Follow-Up of Venom Immunotherapy on Flow Cytometry and Definition of a Protective Index.

BACKGROUND: A major problem of venom-specific immunotherapy (VIT) is the absence of reliable parameters for deciding treatment discontinuation. AIM OF THE STUDY: Intracutaneous tests (ICTs), the basophil activation test (BAT), specific IgEs (sIgEs) and blocking factor (BF) activity were measured during VIT. We made an evaluation by means of a protective index (PI) including ICT, BAT and BF values. MATERIAL AND METHODS: A population of 45 patients who had experienced a systemic reaction after an insect sting were tested before VIT (T0), at 1 week (T1w), at 10 weeks (T10w) and at 21 weeks (T21w), and, for a subgroup of 17 patients, at 3-5 years (T3-5y). Basophil activation (expressed in % CD63 and in the area under the curve) and BF activity were measured by flow cytometry using the CCR3/CD63 protocol. RESULTS: The first 21 weeks of follow-up showed no significant variation in the ICT, sIgE and BAT measurements, except for BAT, by eliminating weak negative anti-IgE responses. In these conditions, the decrease in basophil activation was significant at T10w (p = 0.009) and T21w (p = 0.009). Increased BF activity was also significant at T10w (p = 0.008) and T21w (p = 0.002). The PI threshold calculated from the mean ± 3 standard errors (SE) was 64.8 (14.7 ± 16.7, n = 25) at T0. PI increase was significant at T3-5y (3,430 ± 6,282; p < 0.001). CONCLUSION: VIT induced a significant decrease in ICT values and basophil activation, along with an increase in serum BF activity, significant after 10 weeks of VIT. Evaluated in a larger population, the PI could represent a new tool for the clinico-biological follow-up of VIT efficacy.

Use of lipid rafting for the analysis of human basophil activation by flow cytometry.

BACKGROUND: Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization. OBJECTIVES: Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation. METHODS: Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells. RESULTS: Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium. CONCLUSIONS: Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.

Fractal structures and silica films formed by the Treignac water on inert and biological surfaces.

The Treignac water is a natural mineral water containing mainly orthosilicic acid. On inert substrates, it forms a silica film with fractal structures which cannot be reproduced in laboratory-reconstituted water. These structures form by condensation of orthosilicic acid monomers, following the Witten-Sander model of diffusion-limited aggregation. On biological surfaces, such as tomato leaves, the Treignac water forms a silica film with a different morphology and devoid of fractal structures. The filmogenic properties of this natural mineral water are here discussed in the context of crop protection, as the silica film can provide a barrier and a platform for the immobilization of elicitors of plant defense responses.

Constitutive nitric oxide synthase inhibition combined with histamine and serotonin receptor blockade improves the initial ovalbumin-induced arterial hypotension but decreases the survival time in brown norway rats anaphylactic shock.

Anaphylactic shock accidents after allergen exposure are frequent. After immunization with ovalbumin (OVA), a common dietary constituent, we evaluated the efficacy of pretreatment with histamine-receptor or serotonin-receptor blockers administered alone or in combination with a nitric oxide synthase inhibitor (L-NAME) on OVA-induced anaphylactic shock in Brown Norway rats. Animals were allocated to the following groups (n = 6 each): control (0.9% saline); diphenydramine (15 mg kg(-1)); cimetidine (20 mg kg(-1)); diphenydramine + cimetidine; dihydroergotamine (50 microg kg(-1)); diphenydramine + cimetidine + dihydroergotamine; L-NAME (100 mg/kg) alone or associated with diphenydramine, cimetidine, diphenydramine + cimetidine, dihydroergotamine, or diphenydramine + cimetidine + dihydroergotamine. Mean arterial blood pressure (MABP), heart rate (HR), and survival time were monitored for 60 min following treatment. The shock was initiated with i.v. OVA. The MABP drop after i.v. OVA was worsened by diphenydramine and was modestly attenuated by cimetidine, dihydroergotamine, or both together. L-NAME potentiated slightly the effects of cimetidine and dihydroergotamine by lessening the initial MABP decrease, but this transient effect was not sufficient to prevent the final collapse or to improve survival time. Decreased vasodilatory (prostaglandins E2), increased vasoconstrictory (thromboxane B2) prostaglandins, and unchanged leukotriene C4 concentrations were contributory to the overall hemodynamic changes. Thus, the combined blockade of vasodilator mediators (histamine, serotonin, and nitric oxide) slowed the MABP drop in anaphylactic shock, but did not improve survival. More studies are needed to understand these discordant effects.

Binding of canine anaphylactic antibodies on human basophils: application to canine allergy diagnosis.

