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Resume But de l’etude Etudier, dans une population pediatrique presentant des signes cliniques et biologiques d’allergie alimentaire IgE dependante, l’expression differentielle de marqueurs d’activation par les basophiles, les neutrophiles et les eosinophiles. Patients et methodes Analyse de l’activation cellulaire par cytometrie en flux (CMF) pour les allergenes les plus frequents (arachide, lait, œuf, noisette et crevette). Cette activation a ete analysee avec plusieurs marqueurs : le CD63 et le CD11b pour les basophiles et le CD11b pour les eosinophiles et les neutrophiles. Resultats Au total 55/79 enfants avaient une CMF positive pour au moins un allergene. L’expression du marqueur d’activation CD11b impliquait plus frequemment l’ensemble des polynucleaires (p = 0,0036). L’expression du marqueur CD11b etait plus frequemment observee dans le groupe eczema que dans le groupe urticaire (p = 0,01). Pour un seuil de positivite de 12 % CD63, il existait un lien significatif (p Conclusion Le CD11b est une integrine non encore etudiee en tant que marqueur d’activation allergene dependante pour les polynucleaires. Notre etude a montre que l’expression du marqueur CD11b par les eosinophiles et les neutrophiles peut-etre le resultat d’une activation par l’allergene et IgE dependante confirmee par la surexpression du marqueur CD11b. La relation inverse entre l’expression du CD63 et du CD11b dans l’urticaire et la dermatite atopique permet de faire l’hypothese d’une eventuelle migration tissulaire des leucocytes et en particulier des basophiles.

Introduction La recherche d’une hypersensibilite aux PCI est une demarche frequente, souvent negative, parfois decevante. Methodes Il s’agit d’une etude retrospective sur 4 ans (2014–2017) avec 138 patients (dont un adolescent), par laquelle nous voulons souligner les difficultes de ce type de bilan. Pour tous les patients, des tests cutanees (IDR) et biologiques (IgE specifiques, test d’activation des basophiles, test d’activation des lymphocytes, tryptase serique) ont ete realises avec 3 a 8 produits en fonction de la clinique. Le but etait de guider le choix du radiologue. Tous les dossiers ont ete discutes en reunion pluridisciplinaire (allergologue, immunologue, pharmacologue). Chaque patient a recu ensuite un certificat mentionnant le bilan et les conseils. Resultats Dans 6 cas, le bilan est predictif, toujours negatif. Les reactions rapportees sont toujours benignes, majoritairement cutanees. L’accident date souvent de plusieurs annees, les souvenirs imprecis, avec un manque d’information d’ordre medical (lettre, compte rendu). Pour 40 patients seulement le produit suspect est connu. Les IDR (intradermoreaction) sont negatives chez 102 personnes et positives en lecture immediate chez 23 patients, mais lorsque le produit est connu, elles sont positives pour 7 patients sur 37. En cas de reaction retardee (16 patients), les IDR sont 7 fois positives avec de frequentes reactions croisees si toxidermie. La biologie aide au diagnostic pour 4 patients, en cas de reaction retardee. La tryptase serique est elevee 3 fois avec mastocytose confirmee pour un cas. Conclusion Le diagnostic d’hypersensibilite allergique aux PCI est difficile meme si l’allergologue a maintenant plus d’informations sur la reaction et le produit en cause. Dans cette serie, la biologie a ete utile surtout en cas de reaction retardee.

Resume But de l’etude Nous avons compare l’efficacite du test d’activation des basophiles par cytometrie en flux (TAB) au test de provocation oral (TPO) en simple insu. Patients et methodes Quatre-vingt-sept tests de provocation ont ete realises chez 72 patients âges de huit mois a 15 ans. Les aliments testes sont principalement l’arachide, l’œuf, le poisson et le lait de vache. Le TAB a ete pratique selon le protocole IgE/CD63 et les resultats exprimes en pourcentage CD63 et en index d’activation (IA) calcule par un algorithme dedie. Resultats Les resultats obtenus pour les TPO negatifs nous ont permis de definir des seuils de positivite clinique du TAB (30 % CD63 et 100 pour l’IA), les seuils de positivite statistiques etant respectivement, de 5 % et de 16. Dans ces conditions, les valeurs predictives positives et negatives obtenues (VPP et VPN) sont respectivement, pour le pourcentage CD63 0,95 et 0,77 et pour l’IA 0,82 et 0,96. A partir de ces resultats et des resultats des tests cutanes et des IgE specifiques obtenus parallelement, nous avons etabli un arbre decisionnel qui permettrait de reduire de deux tiers le nombre de TPO necessaires. Conclusions Parmi les differents tests pratiques dans cette etude, la cytometrie en flux presente la meilleure efficacite, en reference aux tests de provocation pratiques en simple insu.

