Overexpression of FcεRI on Bone Marrow Mast Cells, but Not MRGPRX2, in Clonal Mast Cell Disorders With Wasp Venom Anaphylaxis.

Background: Uncertainties remain about the molecular mechanisms governing clonal mast cell disorders (CMCD) and anaphylaxis. Objective: This study aims at comparing the burden, phenotype and behavior of mast cells (MCs) and basophils in patients with CMCD with wasp venom anaphylaxis (CMCD/WVA+), CMCD patients without anaphylaxis (CMCD/ANA-), patients with an elevated baseline serum tryptase (EBST), patients with wasp venom anaphylaxis without CMCD (WVA+) and patients with a non-mast cell haematological pathology (NMHP). Methods: This study included 20 patients with CMCD/WVA+, 24 with CMCD/ANA-, 19 with WVA+, 6 with EBST and 5 with NMHP. We immunophenotyped MCs and basophils and compared baseline serum tryptase (bST) and both total and venom specific IgE in the different groups. For basophil studies, 13 healthy controls were also included. Results: Higher levels of bST were found in CMCD patients with wasp venom anaphylaxis, CMCD patients without anaphylaxis and EBST patients. Total IgE levels were highest in patients with wasp venom anaphylaxis with and without CMCD. Bone marrow MCs of patients with CMCD showed lower CD117 expression and higher expression of CD45, CD203c, CD63, CD300a and FcεRI. Within the CMCD population, patients with wasp venom anaphylaxis showed a higher expression of FcεRI as compared to patients without anaphylaxis. Expression of MRGPRX2 on MCs did not differ between the study populations. Basophils are phenotypically and functionally comparable between the different patient populations. Conclusion: Patients with CMCD show an elevated burden of aberrant activated MCs with a significant overexpression of FcεRI in patients with a wasp venom anaphylaxis.

Culturing cells with mast cell phenotype and function: Comparison of peripheral blood and bone marrow as a source.

BACKGROUND: Studies on the mechanisms that govern mast cell (MC) functions are hindered by the difficulties in isolating sufficient numbers of these tissue-resident cells. Therefore, many research groups use cultured human MCs obtained out of progenitor cells. However, these culture methods significantly differ regarding primary source material, culture durations and conditions. Consequently, the finally obtained cells are likely to exhibit morphological, phenotypical and/or functional heterogeneity. OBJECTIVE: To compare the phenotype and functionality of cells cultured from peripheral blood and bone marrow progenitor cells from patients with suspected clonal MC disease. These cells are designated as PBCMCs and BMCMCs, respectively. METHODS: Twenty paired PBCMCs and BMCMCs cultures starting from CD34+ progenitor cells were compared. Cells were cultured for 4 weeks. Phenotyping included Giemsa and CD117 staining and flow cytometric staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a, CD32, CD63 and CD25. Functional assessment included measurement of the up-regulation of CD63 after cross-linking of the high affinity receptor for IgE (FcεRI) with anti-FcεRI and ligation of MRGPRX2 with substance P. RESULTS: PBCMCs and BMCMCs are phenotypically comparable. Functionally, after activation with anti-FcεRI and substance P, PBCMCs and BMCMCs show similar up-regulation of the lysosomal degranulation marker CD63. However, the yield of PBCMCs is higher than BMCMs and peripheral blood cultures are purer than bone marrow cultures. CONCLUSION: PBCMCs are an attractive alternative to the more difficult to obtain BMCMCs for the exploration of the complex mechanisms that govern IgE- and MRGPRX2-dependent MC activation and degranulation. Unlike BMCMCs, PBCMCs are easily accessible and enable repetitive analyses.

Effectiveness and safety of 12-month certolizumab pegol treatment for axial spondyloarthritis in real-world clinical practice in Europe.

