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.

Long-chain fatty acids block allergic reaction against lipid transfer protein Sola l 7 from tomato seeds.

Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy.

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.

Granulocytes and mast cells in AllergoOncology-Bridging allergy to cancer: An EAACI position paper.

Derived from the myeloid lineage, granulocytes, including basophils, eosinophils, and neutrophils, along with mast cells, play important, often disparate, roles across the allergic disease spectrum. While these cells and their mediators are commonly associated with allergic inflammation, they also exhibit several functions either promoting or restricting tumor growth. In this Position Paper we discuss common granulocyte and mast cell features relating to immunomodulatory functions in allergy and in cancer. We highlight key mechanisms which may inform cancer treatment and propose pertinent areas for future research. We suggest areas where understanding the communication between granulocytes, mast cells, and the tumor microenvironment, will be crucial for identifying immune mechanisms that may be harnessed to counteract tumor development. For example, a comprehensive understanding of allergic and immune factors driving distinct neutrophil states and those mechanisms that link mast cells with immunotherapy resistance, might enable targeted manipulation of specific subpopulations, leading to precision immunotherapy in cancer. We recommend specific areas of investigation in AllergoOncology and knowledge exchange across disease contexts to uncover pertinent reciprocal functions in allergy and cancer and allow therapeutic manipulation of these powerful cell populations. These will help address the unmet needs in stratifying and managing patients with allergic diseases and cancer.

Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis.

Background: Immunopathology in food allergy is characterized by an uncontrolled type 2 immune response and specific-IgE production. Recent studies have determined that group 2 innate lymphoid cells (ILC2) participate in the food allergy pathogenic mechanism and their severity. Our objective was to investigate the role of ILC2 in peach-allergic patients due to non-specific lipid transfer protein (Pru p 3) sensitization. Methods: The immune response in peripheral blood mononuclear cells was characterized in lipid transfer protein-allergic patients and healthy controls. We have analyzed the Pru p 3 uptake on ILC2, the expression of costimulatory molecules, and their involvement on the T-cell proliferative response and cytokine production under different experimental conditions: cytokines involved in group 2 innate lymphoid cell activation (IL-33 and IL-25), Pru p 3 as main food allergen, and the combination of both components (IL-33/IL-25+Pru p 3) using cell sorting, EliSpot, flow cytometry, and confocal microscopy. Results: Our results show that Pru p 3 allergen is taken up by group 2 innate lymphoid cells, regulating their costimulatory molecule expression (CD83 and HLA-DR) depending on the presence of Pru p 3 and its combination with IL-33/IL-25. The Pru p 3-stimulated ILC2 induced specific GATA3+Th2 proliferation and cytokine (IL-4, IL-5, and IL-13) production in lipid transfer protein-allergic patients in a cell contact-dependent manner with no changes in Tbet+Th1- and FOXP3+Treg cell differentiation. Conclusions: The results indicate that in lipid transfer protein-allergic patients, the responsible allergen, Pru p 3, interacts with group 2 innate lymphoid cells, promoting a Th2 cell response. Our results might be of interest in vivo, as they show a role of group 2 innate lymphoid cells as antigen-presenting cells, contributing to the development of food allergy. Consequently, group 2 innate lymphoid cells may be considered as potential therapeutic targets.

Immunomodulatory potential of rapamycin-loaded mesoporous silica nanoparticles: pore size-dependent drug loading, release, and in vitro cellular responses.

