Nanoparticle size influences the proliferative responses of lymphocyte subpopulations

International audience12 nm gold nanoparticles induce cell mediated responses accompanied by inflammatory natural killer cell stimulation, whereas 2 nm gold nanoparticles are more efficiently uptaken without inducing dendritic cell maturation or lymphocyte proliferation

Direct characterization of functional materials by haptenized fluorescent dendrimers for in vitro allergic drug diagnose

β-lactams are the most widely drug prescribed against infections, but they are the most commonly reported medication allergy too. A correct diagnosis of these allergic reactions is crucial to avoid rejecting them by other more expensive broad-spectrum antibiotics, with potential risk factors for the development of multiple drug-resistant bacteria. [1] Skin testing is the most consensual approach to diagnose β-lactam allergy, but this in vivo test is not risky free and should be performed under strict hospital surveillance.[2] In vitro testing is not still widely used on account of their low sensitivity. We report the use of already haptenized fluorescent dendrimers [3] to control the preparation of materials for in vitro test, and their verification by testing on patient sera samples. This fluorescent dendrimer is obtained from a dye with two orthogonal functional groups suitable for click chemistry. [4] Acknowledgments: This work was supported by: MINECO CTQ2016-75870P, Andalusian Regional Ministry Health (PI-0250-2016); European Regional Development Fund and “Plan Propio Universidad de Málaga” (UMA-Andalucía-TECH).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

New Approaches in the Manufacture of Biomaterials for Betalactam Allergic Diagnose

Betalactams are the most widely utilized drugs against infections but are the primary cause of allergic reactions to antibiotic drugs. REF1 An accurate diagnosis of these allergic reactions to betalactams is crucial to avoid the use of unnecessary alternative antibiotics that may reduce efficacy, lead to prolonged treatments, have a higher toxicity or induce bacterial resistance. The most consensual approach to diagnose betalactam allergy are in vivo tests. However, they are not risky free, require experienced personnel and are both time-consuming and expensive for health-care systems, being so in vitro test more appropriate or complementary to the in vivo tests. In vitro tests are not still widely used on account of their low sensitivity. Current efforts are in progress to improve these assays, thus allowing for better diagnosis of allergic responses within patients. REF 2 We report progress in the preparation of new functional materials for in vitro allergic diagnosis testing. In particular, the application of new approaches employing orthogonally functionalised fluorescent dyes based upon 4-amino-1,8 naphthalimide joined with the multivalence of polyamide dendrimers. REF 3 The in vitro diagnosis capabilities of these functional materials was verified by testing on patient sera samples, with results demonstrating their potential for application within the healthcare industry. Acknowledgments: The present study has been supported by MINECO CTQ2016-75870P; by Andalusian Regional Ministry Health (grants: PI-0250-2016); by the European Regional Development Fund (ERDF) and “Plan Propio Universidad de Málaga” (UMA-Andalucía-TECH).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Usefulness of myeloid dendritic cells in cellular in vitro assays for evaluating immediate hypersensitivity reactions to betalactams.

Introduction Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) with an important role detecting, processing, and presenting antigens to T cells. The analysis of maturation after in vitro stimulation with the culprit drug, and their ability to trigger the proliferation of specific T cell populations in patients with immediate drug hypersensitivity reactions (IDHRs) could serve to prove the sensitization to a drug. Most approaches used monocyte-derived DCs (moDCs), although their sensitivity is not optimal. The different nature of moDCs and myeloid DCs (mDCs), main DC population involved during the in vivo development of IDHRs, could influence the sensitivity of these in vitro tests. Therefore, we evaluated the effect of two betalactams (BLs), amoxicillin (AX) and clavulanic acid (CLV) in moDCs and mDCs from selective-allergic patients (AP) to each BL, as well as to assess their capacity to stimulate different T cell populations. Methods mDCs and moDCs were obtained from 14 AX-AP, 14 CLV-AP and 10 Healthy controls (HC). After stimulating with the culprit BL, maturation and their capacity to stimulate different T cell populations was assessed by flow cytometry. Results Higher maturation was observed in both AX- and CLV-AP compared with HC when using BL-stimulated-mDCs, whereas with moDCs, only higher was observed in AX-AP, but not in CLV-AP. The % of positive maturation cases was higher with mDCs than moDCs, with higher % with AX compared to CLV. The most relevant T cell population proliferative response was obtained in CD4+Th2 cells, reaching to 67% of positivity when using mDCs, followed by 50% with the traditional LTT, and only of 22% with moDCs from AX-AP. The specificity was higher than 80% in all cases. Conclusions mDCs from selective AP efficiently recognised the culprit drug and triggered the proliferation of T-cells, mainly those with a Th2 cytokine pattern, although these responses depended on the drug.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Influence of pore size in protein G'-grafted mesoporous silica nanoparticles as a serum pretreatment system for in vitro allergy diagnosis