Abstract Using the technique of human basophil passive sensitization, as employed for human allergy díagnosis, we checked the ability of canine anaphylactic antibodies to sensitize human basophils. Therefore, by sensitizing human basophils with sera taken from dogs allergic to house dust mite, we demonstrated basophil activation as measured by alcian blue staining. Basophil activation was inhibited by heating dog sera at 56 °C for 6 h and by a human myeloma IgE. Basophil activation was also shown by histamine and leukotriene (LTC4) release. These results indicate canine anaphylactic antibodies bind to human basophil IgE receptors and also that they are IgE. The three methods described here for measuring basophil activation may lead to díagnostic methods applicable to canine allergy díagnosis. Resumen Mediante el método de la sensibilización pasiva de basófilos humanos como se utiliza para el díagnóstico de la alergia humana, evaluamos la capacidad de los anticuerpos anafilácticos caninos de sensibilizar basófilos humanos. Asi, sensibilizando basófilos humanos con suero extraido de perros con alergia al ácaro del polvo, demostramos la activación de basófilos medíante la tinción de Azul de Alcián. Se inhibió calentando suero canino a 56 °C durante 6 h. y por IgE de mieloma humano. La activación de los basófilos se mostró también por la liberación de histamina y leucotrieno (LTC4). Estos resultados indican que los anticuerpos caninos anafilácticos se unen a los receptores de IgE en basófilos humanos y también que son IgE. Los tres métodos descritos aqui para medir la activación de basófilos pueden llevar a métodos de díagnóstico aplicables al díagnóstico de la alergia canina. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Union de anticuerpos anafilácticos caninos a basófilos humanos: aplicacion al díagnóstico de alergia canina). Veterinary Dermatology 1996; 7: 185-91.] Résumé Utilisant une technique de sensibilisation passive de basophiles humains, employee pour le díagnostic allergologique chez l'homme, nous avons testé la capacité des anticorps anaphylactiques canins à sensibiliser des basophiles humains. Ainsi, par sensibilisation de basophiles humains avec des sérums provenant de chiens allergiques aux acariens de la poussière de maison, nous avons démontré l'activation des basophiles mesurée par coloration au bleu alcian. Celle-ci est inhibée par des sérums canins chauffés à 56 °C pendant 6 heures et par un myélome IgE humain. L'activation des basophiles a été aussi démontrée par libération d'histamine et de leucotriénes (LTC4). Ces résultats prouvent la présence d'anticorps anaphylactiques canins fixés à des récepteurs IgE de basophiles humains et que ceux-ci sont des IgE. Les trois méthodes décrites ici pour mesurer l'activation des basophiles peuvent être utilisées pour le díagnostic allergologique chez le chien. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Fixation d'anticorps anaphylactiques canins sur des basophiles humains). Veterinary Dermatology 1996; 7: 185-91.] Zusammenfassung Mit der Technik der passiven Basophilensensibilisierung beim Menschen, wie man sie für die Allergiedíagnose beim Menschen anwendet, untersuchten wir die Möglichkeit, menschliche Basophile durch kanine anaphylaktische Antikörper zu sensibilisieren. Dazu wurden humane Basophile mit Sera von Hunden sensibilisiert, die allergisch auf Hausstaubmilben reagierten. Dabei demonstrierten wir eine Basophilenaktivierung, die durch Elsässerblau-Färbung gemessen werden konnte. Der Vorgang wurde verhindert durch Erhitzen der Hundesera auf 56 °C für 6 Stunden und durch humanes Myelom-IgE. Basophilenaktivierung wurde auch durch Histamin-und Leukotrien(LTC4)-Ausschüttung gezeigt. Diese Ergebnisse zeigen, daß kanine anaphylaktische Antikörper sich an humane basophile IgE-Rezeptoren binden und auch IgEs darstellen. Die drei hier beschriebenen Methoden zur Messung der Basophilenaktivierung können zu einer díagnostischen Methode führen, die für die Diagnostik kaniner Allergie anwendbar ist. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Die Bindung von anaphylaktischen Antikörpern des Hundes an Basophile Zellen des Menschen: Anwendung für die Allergiedíagnose beim Hund). Veterinary Dermatology 1996; 7: 185-91.].

[For an efficient and reasonable accreditation of allergen specific IgE]. / Pour une accréditation efficace et raisonnable du dosage des IgE spécifiques d'allergènes.

French medical laboratories must be accredited before November 2016 according to NF/EN/ISO 15189 standard. However, technical accreditation guidelines cannot be applied literally for the determination of specific IgE for several reasons: more than 600 allergen tests, lack of international gold standard, limited external quality controls. Furthermore, the technique for determination of specific IgE is CE DM-IVD marked, common to all specificities, automatised, standardized according to a single calibration curve. Thus, we propose an efficient but reasonable solution conform to the idea of the accreditation by validating the process. We recommend: a flexible extend type A; choice of only one representative allergen (Dermatophagoides pteronyssinus) for repeatability and precision (20 tests, 2 levels 0.5-1 and 8-12 kUA/L) performed on patients sera, reproducibility (30 consecutive determinations using an Internal Quality Control/IQC), accuracy (IQC and rare External Quality Controls) compared with peers. Sensitivity, specificity, dynamic range, detection threshold are determinated by the provider. Linearity may be checked if the laboratory practices sample dilution for values higher than the upper limit guaranteed by the provider. In the absence of international gold standard, the uncertainty is not measurable. In case of change of instrument, the results obtained by the systems must be compared through 35 tests of different specificities distributed across the range of calibration and including 5 values close to the detection limit. This methodology allows a scientifically effective verification, technically and financially reasonable, to ensure the excellence of the performance of the laboratory with regard to peers and users (allergologists and patients).