Nanoarchitectures for efficient IgE cross-linking on effector cells to study amoxicillin allergy.

BACKGROUND: Amoxicillin (AX) is nowadays the ß-lactam that more frequently induces immediate allergic reactions. Nevertheless, diagnosis of AX allergy is occasionally challenging due to risky in vivo tests and non-optimal sensitivity of in vitro tests. AX requires protein haptenation to form multivalent conjugates with increased size to be immunogenic. Knowing adduct structural features for promoting effector cell activation would help to improve in vitro tests. We aimed to identify the optimal structural requirement in specific cellular degranulation to AX using well-precised nanoarchitectures of different lengths. METHOD: We constructed eight Bidendron Antigens (BiAns) based on polyethylene glycol (PEG) linkers of different lengths (600-12,000 Da), end-coupled with polyamidoamine dendrons that were terminally multi-functionalized with amoxicilloyl (AXO). In vitro IgE recognition was studied by competitive radioallergosorbent test (RAST) and antibody-nanoarchitecture complexes by transmission electron microscopy (TEM). Their allergenic activity was evaluated using bone marrow-derived mast cells (MCs) passively sensitized with mouse monoclonal IgE against AX and humanized RBL-2H3 cells sensitized with polyclonal antibodies from sera of AX-allergic patients. RESULTS: All BiAns were recognized by AX-sIgE. Dose-dependent activation responses were observed in both cellular assays, only with longer structures, containing spacers in the range of PEG 6000-12,000 Da. Consistently, greater proportion of immunocomplexes and number of antibodies per complex for longer BiAns were visualized by TEM. CONCLUSIONS: BiAns are valuable platforms to study the mechanism of effector cell activation. These nanomolecular tools have demonstrated the importance of the adduct size to promote effector cell activation in AX allergy, which will impact for improving in vitro diagnostics.

Penicillin and cephalosporin cross-reactivity: role of side chain and synthetic cefadroxil epitopes.

BACKGROUND: Analysis of cross-reactivity is necessary for prescribing safe cephalosporins for penicillin allergic patients. Amoxicillin (AX) is the betalactam most often involved in immediate hypersensitivity reactions (IHRs), and cefadroxil (CX) the most likely cephalosporin to cross-react with AX, since they share the same R1 side chain, unlike cefuroxime (CO), with a structurally different R1. We aimed to analyse cross-reactivity with CX and CO in patients with confirmed IHRs to AX, including sIgE recognition to AX, CX, CO, and novel synthetic determinants of CX. METHODS: Fifty-four patients with confirmed IHRs to AX based on skin test (ST) and/or drug provocation test (DPT) were included. Serum sIgE to AX and benzylpenicillin was determined by Radioallergosorbent test (RAST). Two potential determinants of CX, involving intact or modified R1 structure, with open betalactam ring, were synthesised and sIgE evaluated by RAST inhibition assay. RESULTS: Tolerance to CX (Group A) was observed in 64.8% cases and cross-reactivity in 35.2% cases (Group B). Cross-reactivity with CO was only found in 1.8% cases from Group B. ST to CX showed a negative predictive value of 94.6%. RAST inhibition assays showed higher recognition to CX as well as to both synthetic determinants (66% of positive cases) in Group B. CONCLUSIONS: Cross-reactivity with CX in AX allergic patients is 35%, being ST not enough for prediction. R1, although critical for recognition, is not the unique factor. The synthetic determinants of CX, 1-(HOPhG-Ser-Bu) and 2-(pyrazinone) are promising tools for determining in vitro cross-reactivity to CX in AX allergic patients.