A Rapid Sputum-based Lateral Flow Assay for Airway Eosinophilia using an RNA-cleaving DNAzyme Selected for Eosinophil Peroxidase.

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.

Posttranslational modification and heme cavity architecture of human eosinophil peroxidase-insights from first crystal structure and biochemical characterization.

Eosinophil peroxidase (EPO) is the most abundant granule protein exocytosed by eosinophils, specialized human phagocytes. Released EPO catalyzes the formation of reactive oxidants from bromide, thiocyanate, and nitrite that kill tissue-invading parasites. However, EPO also plays a deleterious role in inflammatory diseases, making it a potential pharmacological target. A major hurdle is the high similarity to the homologous myeloperoxidase (MPO), which requires a detailed understanding of the small structural differences that can be used to increase the specificity of the inhibitors. Here, we present the first crystal structure of mature leukocyte EPO at 1.6 Å resolution together with analyses of its posttranslational modifications and biochemical properties. EPO has an exceptionally high number of positively charged surface patches but only two occupied glycosylation sites. The crystal structure further revealed the existence of a light (L) and heavy (H) chain as a result of proteolytic cleavage. Detailed comparison with the structure of human MPO allows us to identify differences that may contribute to the known divergent enzymatic properties. The crystal structure revealed fully established ester links between the prosthetic group and the protein, the comparably weak imidazolate character of the proximal histidine, and the conserved structure of the catalytic amino acids and Ca2+-binding site. Prediction of the structure of unprocessed proeosinophil peroxidase allows further structural analysis of the three protease cleavage sites and the potential pro-convertase recognition site in the propeptide. Finally, EPO biosynthesis and its biochemical and biophysical properties are discussed with respect to the available data from the well-studied MPO.

Biosynthesis of the Novel Endogenous 15-Lipoxygenase Metabolites N-13-Hydroxy-octodecadienoyl-ethanolamine and 13-Hydroxy-octodecadienoyl-glycerol by Human Neutrophils and Eosinophils.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis inhibition with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15-30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Image Analysis of Eosinophil Peroxidase Immunohistochemistry for Diagnosis of Eosinophilic Esophagitis.

BACKGROUND: Diagnosis of eosinophilic esophagitis (EoE) requires manual quantification of tissue eosinophils. Eosinophil peroxidase (EPX) is an eosinophil-specific, cytoplasmic granule protein released during degranulation. AIMS: The objective of this study was to evaluate image analysis of EPX immunohistochemistry as an automated method for histologic diagnosis of EoE. METHODS: We performed a secondary analysis of prospectively collected esophageal biopsies obtained from adult subjects with EoE and controls. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated for peak eosinophils per high power field (eos/hpf). The same slides were de-stained and re-stained to detect EPX for direct comparison. Slides were digitized, and EPX staining area/mm2 was quantified using image analysis. Paired samples were compared for changes in EPX staining in treatment responders and non-responders. RESULTS: Thirty-eight EoE cases and 49 controls were analyzed. Among EoE subjects, matched post-treatment biopsies were available for 21 responders and 10 non-responders. Baseline EPX/mm2 was significantly increased in EoE subjects and decreased in treatment responders. EPX quantification correlated strongly with eos/hpf (r = 0.84, p < 0.0001) and identified EoE subjects with high diagnostic accuracy (AUC 0.95, p < 0.0001). The optimal diagnostic EPX-positive pixel/area threshold was 17,379 EPX/mm2. Several controls (5/49) with < 15 eos/hpf on H&E staining exceeded this cutoff. CONCLUSIONS: EPX/mm2 correlates strongly with eos/hpf, accurately identifies subjects with EoE, and decreases in treatment responders. Automated quantification of intact eosinophils and their degranulation products may enhance pathologic assessment. Future studies are needed to correlate EPX/mm2 with symptoms, endoscopic findings, and esophageal distensibility.

Antimicrobials offered from nature: Peroxidase-catalyzed systems and their mimics.

The control of antimicrobial resistance requires the development of novel antimicrobial alternatives and naturally occurring peroxidase-catalyzed systems may be of great value in this era of emerging antimicrobial resistance. In the peroxidase system, a peroxidase enzyme catalyzes the oxidation of a halide/pseudohalide, at the expense of hydrogen peroxide, to generate reactive products with broad antimicrobial properties. The appropriate use of peroxidase systems needs a better understanding of the identities and properties of the generated antimicrobial oxidants, specific targets in bacterial cells, their mode of action and the factors favoring or limiting their activity. Here, the ABCs (antibacterial activity, bacterial "backtalk" and cytotoxicity) of these systems and their mimics are discussed. Particular attention is paid to the concomitant use of thiocyanate and iodide dual substrates in peroxidase/peroxidase-free systems with implications on their antimicrobial activity. This review also provides a summary of actual applications of peroxidase systems as bio-preservatives in oral healthcare, milk industry, food/feed specialties and related products, mastitis and wound treatment; lastly, this review points to opportunities for further research and potential applications.

