Long-term hematopoietic stem cells trigger quiescence in Leishmania parasites.

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2-3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits.

Mast cell versus basophil activation test in allergy: Current status.

In the past two decades, we witnessed the evolution of the basophil activation test (BAT) from mainly research applications to a potential complementary diagnostic tool to document IgE-dependent allergies. However, BAT presents some technical weaknesses. Around 10%-15% of tested patients are non-responders, BAT can be negative immediately post-reaction and the use of fresh basophils, ideally analysed within 4 h of collection, restricts the number of tests that can be performed per sample. The need for fresh basophils is especially limiting when conducting batch analyses and interlaboratory comparisons to harmonize BAT methodology. These limitations significantly hinder the wider application of BAT and urge the development of alternative testing, such as the mast cell activation test (MAT). The essential difference between BAT and MAT is the heterogeneity of the starting material used to perform the assays. Mast cells are tissue-resident, so cannot be easily accessed. Current alternative sources for functional studies are generating primary human mast cells, differentiated from donor progenitor cells, or using immortalized mast cell lines. Hence, the methodological approaches for MAT are not only vastly different from BAT, but also different among MAT protocols. This review summarizes the advantages and disadvantages of BAT and MAT assays, dedicating special attention to elucidating the key differences between the cellular sources used and provides an overview of studies hitherto performed comparing BAT and MAT in the diagnosis of IgE-mediated food and drug allergies.

Mast Cell-Targeting Therapies in Mast Cell Activation Syndromes.

PURPOSE OF REVIEW: Provide an overview of the expanding landscape of mast cell (MC)-targeting treatments in mast cell activation syndromes (MCAS). RECENT FINDINGS: Tyrosine-kinase inhibitors (TKIs) targeting wild-type and mutated KIT can efficiently induce MC depletion. Avapritinib and midostaurin can also temper IgE-mediated degranulation. Avapritinib has been recently approved by the FDA for the treatment of indolent systemic mastocytosis (ISM). Targeting activation pathways and inhibitory receptors is a promising therapeutic frontier. Recently, the anti Siglec-8 antibody lirentelimab showed promising results in ISM. MCAS is a heterogeneous disorder demanding a personalized therapeutic approach and, especially when presenting as anaphylaxis, has not been formally captured as outcome in prospective clinical trials with TKI. Long-term safety of TKI needs to be addressed. New drugs under investigation in diseases in which non-neoplastic MCs play a pivotal role can provide important inputs to identify new efficient and safe treatments for MCAS.

Comparison of the passive mast cell activation test with the basophil activation test for diagnosis of perioperative rocuronium hypersensitivity.

BACKGROUND: Rocuronium is a major cause of perioperative hypersensitivity (POH). Skin tests (STs) and quantification of specific immunoglobulin E antibodies (sIgEs) can yield incongruent results. In such difficult cases, the basophil activation test (BAT) can be helpful. Here, we evaluated the passive mast cell activation test (pMAT) as a substitute of BAT as part of the diagnostic tests for rocuronium allergy. METHODS: Sera from patients with a suspected POH reaction potentially related to rocuronium were included. All patients had a complete diagnostic investigation, including STs, quantification of sIgEs to morphine and rocuronium, and BAT. For execution of pMAT, human mast cells were generated from healthy donor peripheral blood CD34+ progenitor cells and sensitised overnight with patient sera. RESULTS: In total, 90 sera were studied: 41 from ST+sIgE+ patients, 13 from ST-sIgE- patients, 20 from ST+sIgE- patients, and 16 from ST-sIgE+ patients. According to BAT results, patients were further allocated into subgroups. Of the 38 BAT+ patients, 25 (66%) showed a positive pMAT as well. Of the 44 BAT- patients, 43 (98%) also showed a negative pMAT. Mast cells that were not passively sensitised did not respond to rocuronium. CONCLUSIONS: We show that the pMAT, in many cases, can substitute for BAT in the diagnosis of rocuronium hypersensitivity and advance diagnosis in difficult cases with uncertain ST or sIgE results when BAT is not locally available.

