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.

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

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.

Dendrimeric Antigens for Drug Allergy Diagnosis: A New Approach for Basophil Activation Tests.

Dendrimeric Antigens (DeAns) consist of dendrimers decorated with multiple units of drug antigenic determinants. These conjugates have been shown to be a powerful tool for diagnosing penicillin allergy using in vitro immunoassays, in which they are recognized by specific IgE from allergic patients. Here we propose a new diagnostic approach using DeAns in cellular tests, in which recognition occurs through IgE bound to the basophil surface. Both IgE molecular recognition and subsequent cell activation may be influenced by the tridimensional architecture and size of the immunogens. Structural features of benzylpenicilloyl-DeAn and amoxicilloyl-DeAn (G2 and G4 PAMAM) were studied by diffusion Nuclear Magnetic Resonance (NMR) experiments and are discussed in relation to molecular dynamics simulation (MDS) observations. IgE recognition was clinically evaluated using the basophil activation test (BAT) for allergic patients and tolerant subjects. Diffusion NMR experiments, MDS and cellular studies provide evidence that the size of the DeAn, its antigen composition and tridimensional distribution play key roles in IgE-antigen recognition at the effector cell surface. These results indicate that the fourth generation DeAns induce a higher level of basophil activation in allergic patients. This approach can be considered as a potential complementary diagnostic method for evaluating penicillin allergy.

Disulfide-Functionalized Unimolecular Micelles as Selective Redox-Responsive Nanocarriers.

Redox-sensitive hyperbranched dendritic-linear polymers (HBDLPs) were prepared and stabilized individually as unimolecular micelles with diameters in the range 25-40 nm. The high molecular weight (500-950 kDa), core-shell amphiphilic structures were synthesized through a combination of self-condensing vinyl copolymerization (SCVCP) and atom transfer radical polymerization (ATRP). Cleavable disulfide bonds were introduced, either in the backbone, or in pendant groups, of the hyperbranched core of the HBDLPs. By triggered reductive degradation, the HBDLPs showed up to a 7-fold decrease in molecular weight, and the extent of degradation was tuned by the amount of incorporated disulfides. The HBDLP with pendant disulfide-linked functionalities in the hyperbranched core was readily postfunctionalized with a hydrophobic dye, as a mimic for a drug. An instant release of the dye was observed as a response to a reductive environment similar to the one present intracellularly. The proposed strategy shows a facile route to highly stable unimolecular micelles, which attractively exhibit redox-responsive degradation and cargo release properties.

Recognition of multiepitope dendrimeric antigens by human immunoglobulin E.

In vitro drug allergy tests have limited sensitivity, partly due to a poor understanding of the immunological recognition of in vitro drug-protein conjugates. We have designed and synthesized multivalent mono- and bi-epitope dendrimeric antigen (DeAn) conjugates and studied their chemical and tridimensional structures. We describe differences in the spatial distribution and conformation of these conjugated epitopes for the first time: a partially hidden benzylpenicilloyl and a more exposed amoxicilloyl. Our data suggest that DeAn conjugates provide a useful model for studying IgE recognition in patients who suffer from an allergic reaction to benzylpenicillin and/or amoxicillin. 1D and 2D NMR, MDS and immunochemical studies provide evidence that both antigen composition and tridimensional distribution play key roles in IgE-antigen recognition. Bi-epitope DeAn conjugates could potentially allow the diagnosis of patients allergic to any of these two drugs with a single test and represent the basis for a broadly-applicable in vitro assay. From the clinical editor: The prevalence of allergic drug reactions is rising and there is an imperative need to identify patients at risk. In this interesting and important article, the authors developed a novel method for detecting drug specific IgE antibodies, responsible for allergic reactions, by using multivalent mono- and bi-epitope Dendrimeric Antigen (DeAn) conjugates. The continued success of this research may pave way of eventual development of a simple diagnostic test.

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.

Bifunctional dendronized cellulose surfaces as biosensors.