Resume But de l’etude Nous evaluons ici une technique utilisant deux marqueurs d’activation (CD63 et IgE) et une expression des resultats en index d’activation (IA) calcule par un algorithme dedie. Materiel et methodes Vingt-six patients allergiques et 23 temoins ayant recu des curares ont ete selectionnes (histoire clinique, tests cutanes et/ou IgE specifiques). Les curares ont ete testes soit en melange (n = 26), soit separement en utilisant, soit les allergenes natifs (n = 30), soit les allergenes commercialises pour cytometrie (n = 9). Les concentrations utilisees sont celles recommandees par la SFAR pour les IDR. Le protocole cytometrique utilise est le protocole IgE/CD63, les resultats etant exprimes en pourcentage de CD63 et en index d’activation (IA), calcule par un algorithme specifique incluant les moyennes d’intensite de fluorescence (MIF) des populations IgE++ et CD63+ et −. Resultats Les curares testes ne montrent pas de toxicite pour les basophiles, y compris a des concentrations dix fois superieures aux concentrations diagnostiques. Avec les allergenes natifs les sensibilites respectives pour le marqueur CD63 seul ou pour l’IA sont de 68 et 100 % pour les melanges, 67 et 100 % pour les curares specifiques. Avec les allergenes commerciaux, la sensibilite pour les curares specifiques est de 47 et 100 %. La specificite est de 100 % pour le CD63 seul et de 98 % pour l’IA. La liaison avec les tests cutanes (Khi2 calcule pour la population des allergiques) est significative pour les deux marqueurs et pour les deux types d’allergenes. Enfin, la correlation index/IgE specifique n’est pas significative. Conclusion Ce nouveau protocole cytometrique permet d’envisager une utilisation plus large de cette methode en allergoanesthesie.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, β-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

INTRODUCTION: We report two cases occurring in 2004 of patients being treated for pleural mesothelioma with a combination of cisplatin or carboplatin and pemetrexed. Investigation by skin tests and flow cytometry confirmed the clinical diagnosis in both cases. CASE REPORTS: Case 1: a man of 62 developed, after 12 courses of cisplatin-pemetrexed, an anaphylactic reaction 5 minutes after the infusion of cisplatin. Treatment was withdrawn permanently. Case 2: a man of 66 developed, after 7 courses of cisplatin-pemetrexed, an anaphylactic reaction within the first minute of the infusion of cisplatin. Subsequently, in March 2004, he received pemetrexed alone without any problems. In August 2004 he was prescribed carboplatin-pemetrexed. Within 5 minutes he developed urticaria, pruritus and abdominal pain. He was treated later with pemetrexed alone with no problems. CONCLUSION: Hypersensitivity to platinum salts usually occurs after several courses of treatment. Skin tests and flow cytometry are a simple, concordant, and reliable way of confirming the diagnosis.

Physicians predominantly rely upon quantification of serum-specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE-mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross-reactive structures is not always straightforward. Flow-assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE-mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non-IgE-mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow-assisted analysis of in vitro-activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow-assisted analysis of in vitro-activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.

Resume L'allergie alimentaire au porc est rare. Cependant, nous en rapportons 13 cas, concernant surtout l'allergie aux rognons. Les reactions peuvent etre severes. Les sensibilisations croisees sont nombreuses avec d'autres mammiferes. Une meilleure connaissance des allergenes en cause est necessaire pour mieux identifier les risques chez ces patients.

