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Chronic spontaneous urticaria (CSU) is associated with skin mast cell activation, and its triggering mechanisms are not completely elucidated. Evidence suggests an autoimmune component of CSU. Our aim was to assess the usefulness of an autoimmune mast cell activation test (aiMAT) for diagnosing and differentiating CSU into different subtypes. We enrolled 43 patients with active, uncontrolled CSU before starting treatment with omalizumab and 15 controls. Patients were evaluated based on omalizumab response. aiMATs were performed using non-IgE-sensitized (NS) or myeloma IgE-sensitized (S) LAD2 cells, which were then stimulated with CSU/control sera (25 µL and 10 µL). The expression of CD63 was assessed with flow cytometry. CD63 response on NS-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.0007) and with 10 µL CSU/control sera (p = 0.0001). The ROC curve analysis demonstrated an area under the curve (AUC) of 0.82. The cutoff for autoimmune-non-IgE-sensitized-MAT was 40.3% CD63+ LAD2, which resulted in 73.3% sensitivity and 81.4% specificity. CD63 response on S-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.03). The ROC curve analysis demonstrated an AUC of 0.66. The cutoff for the autoimmune-myeloma IgE-sensitized-MAT was 58.4% CD63+ cells, which resulted in 62.8% sensitivity and 66.7% specificity. Overall, 36 out of 43 (84%) patients responded to omalizumab, and 7 (16%) were nonresponders. We found no differences between LAD2 CD63 response and response to omalizumab. In conclusion, aiMAT could represent a new diagnostic tool in CSU. Additional studies are needed to evaluate the potential benefits during omalizumab therapy. [ABSTRACT FROM AUTHOR]

Upon first exposure to cetuximab, hypersensitivity reactions can occur. We aimed to assess the utility of the basophil activation test (BAT) to alpha-gal and cetuximab for predicting severe reactions. We prospectively recruited 38 patients and evaluated sIgE to alpha-gal in all patients before the first application of cetuximab. In all alpha-gal-sensitized patients, we evaluated skin tests to meat extracts, gelatine, and cetuximab and performed BAT with alpha-gal and cetuximab. In 24% (9/38) of patients, sIgE to alpha-gal was >0.10 kUA/L, and 8/9 reacted to the cetuximab. Basophil activation tests with alpha-gal were positive in all sensitized patients and were higher in those with severe reactions (18.3% in grade 4 [n = 4] vs. 1.8% in grade 2 [n = 3] or no reaction [n = 1] at 3.3 ng/mL of alpha-gal; p = 0.03). All patients with severe grade 4 reactions had a positive CD63 BAT response to cetuximab compared to patients with moderate or no reaction, who all had negative BAT (57.7% vs. 0.9% at 500 µg/mL, 63.2% vs. 4.1% at 100 µg/mL, 58.2% vs. 2.7% at 10 µg/mL, and 32.1% vs. 3.3% at 1 µg/mL of cetuximab, respectively; p ≤ 0.001). In summary, before initiating cetuximab treatment, sIgE to alpha-gal should be assessed in all patients. To predict the severity of the reaction and to assess the risk of cetuximab-induced anaphylaxis, we should perform BATs with alpha-gal or more discriminative BATs with cetuximab. [ABSTRACT FROM AUTHOR]

