Allergic reactions to β-lactam antibiotics: chemical approaches for improving in vitro diagnosis

Moreover, conjugation of CLV with human serum albumin (HSA) was studied by liquid chromatography mass-spectrometry (LC/MS-MS) and a biotinylated derivative of CLV was used as tool for identification of serum proteins target of modification by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting. By the other hand, we pursued the amplification of immunoassays detection signal. With this objective, fluorescent dendrons allowing site-specific conjugation to biomolecules and bearing 1, 2 or 8 fluorescent units were designed and synthesized, and their suitability for labeling antibodies and fluorescence amplification capacity evaluated [6]. The main conclusions of this work have been that synthesized cyclized structures derived from cefaclor and cefadroxil (Cef1 and Cef2, respectively) are specifically recognised by sIgE from patients allergic to the corresponding cephalosporin or to penicillins containing the same R1 side chain, shedding light into the mechanism involved in allergy to α-aminocephalosporins and complementing studies previously reported [7, 8]. Also, the ability of our synthetic determinants of CLV to activate basophils from patients with immediate reactions to this drug has been demonstrated to be influenced by the chemical structure and their reactivity to proteins. Clav2, coming from AD-I, activates basophils in a higher proportion of patients compared to CLV itself, and thus represents a promising structure to improve the sensitivity of in vitro diagnosis. Furthermore, a 70 Da mass structure was found to modify Lys 195 and Lys 475 of HSA, being this mass compatible with that of AD-I, which confirms that Clav2 is part of the drug-protein conjugate. Moreover, a water soluble biotinylated derivative of CLV (CLV-TEG-B) showed concentration-dependent protein haptenation capacity in vitro, and made possible the identification of HSA, haptoglobin and heavy and light chains of immunoglobulins as potential serum proteins target of modification by CLV. Finally, synthesized fluorescent dendrons G0-Cy5, G1-Cy5 and G3-Cy5 showed an increment in fluorescence with the number of fluorescent units and they were proved to be suitable for site-specific labeling of a model antibody. The labeling with G1-Cy5 dendron (2 fluorescent units) resulted to be the most suitable for immunoassays signal amplification applications, since it was found to enhance fluorescence by one order of magnitude when compared with the antibody labeled with the monovalent probe G0-Cy5 [6]. Fecha de lectura Tesis Doctoral: 30 octubre 2018.Drug hypersensitivity reactions are a significant public health problem with important consequences on patient health and healthcare costs. It has been reported that only a low percentage of initial cases suspected of allergy to antibiotics are finally confirmed [1, 2], and betalactam antibiotics (BLs) are the drugs most frequently involved [3, 4]. In vivo tests (skin test and drug provocation test) are often the first and only option for diagnosis, nevertheless they could be risky for patients. Thus, in vitro tests are a more convenient and safer alternative for diagnosis, however, a sensitive and specific detection of specific IgE (sIgE), crucial for in vitro allergy diagnosis, is difficult to achieve in cases of allergy to drugs due to the extremely low sIgE concentration present in patients serum [5]. The sensitivity of in vitro tests for diagnosing allergy to BLs depends, among other factors, on: (i) the similarity between the structure used in the assay as emulator of the antigenic determinant (AD) formed in vivo and the structure actually formed after the BL intake, which is related to the mechanisms involved in the allergic process, and (ii) the intensity of the detection signal (radioactivity, enzimatic proccess or fluorescence) at low concentrations of sIgE to drugs. The general objective of this thesis is to carry out studies directed to improve current in vitro tests for diagnosing immediate allergic reactions to BLs. More specifically, by one hand, we aimed to research, from a chemical approach, the structures recognized by sIgE for cephalosporins and clavulanic acid (CLV) since there has been an increase of cases of allergy to these BLs in the last years, due to the change in prescription patterns [1]. For this purpose, structures derived from these BLs were designed and synthesized and their immunological evaluation performed using RadioAllergoSorbent Test (RAST) or Basophil Activation Test (BAT)

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

Potential anti-adhesion activity of novel carbosilane zwitterionic dendrimers against eukaryotic and prokaryotic pathogenic microorganisms

The development of biofilms on different surfaces continues to be a major public health problem. The antimicrobial resistance and the difficulty of finding drugs capable of combating these established biofilms generates the urgent need to find compounds that prevent cells from settling and establishing of these complex communities of microorganisms. Zwitterionic modification of nanomaterials allows the formation of a hydration layer, and this highly hydrophilic surface provides antifouling properties as well as a good biocompatibility by preventing non-specific interactions. Thus, they are appropriate candidates to prevent microbial adhesion to different surfaces and, in consequence, avoid biofilm formation. For this reason, we have incorporated zwitterionic moieties in multivalent systems, as are carbosilane dendrimers. Characterization of these systems was performed using nuclear magnetic resonance and mass spectrometry. It has been analysed if the new molecules have capacity to inhibit the biofilm formation in Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that they were more effective against S. aureus, observing a biofilm reduction of 81.5% treating with 32 mg/L of G2SiZWsf dendrimer and by 72.5% using 32 mg/L of the G3SiZWsf dendrimer. Finally, the absence of cytotoxicity was verified by haemolysis and cytotoxicity studies in human cells lines.Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), SpainMinisterio de Economía, Comercio y EmpresaComunidad de Madri

Diseño de un huerto escolar en un centro de educación infantil y primaria para la implementación de metodologías activas de aprendizaje en el marco de un programa interdisciplinar de docencia práctica en la Facultad de Educación

Memoria ID12-0283. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2012-2013

Conformación de una red de huertos escolares comunitarios: formación transversal para la sostenibilidad

Memoria ID-0093. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Diseño de mapas conceptuales como estrategia de representación y organización de conocimiento pedagógico. aplicación Grado de pedagogía.

Memoria ID-230. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2013-2014

Tutoría de titulación en el Grado de Pedagogía (curso 2013/2014): Programa de Orientación personal, académica y profesional de los estudiantes

Memoria ID 171. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2013-2014

Aplicación de la herramienta Socrative® para la evaluación en la asignatura "Repercusiones Bucales de las Enfermedades Sistémicas" en los alumnos de Odontología a través de dispositivos móviles. Fase I. Examen tipo test de preguntas dicotómicas

Incorporación de la herramienta interactiva Socrative® para la gestión de la participación de los alumnos y la evaluación rápida en tiempo real por parte del profesor en la asignatura "Repercusiones Bucales de las Enfermedades Sistémicas" en 3er Curso de Grado de Odontología.Se realizan preguntas y los alumnos contestan a través de sus dispositivos móviles, obteniendo un feedback inmediato de sus contestaciones