12/15-Lipoxygenase choreographs the resolution of IgG-mediated skin inflammation.

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease "bullous pemphigoid-like epidermolysis bullosa acquisita" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (Tregs) into lesional skin. Intriguingly, Alox15-/- mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15-/- eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and Tregs, which in turn inhibit neutrophils.

In utero exposure to genistein decreased intranasal house dust mite-induced respiratory allergy in middle-aged male B6C3F1 offspring.

Despite many hypothesized benefits of dietary isoflavone genistein (GEN) deriving from soy-based products, questions surrounding GEN's developmental effects are increasing. To understand if in utero GEN exposure modulated postnatal respiratory allergies in the middle age, we conducted a time course study in the B6C3F1 offspring (PND 240-330) using a common household allergen (house dust mites: HDM; 10 µg/mouse for PND 240 and 290, and 50 µg/mouse for PND 330, a middle age in mice) following intranasal instillation, a physiological route of allergen exposure. GEN was administered to dams by gavage from gestational day 14 to parturition at a physiologically relevant dose (20 mg/kg body weight). Female and male offspring were sensitized with HDM allergens beginning about one month prior to sacrifice followed by challenges with three weekly dosings of HDM extracts, and they were euthanized at day 3 following the final HDM exposure. In utero exposure to GEN decreased HDM allergen-induced respiratory allergy in male B6C3F1 offspring at PND 330 as reflected by decreases in airway hyperresponsiveness (e.g., Penh value), HDM-specific IgG1 (a Th2 type Ab) and the activity of eosinophil peroxidase in the lung (an indication of eosinophil recruitment to the lungs). However, in utero exposure to GEN had minimal effects on HDM allergen-induced respiratory allergy in the middle-aged female offspring. Changes in serum total IgE, HDM-specific IgE, and lung histopathology scores in both male and female offspring were not biologically significant. Overall, in utero GEN exposure exerted a protective effect on respiratory allergy in the middle-aged male, but not female, B6C3F1 offspring following later-life HDM exposures.

Eosinophils promote corneal wound healing via the 12/15-lipoxygenase pathway.

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.

Functional role of phosphatidylcholine-specific phospholipase C in regulating leukotriene synthesis and degranulation in human eosinophils.

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A2 (cPLA2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) secretion. However, at concentrations that blocked both AA release and LTC4 secretion, D609 had no significant inhibitory effect on stimulated cPLA2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC4 secretion by D609 is due to inhibition of calcium-mediated cPLA2 translocation to intracellular membranes, not inhibition of cPLA2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+]i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC4 synthesis through calcium-mediated cPLA2 activation.

The Charcot-Leyden crystal protein revisited-A lysopalmitoylphospholipase and more.

The Charcot-Leyden crystal protein (CLC-P), a constituent of human and not mouse eosinophils, is one of the most abundant proteins within human eosinophils. It has a propensity to form crystalline structures, Charcot-Leyden crystals, which are hallmarks in their distinctive extracellular crystalline forms as markers of eosinophilic inflammation. The functions of CLC-P within eosinophils have been uncertain. Although the action of CLC-P as a lysophospholipase has been questioned, assays of chromatographically purified CLC-P and crystal-derived CLC-P as well as studies of transfected recombinant CLC-P have consistently documented that CLC-P endogenously expresses lysophospholipase activity, releasing free palmitate from substrate lysopalmitoylphosphatidylcholine. Rather than acting solely as a hydrolytic enzyme to release palmitate from a lysolipid substrate, some other lysophospholipases function more dominantly as acyl-protein thioesterases (APTs), enzymes that catalyze the removal of thioester-linked, long chain fatty acids, such as palmitate, from cysteine residues of proteins. As such APTs participate in palmitoylation, a post-translational modification that can affect membrane localization, vesicular transport, and secretion. CLC-P has attributes of an APT. Thus, whereas CLC-P expresses inherent lysophospholipase activity, like some other lysophospholipase enzymes, it likely also functions in regulating the dynamic palmitoylation cycle, including, given its dominant subplasmalemmal location, at the human eosinophil's plasma membrane.

Functional characteristics of circulating granulocytes in severe congenital neutropenia caused by ELANE mutations.

