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.

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.

Diagnosis of immediate reactions to amoxicillin: Comparison of basophil activation markers CD63 and CD203c in a prospective study.

BACKGROUND: Amoxicillin (AX) combined or not with clavulanic acid (CLV) is frequently involved in IgE-mediated reactions. Drug provocation test (DPT) is considered as the gold standard for diagnosis, although contraindicated in high-risk patients. Basophil activation test (BAT) can help diagnose immediate reactions to beta-lactams, although controversy exists regarding the best activation marker. We have performed a real-life study in a prospective cohort to analyze the real value of BAT as diagnostic tool and the best activation marker, CD63 and CD203c, for the evaluation of immediate reactions to these drugs. METHODS: We prospectively evaluated patients with a clinical suspicion of immediate reactions after AX or AX-CLV administration during a 6-year period. The allergological work-up was done following the EAACI recommendations. BAT was performed in all patients using CD63 and CD203c as activation markers. RESULTS: In AX-allergic patients, both activation markers, CD63 and CD203c, showed similar SE values (48.6% and 46.7%, respectively); however, specificity was of 81.1% and 94.6%, respectively, with CD203c showing good positive predictive value and like-hood ratio. In CLV-allergic patients, CD203c showed higher SE (50%) than CD63 (42.9%), maintaining the same value of SP (80%). Combining the results of both markers can slightly increase the sensitivity (51.4% for AX and 54.8% for CLV), although decreasing the specificity (79.7% and 73%, respectively). Interestingly, all patients with an anaphylactic shock showed a positive BAT to CLV using CD203c. CONCLUSIONS: BAT using CD203c showed a good confirmatory power, especially for AX allergy. Placing BAT as a first step in the diagnostic procedure can help reduce the need of performing a complete allergological work-up in 46.6% of patients, diminishing the risk of reinducing allergic reactions.

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.

Clinical impact and immunological alterations in asthmatic patients allergic to grass pollen subjected to high urban pollution in Madrid.

BACKGROUND: The prevalence of asthma has increased in recent decades. Among the reasons for this increase is environmental pollution. Pollutants cause bronchial inflammation and introduce modifications in the pollen, making it more allergenic. OBJECTIVE: Assess symptoms and medication requirements of asthmatic patients with grass allergies in Madrid (high urban pollution) and Ciudad Real (low pollution), and simultaneously evaluate the in vitro effects that pollen collected in both areas has on the immune cells of patients. METHODS: During two pollen seasons, patients from both cities were included. The patients recorded their symptoms and the asthma medication they took daily. In both cities, pollen data, pollutants and meteorological variables were evaluated. The response to different cell populations from patients in both areas were analysed after "in vitro" stimulation with pollen from both cities. RESULTS: The symptoms and medication use of the patients in Madrid was 29.94% higher. The NO2 concentration in Madrid was triple that of Ciudad Real (33.4 vs. 9.1 µg/m3 of air). All other pollutants had very similar concentrations during the study period. Pollen from the high pollution area caused a significant enhancement of T-CD8+ and NK cells proliferation compared with pollen of low pollution area, independently of the patient's origin. CONCLUSION: Asthmatic patients from Madrid have a worse clinical evolution than those from Ciudad Real because of higher levels of urban pollution, and this could be driven by the higher capacity of pollen of Madrid to activate T-CD8+ and NK cells.

The value of the basophil activation test in the evaluation of patients reporting allergic reactions to the BNT162b2 mRNA COVID-19 vaccine.