Allergen-specific venom immunotherapy (VIT) is a well-established therapy for Hymenoptera venom allergy (HVA). However, the precise mechanism underlying its clinical effect remains uncertain. Our study aimed to identify the molecular mechanisms associated with VIT efficiency. We prospectively included 19 patients with HVA undergoing VIT (sampled before the beginning of VIT, after reaching the maintenance dose, one year after finishing VIT, and after a sting challenge) and 9 healthy controls. RNA sequencing of whole blood was performed on an Illumina sequencing platform. Longitudinal transcriptomic profiling revealed the importance of the inhibition of the NFκB pathway and the downregulation of DUX4 transcripts for the early protection and induction of tolerance after finishing VIT. Furthermore, successful treatment was associated with inhibiting Th2, Th17, and macrophage alternative signalling pathways in synergy with the inhibition of the PPAR pathway and further silencing of the Th2 response. The immune system became activated when reaching the maintenance dose and was suppressed after finishing VIT. Finally, successful VIT restores the immune system's balance to a state similar to that of healthy individuals. Our results underline the important role of the inhibition of four pathways in the clinical effect of VIT: Th2, Th17, NFκB, and macrophage signalling. Two biomarkers specific for successful VIT, regardless of the time of sampling, were C4BPA and RPS10-NUDT3 and should be further tested as potential biomarkers. [ABSTRACT FROM AUTHOR]

Hymenoptera venom–triggered anaphylaxis (HVA) affects up to 8.9% of the general population and is the most frequent cause of anaphylaxis in adults, accounting for approximately 20% of all fatal anaphylaxis cases. Quite often, a fatal reaction is a victim's first manifestation of HVA. Mastocytosis represents one of the most important risk factors for severe HVA. We analyzed patients with documented fatal HVA for the presence of underlying clonal mast cell disorder (cMCD). Here, we report three cases of fatal HVA, with undiagnosed underlying cMCD identified by the presence of the peripheral blood and/or bone marrow KIT p.D816V missense variant postmortem. In the first case, anaphylaxis was the initial episode and was fatal. In the other two cases, both patients were treated with specific venom immunotherapy (VIT), nevertheless, one died of HVA after VIT discontinuation, and the other during VIT; both patients had cardiovascular comorbidities and were taking beta-blockers and/or ACE inhibitors. Our results point to the importance of screening all high-risk individuals for underlying cMCD using highly sensitive molecular methods for peripheral blood KIT p.D816V variant detection, including severe HVA and possibly beekeepers, for proper management and the need for lifelong VIT to prevent unnecessary deaths. Patients at the highest risk of fatal HVA, with concomitant cardiovascular and cMCD comorbidities, might not be protected from field stings even during regular VIT. Therefore, two adrenaline autoinjectors and lifelong VIT, and possibly cotreatment with omalizumab, should be considered for high-risk patients to prevent fatal HVA episodes. [ABSTRACT FROM AUTHOR]

Histamine intolerance (HIT) is a clinical condition caused by decreased intestinal degradation of ingested histamine, primarily due to reduced enzyme diamine oxidase (DAO) activity, leading to histamine accumulation and causing various clinical manifestations. The measurement of serum DAO is commonly used as the main diagnostic test for HIT, although its diagnostic use is still uncertain. In this retrospective study, we aimed to assess the validity of DAO determination in patients with clinically suspected HIT. We measured DAO levels in 249 patients with suspected HIT and 50 healthy adult controls without HIT-related problems. Based on five clinical criteria, we divided patients into two groups: high (all five inclusion criteria; 41 patients) and low probability of HIT (≤4 inclusion criteria; 208 patients). Patients with a "high probability of HIT" had the lowest DAO (median: 8 U/mL, IQR: 6–10) in comparison to patients with a "low probability of HIT (median: 10 U/mL, IQR: 7–16, p = 0.0006) and healthy controls (median: 18 U/mL, IQR: 14–22, p < 0.0001). The specificity and sensitivity for DAO levels < 3/< 10 U/mL (manufacturer's set cut-off) to discriminate between patients with "high probability of HIT" and healthy controls were 100%/92% and 2%/71%. On the other hand, the specificity and sensitivity to discriminate between patients with "high probability of HIT" and "low probability of HIT" were 97%/61% and 2%/71%, respectively. Serum DAO determination represents an additional asset to the diagnosis of HIT based on clinical evaluation and assessment, but the diagnosis should not solely rely on DAO measurements. [ABSTRACT FROM AUTHOR]