Particles with the capacity to bind to immunoglobulin G (IgG) can be used for the purification of IgG or to process clinical samples for diagnostic purposes. For in vitro allergy diagnosis, the high IgG levels in serum can interfere with the detection of allergen-specific IgE, the main diagnostic biomarker. Although commercially available, current materials present a low IgG capture capacity at large IgG concentrations or require complex protocols, preventing their use in the clinic. In this work, mesoporous silica nanoparticles are prepared with different pore sizes, to which IgG-binding protein G’ is grafted. It is found that for one particular optimal pore size, the IgG capture capacity of the material is greatly enhanced. The capacity of this material to efficiently capture human IgG in a selective way (compared to IgE) is demonstrated in both solutions of known IgG concentrations as well as in complex samples, like serum, from healthy controls and allergic patients using a simple and fast incubation protocol. Interestingly, IgG removal using the best-performing material enhances in vitro IgE detection in sera from patients allergic to amoxicillin. These results highlight the great translation potential of this strategy to the clinic in the context of in vitro allergy diagnosis.Funding for Open Access charge: Universidad de Málaga/CBUA. TEM experiments were performed in the ICTS “NANBIOSIS,” more specifically in the U28 Unit at IBIMA Plataforma BIONAND

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

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.We thank Claudia Corazza for her invaluable English language support; Verónica Prados and Ana Molina for their help in technical support in flow cytometry methods. This work has been supported by Institute of Health “Carlos III” (ISCIII) of the Ministry of Economy and Competitiveness (MINECO; grants co-funded by European Regional Development Fund: PI15/01206, PI17/01237, PI18/00095, PI20/01734, RETICS ARADYAL RD16/0006/0001, and RICORS REI (RD21/0002/0008); Andalusian Regional Ministry of Health (grants PI-0241-2016, PE-0172-2018, and PI-0127-2020); Spanish Ministerio de Ciencia e Innovación Proyectos de I + D + I «Programación Conjunta Internacional», EuroNanoMed 2019 (PCI2019-111825-2)). AA holds a Senior Postdoctoral Contract (RH-0099-2020) with the Andalusian Regional Ministry of Health (cofunded by European Social Fund (ESF): "Andalucía se mueve con Europa". GB holds a “Juan Rodes” contract (JR18/00054) by ISCIII of MINECO (cofounded by ESF). ID holds a clinical research stabilization contract by Andalusian Regional Ministry of Health (RB-0001-2022). ML holds a “Rio Hortega” contract (CM20/00210) by ISCIII of MINECO (cofounded by ESF). CF holds a Marie Skłodowska-Curie Individual Fellowship by the European Union's Horizon 2020 research and innovation program (agreement n° PI-0241-2016101027955). CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry of Health (RC-0004-2021). // Funding for open access charge: Universidad de Málaga / CBUA

Silica Particle based nanocomposites for Specific IgE Determination to Betalactams

Current immunoassays for diagnosing betalactam (BL) allergy have limited sensitivity and poor specificity. These include ImmunoCAP, the commercial immunoassay, and the radioallergosorbent test (RAST), a handmade test performed only in specialized laboratories. Both are based on cellulose polymers modified with covalently attachedbetalactam-poly-L-lysine conjugates, which are able to interact with specific IgE (sIgE). In RAST, dendrimers have been used instead of poly-L-lysine in the formation of penicilloyl- conjugates, showing specific recognition by penicillin-sIgE. In this work, we propose the use of nanomaterials consisting on silica particles highly functionalized with drug-dendrimer conjugates (including mono- and bi-epitopic structures), as precise and controlled solid phases able to improve the tests’ reliability. The homogeneous composition of the nano-composites provides high reproducibility and quality, which is critical for hospital applications, while silica particles possess promising properties as biocompatibility, tuneable size, and functionalisation, and their preparation is affordable and reproducible. The use of nano-scaled silica particles improved the results of available immunoassays techniques in terms of sensitivity and specificity, providing the possibility of testing different betalactams, simultaneously.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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 penicillinsThis research was funded by the Institute of Health ‘Carlos III’ (ISCIII) of the Ministry of Economy and Competitiveness (MINECO) (grants cofunded by European Regional Development Fund: PI15/01206, PI17/01237, PI18/00095, RETICS ARADYAL RD16/0006/0001). Andalusian Regional Ministry of Health (grants PE-0172-2018, PI-0127-2020). DrNanoDall project by ISCIII thorough AES 2019 within the ERANET-EuroNanoMed-III framework (AC19/00082). AA holds a Senior Postdoctoral Contract (RH-0099-2020) with the Andalusian Regional Ministry of Health (cofunded by European Social Fund (ESF): “Andalucía se mueve con Europa”). ML holds a “Rio Hortega” contract (CM20/00210), GB and N.P.-S. hold a “Juan Rodés” (JR18/00054 and JR21/00024, respectively) with ISCIII of MINECO (cofunded by ESF). CM holds a ‘Nicolas Monardes’ research contract with the Andalusian Regional Ministry Health (RC-0004-2021). Partial funding for open access charge: Universidad de Málag