BACKGROUND: mRNA-based COVID-19 vaccines have been reported to induce hypersensitivity reactions (HSR) in a small number of individuals. We aimed to evaluate the real-world incidence of the BNT162b2 mRNA COVID-19 vaccine HSR and to determine the value of the basophil activation test (BAT) in the allergological workup of patients reporting these reactions. METHODS: We prospectively enrolled patients with a clinical history indicative of HSR to the BNT162b2 mRNA COVID-19 vaccine. The allergological workup included skin testing (STs) and BAT with polyethylene glycol (PEG) and the vaccine. In those with negative allergy assessments, the administration of the second dose of the BNT162b2 mRNA COVID-19 vaccine was offered. RESULTS: Seventeen adults were included. Eleven cases (64.7%) tested negative in the allergological workup and tolerated the re-administration of the second dose of the vaccine and considered non-allergic. Six cases (35.3%) were considered allergic and classified into three groups: 2 subjects displayed positive STs and/or BAT to PEG (Group A), two individuals displayed positive BAT to the vaccine (Group B), and in 2 patients with moderate or severe reactions, the culprit was not identified, tested negative to STs and BAT to both PEG and vaccine (Group C). We further evaluated the value of BAT when the results were positive to the vaccine and negative to PEG by performing BAT in controls groups, finding positive BAT results in 50% of controls, all of them recovered from COVID-19 infection. In contrast, BAT was negative in patients who had not suffered from COVID-19 disease. CONCLUSIONS: BAT can be used as a potential diagnostic tool for confirming allergy to PEG excipient but not to the vaccine as a positive result in BAT may indicate a past COVID-19 infection instead of an allergy.

Drug hypersensitivity, in vitro tools, biomarkers, and burden with COVID-19 vaccines.

Hypersensitivity reactions to drugs are increasing worldwide. They display a large degree of variability in the immunological mechanisms involved, which impacts both disease severity and the optimal diagnostic procedure. Therefore, drug hypersensitivity diagnosis relies on both in vitro and in vivo assessments, although most of the methods are not well standardized. Moreover, several biomarkers can be used as valuable parameters for precision medicine that provide information on the endotypes, diagnosis, prognosis, and prediction of drug hypersensitivity development, as well on the identification of therapeutic targets and treatment efficacy monitoring. Furthermore, in the last 2 years, the SARS-CoV-2 (severe acute respiratory syndrome-coronavirus) pandemic has had an important impact on health system, leading us to update approaches on how to manage hypersensitivity reactions to drugs used for its treatment and on COVID-19 (Coronavirus disease) vaccines used for its prevention. This article reviews recent advances in these 3 areas regarding drug hypersensitivity: in vitro tools for drug hypersensitivity diagnosis, recently identified biomarkers that could guide clinical decision making and management of hypersensitivity reactions to drugs and vaccines used for COVID-19.

Advances and highlights in T and B cell responses to drug antigens.

The immunological mechanisms involved in drug hypersensitivity reactions (DHRs) are complex, and despite important advances, multiple aspects remain poorly understood. These not fully known aspects are mainly related to the factors that drive towards either a tolerant or a hypersensitivity response and specifically regarding the role of B and T cells. In this review, we focus on recent findings on this knowledge area within the last 2 years. We highlight new evidences of covalent and non-covalent interactions of drug antigen with proteins, as well as the very first characterization of naturally processed flucloxacillin-haptenated human leukocyte antigen (HLA) ligands. Moreover, we have analysed new insights into the identification of risk factors associated with the development of DHRs, such as the role of oxidative metabolism of drugs in the activation of the immune system and the discovery of new associations between DHRs and HLA variants. Finally, evidence of IgG-mediated anaphylaxis in humans and the involvement of specific subpopulations of effector cells associated with different clinical entities are also topics explored in this review. All these recent findings are relevant for the underlying pathology mechanisms and advance the field towards a more precise diagnosis, management and treatment approach for DHRs.

Sequential class switch recombination to IgE and allergen-induced accumulation of IgE+ plasmablasts occur in the nasal mucosa of local allergic rhinitis patients.

BACKGROUND: The involvement of allergen-specific (s)IgE in local allergic rhinitis (LAR) has been debated. Here, we investigate the effect of nasal allergen challenge with Dermatophagoides pteronyssinus (NAC-DP) in mucosal and peripheral B-cell subpopulations in LAR patients. METHODS: Nine LAR, 5 allergic rhinitis (AR), and 5 non-atopic healthy control (HC) individuals were subjected to a 3-day NAC-DP protocol, and nasal biopsies and blood samples were collected before and after provocation. Nasal biopsies were used for immunohistochemistry and gene expression studies, whereas the frequency of lymphocyte subsets and basophil activation test (BAT) were analyzed in blood samples by flow cytometry. sIgG was measured in sera. RESULTS: NAC-DP induced an increase in IgE+ CD38+ plasmablasts in the nasal mucosa of LAR patients, but not in AR or HC individuals. Markers of sequential recombination to IgE (εCSR) (from IgG) were observed in 33% of LAR, 20% of AR, and 0% of HC subjects. NAC-DP increased the proportion of peripheral CD19+ CD20+ CD38+ plasmablasts in AR and LAR patients, but not in HC. Expression of the mucosal homing receptor CXCR3 in peripheral CD19+ CD20+ CD38+ plasmablasts from LAR, AR, and HC individuals was 7%, 5%, and 0.5%, respectively. In vitro DP stimulation increased proliferating CD19+ CD20+ CD38+ plasmablasts in LAR and AR patients, but not in HC. Serum DP-sIgG was higher in LAR and AR patients as compared to HC. BAT was positive in 33%, 100%, and 0% of LAR, AR, and HC subjects, respectively. CONCLUSION: These results suggest that allergen exposure induces the sequential εCSR of IgG+ CD19+ CD20+ CD38+ plasmablasts in the nasal mucosa of LAR patients.

