New Approaches for Basophil Activation Tests Employing Dendrimeric Antigen-Silica Nanoparticle Composites.

In vitro cell activation through specific IgE bound to high-affinity receptors on the basophil surface is a widely used strategy for the evaluation of IgE-mediated immediate hypersensitivity reactions to betalactams. Cellular activation requires drug conjugation to a protein to form a large enough structure displaying a certain distance between haptens to allow the cross-linking of two IgE antibodies bound to the basophil's surface, triggering their degranulation. However, no information about the size and composition of these conjugates is available. Routine in vitro diagnosis using the basophil activation test uses free amoxicillin, which is assumed to conjugate to a carrier present in blood. To standardize the methodology, we propose the use of well-controlled and defined nanomaterials functionalized with amoxicilloyl. Silica nanoparticles decorated with PAMAM-dendrimer-amoxicilloyl conjugates (NpDeAXO) of different sizes and amoxicilloyl densities (50-300 µmol amoxicilloyl/gram nanoparticle) have been prepared and chemically characterized. Two methods of synthesis were performed to ensure reproducibility and stability. Their functional effect on basophils was measured using an in-house basophil activation test (BAT) that determines CD63+ or CD203chigh activation markers. It was observed that NpDeAXO nanocomposites are not only able to specifically activate basophils but also do so in a more effective way than free amoxicillin, pointing to a translational potential diagnosis.

Basophil activation test is a complementary tool in the diagnosis of immediate reactions to platinum salts and taxanes.

BACKGROUND: Delabelling pathways offer confirmatory diagnosis and can prevent unnecessary second-line therapies or drug desensitization procedures after chemotherapeutic hypersensitivity reactions (CHT-HSRs). However, these pathways rely on risky in vivo tests. Data on whether in vitro tests could be helpful are scarce. We assessed the role of basophil activation test (BAT) in the diagnosis of HSRs to platin salts (PSs) and taxanes (TXs) in a well-defined population featuring varied endophenotypes and severities of HSRs. METHODS: We conducted a 3-year-long multicentric, prospective study with 121 suspected-immediate CHT-HSR patients. The allergy workup included clinical history (initial reaction based on Type I, cytokine release syndrome, and mixed phenotype's symptoms and if unable to fit in any of these, as "indeterminate"), skin testing (ST), and drug provocation testing (DPT), provided risk assessment was favorable. Final diagnosis classified patients as "hypersensitive," "non-hypersensitive," or "inconclusive." We performed BAT using CD63 and CD203c as activation markers in patients and controls. Patients underwent DPT regardless of BAT results to prevent bias. RESULTS: ST positivity significantly correlated with skin involvement, Type I phenotype, cancer recurrence, and lifetime exposures before reactions. DPTs were negative in all indeterminate phenotype patients (p = .02) and those considered low-risk, whereas they were negative in 62% moderate-risk patients. 55% were confirmed as hypersensitive (mainly Type I reactions, p < .0001), 24% as non-hypersensitive (mainly TXs and indeterminate phenotypes), and 21% as inconclusive. BAT showed 79% sensitivity in Type I IgE-mediated reactions to PSs with a high correlation to ST. CONCLUSIONS: BAT is a promising tool for delabelling and endotyping CHT-HSRs, especially Type I reactions to PSs, possibly identifying patients at risk of positive DPT. ST seems useful in confirming CHT-HSRs, especially PS-induced reactions, and DPT remains the gold standard, being essential even in moderate-risk patients.

Involvement of autologous myeloid dendritic cells in the evaluation of immediate hypersensitivity reactions to betalactams.

BACKGROUND: Amoxicillin (AX) and clavulanic acid (CLV) are the betalactam antibiotics (BLs) most used to treat bacterial infections, although they can trigger immediate hypersensitivity reactions (IDHRs). The maturation analysis of monocyte-derived dendritic cells (moDCs) and their capacity to induce proliferative response of lymphocytes are useful to test the sensitisation to a drug, although without optimal sensitivity. Nevertheless, this can be improved using directly isolated DCs such as myeloid DCs (mDCs). METHODS: mDCs and moDCs were obtained from 28 allergic patients (AP), 14 to AX, 14 to CLV and from 10 healthy controls (HC). The expression of CCR7, CD40, CD80, CD83, and CD86 was analysed after stimulation with both BLs. We measured the capacity of these pre-primed DCs to induce drug-specific activation of different lymphocyte subpopulations, CD3+, CD4+, CD8+, CD4+Th1, and CD4+Th2, by flow cytometry. RESULTS: Higher expression of CCR7, CD40, CD80, CD83, and CD86 was observed on mDCs compared to moDCs from AP after stimulating with the culprit BL. Similarly, mDCs induced higher proliferative response, mainly of CD4+Th2 cells, compared to moDCs, reaching up to 67% of positive results with AX, whereas of only 25% with CLV. CONCLUSIONS: mDCs from selective AP efficiently recognise the culprit drug which trigger the IDHR. mDCs also trigger proliferation of lymphocytes, mainly those with a Th2 cytokine pattern, although these responses depend on the nature of the drug, mimicking the patient's reaction.