Cellular in vitro assays in the diagnosis of Hymenoptera venom allergy

BACKGROUND: The current diagnostic procedures of anaphylactic reactions to hymenoptera stings include intradermal tests, venom-specific IgE (sIgE) and possibly sting challenge tests. Sometimes, the culprit insect remains unidentified. The usefulness of the cellular assays CAST-ELISA and Flow-CAST in the management of hymenoptera venom allergy was investigated. METHODS: 134 patients with systemic reactions after a yellow jacket wasp and/or honey bee sting and 44 healthy controls underwent skin tests, as well as determination of sIgE (CAP-FEIA), leukocyte sulfidoleukotriene release (CAST-ELISA) and basophil CD63 expression (Flow-CAST) upon insect venom stimulation. The clinical diagnosis based on the history alone served as reference. Sensitivity, specificity, and positive and negative predictive value of all methods were compared. Concordance and correlations among methods were calculated. RESULTS: Sensitivity and specificity of all in vitro tests were consistently high. The combination of all tests (skin tests, sIgE, combined cellular assays) yielded a positive predictive value of 100% for both venoms, if all 3 were positive, and a negative predictive value of 100%, if at least 1 test was positive. Relative specificities were considerably higher for the cellular assays (honey bee: CAST 91.1%, Flow-CAST 85.7%; yellow jacket wasp: CAST 98.4%, Flow-CAST 92.1%) and allow the detection of the culprit insect in patients with reactivity to both insects. The concordance between methods was good. There is no correlation between severity of clinical reaction and cellular assays. CONCLUSION: CAST-ELISA and Flow-CAST are valuable additional diagnostic tools for establishing the true culprit insect in patients with unclear clinical history or sensitization to both insects

Separated hemocyte populations from the ascidian Ciona intestinalis contain and release in vitro opsonizing Ca2+-independent and β-galactoside specific lectins

Cytosolic lectins, Ca2+-independent and β-galactoside-specific, were determined to be containedin hemocyte and pharynx lysate supernatants of Ciona intestinalis, as revealed by hemagglutinationassay with trypsinized rabbit erythrocytes. Ca2+-independence and decreasing β-galactosidesinhibitory capacity (TDG > LacNAc ≥ Lactose > Galactose) have been considered properties typical ofgalectins. These lectins can be promptly released by hemocytes maintained in vitro suggesting theirinvolvement in defense responses including inflammatory reactions. Both cell lysate supernatants andhemocyte culture medium presented β-galactoside-inhibitable opsonizing activity versus yeast.Although a Percoll density gradient separation method showed that several hemocyte types containand release β-galactoside-specific molecules, results suggest that hyaline and granular amoebocytesare the primary source of these molecules

Putative rhamnose-binding lectin in the solitary ascidian Ciona intestinalis

Lectins are sugar-binding proteins involved in cell-cell interaction and the recognition of carbohydrate-containing molecules. They act as humoral factors in non-self recognition, are a key component of the innate immune systems of many metazoans and are involved in phagocyte activation through their opsonizing activity. In recent years, a new lectin family, the rhamnose-binding lectin (RBL) family, has been described and its members can modulate the inflammatory response in fish (Watanabe et al., 2009) as well as in various invertebrates, including the colonial tunicate Botryllus schlosseri. Recently, we succeeded in purifying, by affinity chromatography using a rhamnose column, a putative RBL, from the hemolymph of the solitary ascidian Ciona intestinalis. The molecule is Ca2+-independent and promptly (within 4h) inducible, after LPS inoculation. The eluted fractions, when examined by 15 % SDS-PAGE under reducing condition, showed four bands with apparent molecular masses of 65, 54, 30 and 19 kDa. The agglutinating activity of the isolated fraction was demonstrated using trypsinized rabbit erythrocytes and it was inhibited by glycosides such as rhamnose, galactose and lactose. Moreover, by immunocytochemical analysis using antibodies produced against B. schlosseri RBL and in situ hybridization with a riboprobe for the annotated C. intestinalis RBL, we have observed a positive signal in hyaline and granular amoebocytes and in the endothelium of the pharynx vessels