Next-generation sequencing and genotype association studies reveal the association of HLA-DRB3*02:02 with delayed hypersensitivity to penicillins.

BACKGROUND: Nonimmediate (delayed)-allergic reactions to penicillins are common and some of them can be life-threatening. The genetic factors influencing these reactions are unknown/poorly known/poorly understood. We assessed the genetic predictors of a delayed penicillin allergy that cover the HLA loci. METHODS: Using next-generation sequencing (NGS), we genotyped the MHC region in 24 patients with delayed hypersensitivity compared with 20 patients with documented immediate hypersensitivity to penicillins recruited in Italy. Subsequently, we analyzed in silico Illumina Immunochip genotyping data that covered the HLA loci in 98 Spanish patients with delayed hypersensitivity and 315 with immediate hypersensitivity compared to 1,308 controls. RESULTS: The two alleles DRB3*02:02:01:02 and DRB3*02:02:01:01 were reported in twenty cases with delayed reactions (83%) and ten cases with immediate reactions (50%), but not in the Allele Frequency Net Database. Bearing at least one of the two alleles increased the risk of delayed reactions compared to immediate reactions, with an OR of 8.88 (95% CI, 3.37-23.32; p < .0001). The haplotype (ACAA) from rs9268835, rs6923504, rs6903608, and rs9268838 genetic variants of the HLA-DRB3 genomic region was significantly associated with an increased risk of delayed hypersensitivity to penicillins (OR, 1.7; 95% CI: 1.06-1.92; p = .001), but not immediate hypersensitivity. CONCLUSION: We showed that the HLA-DRB3 locus is strongly associated with an increased risk of delayed penicillin hypersensitivity, at least in Southwestern Europe. The determination of HLA-DRB3*02:02 alleles in the risk management of severe delayed hypersensitivity to penicillins should be evaluated further in larger population samples of different origins.

Synthetic antigenic determinants of clavulanic acid induce dendritic cell maturation and specific T cell proliferation in patients with immediate hypersensitivity reactions.

BACKGROUND: Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests. METHODS: Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry. RESULTS: The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4+ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%. CONCLUSIONS: Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4+ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.

Omics technologies in allergy and asthma research: An EAACI position paper.

Allergic diseases and asthma are heterogenous chronic inflammatory conditions with several distinct complex endotypes. Both environmental and genetic factors can influence the development and progression of allergy. Complex pathogenetic pathways observed in allergic disorders present a challenge in patient management and successful targeted treatment strategies. The increasing availability of high-throughput omics technologies, such as genomics, epigenomics, transcriptomics, proteomics, and metabolomics allows studying biochemical systems and pathophysiological processes underlying allergic responses. Additionally, omics techniques present clinical applicability by functional identification and validation of biomarkers. Therefore, finding molecules or patterns characteristic for distinct immune-inflammatory endotypes, can subsequently influence its development, progression, and treatment. There is a great potential to further increase the effectiveness of single omics approaches by integrating them with other omics, and nonomics data. Systems biology aims to simultaneously and longitudinally understand multiple layers of a complex and multifactorial disease, such as allergy, or asthma by integrating several, separated data sets and generating a complete molecular profile of the condition. With the use of sophisticated biostatistics and machine learning techniques, these approaches provide in-depth insight into individual biological systems and will allow efficient and customized healthcare approaches, called precision medicine. In this EAACI Position Paper, the Task Force "Omics technologies in allergic research" broadly reviewed current advances and applicability of omics techniques in allergic diseases and asthma research, with a focus on methodology and data analysis, aiming to provide researchers (basic and clinical) with a desk reference in the field. The potential of omics strategies in understanding disease pathophysiology and key tools to reach unmet needs in allergy precision medicine, such as successful patients' stratification, accurate disease prognosis, and prediction of treatment efficacy and successful prevention measures are highlighted.