Background: Drug allergy is an increasingly important clinical problem. About 5-10% of patients consulting allergy centers have some form of drug allergy or pseudo allergy. At present there is no consensus concerning the best way to study and diagnose hypersensitivity reactions to drugs. This leads both to practical difficulties in diagnosing individual patients and to more general problems in comparing protocols and results from different centers. Methods/Data base: The European Network for Drug Allergy (ENDA) Drug Ambassador Project aimed to collect information on the ways that different European centers approach these patients. Results: The present report summarizes data collected during this project, focussing on the organization of drug allergy consultations, diagnostic protocols and the various in vitro and in vivo investigation procedures in current use. The study revealed wide variation in the diagnostic procedures used for drug allergy in clinical practice, despite the apparent consensus achieved in recommendations and position papers. Conclusion: Reciprocal exchange of information and discussions based on these observations may help to achieve standardization and harmonization in this sector with significant potential benefits for patients with drug allergies.

Background Allergen-specific serum immunoglobulin E detection and quantification have become an important step in allergy diagnosis and follow-up. In line with the current trend of laboratory test accreditation to international standards, we set out to design and assess an accreditation procedure for allergen-specific serum IgE. Methods Method validation according to the accreditation procedure under the EN ISO 15189 standard was carried out for allergen-specific immunoglobulin E determination using the fluoroimmunoenzymatic method ImmunoCAP® (ThermoFisher). Data were produced by 25 hospital laboratories in France. A total of 29 allergen specificities including mixes, extracts, and molecular allergens were assayed. Allergen-specific serum immunoglobulin E concentrations ranged from 0.1 to 100 kUA/l. Results Repeatability, reproducibility, and accuracy results fulfilled method validation criteria for automated laboratory tests and proved similar irrespective of the allergen specificity, allergen-specific serum immunoglobulin E concentration, or individual laboratory. Conclusion Allergen-specific serum immunoglobulin E determination with the fluoroimmunoenzymatic method ImmunoCAP® is a highly repeatable, reproducible, and accurate method which may be considered as a single analyte assay in view of the EN ISO 15189 accreditation procedure.

Introduction La detection et la quantification des immunoglobulines (Ig) E specifiques d’allergene est une etape importante du diagnostic et du suivi en allergologie. Elle est soumise actuellement, au meme titre que l’ensemble des examens de biologie medicale, a l’obligation d’accreditation selon la norme internationale EN ISO 15189. Le reseau des biologistes de l’allergie AllergoBioNet propose une procedure d’accreditation du dosage des IgE specifiques d’allergene fondee sur le principe de l’analyte unique utilisant une courbe de calibration commune. Cette strategie genere un surcout raisonnable compte tenu de la diversite des allergenes. Methodes La validation de methode selon la norme EN ISO 15189 a ete realisee pour la determination des IgE specifiques d’allergene avec la methode immunofluoroenzymatique ImmunoCAP® (Thermo Fisher Scientific, Phadia). Resultats Les donnees ont ete produites par 25 laboratoires de CH et CHU francais. Au total, les IgE specifiques de 29 allergenes incluant des melanges, des extraits et des composants moleculaires ont ete analysees pour des concentrations comprises entre 0,1 et 100 kUA/L. Discussion Les performances analytiques (repetabilite, reproductibilite et justesse) du dosage des IgE specifiques respectent les criteres de validation de methode attendus pour les examens de laboratoire automatises et sont comparables quelles que soient la specificite allergenique testee, la concentration serique des IgE ou le laboratoire executant l’analyse. Conclusion Le dosage des IgE specifiques d’allergene par la methode immunofluoroenzymatique ImmunoCAP® presente des performances analytiques permettant de satisfaire aux exigences de la norme EN ISO 15189 et d’assurer aux prescripteurs et aux patients la fiabilite des resultats fournis, via une strategie d’accreditation « analyte unique ». La procedure d’accreditation validee par notre etude permet d’eviter un surcout en reactifs estime a un demi-million d’euros par automate.