Well-defined dendronized cellulose substrates displaying multiple representations of dual-functionality were constructed by taking advantage of the efficiency of the click reaction combined with traditional anhydride chemistry. First, activated cellulose surfaces were decorated with several generations of dendrons, and their peripheral reactive groups were subsequently reacted with a trifunctional orthogonal monomer. The generated substrate tool box was successfully explored by accurately tuning the surface function using a versatile orthogonal dual postfunctionalization approach. In general, the reactions were monitored by using a click-dye reagent or a quartz crystal microbalance (QCM) technique, and the resulting surfaces were well-characterized using XPS, FT-IR, and contact angle measurements. Utilizing this approach two different surfaces have been obtained; that is, triethylenglycol oligomers and amoxicillin molecules were efficiently introduced to the dendritic surface. As a second example, mannose-decorated hydroxyl functional surfaces illustrated their potential as biosensors by multivalent detection of lectin protein at concentration as low as 5 nM.

Methodologies to anchor dendrimeric nanoconjugates to solid phase: toward an efficient in vitro detection of allergy to ß-lactam antibiotics.

ß-lactam antibiotics are the most common cause of adverse drug reactions, and in vitro immunoassays are often employed to diagnose this allergy. Cellulose solid supports displaying hapten-carrier conjugates are traditionally used for quantifying specific IgE antibodies to determine whether a patient is allergic to the hapten. Our approach makes use of polyamidoamine dendrimers as carriers, and differs from traditional solid-phase activation methods by using two different haloalkanoyl halides as reagents instead of cyanogen bromide. Evaluation of the functionalization in the three different surfaces with benzylpenicilloyl-dendrimer conjugates showed a higher density of conjugates when using haloalkanoyl halides. The resulting surfaces were evaluated in vitro by radioimmunoassay using sera from patients allergic to penicillins. Selective and specific IgE recognition was observed in the different activated disks, though those activated with haloalkanoyl halides presented better sensitivity. FROM THE CLINICAL EDITOR: A novel and sensitive method of dendrimeric nanoconjugate-based in vitro detection of allergy to ß-lactam antibiotics is presented in this basic science paper.

Multiepitope Dendrimeric Antigen-Silica Particle Composites as Nano-Based Platforms for Specific Recognition of IgEs.

ß-lactam antibiotics (BLs) are the drugs most frequently involved in drug hypersensitivity reactions. However, current in vitro diagnostic tests have limited sensitivity, partly due to a poor understanding of in vivo drug-protein conjugates that both induce the reactions and are immunologically recognized. Dendrimeric Antigen-Silica particle composites (DeAn@SiO2), consisting on nanoparticles decorated with BL-DeAns are promising candidates for improving the in vitro clinical diagnostic practice. In this nano-inspired system biology, the synthetic dendrimer plays the role of the natural carrier protein, emulating its haptenation by drugs and amplifying the multivalence. Herein, we present the design and synthesis of new multivalent mono- and bi-epitope DeAn@SiO2, using amoxicillin and/or benzylpenicillin allergenic determinants as ligands. The homogeneous composition of nanoparticles provides high reproducibility and quality, which is critical for in vitro applications. The suitable functionalization of nanoparticles allows the anchoring of DeAn, minimizing the nonspecific interactions and facilitating the effective exposure to specific IgE; while the larger interaction area increments the likelihood of capturing specific IgE. This achievement is particularly important for improving sensitivity of current immunoassays since IgE levels in BL allergic patients are very low. Our data suggest that these new nano-based platforms provide a suitable tool for testing IgE recognition to more than one BL simultaneously. Immunochemical studies evidence that mono and bi-epitope DeAn@SiO2 composites could potentially allow the diagnosis of patients allergic to any of these drugs with a single test. These organic-inorganic hybrid materials represent the basis for the development of a single screening for BL-allergies.

Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation.

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.

Diagnostic Approach of Hypersensitivity Reactions to Cefazolin in a Large Prospective Cohort.

