells than for CD117- CD203c+low cells, respectively 994 (695-6815) Molecules of Equivalent Specific Fluorochrome V500 per cell (MESF-V500/cell) and 797 (629-4978) MESF-V500/cell (P = 0.02). After activation, CD117+ CD203c+hi cells showed significant histamine release of 578 (366-1521) MESF-V500/cell, whilst CD117- CD203c+low cells resulted in 310 (217-366) MESF-V500/cell histamine release. CONCLUSION: This study discloses that culturing HuMC from CD34+ progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

In Vitro Diagnosis of Immediate Drug Hypersensitivity Anno 2017: Potentials and Limitations.

BACKGROUND: For most physicians, quantification of drug-specific immunoglobulin E (drug-sIgE) antibodies constitutes the primary in vitro measure to document immediate drug hypersensitivity reactions (IDHR). Unfortunately, this is often insufficient to correctly identify patients with IgE-mediated IDHR and impossible for non-IgE-mediated IDHR that result from alternative routes of basophil and mast cell activation. In these difficult cases, diagnosis might benefit from cellular tests such as basophil activation tests (BAT). AIM: The aim was to review the potential and limitations of quantification of sIgE and BAT in diagnosing IDHR. The utility of quantification of serum tryptase is discussed. METHODS: A literature search was conducted using the key words allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry, specific IgE antibodies; this was complemented by the authors' own experience. RESULTS: The drugs that have been most studied with both techniques are ß-lactam antibiotics and curarizing neuromuscular blocking agents (NMBA). For sIgE morphine, data are available on the value of this test as a biomarker for sensitization to substituted ammonium structures that constitute the major epitope of NMBA, especially rocuronium and suxamethonium. For the BAT, there are also data on non-steroidal anti-inflammatory drugs (NSAIDs) and iodinated radiocontrast media. For ß-lactam antibiotics, sensitivity and specificity of sIgE varies between 0 and 85% and 52 and 100%, respectively. For NMBA, sensitivity and specificity varies between 38.5 and 92% and 85.7 and 100%, respectively. Specific IgE to morphine should not be used in isolation to diagnose IDHR to NMBA nor opiates. For the BAT, sensitivity generally varies between 50 and 60%, whereas specificity attains 80%, except for quinolones and NSAIDs. CONCLUSIONS: Although drug-sIgE assays and BAT can provide useful information in the diagnosis of IDHR, their predictive value is not absolute. Large-scale collaborative studies are mandatory to harmonize and optimize test protocols and to establish drug-specific decision thresholds.

Influence of anti-tumor necrosis factor therapy (Adalimumab) on regulatory T cells and dendritic cells in rheumatoid arthritis.

OBJECTIVE: To investigate whether anti-TNF therapy could have an effect on dendritic cells (DCs) and regulatory T cells in rheumatoid arthritis (RA) patients. METHODS: A four-colour flow cytometric technique was used to measure CD4+CD25+ T cells i.e. CD4+CD25high+ (regulatory T cells) and CD4+CD25low+ (activated T cells)), DCs as well as the in vitro, intracellular, lipopolysaccharide-stimulated cytokine production of DCs. RESULTS: Clinical and laboratory parameters of disease activity decreased after anti-TNF treatment. Before anti-TNF therapy, RA patients demonstrated a decreased count of Th2-promoting lymphoid DCs as compared to controls and after anti-TNF therapy this decrease was sustained. Intracellular cytokine production was only found in the myeloid DCs population and there was a higher production of TNF-alpha and IL1-b as compared to healthy controls. Treatment did not alter this cytokine production. Before anti-TNF therapy, the percentage CD4+CD25+ T cells was significantly elevated in RA patients than in healthy controls. CONCLUSION: These results demonstrate anti-TNF to be a potent anti-inflammatory drug, as mirrored by the decrease in clinical and biological parameters as well as the decrease in activated CD4+ T cells. However, in this study no demonstrable effect on DCs and regulatory T cells was found.