Resume Les auteurs rapportent 12 cas de chocs anaphylactiques survenus apres morsure de tiques de pigeon (Argas reflexus). La symptomatologie soudaine et severe associait des manifestations cutanees ou muqueuses (12 cas), respiratoires (8 cas), digestives (6 cas), cardio-circulatoires (12 cas), aboutissant dans 8 cas a une perte de connaissance et necessitant dans 8 cas un traitement en service de reanimation. Deux types de reactions secondaires aux morsures d'Argas reflexus ont ete decrites : d'une part des reactions d'envenimation, d'autre part des reactions anaphylactiques a mediation IgE (urticaire, angio-oedeme, chocs anaphylactiques souvent recidivants). Le diagnostic repose sur l'anamnese : notion de piqure ou de morsure, survenant le plus souvent la nuit, a la saison chaude, presence de pigeons dans l'environnement immediat, identification du parasite. Il est confirme par le dosage des IgE seriques specifiques d'Argas reflexus (classe ≥ 3 dans 8 des 9 observations ou il a ete effectue). Argas reflexus est une tique molle appartenant a l'ordre des acariens. Il s'agit d'un parasite hematophage specifique des pigeons, principalement des pigeons des villes. Se refugiant a l'interieur des habitations, surtout dans l'habitat urbain ancien et dans les etages superieurs ou sont presentes des colonies de pigeons, il peut s'attaquer a l'homme, le mordre et induire des accidents anaphylactiques. Les allergenes sont presumes etre des produits anticoagulants d'origine salivaire. Le traitement curatif est celui du choc anaphylactique; le traitement preventif repose sur la suppression des pigeons, de leurs nids et des Argasides qui les parasitent.

Resume Un dosage de tryptase serique post-mortem est realise dans 10 cas dont la mort est due a une cause cardiaque (6), liee a une neoplasie (2), un choc septique (1) et une mort subite du nourrisson (1). Le sang est preleve par ponction cardiaque (7), carotidienne (5) et veineuse peripherique (1). Le dosage est effectue par test radio-immunologique en phase solide Pharmacia. Sur 13 dosages 3 sont superieurs a 4 ng/ml (N

In this paper we present a model for the histamine negative feedback on the human basophil degranulation and another one for the feed-forward of these basophils on the extra-cellular histamine. These two models allow us to suggest a characterisation of the incubation time.

We first present a linear compartmental model describing human basophiol degranulation by means of an antigen concentration. Then we study an optimal control problem associated to this linear compartmental model in presence of high dilutions. For solving this control problem we used the LAPLACE transform.

Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs.We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes).In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique.The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

Carboxymethylcellulose (CMC) is used extensively in the pharmaceutical and food industries on account of its various properties. Anaphylactic reactions are rare. It has been reported principally after intra-articular infiltration of sustained-release corticosteroids containing CMC and, very rarely, after barium enema.A case of pre-lethal anaphylactic shock after barium enema was studied by prick-test, intra-dermal reaction (IDR), leukocyte histamine release test (LHRT), basophil activation test (BAT), cystein-leukotriene release test (CAST) and dot-blot analysis.IDR to CMC was positive at a concentration of 10 microg/ml. BAT and CAST were positive. Specific IgE were identified using dot-blot analysis.This is the third report of CMC-specific IgE and the second of anaphylaxis to CMC associated with a barium suspension in contact with GI tract mucosa. CMC as an excipient in medicinal products may therefore be a risk factor for severe anaphylaxis after injection or following contact with GI tract mucosa. Sensitization and allergic reactions by CMC in food additives have to be considered.

We first present a global simulation model describing inhibition of human basophil degranulation by means of high dilutions. Then we study an optimal control problem associated to a non-linear compartmental model. This control is associated to an antigen concentration. For solving this control problem we used a dynamic programming method.