OBJECTIVES: The efficacy and safety of certolizumab pegol (CZP), an Fc-free, PEGylated anti-TNF, in axial spondyloarthritis (axSpA) has been established in clinical trial settings. We report CZP effectiveness and safety in European clinical practice in patients with axSpA, including radiographic (r-) and non-radiographic (nr-) axSpA. METHODS: CIMAX (NCT02354105), a European non-interventional multicentre prospective study, observed CZP treatment response and safety over 12 months in a real-world axSpA cohort. The primary outcome was change from baseline in BASDAI to week 52, with additional outcomes pertaining to effectiveness and safety. Patients who received ≥1 dose CZP were followed up for adverse events, and those with baseline and ≥1 post-baseline BASDAI assessment were included in effectiveness analyses. RESULTS: A total of 672 patients (r-axSpA: 469; nr-axSpA: 201; unconfirmed diagnosis: 2) from 101 sites received ≥1 dose of CZP, of whom 564 (r-axSpA: 384; nr-axSpA: 179; unconfirmed: 1) were included in the effectiveness analyses. The mean baseline BASDAI was 6.1 in the overall axSpA population and r-axSpA and nr-axSpA subpopulations. At week 52, the mean (s.d.) change in BASDAI was -2.9 (2.3; n = 439); for r-axSpA and nr-axSpA, it was -2.9 (2.2; n = 301) and -2.8 (2.4; n = 137), respectively (P <0.0001 for all). Similar improvements were seen across other axSpA disease measures. In total, 37.9% (255/672) patients experienced adverse events, and 1.8% (12/672) experienced ≥1 serious adverse events. CONCLUSION: Improvements observed in signs and symptoms of axSpA following one year of CZP treatment in real-world clinical practice were similar to those from previous randomized clinical trials, with no new safety concerns.

Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques.

The basis of flow cytometric allergy diagnosis is the quantification of changes in the expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow the detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

Basophil Activation Experiments in Immediate Drug Hypersensitivity: More Than a Diagnostic Aid.

BACKGROUND: Correct diagnosis of immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge, mainly because of the absence of reliable in vitro tests, uncertainties associated with skin testing, and incomplete understanding of the underlying mechanisms. AIM: To summarize and hypothesize on the potential of basophil activation test (BAT) as a safe aid to explore the mechanistic endotypes of IDHR, to identify antibody recognition sites, and to monitor drug desensitization. METHODS: A literature search was conducted using the keywords "allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry"; this was complemented by the authors' own expertise. RESULTS: At present BAT has mainly been employed as a diagnostic aid. However, evidence is emerging that the technique might also deepen our insights in immune (allergic) and nonimmune (nonallergic) mechanistic processes of IDHR. It is anticipated that BAT might also benefit the identification of antibody recognition sites and benefit our understandings of desensitization strategies. CONCLUSION: Although the nondiagnostic application of BAT in IDHR is still in its infancy, with increasing employment, we can expect the technique to become a valuable asset to study many domains of IDHR that remain poorly understood.

Performance of basophil activation test and specific IgG4 as diagnostic tools in nonspecific lipid transfer protein allergy: Antwerp-Barcelona comparison.

BACKGROUND: Recent studies show that nsLTP sensitization is not limited to the Mediterranean basin and can present diverse clinical phenotypes. It remains challenging to predict clinical outcome when specific IgE antibodies (sIgE) to nsLTPs are present. This study compares both clinical and in vitro allergy characteristics but also diagnostic performance of a basophil activation test (BAT) and sIgG4 in nsLTP-sensitized patients from Antwerp (ANT, Belgium) and Barcelona (BCN, Spain). METHODS: Adult subjects with positive sIgE rPru p 3 and/or rMal d 3 ≥ 0.10 kUA /L (n = 182) and healthy controls (n = 37) were included. NsLTP-sensitized individuals were stratified according to clinical symptoms with peach/apple, respectively. BAT rPru p 3 and rMal d 3 were performed and sIgG4 antibodies to both components quantified. RESULTS: In BCN, only ratios of sIgG4/sIgE rMal d 3 and BAT rMal d 3 (0.001 µg/mL) can identify clinically relevant Mal d 3 sensitization (sensitivity of 60%-63% and a specificity of 75%-67%, respectively). In ANT, only the sIgE/total IgE rPru p 3 ratio shows added value (sensitivity 60% and specificity 83%). Finally, it appears that symptomatic patients in BCN are more sensitive to lower allergen concentrations compared to ANT. In addition, it was shown that ANT patients were more often sensitized to pollen and that specific pollen sources differed between regions. CONCLUSIONS: NsLTP-related allergy profiles and diagnostic performance differ significantly between regions and are component-specific, which makes extrapolation of data difficult to do. In addition, it seems that basophil sensitivity might show geographical differences. Additional research is needed to confirm these findings.