Allergies and COVID-19 vaccines: An ENDA/EAACI Position paper.

BACKGROUND: Anaphylaxis, which is rare, has been reported after COVID-19 vaccination, but its management is not standardized. METHOD: Members of the European Network for Drug Allergy and the European Academy of Allergy and Clinical Immunology interested in drug allergy participated in an online questionnaire on pre-vaccination screening and management of allergic reactions to COVID-19 vaccines, and literature was analysed. RESULTS: No death due to anaphylaxis to COVID-19 vaccines has been confirmed in scientific literature. Potential allergens, polyethylene glycol (PEG), polysorbate and tromethamine are excipients. The authors propose allergy evaluation of persons with the following histories: 1-anaphylaxis to injectable drug or vaccine containing PEG or derivatives; 2-anaphylaxis to oral/topical PEG containing products; 3-recurrent anaphylaxis of unknown cause; 4-suspected or confirmed allergy to any mRNA vaccine; and 5-confirmed allergy to PEG or derivatives. We recommend a prick-to-prick skin test with the left-over solution in the suspected vaccine vial to avoid waste. Prick test panel should include PEG 4000 or 3500, PEG 2000 and polysorbate 80. The value of in vitro test is arguable. CONCLUSIONS: These recommendations will lead to a better knowledge of the management and mechanisms involved in anaphylaxis to COVID-19 vaccines and enable more people with history of allergy to be vaccinated.

Basophil Activation Test Utility as a Diagnostic Tool in LTP Allergy.

Plant-food allergy is an increasing problem, with nonspecific lipid transfer proteins (nsLTPs) triggering mild/severe reactions. Pru p 3 is the major sensitizer in LTP food allergy (FA). However, in vivo and in vitro diagnosis is hampered by the need for differentiating between asymptomatic sensitization and allergy with clinical relevance. The basophil activation test (BAT) is an ex vivo method able to identify specific IgE related to the allergic response. Thus, we aimed to establish the value of BAT in a precise diagnosis of LTP-allergic patients. Ninety-two individuals with peach allergy sensitized to LTP, Pru p 3, were finally included, and 40.2% of them had symptoms to peanut (n = 37). In addition, 16 healthy subjects were recruited. BAT was performed with Pru p 3 and Ara h 9 (peanut LTP) at seven ten-fold concentrations, and was evaluated by flow cytometry, measuring the percentage of CD63 (%CD63+) and CD203c (%CD203chigh) cells, basophil allergen threshold sensitivity (CD-Sens), and area under the dose−response curve (AUC). Significant changes in BAT parameters (%CD63+ and %CD203chigh) were found between the controls and patients. However, comparisons for %CD63+, %CD203chigh, AUC, and CD-Sens showed similar levels among patients with different symptoms. An optimal cut-off was established from ROC curves, showing a significant positive percentage of BAT in patients compared to controls and great values of sensitivity (>87.5%) and specificity (>85%). In addition, BAT showed differences in LTP-allergic patients tolerant to peanut using its corresponding LTP, Ara h 9. BAT can be used as a potential diagnostic tool for identifying LTP allergy and for differentiating peanut tolerance, although neither reactivity nor sensitivity can distinguish the severity of the clinical symptoms.

Detection of Serum-Specific IgE by Fluoro-Enzyme Immunoassay for Diagnosing Type I Hypersensitivity Reactions to Penicillins.

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins.

Role of nanostructures in allergy: Diagnostics, treatments and safety.