Background At the beginning of this series of experiments we were looking for a model based on the use of purified commercially available compounds based on a fully described and accepted pharmacological model to study of the biological effect of high dilutions. Negative feedback induced by histamine, a major pro-inflammatory mediator, on basophils and mast cells activation via an H2 receptor me these criteria. The simplest way of measuring basophil activation in the early 1980's was the human basophil activation test (HBDT). Objectives Our major goal was first to study the biological effect of centesimal histamine dilutions beyond the Avogadro limit, on the staining properties of human basophils activated by an allergen extract initially house dust mite, then an anti-IgE and N-formyl-Met-Leu-Phe (fMLP). Technical development over the 25 years of our work led us to replace the manual basophil counting by flow cytometry. The main advantages were automation and observer independence. Using this latter protocol our aim was to confirm the existence of this phenomenon and to check its specificity by testing, under the same conditions, inactive analogues of histamine and histamine antagonists. More recently, we developed an animal model (mouse basophils) to study the effect of histamine on histamine release. Methods and results For the HBDT model basophils were obtained by sedimentation of human blood taken on EDTA and stained with Alcian blue. Results were expressed in percentage activation. Histamine dilutions tested were freshly prepared in the lab by successive centesimal dilutions and vortexing. Water controls were prepared in the same way. For the flow cytometric protocol basophils were first labeled by an anti-IgE FITC (basophil marker) and an anti-CD63 (basophil activation marker). Results were expressed in percentage of CD63 positive basophils. Another flow cytometric protocol has been developed more recently, based on basophil labeling by anti-IgE FITC (fluorescein isothiocyanate) and anti-CD203 PE (another human basophil activation marker). Results were expressed in mean fluorescence intensity of the CD203c positive population (MFI-CD203c) and an activation index calculated by an algorithm. For the mouse basophil model, histamine was measured spectrofluorimetrically. The main results obtained over 28 years of work was the demonstration of a reproducible inhibition of human basophil activation by high dilutions of histamine, the effect peaks in the range of 15–17CH. The effect was not significant when histamine was replaced by histidine (a histamine precursor) or cimetidine (histamine H2 receptor antagonist) was added to the incubation medium. These results were confirmed by flow cytometry. Using the latter technique, we also showed that 4-Methyl histamine (H2 agonist) induced a similar effect, in contrast to 1-Methyl histamine, an inactive histamine metabolite. Using the mouse model, we showed that histamine high dilutions, in the same range of dilutions, inhibited histamine release. Conclusions Successively, using different models to study of human and murine basophil activation, we demonstrated that high dilutions of histamine, in the range of 15–17CH induce a reproducible biological effect. This phenomenon has been confirmed by a multi-center study using the HBDT model and by at least three independent laboratories by flow cytometry. The specificity of the observed effect was confirmed, versus the water controls at the same dilution level by the absence of biological activity of inactive compounds such as histidine and 1-Methyl histamine and by the reversibility of this effect in the presence of a histamine receptor H2 antagonist.

This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result.In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay.With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%).A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

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.

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We compared by basophil degranulation and histamine release the reactivity to protein A and anti-IgE of the basophils from 3 groups of patients: group I (blood donors), group II (rhinitis) and group III (atopic dermatitis). We demonstrated that histamine release and basophil degranulation gave tightly correlated results and that by both methods group III was significantly (p less than 0.01) more sensitive to the effectors than the two other groups. Protein A and anti-IgE stimulation curves showed a rebound effect at low concentrations of effectors. This was, however, only significant (p less than 0.01) for group III. Bacterial hypersensitivity may be IgE-mediated and may be related to the increased sensitivity to bacterial antigens and the severity of atopic dermatitis in patients infected by Staphylococcus aureus.

We present a global simulation model applied to the HBDT and a non-linear compartmental model which yield evidence on the negative feedback of the histamine on the degranulation of human basophils in the presence of an antigen dilution and a dilution of some homeopathetic preparation. The compartmental system's control problem was set and led us to the computation of the optimal homeopathetic dilution.

We report two cases occurring in 2004 of patients being treated for pleural mesothelioma with a combination of cisplatin or carboplatin and pemetrexed. Investigation by skin tests and flow cytometry confirmed the clinical diagnosis in both cases.Case 1: a man of 62 developed, after 12 courses of cisplatin-pemetrexed, an anaphylactic reaction 5 minutes after the infusion of cisplatin. Treatment was withdrawn permanently. Case 2: a man of 66 developed, after 7 courses of cisplatin-pemetrexed, an anaphylactic reaction within the first minute of the infusion of cisplatin. Subsequently, in March 2004, he received pemetrexed alone without any problems. In August 2004 he was prescribed carboplatin-pemetrexed. Within 5 minutes he developed urticaria, pruritus and abdominal pain. He was treated later with pemetrexed alone with no problems.Hypersensitivity to platinum salts usually occurs after several courses of treatment. Skin tests and flow cytometry are a simple, concordant, and reliable way of confirming the diagnosis.