The Limited Value of Prolonged Drug Challenges in Nonimmediate Amoxicillin (Clavulanic Acid) Hypersensitivity.

BACKGROUND: Misdiagnosis of amoxicillin (clavulanic acid) (AX(/CL)) hypersensitivity has serious consequences. A drug challenge (DC) is the final diagnostic to affirm or infirm AX(/CL) hypersensitivity. However, uncertainties remain whether a prolonged drug challenge (pDC) should benefit the diagnosis of a nonimmediate AX(/CL) hypersensitivity. OBJECTIVE: To assess the added value of a standardized 7-day pDC in the diagnosis of nonimmediate or unclear penicillin hypersensitivity. METHODS: A total of 132 patients with a history of a nonimmediate hypersensitivity reaction or an unclear reaction to AX(/CL) or an undefined penicillin with a negative diagnostic workup including a single-day DC (DC) with AX(/CL) were selected. In all these patients, an additional pDC with AX(/CL) was planned. Thirteen patients started the pDC immediately after the DC. To ensure that hypersensitivity symptoms manifesting during the pDC course do not result from the DC, in the remaining 119 patients, the pDC was scheduled after a washout of 1 week. RESULTS: A total of 128 patients (12 without washout, 116 with washout) completed the pDC. Three patients reacted with a mild maculopapular exanthema. However, the value of a pDC was evidenced in only 1 patient who reacted during her pDC after an uneventful washout. In 2 patients pDC was cancelled because they reacted during the washout. CONCLUSIONS: A pDC is of limited added value to the diagnostic algorithms of nonimmediate hypersensitivity reaction or unclear hypersensitivity reactions to AX(/CL). In our hands, the traditionally recommended diagnostic algorithm that offers a 1-day DC as a final diagnostic in patients with negative workup for AX(/CL) is appropriate.

Cannabis allergy: what the clinician needs to know in 2019.

INTRODUCTION: Although the use of cannabis dates back millennia, the first description of cannabis allergy is relatively recent (1971). Recent large-scale data show that cannabis allergy can manifest severe and generalized symptoms with extensive cross-reactions. Thus, it is essential to become familiarized with its clinical presentation, diagnostic aids, and adequate therapeutic guidance. Areas covered: Here we provide a hands-on overview on cannabis allergy focusing on symptomatology and the reliability of diagnostic options. Recent advances in proteomics are discussed in detail, elucidating the link with nsLTP-related allergies. The proteomics advancements have paved the way for more reliable diagnostics, especially component-based tools. Finally, the current experience in treatment options is highlighted. Expert opinion: Cannabis allergy is an allergy entity which can significantly impact the quality of life. For optimal diagnosis, we advise to start with a validated and standardized crude-extract based test such as sIgE hemp complemented by component-based diagnostics such as sIgE Can s 3 quantifications where available. Future research should lift the veil on the true prevalence of cannabis allergy and the importance of other cannabis allergens to further guide our practice.

Acute Management, Diagnosis, and Follow-Up of Suspected Perioperative Hypersensitivity Reactions in Flanders 2001-2018.

BACKGROUND: Despite numerous efforts to describe the clinical manifestations and the epidemiology of perioperative hypersensitivity (POH), there remains room to increase awareness among anesthetists and immunologists/allergists. OBJECTIVE: To report the findings of a 17-year survey of suspected POH in Antwerp, Belgium. METHODS: We analyzed clinical and diagnostic data from 715 patients referred because of a suspected POH reaction, between January 1, 2001, and May 31, 2018. A total of 456 patients demonstrating a POH could be queried about subsequent anesthesia. RESULTS: A total of 608 cases formed the final dataset; 208 had a non-life-threatening reaction and 400 a life-threatening reaction. In life-threatening reactions, hypotension was predominating. In the non-life-threatening reactions, 83.9% of the patients displayed cutaneous manifestations. In life-threatening reactions, intravenous adrenaline and fluids were administered in 75.7% and 31%, respectively, and 41.3% had their intervention abandoned. Mast cell activation (MCA) was mainly, but not exclusively, observed in severe grades but did not predict the mechanistic process nor the culprit. A cause was identified in 77.8% of severe and 48.6% of milder cases. Main culprits were neuromuscular blocking agents, latex, cefazolin, and dyes. A total of 156 cases had uneventful anesthesia, except 1 patient who was inadvertently re-exposed to hidden chlorhexidine. CONCLUSIONS: This study highlights that there is room for an improved acute management and an optimized diagnostic workup that should not be restricted to patients with severe reactions and/or showing MCA.