A review of cannabis allergy in the early days of legalization.

Cannabis allergy is a burgeoning field; consequently, research is still in its infancy and allergists' knowledge surrounding this topic is limited. As cannabis legalization expands across the world, it is anticipated that there will be an increase in cannabis use. Thus, we hypothesize that a concomitant rise in the incidence of allergy to this plant can be expected. Initiatives aimed at properly educating health care professionals are therefore necessary. This review presents the most up-to-date information on a broad range of topics related to cannabis allergy. Although the clinical features of cannabis allergy are becoming more well described and recognized, the tools available to make a correct diagnosis are meager and often poorly accessible. In addition, research on cannabis allergy is still taking its first steps, and new and potentially groundbreaking findings in this field are expected to occur in the next few years. Finally, although therapeutic approaches are being developed, patient and physician education regarding cannabis allergy is certainly needed.

Suspected perioperative anaphylaxis: are we making the correct diagnosis?

We provide a commentary on aspects of a prospective study of the epidemiology of perioperative anaphylaxis in Japan (Japanese Epidemiologic Study for Perioperative Anaphylaxis [JESPA]). Accurate diagnosis of perioperative anaphylaxis is important for research but essential for clinical safety. We evaluate the diagnostic approach used in the JESPA study and caution against over-reliance on diagnostic tests that lack sensitivity and specificity when clinical data suggest an immediate perioperative hypersensitivity reaction is likely.

Mast cell activation test: A new asset in the investigation of the chlorhexidine cross-sensitization profile.

BACKGROUND: Insights into the IgE cross-sensitization and possible cross-reactivity patterns of sera reactive to chlorhexidine (CHX) are still incomplete and are likely to benefit from a functional exploration using a passive mast cell activation test (pMAT). Therefore, we want to study whether the pMAT with CHX-specific IgE (sIgE) enables to depict effector cell degranulation in response to alexidine (ALX), octenidine (OCT) and/or polyhexamethylene biguanide (PHMB) indicative of cross-reactivity between these compounds and CHX. METHODS: Serum of 10 CHX-allergic patients, nine individuals with an isolated sIgE CHX and five healthy controls were included. Human cultured mast cells (MCs) were, before and after sensitization, challenged with CHX, ALX, OCT or PHMB. Degranulation was measured via quantification of upregulation of CD63. RESULTS: Mast cell responsiveness to ALX and OCT was demonstrable with 4/10 and 3/10 of the sera of CHX-allergic patients respectively. Percentage of degranulation varied between 12 and 34% for ALX-reactive MCs and between 4 and 22% for OCT-reactive MCs. No reactivity to ALX or OCT was demonstrable when using sera obtained from individuals with an isolated sIgE CHX or from healthy controls. Unlike CHX, ALX and OCT, PHMB turned out to be a direct MC activator via occupation of MRGPRX2. PHMB-reactive sIgEs were demonstrable in some patients with an isolated sIgE CHX but were unable to trigger PHMB-induced degranulation in MRGPRX2 knockdown MCs. CONCLUSION: Mast cells constitute an attractive tool to explore cross-reactivity between structurally similar compounds. Along with the identification of safe alternatives for the individual patient, the pMAT can advance our insights into sIgE cross-reactivity patterns including assessment of molecules not yet approved for human use.

Cannabis-related allergies: An international overview and consensus recommendations.

Cannabis is the most widely used recreational drug in the world. Cannabis sativa and Cannabis indica have been selectively bred to develop their psychoactive properties. The increasing use in many countries has been accelerated by the COVID-19 pandemic. Cannabis can provoke both type 1 and type 4 allergic reactions. Officially recognized allergens include a pathogenesis-related class 10 allergen, profilin, and a nonspecific lipid transfer protein. Other allergens may also be relevant, and recognition of allergens may vary between countries and continents. Cannabis also has the potential to provoke allergic cross-reactions to plant foods. Since cannabis is an illegal substance in many countries, research has been hampered, leading to challenges in diagnosis since no commercial extracts are available for testing. Even in countries such as Canada, where cannabis is legalized, diagnosis may rely solely on the purchase of cannabis for prick-to-prick skin tests. Management consists of avoidance, with legal issues hindering the development of other treatments such as immunotherapy. Education of healthcare professionals is similarly lacking. This review aimed to summarize the current status of cannabis allergy and proposes recommendations for the future management of this global issue.