Histamine is known to elicit a negative feedback effect on anti-IgE and allergen-induced basophil activation. A series of experiments performed between 1981 and 1995 using a manual method showed biological activity of highly diluted histamine. Most of the experiments used histamine in the range 10(-30) (15C)-10(-36) M (18C). These results were confirmed by automated flow cytometry, but this method is based on the selection of basophils by anti-IgE and analysis of basophil activation by anti-CD 63, showing significant but relatively low inhibition (approximately 14%), insufficient to convince the scientific community of the reality of the phenomenon.We investigated if the use of CD 203c, a basophil specific, earlier marker than CD 63 of the activation cascade, increased the sensitivity of the method, testing two target histamine dilutions, 10(-4) (2C) and 10(-32) M (16C).Basophils, obtained from buffy coats, were pre-incubated with the histamine dilutions and activated by two agonists: anti-IgE and fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Basophil activation was stopped with EDTA. The cells were labelled with anti-IgE, anti-CD 13 and anti-CD 14 for basophil selection, and anti-CD 63 and anti-CD 203c for basophil activation. Results were expressed in up-regulation percentage for CD 63 or mean intensity of fluorescence (MFI) for CD 203c.Histamine 10(-4) M (2C) and histamine 10(-32) M (16C) were capable of inhibiting both IgE-dependent (anti-IgE) and IgE-independent (fMLP) basophil activation. The percentage inhibition depended on the activation marker used. The highest inhibition for histamine dilution 16C was observed with CD 203c (38%, P0.001), approximately half the inhibition observed with histamine 2C (73%).These new flow cytometric protocols confirmed that high dilutions of histamine may inhibit basophil activation and that the inhibitory effect is not restricted to IgE-dependent activation. The use of CD 203c instead of CD 63 increased the magnitude of the response.

A case of oral allergy to dental alloys is presented, highlighting the interest of dosage of salivary nickel and of flow cytometry showing a selective CD4+ activation. The discrepancy between the rarity of oral allergy to dental alloys and the frequency of nickel sensitization and nickel-induced contact dermatitis leads to discuss the mechanisms of oral tolerance.

BACKGROUND: In order to demonstrate that high dilutions of histamine are able to inhibit basophil activation in a reproducible fashion, several techniques were used in different research laboratories. OBJECTIVE: The aim of the study was to investigate the action of histamine dilutions on basophil activation. METHODS: Basophil activation was assessed by alcian blue staining, measurement of histamine release and CD63 expression. Study 1 used a blinded multi-centre approach in 4 centres. Study 2, related to the confirmation of the multi-centre study by flow cytometry, was performed independently in 3 laboratories. Study 3 examined the histamine release (one laboratory) and the activity of H(2) receptor antagonists and structural analogues (two laboratories). RESULTS: High dilutions of histamine (10(-30)-10(-38) M) influence the activation of human basophils measured by alcian blue staining. The degree of inhibition depends on the initial level of anti-IgE induced stimulation, with the greatest inhibitory effects seen at lower levels of stimulation. This multicentre study was confirmed in the three laboratories by using flow cytometry and in one laboratory by histamine release. Inhibition of CD63 expression by histamine high dilutions was reversed by cimetidine (effect observed in two laboratories) and not by ranitidine (one laboratory). Histidine tested in parallel with histamine showed no activity on this model. CONCLUSIONS: In 3 different types of experiment, it has been shown that high dilutions of histamine may indeed exert an effect on basophil activity. This activity observed by staining basophils with alcian blue was confirmed by flow cytometry. Inhibition by histamine was reversed by anti-H2 and was not observed with histidine these results being in favour of the specificity of this effect We are however unable to explain our findings and are reporting them to encourage others to investigate this phenomenon.

Immuno-allergology of iodine contrast products is much discussed, the main questions posed by these molecules particularly concern the existence of specific IgE and their frequency, the specificity of the antibodies, involvement of the iodine atoms and their routes of sensitisation. Our preliminary study on 6 cases of urticaria (5) and erythema (1) after use of ioxaglate and with positive search for ioxaglate-specific IgE (2.3 to 6.7) after coupling to Sepharose (Baldo and Fisher technique). The results of this technique are expressed with reference to a pool of serum of subjects who had no contact with these contrast products. The threshold of positivity was 2, the mean of the results from 10 subjects who were exposed lay between 1.37 +/- 0.42. Inhibition of specific IgE by ioxaglate was positive in 6 cases. In 2/3 cases inhibition by potassium iodide was also observed. Several published studies in favour of the existence of specific IgE of close specificity, the inhibitions of specific IgE presented were mostly only observed for the contrast product used. An hypothesis would be that the ionic molecules have a greater capacity to induce the synthesis of specific IgE than non-ionic molecules because of their affinity for carrier proteins. The inhibitions seen with mineral iodine in this short study do not permit total elimination of the participation of the iodine atom at the antigenic site.

