basophil-activation-test-for-allergy-diagnosis

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Instituto de Investigación Biomédica de Málaga-IBIMA

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in allergic reactions of which two main types exist: IgE-mediated and crossintolerance. The diagnosis of crossintolerance reactions is often based on the drug provocation test. The potential value of the basophil activation test (BAT) was evaluated using different basophil markers in the diagnosis of patients with crossintolerance to NSAIDs and cutaneous symptoms. We studied 46 patients with crossintolerance to NSAIDs and 45 tolerant controls. BAT was performed with acetyl salicylic acid, paracetamol, diclofenac, dipyrone, naproxen, and ibuprofen at four different concentrations using CD193 and CD203c as basophil markers and CD63 as activation marker. We compared BAT results using CD193⁺ or CD193⁺ CD203c⁺ for basophil selection and found a significant increase in the stimulation index when using CD193⁺ CD203c⁺ in both patients and controls (P = 0.004 and P = 0.017, respectively). Selection of living cells only produced an increase in basophil stimulation in patients for both CD193⁺ and CD193⁺ CD203c⁺ (P < 0.001 for both), whereas in controls there was no change with CD193⁺ and a decrease with CD193⁺ CD203c⁺ (P = 0.001). We found that CD193⁺ CD203c⁺ increased the percentage of positive cases in patients and controls when compared with CD193⁺. When excluding dead cells, there was an increase of 21.7% in patients and 10% in controls. These results indicate that using CD193⁺ CD203⁺, excluding dead cells, is the best approach for BAT although this test is not recommended for the diagnosis of patients with crossintolerance to NSAIDs owing to its low sensitivity and specificity.

Basophil activation after nonsteroidal anti-inflammatory drugs stimulation in patients with immediate hypersensitivity reactions to these drugs.

Drug-induced maculopapular exanthemas (MPEs) are mediated by Th1 CD4 T cells. One of the mechanisms of control of Th1 cells in homeostasis is the interaction between the checkpoint inhibitor Tim3 and its physiological ligand galectin-9 (Gal9). Disorders affecting this axis may be responsible for various autoimmune and immunological diseases. The aim of this study was to determinate the influence of the Tim3-Gal9 axis on the development of MPE induced by drugs.

Expression of the Tim3-galectin-9 axis is altered in drug-induced maculopapular exanthema.

Drug hypersensitivity reactions (DHRs) are typically classified into immediate and delayed reactions based on the time interval between drug exposure and onset of symptoms. Clinical manifestations range from mild to severe and life-threatening reactions. The most severe clinical entities are anaphylaxis and anaphylactic shock for immediate reactions, and severe cutaneous adverse reactions such as Steven Johnson Syndrome and Toxic Epidermal Necrolysis for delayed reactions. The diagnosis is complex and challenging, as drug provocation tests and even skin tests can be very risky procedures, which makes them not recommended. Therefore, it is necessary to search for useful early biomarkers to manage the diagnosis of these reactions. These biomarkers could be useful to determine the clinical entity, but not to identify the culprit drug. Some of the currently available biomarkers are few genetic associations of drug allergy with polymorphisms of human leukocyte antigen (HLA), the detection of inflammatory and lipid mediators in serum, or the detection of cytokines, chemokines, and cytotoxic markers in skin biopsies. In this literature review, it has been summarize the immunological mechanisms involved in severe reactions, both immediate and delayed, and different early biomarkers: those currently used for the diagnosis of these reactions as well as possible early biomarkers that could be useful with further studies to standardize their clinical use.

Early Biomarkers for Severe Drug Hypersensitivity Reactions.

Design of an antigenic determinant of cefaclor: Chemical structure-IgE recognition relationship.

Allergic rhinitis (AR) and local allergic rhinitis (LAR) are defined by nasal reactivity to aeroallergens with and without positive skin prick test (SPT), respectively. In this study, we aimed to investigate whether both types of allergen-specific reactivity can coexist in the same individual.

Coexistence of nasal reactivity to allergens with and without IgE sensitization in patients with allergic rhinitis.

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.

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

Betalactam (BL) antibiotics are the most common cause of drug hypersensitivity. Amoxicillin (AX), which is often prescribed alongside clavulanic acid (Clav), is the most common elicitor. The aim of this study was to determine whether AX and Clav-responsive T-cells are detectable in patients with immediate hypersensitivity to AX-Clav, to assess whether these T-cells display the same specificity as that detected in skin and provocation testing, and to explore T-cell activation pathways.

Characterization of amoxicillin and clavulanic acid specific T-cell clones from patients with immediate drug hypersensitivity.

Reply.

Comparison of diagnostic accuracy of acoustic rhinometry and symptoms score for nasal allergen challenge monitoring.

