The influence of the carrier molecule on amoxicillin recognition by specific IgE in patients with immediate hypersensitivity reactions to betalactams

10 p.-4 fig.-1 tab.The optimal recognition of penicillin determinants, including amoxicillin (AX), by specific IgE antibodies is widely believed to require covalent binding to a carrier molecule. The nature of the carrier and its contribution to the antigenic determinant is not well known. Here we aimed to evaluate the specific-IgE recognition of different AX-derived structures. We studied patients with immediate hypersensitivity reactions to AX, classified as selective or cross-reactors to penicillins. Competitive immunoassays were performed using AX itself, amoxicilloic acid, AX bound to butylamine (AXO-BA) or to human serum albumin (AXO-HSA) in the fluid phase, as inhibitors, and amoxicilloyl-poli-L-lysine (AXO-PLL) in the solid-phase. Two distinct patterns of AX recognition by IgE were found: Group A showed a higher recognition of AX itself and AX-modified components of low molecular weights, whilst Group B showed similar recognition of both unconjugated and conjugated AX. Amoxicilloic acid was poorly recognized in both groups, which reinforces the need for AX conjugation to a carrier for optimal recognition. Remarkably, IgE recognition in Group A (selective responders to AX) is influenced by the mode of binding and/or the nature of the carrier; whereas IgE in Group B (cross-responders to penicillins) recognizes AX independently of the nature of the carrier.The present study has been supported by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF): PI12/02529, PI15/01206, CP15/00103,Red de Reacciones Adversas a Alergenos y Farmacos RD12/0013/0001, RD12/0013/0003 and RD12/0013/0008,RD09/0076/00112 for the Biobank network and PT13/0010/0006 for the Biobank platform) and by State Secretariat for Research, Development and Innovation of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF): MINECO SAF2012-36519, SAF2015-68590-R/FEDER and CTQ2013-41339-P). Andalusian Regional Ministry of Economy and Knowledge (grants cofunded by European Regional Development Fund (ERDF): CTS-06603); Andalusian Regional Ministry Health (grants:PI-0699-2011, PI-0159-2013 and PI-0179-2014) and Merck-Serono Research Grant from Fundación Salud 2000. CM holds a ‘Nicolas Monardes’ research contract by Andalusian Regional Ministry Health: C-0044-2012 SAS 2013. MIM holds a ‘Miguel Servet I’ research contract by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Social Fund (ESF)): CP15/00103. AA thanks “pFIS fellowship” (FI08/00385) from ISCIII and Andalucia “Talent Hub Fellowship” (TAHUB/II-004) cofunded by the Junta de Andalucia and the European Union, VII Framework Programme of the European Commission (grant agreement No. 291780).Peer reviewe

Radiographers supporting radiologists in the interpretation of screening mammography: a viable strategy to meet the shortage in the number of radiologists.

BackgroundAn alternative approach to the traditional model of radiologists interpreting screening mammography is necessary due to the shortage of radiologists to interpret screening mammograms in many countries.MethodsWe evaluated the performance of 15 Mexican radiographers, also known as radiologic technologists, in the interpretation of screening mammography after a 6 months training period in a screening setting. Fifteen radiographers received 6 months standardized training with radiologists in the interpretation of screening mammography using the Breast Imaging Reporting and Data System (BI-RADS) system. A challenging test set of 110 cases developed by the Breast Cancer Surveillance Consortium was used to evaluate their performance. We estimated sensitivity, specificity, false positive rates, likelihood ratio of a positive test (LR+) and the area under the subject-specific Receiver Operating Characteristic (ROC) curve (AUC) for diagnostic accuracy. A mathematical model simulating the consequences in costs and performance of two hypothetical scenarios compared to the status quo in which a radiologist reads all screening mammograms was also performed.ResultsRadiographer's sensitivity was comparable to the sensitivity scores achieved by U.S. radiologists who took the test but their false-positive rate was higher. Median sensitivity was 73.3 % (Interquartile range, IQR: 46.7-86.7 %) and the median false positive rate was 49.5 % (IQR: 34.7-57.9 %). The median LR+ was 1.4 (IQR: 1.3-1.7 %) and the median AUC was 0.6 (IQR: 0.6-0.7). A scenario in which a radiographer reads all mammograms first, and a radiologist reads only those that were difficult for the radiographer, was more cost-effective than a scenario in which either the radiographer or radiologist reads all mammograms.ConclusionsGiven the comparable sensitivity achieved by Mexican radiographers and U.S. radiologists on a test set, screening mammography interpretation by radiographers appears to be a possible adjunct to radiologists in countries with shortages of radiologists. Further studies are required to assess the effectiveness of different training programs in order to obtain acceptable screening accuracy, as well as the best approaches for the use of non-physician readers to interpret screening mammography

Jardins per a la salut

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia. Assignatura: Botànica farmacèutica. Curs: 2014-2015. Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són el recull de les fitxes botàniques de 128 espècies presents en el Jardí Ferran Soldevila de l’Edifici Històric de la UB. Els treballs han estat realitzats manera individual per part dels estudiants dels grups M-3 i T-1 de l’assignatura Botànica Farmacèutica durant els mesos de febrer a maig del curs 2014-15 com a resultat final del Projecte d’Innovació Docent «Jardins per a la salut: aprenentatge servei a Botànica farmacèutica» (codi 2014PID-UB/054). Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pels professors de l’assignatura. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica. També s’ha pretès motivar els estudiants a través del retorn de part del seu esforç a la societat a través d’una experiència d’Aprenentatge-Servei, deixant disponible finalment el treball dels estudiants per a poder ser consultable a través d’una Web pública amb la possibilitat de poder-ho fer in-situ en el propi jardí mitjançant codis QR amb un smartphone

Hypersensitivity to fluoroquinolones: The expression of basophil activation markers depends on the clinical entity and the culprit fluoroquinolone.