Exploring the Diagnosis and Profile of Cannabis Allergy.

BACKGROUND: Cannabis allergy (CA) has mainly been attributed to Can s 3, the nonspecific lipid transfer protein (nsLTP) of Cannabis sativa. Nevertheless, standardized diagnostic tests are lacking and research on CA is scarce. OBJECTIVE: To explore the performance of 5 cannabis diagnostic tests and the phenotypic profile of CA. METHODS: A total of 120 patients with CA were included and stratified according to the nature of their cannabis-related symptoms; 62 healthy and 189 atopic controls were included. Specific IgE (sIgE) hemp, sIgE and basophil activation test (BAT) with a recombinant Can s 3 protein from Cannabis sativa (rCan s 3), BAT with a crude cannabis extract, and a skin prick test (SPT) with an nCan s 3-rich cannabis extract were performed. Clinical information was based on patient history and a standardized questionnaire. RESULTS: First, up to 72% of CA reporting likely-anaphylaxis (CA-A) are Can s 3 sensitized. Actually, the Can s 3-based diagnostic tests show the best combination of positive and negative predictive values, 80% and 60%, respectively. sIgE hemp displays 82% sensitivity but only 32% specificity. Secondly, Can s 3+CA reported significantly more cofactor-mediated reactions and displayed significantly more sensitizations to other nsLTPs than Can s 3-CA. Finally, the highest prevalence of systemic reactions to plant-derived foods was seen in CA-A, namely 72%. CONCLUSIONS: The most effective and practical tests to confirm CA are the SPT with an nCan s 3-rich extract and the sIgE rCan s 3. Can s 3 sensitization entails a risk of systemic reactions to plant-derived foods and cofactor-mediated reactions. However, as Can s 3 sensitization is not absolute, other cannabis allergens probably play a role.

Rocuronium Hypersensitivity: Does Off-Target Occupation of the MRGPRX2 Receptor Play a Role?

BACKGROUND: The neuromuscular blocking agent (NMBA) rocuronium is a relevant cause of perioperative hypersensitivity (POH) with a significant risk of diagnostic error. Recently, it has been suggested to reclassify hypersensitivity to NMBA as type A reactions resulting from off-target occupation of the nonimmune MRGPRX2 receptor. OBJECTIVE: To investigate whether basophil activation experiments can benefit diagnosis and add to the insights in the pathomechanisms of rocuronium hypersensitivity. METHODS: A total of 140 patients with a suspected POH to rocuronium in whom peak tryptase was available had complete diagnostic workup for all potential culprits including triple confirmatory testing with skin tests, basophil activation test (BAT), and quantification of specific IgE (sIgE) antibodies to rocuronium and morphine. To further analyze the clinical relevance of sIgE antibodies, quantitative basophil inhibition experiments were performed by coincubation of the cells with rocuronium and morphine, an opiate known to harbor a substituted ammonium structure. RESULTS: Diagnosis of rocuronium hypersensitivity was established in 72 of 140 patients (51.4%), of whom 65 (90.3%) demonstrated mast cell activation. Of the 72 patients, 64 displayed a positive skin test, 8 (11.1%) had their diagnosis documented only by BAT. Coincubation of morphine and rocuronium induced a dose-dependent inhibition of BAT with rocuronium that was restricted to 4 of 6 patients with IgE reactivity to rocuronium and/or morphine. CONCLUSIONS: BAT can benefit diagnosis of rocuronium hypersensitivity. As basophils barely express MRGPRX2 and BAT rocuronium can be inhibited by morphine, we believe that hypersensitivity to rocuronium still mainly results from IgE/high-affinity receptor for sIgE (FcεRI)-dependent effector cell activation. However, it cannot be excluded that in a few patients rocuronium hypersensitivity results from off-target occupation of the MRGPRX2 receptor.

