Meglumine gadoterate induces immunoglobulin-independent human mast cell activation and MRGPRX2 internalizationTo the Editor,Gadolinium-based contrast agents (GBCA) are intravenous drugs used to enhance resolution in magnetic resonance imaging. They can induce immediate hypersensitivity reactions, yet their pathogenic mechanisms remain poorly characterized. This hampers the ability to predict which patients are at risk of developing them.1 In fact, affected patients usually show negative skin-tests and can react upon the first known GBCA exposure, which implies that IgE-independent mechanisms might be driving this inflammatory response.The Mas-related G protein-coupled receptor member X2 (MRGPRX2) has been recently associated with non-IgE mediated immediate hypersensitivity reactions.2 Some drugs, such as fluoroquinolones, vancomycin, neuromuscular blockade agents, icatibant, morphine, leuprolide and iodinated contrast media, have been reported to activate MRGPRX2, which is highly expressed in mast cells (MCs).3To assess the ability of GBCA to induce non-IgE-mediated hypersensitivity reactions, we stimulated the human MC line LAD2 with several commercial GBCA, namely, meglumine gadoterate, gadobutrol, gadoxetate disodium and gadoteridol. Then, we determined cell viability and degranulation by flow cytometry4 (see a detailed material and methods section in this article´s online supplementary ).Of the GBCA tested, only meglumine gadoterate was able to induce significant MC activation (Figure 1A ) without compromising cell viability (Figure 1B ), as compared to unstimulated MCs. We further assessed MRGPRX2 expression on LAD2 cells by flow cytometry, as well as changes in its expression following stimulations with either meglumine gadoterate or vancomycin (a known agonist of MRGPRX2).5 Under basal conditions, LAD2 cells expressed high levels of MRGPRX2 (Figure 1C ). Following incubation with vancomycin, the level of MRGPRX2 expression was reduced, as compared to untreated LAD2 cells. Interestingly, we observed a similar decrease in MRGPRX2 expression levels upon meglumine gadoterate and vancomycin challenges, as compared to controls, suggesting both the signaling and the internalization of this receptor (Figure 1D ).Meglumine gadoterate is an ionic macrocyclic paramagnetic contrast media. It is composed by gadolinium, which together with the chelating agent tetraxetan (also known as DOTA), yields gadoteric acid. The base meglumine and gadoteric acid form the salt meglumine gadoterate (Figure 2A ). Given that MRGPRX2 has affinity for cationic amphiphilic compounds,6 we ascertained the ability of meglumine to induce MC activation. Meglumine itself induced MC degranulation without affecting cell viability, as compared to untreated cells (Figure 2B ), although a reduction in MRGPX2 expression could not be confirmed (data not shown). Interestingly, meglumine caused MC activation at lower concentrations than meglumine gadoterate, according to the half maximal effective concentration (EC50) of both substances (Figure 2C ). The logarithmically transformed EC50 for meglumine gadoterate was 2.04 (R2= 0.75), and for meglumine was about one order of magnitude lower (1.06; R2= 0.71). Considering the EC50 for meglumine and its proportion in meglumine gadoterate (~26%), meglumine could be its main component responsible for MC degranulation.In conclusion, our study demonstrates the ability of meglumine gadoterate to induce MC activation, by an immunoglobulin-independent mechanism that is likely mediated by MRGPRX2. Furthermore, we have delved into the meglumine gadoterate components that are involved in MC activation, and identified meglumine as a potential causative of non-IgE mediated hypersensitivity reactions. These data raise the possibility that immediate hypersensitivity reactions following intravascular administration of ionic iodinated contrast media may be at least partly mediated by meglumine. Further studies should be performed to define clinically relevant interactions between diverse radiological contrast media and MRGPRX2.Authors: Paula H. Ruiz de Azcárate,1#Rodrigo Jiménez-Saiz,1-4 #* Celia López-Sanz,1 Azahara López-Raigada,5Francisco Vega,5 Carlos Blanco,5*# First authors* Corresponding authorsAffiliations: 