Although fluoroquinolones (FQs) are generally well-tolerated antibiotics, increasing numbers of hypersensitivity reactions have been reported. These can be evaluated in vitro by basophil activation tests (BATs); however, sensitivity is not optimal. Many factors could influence sensitivity such as basophil activation markers. The objective of this study was to evaluate the influence of 2 different activations markers, CD63 and CD203c, on the sensitivity of BAT to FQ. We studied 17 patients with immediate allergic reactions to FQ. BAT was performed with moxifloxacin and ciprofloxacin using CD193 (CCR3) for basophil selection and CD203c or CD63 as activation markers. Stimulation with ciprofloxacin induced a significantly higher expression of CD63 in ciprofloxacin-allergic patients compared to moxifloxacin-allergic patients (P = 0.002). In patients allergic to moxifloxacin with anaphylactic shock, we have observed an increase in the percentage of cells that upregulate CD203c, whereas patients with anaphylaxis preferentially upregulate CD63. The best sensitivity-specificity was obtained using a cutoff of 3 and the culprit FQ, using CD203c for moxifloxacin-allergic patients (sensitivity = 36.4%; specificity = 94.4%), and CD63 for ciprofloxacin-allergic patients (sensitivity = 83.3%; specificity = 88.9%). A negative correlation was found between the upregulation of CD63 and CD203c and the time interval between the reaction occurrence and the performance of the test (Spearman r = -0.446; P

Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams

Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency

16 p.-7 fig.-2 tab.-1 graph. abst.Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.This work was supported by grant SAF2015-68590-R from MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal from ISCIII/FEDER RD16/0006/0021 to DP-S; RD16/0006/0001 to MT, RD16/0006/0024 to MB; grants CP15/00103 and PI17/ 01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian Regional Ministry Health to MM. AA holds a “Sara Borrell” research contract (CD17/0146) supported by ISCIII from MINECO [confounded by the European Social Fund (ESF)]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Amoxicillin haptenation of α-enolase is modulated by active site occupancy and acetylation

17 p.-8 fig.-1 tab.Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.This work was supported by grants from the Ministerio de Ciencia e Innovación cofunded by ERDF (SAF2015-68590-R and RTI2018-097624-B-I00 to DPS and PCI2019-111825-2 Proyectos de I+D+I “Programación Conjunta Internacional” EuroNanoMed 2019 and PID2019-104293GB-I00 to EI), the Instituto de Salud Carlos III ERDF (RETIC ARADyAL RD16/0006/0021 to DPS, RETIC ARADyAL RD16/0006/0001 to MS and MIM, CPII20/00028 to MIM and RETIC ARADyAL RD16/0006/0012 to EPI, and CPII20/00028 to MIM), Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007 to EPI), Consejería de Transformación Económica, Industria, Conocimiento y Universidades of Junta de Andalucía (PY20_00384 to EPI). The funders had no role in study design, analysis of the data or decission to publish. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

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

Background: Amoxicillin (AX) is nowadays the β-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.European Union's H2020 research and innovation programme under the Marie Skłodowska-Curie, Grant/Award Number: 713721 Andalusian Regional Ministry Health Grant/Award Number: RC-0004-2021, PI-0699-2011 and PI-0179-2014 Institute of Health "Carlos III" Grant/Award Number PI12/02529, PI15/01206, PI18/00095, CP15/00103, PI17/01237, PI20/01734, PI20/01447, ARADYAL RD16/0006/0001 and Euronanomed Program AC19/00082Ye

Identification of an antigenic determinant of clavulanic acid responsible for IgE-mediated reactions

12 p.-6 fig.-1 graf. abst.BACKGROUND: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis.METHODS: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches.RESULTS: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls.CONCLUSION: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound.The present study has been supported by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF): PI12/02529, PI15/01206, CP15/00103, RETICs RIRAAF RD12/0013/0001, RD12/0013/0003 and RD12/0013/0008, RETIC ARADYAL RD16/0006/0001, RD16/0006/0012 and RD16/0006/0021, Biobank Network RD09/0076/00112, and Biobank platform PT13/0010/0006) and by State Secretariat for Research, Development and Innovation of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF), “Una manera de hacer Europa”: MINECO SAF2012-36519, SAF2015-68590R, CTQ2013-41339P, and CTQ2016-75870P). Andalusian Regional Ministry of Economy and Knowledge (grants cofunded by European Regional Development Fund (ERDF), “Andalucía se mueve con Europa”: CTS-06603); Andalusian Regional Ministry Health (grants: PI-0699-2011, PI-0159-2013, PI-0179-2014, PI-0241-2016, and PI-0250-2016), Merck-Serono Research Grant from Fundación Salud 2000 and “Premio UNICAJA a la innovación en biomedicina y salud.” CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry Health: C-0044-2012 SAS 2013. MIM holds a “Miguel Servet I” and GB holds a “Rio Hortega,” both research contracts by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Social Fund (ESF)): CP15/00103 and CM16/00067. TDF hold a “Ramon y Cajal” research contract from Ministry of Economy and Competitiveness (RYC-2013-13138).Peer reviewe