BACKGROUND: Cefazolin is a common trigger of perioperative anaphylaxis. The diagnostic approach is controversial because the optimal concentration for skin testing is uncertain, drug provocation tests (DPTs) are contraindicated in severe reactions, and in vitro tests are not thoroughly validated. OBJECTIVE: We aimed to characterize a large number of patients reporting cefazolin allergic reactions and to analyze the diagnostic role of in vivo and in vitro tests. METHODS: We prospectively evaluated patients with suspicion for allergic reactions to cefazolin by clinical history, skin tests (STs), and, if negative, DPT. In a subgroup of patients, basophil activation test (BAT) and radioallergosorbent test were done before allergologic workup was performed and the final diagnosis was achieved. RESULTS: We evaluated 184 patients, 76 of whom were confirmed as allergic (41.3%), 90 were nonallergic (48.9%), and 18 were nonconfirmed (9.8%). All patients reporting anaphylactic shock and most reporting anaphylaxis were confirmed to be allergic (P < .001). Forty allergic patients (52.6%) were confirmed by STs, 22 by DPT (28.9%), and 14 by clinical history (18.4%). All subjects manifesting exanthemas and pruritus were nonallergic. The BAT sensitivity was 66.7% when CD63 and CD203c were combined as activation markers. Six of 8 patients with negative STs and positive DPT had a positive BAT. CONCLUSIONS: Patients allergic to cefazolin often reported severe immediate-type reactions. Skin tests enabled a diagnosis in half of patients when using cefazolin at 20 mg/mL. Unfortunately, DPT could not be performed in all patients owing to reaction severity, which makes BAT a promising diagnostic tool. Further research is needed to clarify the underlying mechanisms, especially in severe reactions.

The Role of Benzylpenicilloyl Epimers in Specific IgE Recognition.

The high prevalence of allergy to ß-lactam antibiotics is a worldwide issue. Accuracy of diagnostic methods is important to prove tolerance or allergy, with skin test considered the best validated in vivo method for diagnosing immediate reactions to ß-lactams. Although drug provocation test is the reference standard, it cannot be performed in highly risk reactions or in those with positive skin tests. For skin tests, the inclusion of major and minor determinants of benzylpenicillin (BP) is recommended. Commercial skin test reagents have changed along time, including as minor determinants benzylpenicillin, benzylpenicilloate (BPO), and benzylpenilloate (PO). Major determinants consists of multivalent conjugates of benzylpenicilloyl coupled through amide bond to a carrier polymer, such as penicilloyl-polylysine (PPL) or benzylpenicilloyl-octalysine (BP-OL). The chemical stability of such reagents has influenced the evolution of the composition of the commercial kits, as this requirement is necessary for improving the quality and standardization of the product. In this work, we provide a detailed study of the chemical stability of BP determinants. We observed that those structures suffer from an epimerization process in C-5 at different rates. Butylamine-Benzylpenicilloyl conjugates (5R,6R)-Bu-BPO and (5S,6R)-Bu-BPO were selected as a simple model for mayor determinant to evaluate the role of the different epimers in the immunoreactivity with sera from penicillin-allergic patients. In vitro immunoassays indicate that any change in the chemical structure of the antigenic determinant of BP significantly affects IgE recognition. The inclusion of stereochemically pure compounds or mixtures may have important implications for both the reproducibility and sensitivity of in vivo and in vitro diagnostic tests.

Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with ß-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this ß-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through ß-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened ß-lactam was detected bound to the 404-430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in ß-lactams allergy.

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-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 ß-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.

An Update on the Immunological, Metabolic and Genetic Mechanisms in Drug Hypersensitivity Reactions.

