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Background: Biomarkers of eosinophilic disease activity, especially in the context of novel therapies that reduce blood eosinophil counts, are an unmet need. Absolute eosinophil count (AEC) does not accurately reflect tissue eosinophilia or eosinophil activation. Therefore, the aims of this study were to compare the reliability of plasma and urine eosinophil major basic protein 1, eosinophil cationic protein, eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase measurement and to evaluate the usefulness of eosinophil granule protein (EGP) measurement for the assessment of disease activity in patients with eosinophil-associated diseases treated with mepolizumab, benralizumab, or dexpramipexole., Methods: Eosinophil granule protein concentrations were measured in serum, plasma, and urine from healthy volunteers and patients with hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and eosinophilic asthma using a multiplex assay., Results: Urine EGP concentrations remained stable, whereas serum and plasma EGP concentrations increased significantly with delayed processing. Plasma (p) EDN, but not urine (u) EDN, concentration correlated with AEC and negatively correlated with prednisone dose. Both pEDN and uEDN decreased significantly following treatment of HES patients with benralizumab and EGPA patients with mepolizumab. uEDN appeared to increase with clinical relapse in both patient groups., Conclusions: Measurement of EGP in urine is noninvasive and unaffected by cellular lysis. Although plasma and urine EDN concentrations showed a similar pattern following benralizumab and mepolizumab treatment, the lack of correlation between AEC or prednisone dose and uEDN concentrations suggests that measurement of uEDN may provide a potential biomarker of disease activity in patients with HES and EGPA., (© 2022 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Eosinophilia and eosinophil activation are recurrent features in various reactive states and certain hematologic malignancies. In patients with hypereosinophilia (HE), HE-induced organ damage is often encountered and may lead to the diagnosis of a hypereosinophilic syndrome (HES). A number of known mechanisms and etiologies contribute to the development of HE and HES. Based on these etiologies and the origin of eosinophils, HE and HES are divided into primary forms where eosinophils are clonal cells, reactive forms where an underlying reactive or neoplastic condition is detected and eosinophils are considered to be "non-clonal" cells, and idiopathic HE and HES in which neither a clonal nor a reactive underlying pathology is detected. Since 2012, this classification and the related criteria have been widely accepted and regarded as standard. However, during the past few years, new developments in the field and an increasing number of markers and targets have created a need to update these criteria and the classification of HE and HES. To address this challenge, a Working Conference on eosinophil disorders was organized in 2021. In this conference, a panel of experts representing the relevant fields, including allergy, dermatology, hematology, immunology, laboratory medicine, and pathology, met and discussed new markers and concepts as well as refinements in definitions, criteria and classifications of HE and HES. The outcomes of this conference are presented in this article and should assist in the diagnosis and management of patients with HE and HES in daily practice and in the preparation and conduct of clinical trials., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Glucocorticoid resistance has been associated with Th17-driven inflammation, the mechanisms of which are not clear. We determined whether human and mouse Th17 cells are resistant to glucocorticoid-induced apoptosis., Methods: Freshly isolated human blood Th17 cells and in vitro differentiated Th17 cells from IL-17F red fluorescent protein reporter mice were treated with dexamethasone, a potent glucocorticoid. Apoptosis was measured using annexin V and DAPI staining. Screening of apoptosis genes was performed using the apoptosis PCR array. Levels of molecules involved in apoptosis were measured using quantitative RT-PCR, flow cytometry, and Western blotting. Knockdown of BCL-2 in murine Th17 cells was performed via retroviral transduction. Cytokines were measured using ELISA. A murine Th17-driven severe asthma model was examined for Th17 glucocorticoid sensitivity in vivo., Results: Human and mouse Th17 cells and mouse Th2 cells were resistant to glucocorticoid-induced apoptosis. Th17 cells had glucocorticoid receptors levels comparable to those in other T effectors cells. Th17 cells had high levels of BCL-2, knockdown of which sensitized Th17 cells to dexamethasone-induced apoptosis. Production of IL-22, but not IL-17A and IL-17F, was suppressed by glucocorticoids. STAT3 phosphorylation in Th17 cells was insensitive to glucocorticoid inhibition. Lung Th17 cells in the murine severe asthma model were enhanced, rather than suppressed, by glucocorticoids., Conclusion: Th17 cells are resistant to glucocorticoid-induced apoptosis and cytokine suppression, at least in part due to high levels of BCL-2. These findings support a role of Th17 cells in glucocorticoid-resistant inflammatory conditions such as certain endotypes of asthma., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Clara cell 10-kDa protein (CC10) is a multifunction protein with anti-inflammatory and immunomodulatory effects; hence we compared the CC10 expression between chronic rhinosinusitis (CRS) patients with and without nasal polyps (NPs), analyzed its association with disease severity and response to surgery, and explored its regulation via cytokines., Methods: The plasma and tissue CC10 levels were compared between controls and CRS patients with and without NPs by means of quantitative RT-PCR, ELISA, and immunohistochemistry. Computed tomography (CT) scan and endoscopy findings and symptoms were scored. Nasal explant culture was used to explore the effect of TNF-alpha, IL-1beta, IL-4, INF-gamma, and IL-10 on CC10 gene regulation., Results: Compared with controls, the CC10 expression in sinonasal mucosa was significantly inhibited in both CRS patients with and without NPs. There was a significant further decrease of CC10 expression in patients with NPs and asthma. No difference in CC10 plasma levels was found between controls and patients. CC10 levels inversely correlated with preoperative CT scores, and postoperative endoscopy and symptom scores. TNF-alpha, IL-1beta and IL-4 inhibited, whereas INF-gamma and IL-10 promoted CC10 production in nasal mucosa. A significantly faster decay of CC10 transcripts was seen after IL-1beta treatment. IL-1beta and IL-10 induced thyroid transcription factor-1 expression. INF-gamma increased, whereas IL-4 inhibited hepatocyte nuclear factor-3alpha expression., Conclusion: CC10 may take part in the pathogenesis of CRS and correlates with disease severity and response to surgery. Different cytokines can regulate CC10 expression in nasal mucosa differentially through modulating mRNA stability and certain transcriptional factors expression.