Principles, potential, and limitations of ex vivo basophil activation by flow cytometry in allergology: A narrative review.

The major challenge of allergy diagnosis lies in the development of accessible and reliable diagnostics allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures. Since the late nineties, evidence has accumulated that flow-assisted analysis and quantification of ex vivo-activated basophils (according to the basophil activation test [BAT]) might meet this requirement for different IgE-dependent allergies and particular forms of autoimmune urticaria. Other so-called nondiagnostic applications of the BAT involve therapeutic monitoring, follow-up of natural histories, and identification of allergenic recognition sites. However, it has also become clear that appropriate use of the BAT necessitates knowledge about degranulation metrics and guidance to guarantee correct execution and interpretation of the results. Here, we have reviewed the most relevant applications and limitations of the BAT. Some personal statements and views about its perspectives are made.

Mast cell activation tests by flow cytometry: A new diagnostic asset?

Since the late nineties, evidence has accumulated that flow-assisted basophil activation test (BAT) might be an accessible and reliable method to explore the mechanisms governing basophil degranulation and diagnostic allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures for different IgE-dependent allergies and particular forms of autoimmune urticaria. Although the BAT offers many advantages over mediator release tests, it is left with some weaknesses that hinder a wider application. It is preferable to perform the BAT analysis within 4 h of collection, and the technique does not advance diagnosis in patients with non-responsive cells. Besides, the BAT is difficult to standardize mainly because of the difficulty to perform large batch analyses that might span over several days. This article reviews the status of flow cytometric mast cell activation test (MAT) using passively sensitized mast cells (MCs) with patients' sera or plasma (henceforth indicated as passive MAT; pMAT) using both MC lines and cultured MCs in the diagnosis of IgE-dependent allergies. In addition, this paper provides guidance for generating human MCs from peripheral blood CD34+ progenitor cells (PBCMCs) and correct interpretation of flow cytometric analyses of activated and/or degranulating cells. With the recent recognition of the mas-related G protein-coupled receptor X2 (MRGPRX2) occupation as a putative mechanism of immediate drug hypersensitivity reactions (IDHRs), we also speculate how direct activation of MCs (dMAT)-that is direct activation by MRGPRX2 agonists without prior passive sensitization-could advance paradigms for this novel endotype of IDHRs.

The diagnosis and management of allergic reactions in patients sensitized to non-specific lipid transfer proteins.

Sensitization to one or more non-specific lipid transfer proteins (nsLTPs), initially thought to exist mainly in southern Europe, is becoming accepted as a cause of allergic reactions to plant foods across Europe and beyond. The peach nsLTP allergen Pru p 3 is a dominant sensitizing allergen and peaches a common food trigger, although multiple foods can be involved. A frequent feature of reactions is the requirement for a cofactor (exercise, alcohol, non-steroidal anti-inflammatory drugs, Cannabis sativa) to be present for a food to elicit a reaction. The variability in the food and cofactor triggers makes it essential to include an allergy-focused diet and clinical history in the diagnostic workup. Testing on suspected food triggers should also establish whether sensitization to nsLTP is present, using purified or recombinant nsLTP allergens such as Pru p 3. The avoidance of known trigger foods and advice on cofactors is currently the main management for this condition. Studies on immunotherapy are promising, but it is unknown whether such treatments will be useful in populations where Pru p 3 is not the primary sensitizing allergen. Future research should focus on the mechanisms of cofactors, improving diagnostic accuracy and establishing the efficacy of immunotherapy.

IgE-mediated chlorhexidine allergy-Cross-reactivity with other biguanide disinfectants.