Occupational cannabis exposure and allergy risks.

OBJECTIVES: Cannabis allergy has mainly been described following recreational use but some cases also point to cannabis sensitisation as a result of occupational exposure. As a consequence, little is known on the prevalence and clinical phenotype of occupational cannabis allergy. Therefore, this study aims to explore the allergy-associated health risks of occupational cannabis exposure in Belgian police force personnel. METHODS: 81 participants, active in the police force, reporting regular occupational cannabis exposure during the past 12 months, were included. History was combined with a standardised questionnaire on allergies and cannabis exposure.Basophil activation tests (BATs) with a crude cannabis extract and rCan s 3 were performed. In addition, specific (s)IgE rCan s 3 as well as sIgE to house dust mite, six pollen and three mould allergens were quantified. RESULTS: Although 42% of the participants reported respiratory and/or cutaneous symptoms on occupational cannabis exposure, all cannabis diagnostics were entirely negative, except one symptomatic case demonstrating a borderline result. Furthermore, there is no significant difference between the groups with and without symptoms on cannabis exposure in terms of allergenic sensitisations. CONCLUSIONS: The origins of the reported respiratory and cutaneous symptoms during cannabis exposure remain elusive but are probably due to non-immune reactions. It should be noted that the study was volunteer-based possibly reflecting an excessive number of symptomatic individuals. Nevertheless, as only one participant reported using fully protective gear, much improvement is needed for reducing the number of symptoms reported on duty, independent of their origin.

Cross-Reactive Aeroallergens: Which Need to Cross Our Mind in Food Allergy Diagnosis?

Secondary food allergies due to cross-reactivity between inhalant and food allergens are a significant and increasing global health issue. Cross-reactive food allergies predominantly involve plant-derived foods resulting from a prior sensitization to cross-reactive components present in pollen (grass, tree, weeds) and natural rubber latex. Also, primary sensitization to allergens present in fungi, insects, and both nonmammalian and mammalian meat might induce cross-reactive food allergic syndromes. Correct diagnosis of these associated food allergies is not always straightforward and can pose a difficult challenge. As a matter of fact, cross-reactive allergens might hamper food allergy diagnosis, as they can cause clinically irrelevant positive tests to cross-reacting foods that are safely consumed. This review summarizes the most relevant cross-reactivity syndromes between inhalant and food allergens. Particular focus is paid to the potential and limitations of confirmatory testing such as skin testing, specific IgE assays, molecular diagnosis, and basophil activation test.

In Vitro Diagnosis of Immediate Drug Hypersensitivity During Anesthesia: A Review of the Literature.

Quantification of specific IgE (sIgE) antibodies constitutes an important measure to document anesthesia-related immediate hypersensitivity reactions (IHRs). However, only a few drug-specific assays are available and their predictive value is not known. In cases of non-IgE mediated IHRs, diagnosis might benefit from cellular tests such as basophil mediator release tests and basophil activation tests (BATs). To review the potential and limitations of quantification of sIgE, mediator release, and BAT in anesthesia-related IHRs, a literature search was conducted using the key words allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry, MRGPRX2, specific IgE antibodies, leukotrienes, histamine, and tryptase; this was complemented by the authors' experience. The drugs and compounds that have predominantly been studied are neuromuscular blocking agents (NMBAs), ß-lactams, latex, and chlorhexidine. For sIgE NMBA, sensitivity and specificity varies between 38.5% to 92% and 92% to 100%, respectively. For sIgE ß-lactams, sensitivity varies between 0% to 85% and specificity between 52% to 100%. sIgE to morphine should not be used in isolation to diagnose IHRs to NMBAs or opiates. sIgE for latex, and, in difficult cases, molecular diagnosis with quantification of sIgE to Hevea components constitute reliable diagnostics. For drugs, the sensitivity of BAT varies between 50% and 60% and specificity reaches 80% to 90%. Basophil mediator release tests seem to be abandoned and supplanted by BATs.