A correct diagnosis of drug hypersensitivity reactions (DHRs) is very important for both the patient and health system. However, DHRs diagnosis is complex, time consuming, requires trained personnel, is not standardized for many drugs, involves procedures not exempt of risk, and in most cases lacks standardized in vivo and in vitro tests. Thus, there is an urgent need for improving the different approaches to diagnose patients with suspected DHRs. In this review, we have analyzed the advances performed in immediate and nonimmediate DHRs diagnosis during the last two years and obtained several conclusions: the significant heterogeneity in current practice among centers illustrates the need to re-evaluate, update, and standardize in vivo tests and protocols for the diagnosis and management of patients with suspected drug allergy. Regarding in vitro tests, the latest studies have focused on increasing their sensitivity or on establishing the sensitivity and specificity for the tests performed with new drugs. There seems to be a consensus about combining in vivo and in vitro tests as the best way to increase the diagnostic accuracy.

Advances and novel developments in drug hypersensitivity diagnosis.

House dust mites (HDMs) are the allergenic sources most frequently involved in airway allergy. Nevertheless, not every sensitized patient develops respiratory symptoms upon exposure to HDM, and there is a clinical need to differentiate allergic asthmatics (AAs) from atopic non-allergic asthmatics with HDM sensitization. This differentiation sometimes requires in vivo provocations like the bronchial allergen challenge (BAC). Interestingly, recent data demonstrate that non-atopic patients with asthma can also develop positive BAC results. This novel phenotype has been termed local allergic asthma (LAA). The interest in identifying the allergic triggers of asthma resides in the possibility of administering allergen immunotherapy (AIT). AIT is a disease-modifying intervention, the clinical benefit of which persists after therapy discontinuation. Recently, new modalities of sublingual tablets of HDM immunotherapy registered as pharmaceutical products (HDM-SLIT tablets) have become commercially available. HDM-SLIT tablets have demonstrated a robust effect over critical asthma parameters (dose of inhaled corticosteroids, exacerbations, and safety), thus being recommended by international guidelines for patients with HDM-driven AA. In this review, we will summarize the current knowledge on the phenotype and endotype of HDM-driven AA, and LAA, address the difficulties for BAC implementation in the clinic, and discuss the effects of AIT in AA and LAA.

Precision Medicine in House Dust Mite-Driven Allergic Asthma.

Analysis of cross-reactivity is necessary for prescribing safe cephalosporins for penicillin allergic patients. Amoxicillin (AX) is the betalactam most often involved in immediate hypersensitivity reactions (IHRs), and cefadroxil (CX) the most likely cephalosporin to cross-react with AX, since they share the same R1 side chain, unlike cefuroxime (CO), with a structurally different R1. We aimed to analyse cross-reactivity with CX and CO in patients with confirmed IHRs to AX, including sIgE recognition to AX, CX, CO, and novel synthetic determinants of CX.

Penicillin and cephalosporin cross-reactivity: role of side chain and synthetic cefadroxil epitopes.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-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 β-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 β-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 β-lactam was detected bound to the HSA 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 β-lactams allergy.

Article	Year
Basophil activation after nonsteroidal anti-inflammatory drugs stimulation in patients with immediate hypersensitivity reactions to these drugs. Cytometry. Part A : the journal of the International Society for Analytical Cytology\| PubMed ID: 24443418	2014
Expression of the Tim3-galectin-9 axis is altered in drug-induced maculopapular exanthema. Allergy\| PubMed ID: 31034608	2019
Early Biomarkers for Severe Drug Hypersensitivity Reactions. Current pharmaceutical design\| PubMed ID: 31696802	2019
Design of an antigenic determinant of cefaclor: Chemical structure-IgE recognition relationship. The Journal of allergy and clinical immunology\| PubMed ID: 31821817	2020
Coexistence of nasal reactivity to allergens with and without IgE sensitization in patients with allergic rhinitis. Allergy\| PubMed ID: 31995231	2020
Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency. Frontiers in pharmacology\| PubMed ID: 32210804	2020
Characterization of amoxicillin and clavulanic acid specific T-cell clones from patients with immediate drug hypersensitivity. Allergy\| PubMed ID: 32246774	2020
Reply. The Journal of allergy and clinical immunology\| PubMed ID: 32362530	2020
Comparison of diagnostic accuracy of acoustic rhinometry and symptoms score for nasal allergen challenge monitoring. Allergy\| PubMed ID: 32687610	2021
Advances and novel developments in drug hypersensitivity diagnosis. Allergy\| PubMed ID: 32990987	2020
Precision Medicine in House Dust Mite-Driven Allergic Asthma. Journal of clinical medicine\| PubMed ID: 33255966	2020
Penicillin and cephalosporin cross-reactivity: role of side chain and synthetic cefadroxil epitopes. Clinical and translational allergy\| PubMed ID: 33292516	2020
Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies. Frontiers in pharmacology\| PubMed ID: 33442385	2020

Adriana Ariza

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