Drug hypersensitivity reactions (DHRs) represent a major burden on the healthcare system since their diagnostic and management are complex. As they can be influenced by individual genetic background, it is conceivable that the identification of variants in genes potentially involved could be used in genetic testing for the prevention of adverse effects during drug administration. Most genetic studies on severe DHRs have documented HLA alleles as risk factors and some mechanistic models support these associations, which try to shed light on the interaction between drugs and the immune system during lymphocyte presentation. In this sense, drugs are small molecules that behave as haptens, and currently three hypotheses try to explain how they interact with the immune system to induce DHRs: the hapten hypothesis, the direct pharmacological interaction of drugs with immune receptors hypothesis (p-i concept), and the altered self-peptide repertoire hypothesis. The interaction will depend on the nature of the drug and its reactivity, the metabolites generated and the specific HLA alleles. However, there is still a need of a better understanding of the different aspects related to the immunological mechanism, the drug determinants that are finally presented as well as the genetic factors for increasing the risk of suffering DHRs. Most available information on the predictive capacity of genetic testing refers to abacavir hypersensitivity and anticonvulsants-induced severe cutaneous reactions. Better understanding of the underlying mechanisms of DHRs will help us to identify the drugs likely to induce DHRs and to manage patients at risk.

Dendrimerized cellulose as a scaffold for artificial antigens with applications in drug allergy diagnosis.

Cellulose-based dendrimerized material was prepared and its quality was assessed by determining the number of amine functional groups incorporated. Based on the results for a series of preparations, the material was obtained in a highly reproducible manner thanks to the particular chemical construction method used. The number of amine groups incorporated and the amount of dendrimer attached are directly related to the dendrimer generation. The combination of the properties of the cellulose polymer and those of the dendrimeric state provides biocompatible materials amenable to easy chemical characterization. The proposed method provides an effective tool for developing clinically testable materials with a view to studying adverse immunological responses to drugs in humans.

Elemental and optical imaging evaluation of zwitterionic gold nanoclusters in glioblastoma mouse models.

We report in this study the in vivo biodistribution of ultra-small luminescent gold (Au) particles (∼1.5 nm core size; 17 kDa), so-called nanoclusters (NCs), stabilized by bidentate zwitterionic molecules in subcutaneous (s.c.) and orthotopic glioblastoma mice models. Particular investigations on renal clearance and tumor uptake were performed using highly sensitive advanced imaging techniques such as multi-elemental Laser-Induced Breakdown Spectroscopy (LIBS) imaging and in-line X-ray Synchrotron Phase Contrast Tomography (XSPCT). Results show a blood circulation time of 6.5 ± 1.3 min accompanied by an efficient and fast renal clearance through the cortex of the kidney with a 66% drop between 1 h and 5 h. With a similar size range, these Au NCs are 5 times more fluorescent than the well-described Au25GSH18 NCs in the near-infrared (NIR) region and present significantly stronger tumor uptake and retention illustrated by an in vivo s.c. tumor-to-skin ratio of 1.8 measured by non-invasive optical imaging and an ex vivo tumor-to-muscle of 6.1. This work highlights the pivotal role of surface coating in designing optimum Au NC candidates for cancer treatment.

In Vitro Diagnostic Testing for Antibiotic Allergy.

Allergy to antibiotics is an important worldwide problem, with an estimated prevalence of up to 10% of the population. Reaction patterns for different antibiotics have changed in accordance with consumption trends. Most of the allergic reactions to antibiotics have been reported for betalactams, followed by quinolones and macrolides and, to a lesser extent, to others, such as metronidazole clindamycin and sulfonamides. The diagnostic procedure includes a detailed clinical history, which is not always possible and can be unreliable. This is usually followed by in vivo, skin, and drug provocation tests. These are not recommended for severe, potentially lifethreaten reactions or for drugs that are known to produce a high rate of false positive results. Given the limitations of in vivo tests, in vitro test can be helpful for diagnosis, despite having suboptimal sensitivity. The most highly employed techniques for diagnosing immediate reactions to antibiotics are immunoassays and basophil activation tests, while lymphocyte transformation tests are more commonly used to diagnose non-immediate reactions. In this review, we describe different in vitro techniques employed to diagnose antibiotic allergy.