BACKGROUND: Chlorhexidine (CHX) is a widely utilized disinfectant that can cause IgE-mediated urticaria/anaphylaxis. The cross-reactivity of patients with IgE-mediated CHX allergy with other disinfectants, which share structural similarities with CHX like polyhexanide (polyhexamethylene biguanide; PHMB), alexidine (ALX), or octenidine (OCT), is unknown. METHODS: Forty-four patients with anaphylaxis or urticaria upon CHX exposure and positive skin prick test (SPT) and/or positive CHX ImmunoCAP test (Phadia TFS, Uppsala, Sweden) were recruited. IgE to the biguanide and/or hexamethylene structure was investigated with PHMB ImmunoCAP (n = 32) and by basophil activation tests (BAT) with CHX and ALX (n = 37). Inhibition tests of CHX and PHMB ImmunoCAPs by CHX, ALX, PHMB, and OCT were performed. RESULTS: IgE reactivity to PHMB as surrogate marker for biguanide/hexamethylene reactivity was detected in 5/32 sera. Seven of 37 patients showed a positive BAT with ALX, but only under optimized conditions. Binding to CHX ImmunoCAP was inhibited by ALX in 1/32 sera, and binding to PHMB was blocked by ALX (1/5) and by OCT in another (1/5). In SPT, 9/10 patients were positive for CHX and 3 of them with ALX (only at highest concentration at 5 mg/mL). A further patient reacted primarily with OCT and showed IgE cross-reactivity with CHX, ALX, and PHMB. CONCLUSION: The IgE response to CHX seems polyclonal. The chloroguanide ending of CHX is the main epitope for the IgE and is suitable as screening assay to detect CHX reactivity. IgE-reactivities with the biguanide or hexamethylene components of other disinfectants (ALX, PHMB) can be detected by SPT, PHMB ImmunoCAP, and ALX-BAT in 15%-33% of CHX-allergic patients.

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.

Mast cell activation test in chlorhexidine allergy: a proof of concept.

BACKGROUND: Immediate drug hypersensitivity reactions are an increasing public health issue and a frequent cause of life-threatening anaphylaxis. Conventional confirmatory testing include skin tests and, for a few drugs, quantification of drug-specific immunoglobulin E (IgE) antibodies. However, none of these tests are absolutely predictive for the clinical outcome, and can yield false-negative and false-positive results. We performed a proof-of-concept study to assess whether a mast cell activation test could improve diagnosis of IgE-mediated chlorhexidine hypersensitivity, a common cause of perioperative anaphylaxis. METHODS: Human mast cells were generated from CD34+ progenitor cells and sensitised with patients' sera to become IgE+ human mast cells (dMCIgE+), and then incubated with chlorhexidine to assess degranulation. We compared the diagnostic performance of this mast cell activation test with serum from patients with and without positive skin test and basophil activation test to chlorhexidine. RESULTS: In dMC sensitised with sera from patients with a positive skin test and basophil activation test to chlorhexidine showed drug-specific and concentration-dependent degranulation upon stimulation with chlorhexidine, determined by surface upregulation of the degranulation marker CD63. In contrast, dMC sensitised with sera from patients with a negative skin test and basophil activation test to chlorhexidine were unresponsive in the mast cell activation test. CONCLUSIONS: Our study suggests that the mast cell activation test can be used to diagnose IgE/FcεRI-dependent immediate drug hypersensitivity reactions. It also shows potential to assess the clinical relevance of drug-specific IgE antibodies in their ability to elicit mast cell degranulation, and therefore discriminate between allergy and sensitisation. Extended studies are required to verify whether this technique can be used in other causes of perioperative anaphylaxis.

Controversies in drug allergy: In vitro testing.

Despite their low frequency, drug hypersensitivity reactions (DHRs) can be serious and result in lifelong sequelae. The diagnosis is critical to avert future reactions and should identify the culprit drug or drugs and safe alternatives. However, making the diagnosis can be complex and challenging. Reliable in vitro tests can offer the potential to improve a diagnosis of DHR and influence medical decision making. Importantly, in vitro testing is frequently not performed as a test in isolation but rather as a component of a diagnostic algorithm along with additional tests. There are several in vitro approaches for the different endotypes of DHRs. However, only few are available for routine diagnosis, and many are restricted to research laboratories. In vitro tests exhibit varying sensitivity and specificity depending on the drug involved and the clinical phenotype. In vitro tests can complement skin tests, especially in patients with negative or equivocal skin test responses inconsistent with the clinical presentation and in severe reactions in which drug provocation tests are contraindicated. The main unmet need for many in vitro tests for the diagnosis of DHRs is validation in larger studies with standardized controls that could harmonize diagnostic management between the United States, European Union, and other regions of the world.