Nanotechnology is science, engineering and technology conducted at the nanoscale, which is about 1-100 nm. It has led to the development of nanomaterials, which behave very differently from materials with larger scales and can have a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition and functionalization of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers and nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalization with ligands, which provide the desired interaction with the immunological system, that is dendritic cell receptors to activate and/or modulate the response, as well as specific IgE, or effector cell receptors. However, undesired issues related to toxicity and hypersensitivity responses can also happen and would need evaluation. There are wide panels of accessible structures, and controlling their physico-chemical properties would permit obtaining safer and more efficient compounds for clinical applications goals, either in diagnosis or treatment. The application of dendrimeric antigens, nanoallergens and nanoparticles in allergy diagnosis is very promising since it can improve sensitivity by increasing specific IgE binding, mimicking carrier proteins or enhancing signal detection. Additionally, in the case of immunotherapy, glycodendrimers, liposomes, polymers and nanoparticles have shown interest, behaving as platforms of allergenic structures, adjuvants or protectors of allergen from degradation or having a depot capacity. Taken together, the application of nanotechnology to allergy shows promising facts facing important goals related to the improvement of diagnosis as well as specific immunotherapy.

Single-dose prolonged drug provocation test, without previous skin testing, is safe for diagnosing children with mild non-immediate reactions to beta-lactams.

INTRODUCTION: Mild non-immediate reactions (NIRs) to beta-lactams (BLs) are the most frequent manifestation of drug allergy in children. The diagnostic approach is complex as the utility of skin tests (STs) and lymphocyte transformation tests (LTTs) is controversial. Drug provocation test (DPT) is the gold standard, although no standardized protocols exist. We aimed to investigate the utility of DPT in a unique dose without previous STs, and LTTs in the diagnosis of NIRs to BLs in children. METHODS: We prospectively evaluated children 0-14 years old referred to the Regional University Hospital of Málaga during 2017-2020 reporting NIRs to BLs. We performed a DPT with a unique dose followed by regular treatment at home. If positive, STs and LTTs were done after the reaction had disappeared. RESULTS: We included 194 children, having 24 (12.4%) a positive DPT. The main culprit was AX (70.1%) followed by AX-clavulanic acid (CLV) (26.8%) and the main symptoms maculopapular exanthema (MPE) (49.5%) and delayed-urticaria (48.5%). A decrease (p = 0.013) in the interval of days between drug administration and onset of symptoms was observed in positive DPT compared with the original reaction (3.5 vs 6 days), with no differences in the overall percentage of MPE and delayed-appearing urticaria (p = 0.551). No severe reactions occurred during DPT. Moreover, STs were positive in 13.33% and LTTs in 52.9%. CONCLUSIONS: Single-dose DPT without previous STs is a safe and useful way to assess NIRs to BLs in children. LTT has shown to be useful, confirming a T-cell mechanism involved in these reactions.

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.

Dendritic cells inclusion and cell-subset assessment improve flow-cytometry-based proliferation test in non-immediate drug hypersensitivity reactions.

BACKGROUND: Lymphocyte transformation test (LTT) has been widely used to evaluate non-immediate drug hypersensitivity reactions (NIDHRs). However, the lack of standardization and the low sensitivity have limited its routine diagnostic use. The drug presentation by dendritic cells (DCs) and the assessment of proliferation on effector cells have shown promising results. Flow-cytometry-based methods can help apply these improvements. We aimed to assess the added value of using drug-primed-DCs and the determination of the proliferative response of different lymphocyte subpopulations in NIDHRs. METHODS: Patients with confirmed NIDHR were evaluated by both conventional (C-LTT) and with drug-primed-DCs LTT (dDC-LTT)analysing the proliferative response in T cells and other effector cell subpopulations by using the fluorescent molecule, carboxyfluorescein diacetate succinimidyl ester (CFSE). RESULTS: The C-LTT showed a significantly lower sensitivity (29.4%) compared with dDC-LTT (61.8%), which was confirmed analysing each particular clinical entity: SJS-TEN (62.5% vs 87.5%), MPE (15% vs 47.4%) and AGEP (33% vs 80%). When including the effector cell subpopulations involved in each clinical entity, CD3+ +CD4+ Th 1 or CD3+ +NK cells in SJS-TEN, CD3+ +CD4+ Th 1+NK cells in MPE and CD3+ +NK cells in AGEP, we could significantly increase the sensitivity of the in vitro test to 100%, 68.4% and 100%, respectively, with an overall sensitivity of 87% and 85% of specificity in NIDHR. CONCLUSIONS: The use of a flow-cytometry-based test, DCs as drug presenting cells, and focusing on effector cell subpopulations for each clinical entity significantly improved the drug-specific proliferative response in NIDHRs with a unique cellular in vitro test.