BACKGROUND: Neutrophils and eosinophils are multifunctional granulocytes derived from common myelocytic-committed progenitor cells. Severe congenital neutropenia 1 (SCN1) caused by ELANE mutations is a rare disease characterized by very low numbers of circulating neutrophils. Little is known about the functional characteristics of the SCN1 granulocytes, except that eosinophilia has been noticed in both bone marrow and peripheral blood. In this study, we profiled the number and function of granulocytes in patients suffering from SCN1. METHODS: Nine patients diagnosed with SCN1 were enrolled in this study and absolute counts of eosinophils and neutrophils from bone marrow aspirates and peripheral blood samples were analysed. In addition, Ficoll-Paque enriched granulocytes from patients and healthy controls were analysed for specific eosinophil and neutrophil markers using flow cytometry and for NADPH-oxidase activity-profile by chemiluminescence. RESULTS: Our data demonstrate a skewed granulocyte population in SCN1 patients dominated by eosinophils in both bone marrow and peripheral blood. The latter was detected only by blood smear examination, but not by automated blood analysers. Furthermore, we show that the SCN1 eosinophils exerted normal production of reactive oxygen species generated by the NADPH-oxidase, however the response was profoundly different from that of healthy control neutrophils. CONCLUSIONS: SCN1 patients with ELANE mutations suffer from neutropenia yet display eosinophilia in the bone marrow and blood, as revealed by smear examination but not by automatic blood analysers. The SCN1 eosinophils are functionally normal regarding production of reactive oxygen species (ROS). However, the ROS profile produced by eosinophils differs drastically from that of neutrophils isolated from the same blood donor, implying that the eosinophilia in SCN1 cannot compensate for the loss of neutrophils regarding ROS-mediated functions.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Impact of eosinophil-peroxidase (EPX) deficiency on eosinophil structure and function in mouse airways.

Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/- and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted.

Peroxidasin and eosinophil peroxidase, but not myeloperoxidase, contribute to renal fibrosis in the murine unilateral ureteral obstruction model.

Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.

Treatment with galectin-1 eye drops regulates mast cell degranulation and attenuates the severity of conjunctivitis.

Galectin-1 (Gal-1) is a ß-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100 mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3 µg/eye) or sodium cromoglycate (SCG; 40 mg/ml) in both eyes and, after 15 min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3 µg) and SCG. As expected, an increased proportion of degranulated mast cells (62%, P < 0.01) and lower histamine levels were observed after 6 h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31-36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3 µg) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation.

Eosinophils promote inducible NOS-mediated lung allograft acceptance.

Lungs allografts have worse long-term survival compared with other organ transplants. This is most likely due to their unique immunoregulation that may not respond to traditional immunosuppression. For example, local NO generation by inducible NOS (iNOS) is critical for lung allograft acceptance but associates with rejection of other solid organs. The source of NO in accepting lung allografts remains unknown. Here, we report that, unlike the case for other pulmonary processes in which myeloid cells control NO generation, recipient-derived eosinophils play a critical and nonredundant role in iNOS-mediated lung allograft acceptance. Depletion of eosinophils reduces NO levels to that of recipients with global deletion of iNOS and leads to a costimulatory blockade-resistant form of rejection. Furthermore, NO production by eosinophils depends on Th1 polarization by inflammatory mediators, such as IFN-γ and TNF-α. Neutralization of such mediators abrogates eosinophil suppressive capacity. Our data point to what we believe to be a unique and previously unrecognized role of eosinophil polarization in mediating allograft tolerance and put into perspective the use of high-dose eosinophil-ablating corticosteroids after lung transplantation.

Enhanced activation of eosinophils in peripheral blood and implications for eosinophilic esophagitis diagnosis.

BACKGROUND AND AIM: Eosinophils are markers of the eosinophilic esophagitis (EoE) disease, and this work aimed to assess whether activation of eosinophils could be a noninvasive test to contribute for EoE diagnosis. METHODS: The activation state of peripheral blood eosinophils in EoE patients and control subjects was assessed based on the morphological aspects of the eosinophil after adherence to slide. Cyclooxygenase-2 and 5-lipoxygenase expressions were evaluated by means of immunofluorescence microscopy to verify if and which eicosanoid pathway is triggered in eosinophils in blood in EoE. RESULTS: The eosinophils of patients with EoE were significantly more activated than those of control individuals. The lowest percentage of normal eosinophils for control subjects was 40%, while the highest percentage of eosinophils of normal aspect for patients with EoE was 32%. Considering 36% as a cutoff for normal eosinophils, this value differentiated all individuals with EoE from individuals without the disease with a sensitivity of 100%, considering the diagnosis of EoE as currently defined. Eosinophils of EoE patients showed higher expression of cyclooxygenase-2 than those of control subjects. CONCLUSIONS: The quantification of morphological changes in eosinophils is a feasible, easy, and reliable manner to identify EoE patients. Therefore, patients with symptoms of esophageal dysfunction showing higher than 36% activated eosinophils in peripheral blood could be a useful way to help definition and diagnostic criterion for EoE.