Rapamycin is a potent immunosuppressive drug that has been recently proposed for a wide range of applications beyond its current clinical use. For some of these proposed applications, encapsulation in nanoparticles is key to ensure therapeutic efficacy and safety. In this work, we evaluate the effect of pore size on mesoporous silica nanoparticles (MSN) as rapamycin nanocarriers. The successful preparation of MSN with 4 different pore sizes was confirmed by dynamic light scattering, zeta potential, transmission electron microscopy and N2 adsorption. In these materials, rapamycin loading was pore size-dependent, with smaller pore MSN exhibiting greater loading capacity. Release studies showed sustained drug release from all MSN types, with larger pore MSN presenting faster release kinetics. In vitro experiments using the murine dendritic cell (DC) line model DC2.4 showed that pore size influenced the biological performance of MSN. MSN with smaller pore sizes presented larger nanoparticle uptake by DC2.4 cells, but were also associated with slightly larger cytotoxicity. Further evaluation of DC2.4 cells incubated with rapamycin-loaded MSN also demonstrated a significant effect of MSN pore size on their immunological response. Notably, the combination of rapamycin-loaded MSN with an inflammatory stimulus (lipopolysaccharide, LPS) led to changes in the expression of DC activation markers (CD40 and CD83) and in the production of the proinflammatory cytokine TNF-α compared to LPS-treated DC without nanoparticles. Smaller-pored MSN induced more substantial reductions in CD40 expression while eliciting increased CD83 expression, indicating potential immunomodulatory effects. These findings highlight the critical role of MSN pore size in modulating rapamycin loading, release kinetics, cellular uptake, and subsequent immunomodulatory responses.

Nasal allergen-neutralizing antibodies correlate closely with tolerated intranasal allergen challenge dose following grass pollen subcutaneous immunotherapy in patients with local allergic rhinitis.

BACKGROUND: Local allergic rhinitis (LAR) is defined by chronic nasal symptoms, absence of atopy, positive nasal allergen challenge (NAC) and a good response to subcutaneous allergen immunotherapy (SCIT). We sought to investigate SCIT capacity to induce local and systemic blocking antibodies in LAR patients. METHODS: A RDBPC study of grass SCIT was performed, with participants receiving either SCIT (Group A; n = 10) or placebo (Group B; n = 14) in the first 6 months. Both groups subsequently received SCIT for 12 months at Year 2. Nasal and serum antibodies (IgG4, IgA1 and IgA2) and their inhibitory capacity were measured at multiple timepoints. RESULTS: The allergen concentration tolerated increased significantly at 6 months (Group A; p = .047) and 24 months (Group B; p = .049) compared with baseline and persisted until the end of the study. Induction of serum sIgA1 to Phl p was seen in Groups A and B, albeit the former being induced earlier (1.71-fold, p = .027). A significant induction in sIgG4 to Phl p 1 and 5 was observed in serum of Group A (p = .047 and p = .0039) and sIgA2 to Phl p in Group B (p = .032 and p = .0098) at 18 and 24 months, respectively. Both local and systemic blocking antibodies can inhibit allergen-IgE complexes binding to CD23 on B cells, and this correlated with level of allergen tolerated intra-nasally in Group A (serum; 𝜌 = -.47, p = .0006, nasal; 𝜌 = -.38, p = .0294). CONCLUSIONS: Grass pollen SCIT induced functional systemic blocking antibodies that correlate with the concentration of allergen tolerated following NAC, highlighting their potential as a biomarker of SCIT in LAR.

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.

Biomarkers of immediate drug hypersensitivity.

Immediate drug hypersensitivity reactions (IDHRs) are a burden for patients and the health systems. This problem increases when taking into account that only a small proportion of patients initially labelled as allergic are finally confirmed after an allergological workup. The diverse nature of drugs involved will imply different interactions with the immunological system. Therefore, IDHRs can be produced by a wide array of mechanisms mediated by the drug interaction with specific antibodies or directly on effector target cells. These heterogeneous mechanisms imply an enhanced complexity for an accurate diagnosis and the identification of the phenotype and endotype at early stages of the reaction is of vital importance. Currently, several endophenotypic categories (type I IgE/non-IgE, cytokine release, Mast-related G-protein coupled receptor X2 (MRGPRX2) or Cyclooxygenase-1 (COX-1) inhibition and their associated biomarkers have been proposed. A precise knowledge of endotypes will permit to discriminate patients within the same phenotype, which is crucial in order to personalise diagnosis, future treatment and prevention to improve the patient's quality of life.