Protein–peptide interactions are an essential player in cellular processes and, thus, of great interest as potential therapeutic agents. However, identifying the protein's interacting surface has been shown to be a challenging task. Here, we present a methodology for protein–peptide interaction identification, implementing phage panning, next-generation sequencing and bioinformatic analysis. One of the uses of this methodology is identification of allergen epitopes, especially suitable for globular inhaled and venom allergens, where their binding capability is determined by the allergen's conformation, meaning their interaction cannot be properly studied when denatured. A Ph.D. commercial system based on the M13 phage vector was used for the panning process. Utilization of various bioinformatic tools, such as PuLSE, SAROTUP, MEME, Hammock and Pepitope, allowed us to evaluate a large amount of obtained data. Using the described methodology, we identified three peptide clusters representing potential epitopes on the major wasp venom allergen Ves v 5. [ABSTRACT FROM AUTHOR]

Mycoplasma pneumoniae (Mp) can cause several extrapulmonary manifestations, most frequently dermatological ones. It is largely unknown whether Mp genotype determines Mp-induced cutaneous disease. The aim of our study was to assess the association between Mp genotype and this clinical outcome. We performed a retrospective study of children referred with signs of acute Mp infection from 1 January 2014 to 31 December 2014. We compared the characteristics of children presenting as cutaneous disease, upper (URTI) and lower respiratory tract infection (LRTI). In addition, we separately analyzed the data of patients presenting with Mp-induced cutaneous disease. We evaluated data from 435 patients (mean age 7.3 years, SD 3.4 years; 52.0% boys) who had Mp PCR-positive pharyngeal swab, P1 genotype and/or multilocus variable-number tandem-repeat analysis (MLVA) genotype defined and no viral co-detection, presenting as cutaneous disease (38/435), URTI (46/435) or LRTI (351/435). The majority of patients had urticarial (55%, 21/38) or maculopapular eruptions (37%, 14/38). We found no association between Mp genotype and clinical outcome of cutaneous disease, nor any specific dermatological presentation. In the group with cutaneous disease, 18% (7/38) required hospital admission because of rash. We found that infection with MLVA-3,6,6,2 strains was more common in admitted patients than in outpatients (40% vs. 4%, p = 0.017) and significantly affected the likelihood of hospital admission in a logistic regression model. The results of our cohort study suggest that Mp genotype does not determine Mp-induced cutaneous disease or a specific dermatological presentation. Nevertheless, infections with certain MLVA strains could induce more severe cutaneous disease requiring hospitalization. [ABSTRACT FROM AUTHOR]

Hymenoptera venom allergy is one of the most severe allergic diseases, with a considerable prevalence of anaphylactic reaction, making it potentially lethal. In this review, we provide an overview of the current knowledge and recent findings in understanding induced immune mechanisms during different phases of venom immunotherapy. We focus on protection mechanisms that occur early, during the build-up phase, and on the immune tolerance, which occurs later, during and after Hymenoptera venom immunotherapy. The short-term protection seems to be established by the early desensitization of mast cells and basophils, which plays a crucial role in preventing anaphylaxis during the build-up phase of treatment. The early generation of blocking IgG antibodies seems to be one of the main reasons for the lower activation of effector cells. Long-term tolerance is reached after at least three years of venom immunotherapy. A decrease in basophil responsiveness correlates with tolerated sting challenge. Furthermore, the persistent decline in IgE levels and, by monitoring the cytokine profiles, a shift from a Th2 to Th1 immune response, can be observed. In addition, the generation of regulatory T and B cells has proven to be essential for inducing allergen tolerance. Most studies on the mechanisms and effectiveness data have been obtained during venom immunotherapy (VIT). Despite the high success rate of VIT, allergen tolerance may not persist for a prolonged time. There is not much known about immune mechanisms that assure long-term tolerance post-therapy. [ABSTRACT FROM AUTHOR]