1Department of Immunology, Instituto de Investigación Sanitaria Hospital Universitario de La Princesa (IIS-Princesa), Universidad Autónoma de Madrid (UAM), Madrid, Spain.2Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB)-CSIC, Madrid, Spain.3Faculty of Experimental Sciences, Universidad Francisco de Vitoria (UFV), Madrid, Spain.4Department of Medicine, McMaster Immunology Research Centre (MIRC), Schroeder Allergy and Immunology Research Institute (SAIRI), McMaster University, Hamilton, ON, Canada.5Department of Allergy, Instituto de Investigación Sanitaria Hospital Universitario de La Princesa (IIS-Princesa), Universidad Autónoma de Madrid (UAM), Madrid, Spain.*Co-correspondence to :1) Rodrigo Jiménez-Saiz, Department of Immunology, Instituto de Investigación Sanitaria Hospital Universitario de La Princesa (IIS-Princesa), Diego de León 62, 28006, Madrid, Spain. Email address: [email protected]) Carlos Blanco, Department of Allergy, Instituto de Investigación Sanitaria Hospital Universitario de La Princesa (IIS-Princesa), Diego de León 62, 28006, Madrid, Spain. Email address: [email protected] Funding information: RJS reports grants by the FSE/FEDER through the Instituto de Salud Carlos III (CP20/00043; PI22/00236; Spain), The Nutricia Research Foundation (NRF-2021-13; The Netherlands), New Frontiers in Research Fund (NFRFE-2019-00083; Canada) and SEAIC (BECA20A9; Spain). PHR is supported by the INVESTIGO Program of the Community of Madrid (Spain), which is funded by “Plan de Recuperación, Transformación y Resiliencia” and “NextGenerationEU” of the European Union (09-PIN1-00015.6/2022).Conflict of interest : All the authors have no significant conflicts of interest to declare in relation to this manuscript.References1. Vega F, Lopez-Raigada A, Mugica MV, Blanco C. Fast challenge tests with gadolinium-based contrast agents to search for an alternative contrast media in allergic patients. Allergy.2022;77(10):3151-3153.2. Kolkhir P, Ali H, Babina M, et al. MRGPRX2 in drug allergy: What we know and what we do not know. J Allergy Clin Immunol. 2022.3. Foer D, Wien M, Karlson EW, Song W, Boyce JA, Brennan PJ. Patient Characteristics Associated With Reactions to Mrgprx2-Activating Drugs in an Electronic Health Record-Linked Biobank. J Allergy Clin Immunol Pract. 2022.4. López-Sanz C, Sánchez-Martínez E, Jiménez-Saiz R. Protocol to desensitize human and murine mast cells after polyclonal IgE sensitization. STAR Protocols. 2022;3(4):101755.5. Navines-Ferrer A, Serrano-Candelas E, Lafuente A, Munoz-Cano R, Martin M, Gastaminza G. MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia. Sci Rep.2018;8(1):11628.6. Wolf K, Kühn H, Boehm F, et al. A group of cationic amphiphilic drugs activates MRGPRX2 and induces scratching behavior in mice. J Allergy Clin Immunol. 2021;148(2):506-522.e508.

Allergic diseases are allergen-induced immunological disorders characterized by the development of type 2 immunity and IgE responses. The prevalence of allergic diseases has been on the rise alike cardiovascular disease (CVD), which affects arteries of different organs such as the heart, the kidney and the brain. The underlying cause of CVD is often atherosclerosis, a disease distinguished by endothelial dysfunction, fibrofatty material accumulation in the intima of the artery wall, smooth muscle cell proliferation, and Th1 inflammation. The opposed T-cell identity of allergy and atherosclerosis implies an atheroprotective role for Th2 cells by counteracting Th1 responses. Yet, the clinical association between allergic disease and CVD argues against it. Within, we review different phases of allergic pathology, basic immunological mechanisms of atherosclerosis and the clinical association between allergic diseases (particularly asthma, atopic dermatitis, allergic rhinitis and food allergy) and CVD. Then, we discuss atherogenic mechanisms of type 2 immunity and allergic inflammation including acute allergic reactions (IgE, IgG1, mast cells, macrophages and allergic mediators such as vasoactive components, growth factors and those derived from the complement, contact and coagulation systems) and late phase inflammation (Th2 cells, eosinophils, type 2 innate-like lymphoid cells, alarmins, IL-4, IL-5, IL-9, IL-13 and IL-17).