An EAACI position paper on the investigation of perioperative immediate hypersensitivity reactions.

Perioperative immediate hypersensitivity reactions are rare. Subsequent allergy investigation is complicated by multiple simultaneous drug exposures, the use of drugs with potent effects and the many differential diagnoses to hypersensitivity in the perioperative setting. The approach to the investigation of these complex reactions is not standardized, and it is becoming increasingly apparent that collaboration between experts in the field of allergy/immunology/dermatology and anaesthesiology is needed to provide the best possible care for these patients. The EAACI task force behind this position paper has therefore combined the expertise of allergists, immunologists and anaesthesiologists. The aims of this position paper were to provide recommendations for the investigation of immediate-type perioperative hypersensitivity reactions and to provide practical information that can assist clinicians in planning and carrying out investigations.

In vitro diagnostic tests for perioperative hypersensitivity, a narrative review: potential, limitations, and perspectives.

Correct diagnostic management of perioperative hypersensitivity aims to identify the underlying mechanism(s), responsible culprit(s), and safe alternative drugs or techniques. Although drug provocation tests are considered the gold standard, diagnosis of perioperative hypersensitivity mainly relies on skin testing. Use of in vitro tests, such as quantification of specific immunoglobulin E antibodies, serum tryptase, and plasma histamine, as well as basophil activation tests is becoming widespread. These latter tests have the advantage of having no risk of recurrence of immediate hypersensitivity reactions. In this narrative review, we summarise the principles of these in vitro tests, and the possibilities and limitations when these tests are used for testing sensitivity to substances with a high risk of causing perioperative hypersensitivity. Hence, we focus on neuromuscular blocking agents, antibiotics, natural rubber latex, and opiates/opioids. The combination of multiple tests would allow diagnosis of perioperative hypersensitivity with the right balance of safety and accuracy.

Molecular mechanisms and pathophysiology of perioperative hypersensitivity and anaphylaxis: a narrative review.

Perioperative hypersensitivity reactions (POH) constitute a clinical and diagnostic challenge, a consequence of heterogeneous clinical presentations, and multiple underlying pathomechanisms. POH do not necessarily involve an allergen-specific immune response with cross-linking of specific immunoglobulin E (sIgE) antibodies on mast cells and basophils. POH can also result from alternative specific and non-specific effector cell activation/degranulation such as complement-derived anaphylatoxins and off-target occupancy of mast cell, basophil, or both surface receptors. Moreover, POH and anaphylaxis can occur independent from mast cell and basophil degranulation. The manifestations of POH primarily affect the cardiovascular, respiratory, and integumentary systems. POH present within the context of surgical or procedural pathology and the effects of surgical and anaesthetic techniques on pre-existing physiological reserve. The majority of cases of appropriately-treated intraoperative anaphylaxis can be considered a compensated cardiovascular anaphylaxis. With increasing severity of anaphylaxis, maldistribution and hypovolaemia lead to reduced venous return and circulatory failure. Treatment with a combination of epinephrine and i.v. fluid is critical for successful resuscitation, although the excessive use of epinephrine without adequate volume expansion may be deleterious. Neural control of the airways is important in the pathophysiology of bronchospasm. Anticholinergic drug premedication is beneficial in patients with hyperreactive airways. Pulmonary oedema can result from a combination of pulmonary capillary hypertension, incompetence of the alveolocapillary membrane, or both. Angioedema can be distinguished mechanistically into histaminergic and non-histaminergic (e.g. bradykinin-mediated). An understanding of the molecular mechanisms and pathophysiology of POH are essential for the immediate management and subsequent investigation of these cases.