A protocol based on anti-IgE induced human basophil activation was set up to allow the intra-assay calculation of basophil count significance. In 6 successive experiments, we demonstrated the normality of the basophil count distribution through a comparison of the mean values by Student's t-test. We confirm the inhibition of anti-IgE induced human basophil activation by histamine dilutions ranging from 10−10 M to 10−38 M, the most frequent inhibition observed being for 10−18, 10−20, and 10−32 M, in agreement with our previous results.

After a century of therapeutic use of acetyl salicylic acid (ASA), the different mechanisms involved leading to the therapeutic activity and to the various clinical reactions (even life threatening) observed in patients sensitive to ASA or in general to the non steroidal anti-inflammatory drugs (NSAID's) are still to be discovered or confirmed. During the last century a lot of scientific results have been published in favour of a wide panel of mechanism including an humoral (IgE and/or IgG) or a cellular (lymphocytes) immune reaction, inhibition of the cyclooxygenases, a cellular activation (mast cells, basophils, eosinophils and platelets), a competition at the serum albumin level and an activation of the complement cascade. In general the same actors than in the anaphylactic reaction are found (mediators, cellular activation and amplification systems). It is clear today that these reactions for which an accurate diagnosis is mandatory cannot be explained by a single mechanism, which is the case of a lot of other allergens.

This work is devoted to the study of 22 dossiers of persons who have presented with clinical signs following taking a drug. The study was confined to patients in whom the symptoms had as origin a hypersensitivity (true or non-specific by histamine release). This study has allowed analysis of the value of several serological tests (measurement of leukotrienes, activation of basophils and T specific lymphocytes) in diagnosis and so to define the path to take that is best adapted to each case. The conclusion presents an agreement between the suspected diagnosis, after allergy enquiry and prick-tests, and the results of serological tests.

Some adverse reactions to iodinated contrast material (ICM) are considered allergy-like, with cutaneous, cardiovascular, respiratory, and digestive symptoms. Allergy-like reactions are usually unpredictable. Reactions are more frequent with ionic than with nonionic material, but the frequency of deaths is almost identical. In a recent study, 20 severe unexpected reactions to ICM, including 10 life-threatening reactions and one death, were investigated by measuring mediators in blood, within the first minutes or hours of reaction. The responsible ICMs were mostly ionic materials. Histamine and tryptase release correlated with the severity of the reaction. Specific IgE against the responsible ICM was significantly higher in reactors than in controls. A few patients had positive skin tests to the administered ICM, suggesting type-I allergic reaction. Only 2.4% and 3.1% of the cases yielded a positive IgE-RIA, in a second retrospective study which included 165 patients recruited during a 4-year period. In conclusion, IgE-mediated anaphylaxis is rare, but it may be one of the possible mechanisms of severe adverse reactions to ICM.

Adverse effects of medications, most notably antimicrobials, are becoming increasingly common and raise difficult challenges in the area of clinical pattern definition (wide variety of symptoms, polypharmacy in many cases), diagnosis, and methodology (need for a rapid diagnosis, frequent obscurity of causative mechanisms, and less than ideal reliability of laboratory techniques). Sixty patients were treated by rush immunotherapy to one or more antimicrobials. The pretreatment evaluation included oriented history taking, skin tests, blood cell counts, IgE assays, and cell activation tests (basophils and lymphocytes). The results of this study confirm the usefulness of skin tests (intradermal, prick, or patch tests), which provided etiological orientation in 54 of the 60 cases. They also provide additional evidence of the lack of reliability of currently available in vitro tests (only 29 of the 60 tests were positive).