Background: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that bind sialic acid and mostly contain immunoreceptor tyrosine-based inhibitory motifs, suggesting that these molecules possess inhibitory functions. We have recently identified Siglec-8 as an eosinophil-prominent Siglec, and cross-linking of Siglec-8 on human eosinophils induces apoptosis. In this article, we address the in vivo consequences of Siglec engagement. We and others have identified mouse Siglec-F as the closest functional paralog of human Siglec-8, based on shared ligand-binding and expression pattern. We therefore hypothesized that Siglec-F engagement would affect levels and viability of eosinophils in vivo., Methods: Wild type and hypereosinophilic mice were administered Siglec-F antibody and levels of eosinophils in peripheral blood and tissue were measured. Eosinophil apoptosis (in vivo and in vitro) was determined by binding of Annexin-V., Results: Studies in IL-5 transgenic mice, displaying hypereosinophilia, show that administration of a single dose of Siglec-F antibody results in rapid reductions in quantum of eosinophils in the blood. This decrease was accompanied by reductions in tissue eosinophils. Quantum of eosinophils in blood was decreased using two separate antibodies, as well as in other mouse models (wild type mice and in a mouse model of chronic eosinophilic leukemia). Mechanistic studies demonstrated that Siglec-F antibody administration induced apoptosis of eosinophils in vivo and in vitro., Conclusion: These data demonstrate that activation of innate immune receptors, like Siglec-F, can significantly reduce mouse eosinophil viability. As such, targeting Siglec-8/F may be a therapeutic approach for eosinophilic disorders.

Effector mechanisms in anaphylaxis were reviewed. Current approaches to confirmation of the clinical diagnosis were discussed. Improved methods for distinguishing between allergen sensitization (which is common in the general population) and clinical risk of anaphylaxis (which is uncommon) were deliberated. Innovative techniques that will improve risk assessment in anaphylaxis in the future were described.

Using human mast cells (MC) derived by culture of CD34+ peripheral blood precursors, a comprehensive study was performed of expression of 11 known Siglecs. Analysis was initially performed at the mRNA level using gene arrays. Positive results were then validated at the protein level using indirect immunofluorescence and flow cytometry, and for some Siglecs, Western blot analysis was also used. Culture-derived MC expressed mRNA for CD22 (Siglec-2), CD33 (Siglec-3), Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for CD22 and Siglec-10, where levels were low or undetectable. However, Western blotting was able to detect MC expression of CD22 and Siglec-10, suggesting that these proteins were mostly cytoplasmic. CD34+ precursor cells from peripheral blood constitutively expressed surface CD33, Siglec-5 and Siglec-10. As they matured into MC, their constitutive levels of CD33 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 appeared de novo, all in parallel with accumulation of histamine and other MC markers, such as surface expression of FcepsilonRIalpha, and CD51. Phenotypic analysis of LAD-2 MC yielded a similar pattern of Siglec expression except that CD22 expression was particularly prominent. Finally, immunohistochemistry confirmed expression of these same Siglecs by mature tryptase-positive MC in human lung tissues. These data demonstrate an extensive and previously unappreciated pattern of Siglec expression on human MC. Whether engagement and signaling through these inhibitory Siglecs can impact MC biology will require further investigation.