Fast gadolinium-based contrast agent challenge test searching for an alternative contrast mediaTo the Editor,Gadolinium-based contrast agents (GBCA) are used in contrast-enhanced magnetic resonance imaging. Hypersensitivity reactions (HSR) to GBCA are scarce, with an incidence of 0.07% and a recurrence rate of 30%, being urticaria the most common presentation (91%), with 0.52/10000 of severe reactions reported1. Recommendation of an alternative GBCA without checking tolerance is dangerous, due to high cross-reactivity between them2. Moreover, premedication is not enough1, showing an overall rate of breakthrough reactions of 39%3.Allergy studies to achieve a safe recommendation in HSR to GBCA have been performed. Negative predictive value of skin-tests to GBCA has been estimated in 84%1. Therefore, more than 10% of patients could react using an alternative negative skin-tested GBCA, and thus, good tolerance to GBCA should be confirmed through a drug challenge-test (DCT)4. These tests are usually performed at graded administrations, and with observation periods between doses1,5. However, since GBCA is usually given as a bolus during radiologic exams, DCT at slow rates cannot be extrapolated to further administrations. Trying to avoid this limitation, we study the tolerance of an alternative GBCA, by means of a fast DCT, approaching the infusion rates used in clinical practice.In accordance with the safety warnings to avoid linear GBCA, we have only used the macrocyclic drugs gadobutrol (Gb) and gadoteric acid (Ga). After obtaining signed informed consent from the patients, skin pricktests (SPT) with undiluted macrocyclic GBCA commercial solutions were done. When SPT at 20 min yielded negative results, intradermal tests (IDT) with 1:10 dilutions were performed, with subsequent readings at both 20 min and 24 hours.A fast DCT with negative skin-tested GBCA was then performed, following our methodology to study HSR to iodinated contrast media, previously described elsewhere6. Doses were 0.2 mg/kg for Ga and 0.1 mg/kg for Gb. First, one third of the total dose of Ga was administered at a rate of 120 cc/hour and, immediately after, the remaining 2/3 at 80 cc/hour. In case of Gb, infusion rates were half those of Ga, i.e., 1/3 at 60 cc/hour and 2/3 at 40 cc/hour. Total infusion time was 8 minutes for both of them. Well-tolerated GBCA was finally recommended for subsequent examinations, and its tolerance was recorded if it was used later.Study results of sixteen patients that were enrolled are summarized in Table 1. They were 12 women and 4 men, with median age of 45.5 years (range 28-73). Adverse reactions to GBCA were immediate in 13 patients (12 urticaria or exanthema, and 1 anaphylaxis), and delayed exanthema in the remaining 3. Gb was involved in 11 reactions, and unknown GBCA in the other 5. Most of the patients (14/16) had been previously exposed to GBCA.Median delay to perform the allergy study was 10 months (range 2-72 months). All skin-tests were negative, except in one patient who showed an immediate positive SPT to Gb, which had been the GBCA involved in the adverse reaction. In our study, we have estimated a negative predictive value of skintests to GBCA of 89%. DCT were negative in 14 patients (12 with Ga, and 2 with Gb). Finally, 15 out of 16 patients had an alternative GBCA, avoiding the use of premedication. In fact, tolerance has been confirmed in 7 of them in subsequent examinations.Safety of our protocol has been confirmed because our 2 positive DCT showed only mild reactions (delayed exanthema and immediate urticarial, both with Ga), and also by including a patient with previous anaphylaxis to GBCA.Here we present a prospective protocol to identify a safe alternative GBCA, including DCT at high infusion rates. Further studies will be necessary on this item/to check this.