A synthetic glycodendropeptide induces methylation changes on regulatory T cells linked to tolerant responses in anaphylactic-mice.

Introduction: Lipid transfer proteins (LTPs) are allergens found in a wide range of plant-foods. Specifically, Pru p 3, the major allergen of peach, is commonly responsible for severe allergic reactions. The need for new alternatives to conventional food allergy treatments, like restrictive diets, suggests allergen immunotherapy as a promising option. It has been demonstrated that sublingual immunotherapy (SLIT) with synthetic glycodendropeptides, such as D1ManPrup3, containing mannose and Pru p 3 peptides induced tolerance in mice and that the persistence of this effect depends on treatment dose (2nM or 5nM). Moreover, it produces changes associated with differential gene expression and methylation profile of dendritic cells, as well as phenotypical changes in regulatory T cells (Treg). However, there are no works addressing the study of epigenetic changes in terms of methylation in the cell subsets that sustain tolerant responses, Treg. Therefore, in this work, DNA methylation changes in splenic-Treg from Pru p 3 anaphylactic mice were evaluated. Methods: It was performed by whole genome bisulphite sequencing comparing SLIT-D1ManPrup3 treated mice: tolerant (2nM D1ManPrup3), desensitized (5nM D1ManPrup3), and sensitized but not treated (antigen-only), with anaphylactic mice. Results: Most of the methylation changes were found in the gene promoters from both SLIT-treated groups, desensitized (1,580) and tolerant (1,576), followed by the antigen-only (1,151) group. Although tolerant and desensitized mice showed a similar number of methylation changes, only 445 genes were shared in both. Remarkably, interesting methylation changes were observed on the promoter regions of critical transcription factors for Treg function like Stat4, Stat5a, Stat5b, Foxp3, and Gata3. In fact, Foxp3 was observed exclusively as hypomethylated in tolerant group, whereas Gata3 was only hypomethylated in the desensitized mice. Discussion: In conclusion, diverse D1ManPrup3 doses induce different responses (tolerance or desensitization) in mice, which are reflected by differential methylation changes in Tregs.

Allergen exposure boosts peripheral Th9 responses in patients with local allergic rhinitis.

KEY POINTS: Intranasal allergen exposure increases peripheral total Th2 and Th9 cells in patients with local allergic rhinitis (LAR). Peripheral T-cell response seems dominated by Th9 cells in patients with LAR, whereas Th2 responses prevail in patients with allergic rhinitis. Our results identify Th9 cells as potential therapeutic targets for patients with LAR.

In vitro diagnostic testing for drug allergy in children.

Diagnosing Drug Hypersensitivity Reactions (DHRs) could be a complicated process especially in children, since allergic-like manifestation at this age is more often the expression of concomitant infections rather than a actual DHRs. In vivo tests are usually suggested as a first step; however, prick and intradermal tests could be painful and have shown different sensitivity and specificity among published studies. In some cases, in vivo tests such as Drug Provocation test (DPT) could be even contraindicated. Therefore, the need for in vitro testing is compelling, to add useful information along the diagnostic pathway and to limit the need of DPT. In this review, we analyze the different types of in vitro tests, focusing on those used more widely such as specific IgE and on those that are still for research settings, such as basophil activation test and lymphocyte transformation test, but that have shown some useful diagnostic potential.

Microneedle array patches for allergen-specific immunotherapy.

The incidence of allergies has been steadily increasing in recent years. Allergen-specific immunotherapy (AIT) represents the only approach capable of inducing long-term immune tolerance toward allergens. However, the clinical success of AIT is limited by efficacy or safety concerns related to the administration route. Therapeutic delivery in the skin appears promising, given the presence of immune cells in the skin and the relatively low level of systemic distribution that occurs with this delivery method. However, the stratum corneum greatly limits this route. In this regard, the use of microneedles has been proposed to improve the delivery of therapeutics into the skin. In this review, we discuss recent developments in the use of microneedles for AIT, highlighting avenues for future research.