The biological exploration of the anaphylactoid reaction is based on tryptase and plasma histamine determinations. As this reaction may be considered as a particular case of the acute inflammatory reaction, we have compared, on 25 subjects having presented a drug induces anaphylactoid reaction grade 1 to 3 the plasma level of tryptase, histamine and interleukin 6 (IL6) at 30 and 90 min. On a population of patients having presented an increase of plasma histamine and/or tryptase (n = 14), we have shown that the concentrations of tryptase, histamine and IL6 respectively plateaued (63.9 +/- 70 micrograms/l et 74 +/- 102 micrograms/l), decreased (82.5 +/- 102 mmole/l et 20.5 +/- 27 mmole/l), or greatly increased (19.5 +/- 25.3 ng/l et 320 +/- 745 ng/l) at 30 and 90 min. Correlation between IL6 and the two parameters was only significant for tryptase at 30 and 90 min. Due to the longer lasting kinetic of IL6 (48 h) as compared to histamine and tryptase, IL6 could have a place in the immediate exploration of the anaphylactoid reactions, these results having to be completed by studies on a longer delay and on groups of well defined clinical reactions.

A protocol has been produced for the study of anaphylactic accidents that occur post-operatively that allows definition of the anaphylactic origin, and so reactions that are mediated by IgE in post-operative accidents. This protocol occurs in two stages, the first is done in the minutes and hours that follow the anaphylactic accident, and the second a month or 6 weeks afterwards. At first, we evaluate the sequential study of the liberation of the mediators of anaphylaxis, plasma histamine, serum tryptase, urinary methylhistamine and, more recently, leucotriene E4. The second study is devoted to reactions that are mediated by IgE, essentially, specific serum IgE, tests of activation of basophils by flow cytometry, measurement of leucotriene C4 and skin tests. A study on 16 subjects has evaluated and validated the protocol and shown a significant level of correspondence of results between the sequential measurement of mediators on one hand and on the other the search for IgE-mediated reactions every time that there was an anaphylactic reaction.

Among the different methods used for the analysis of human basophil activation as histamine and leukotriene release, flow cytometry presents several methodological advantages. Moreover, preliminary results obtained for a wide allergen panel have demonstrated its high sensitivity and specificity. We present here the results of the methods used to validate the flow cytometric analysis of human basophil activation by double anti-IgE FITC/anti-CD63 PE labelling of human leucocytes. The use of multiple labelling by monoclonals having different specificities have shown that the selected cells express the phenotype CD14-, CD19-, CD41- or +/-, CD45+, CD63- or +. This phenotype is related to human basophils. We have also demonstrated that the expression of CD63 is related to basophil activation and not to aggregated platelets which are also able to express this marker.

Diagnosis of allergy to hymenoptera venom must be precise and depends on indisputable bio-clinical criteria, because of specific immunotherapy for indications such as systemic and/or anaphylactic reactions. Until nowadays, diagnosis was by specific IgE, histamine release and skin tests, most often done for the venoms of wasp, honey bee and hornet at the same time since, in 7-8 cases in 10 the patients had not identified the responsible insect. Basophil activation test (TAB) by Flux cytometry and measurement of leukotriene C4 (LTC4) are new techniques of great reliability. The work shows the correlations between the different immunobiological parameters by reference to TAB by CAF and measurement of LTC4. When the overall results for mixed venoms or those for single venoms are considered, the correlations between TAB, LTC4 and the other parameters are highly significant. It can now be considered objectively that TAB by CTF and measurement of LCT4 are the two highest-performing techniques for diagnosis of hymenoptera venom allergy and so validates them.

Allergy to fish is one of the most common food allergies. Gad c 1 is the only fish allergen which has been purified and characterized. Other allergens have been detected by Western blot in cod extracts. We have now improved the Western-blot procedure in order to characterize fish IgE-reactive proteins from extracts prepared under different conditions: pre-rigor mortis and post-rigor mortis, EDTA addition or not, and DEAE ion-exchange chromatography. Several IgE-reactive protein bands have been identified over a wide molecular-weight range. In particular, the 104- and 130-kDa IgE-reactive protein bands were detected. These new bands may correspond to aggregates, as EDTA increased the relative amount of the 60-, 67-, 104-, and 130-kDa IgE-reactive protein bands in Western blot. All these bands were also detected by antiparvalbumin monoclonal antibody, specific to the first calcium-binding site. The longer period of storage increased the relative amounts of the 41-, 80-, 104-, and 130-kDa IgE-reactive protein bands. The 18-kDa band was detected only in fish stored for several days. In conclusion, we have described IgE-reactive protein bands over a wide molecular-weight range (12-130 kDa) in Western blot of cod extract, and shown that EDTA and storage conditions may influence the relative distribution of IgE-reactive protein bands.