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.

Eosinophil peroxidase promotes bronchial epithelial cells to secrete asthma-related factors and induces the early stage of airway remodeling.

Asthma is a heterogeneous disease characterized by chronic airway inflammation, reversible airflow limitation, and airway remodeling. Eosinophil peroxidase (EPX) is the most abundant secondary granule protein unique to activated eosinophils. In this study, we aimed to illustrate the effect of EPX on the epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Our research found that both EPX and ADAM33 were negatively correlated with FEV1/FVC and FEV1%pred, and positively correlated with IL-5 levels. Asthma patients had relatively higher levels of ADAM33 and EPX compared to the healthy control group. The expression of TSLP, TGF-ß1 and ADAM33 in the EPX intervention group was significantly higher. Moreover, EPX could promote the proliferation, migration and EMT of BEAS-2B cells, and the effect of EPX on various factors was significantly improved by the PI3K inhibitor LY294002. The findings from this study could potentially offer a novel therapeutic target for addressing airway remodeling in bronchial asthma, particularly focusing on EMT.

Reactive Halogen Species: Role in Living Systems and Current Research Approaches.

Reactive halogen species (RHS) are highly reactive compounds that are normally required for regulation of immune response, inflammatory reactions, enzyme function, etc. At the same time, hyperproduction of highly reactive compounds leads to the development of various socially significant diseases - asthma, pulmonary hypertension, oncological and neurodegenerative diseases, retinopathy, and many others. The main sources of (pseudo)hypohalous acids are enzymes from the family of heme peroxidases - myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. Main targets of these compounds are proteins and peptides, primarily methionine and cysteine residues. Due to the short lifetime, detection of RHS can be difficult. The most common approach is detection of myeloperoxidase, which is thought to reflect the amount of RHS produced, but these methods are indirect, and the results are often contradictory. The most promising approaches seem to be those that provide direct registration of highly reactive compounds themselves or products of their interaction with components of living cells, such as fluorescent dyes. However, even such methods have a number of limitations and can often be applied mainly for in vitro studies with cell culture. Detection of reactive halogen species in living organisms in real time is a particularly acute issue. The present review is devoted to RHS, their characteristics, chemical properties, peculiarities of interaction with components of living cells, and methods of their detection in living systems. Special attention is paid to the genetically encoded tools, which have been introduced recently and allow avoiding a number of difficulties when working with living systems.

Safety and Efficacy of Dexpramipexole in Eosinophilic Asthma (EXHALE): A randomized controlled trial.

BACKGROUND: There is a need for new and effective oral asthma therapies. Dexpramipexole, an oral eosinophil-lowering drug, has not previously been studied in asthma. OBJECTIVE: We sought to evaluate the safety and efficacy of dexpramipexole in lowering blood and airway eosinophilia in subjects with eosinophilic asthma. METHODS: We performed a randomized, double-blind, placebo-controlled proof-of-concept trial in adults with inadequately controlled moderate to severe asthma and blood absolute eosinophil count (AEC) greater than or equal to 300/µL. Subjects were randomly assigned (1:1:1:1) to dexpramipexole 37.5, 75, or 150 mg BID (twice-daily) or placebo. The primary end point was the relative change in AEC from baseline to week 12. Prebronchodilator FEV1 week-12 change from baseline was a key secondary end point. Nasal eosinophil peroxidase was an exploratory end point. RESULTS: A total of 103 subjects were randomly assigned to dexpramipexole 37.5 mg BID (N = 22), 75 mg BID (N = 26), 150 mg BID (N = 28), or placebo (N = 27). Dexpramipexole significantly reduced placebo-corrected AEC week-12 ratio to baseline, in both the 150-mg BID (ratio, 0.23; 95% CI, 0.12-0.43; P < .0001) and the 75-mg BID (ratio, 0.34; 95% CI, 0.18-0.65; P = .0014) dose groups, corresponding to 77% and 66% reductions, respectively. Dexpramipexole reduced the exploratory end point of nasal eosinophil peroxidase week-12 ratio to baseline in the 150-mg BID (median, 0.11; P = .020) and the 75-mg BID (median, 0.17; P = .021) groups. Placebo-corrected FEV1 increases were observed starting at week 4 (nonsignificant). Dexpramipexole displayed a favorable safety profile. CONCLUSIONS: Dexpramipexole demonstrated effective eosinophil lowering and was well tolerated. Additional larger clinical trials are needed to understand the clinical efficacy of dexpramipexole in asthma.

An eosinophil peroxidase activity assay accurately predicts eosinophilic chronic rhinosinusitis.

BACKGROUND: A definitive diagnosis of eosinophilic chronic rhinosinusitis (eCRS) requires invasive surgical tissue sampling and histologic enumeration of intact eosinophils. Eosinophil peroxidase (EPX) is an accurate biomarker of sinonasal tissue eosinophilia in CRS regardless of polyp status. A less invasive and rapid method that accurately identifies tissue eosinophilia would be of great benefit to patients. OBJECTIVE: We sought to evaluate a new clinical tool that uses a nasal swab and colorimetric EPX activity assay to predict a diagnosis of eCRS. METHODS: A prospective, observational cohort study was conducted using nasal swabs and sinonasal tissue biopsies obtained from patients with CRS electing endoscopic sinus surgery. Patients were classified as non-eCRS (n = 19) and eCRS (n = 35) on the basis of pathologically determined eosinophil counts of less than 10 or greater than or equal to 10 eosinophils/HPF, respectively. Swab-deposited EPX activity was measured and compared with tissue eosinophil counts, EPX levels, and CRS-specific disease metrics. RESULTS: EPX activity was significantly increased in patients with eCRS than in patients without eCRS (P < .0001). With a relative absorbance unit cutoff value of greater than or equal to 0.80, the assay demonstrated high sensitivity (85.7%) and moderate specificity (79.0%) for confirming eCRS. Spearman correlations between EPX activity and tissue eosinophil counts (rs = 0.424), EPX levels (rs = 0.503), and Lund-Kennedy endoscopy scores (rs = 0.440) in eCRS were significant (P < .05). CONCLUSIONS: This investigation evaluates a nasal swab sampling method and EPX activity assay that accurately confirms eCRS. This method could potentially address the unmet need to identify sinonasal tissue eosinophilia at the point-of-care, as well as to longitudinally monitor eosinophil activity and treatment response.

Eosinophil Granule Proteins Involvement in Acute Appendicitis-An Allergic Disease?

Several pieces of evidence point to an allergic component as a trigger of acute appendicitis. As the Th2 immune response is characterized by eosinophil mobilization to the target organ and release of their cationic granule proteins, it is reasonable to investigate if the degranulation of eosinophils could be associated with the local injury. The primary aim of this study is to evaluate the participation of eosinophils granules proteins in acute appendicitis, both at local and systemic levels and the secondary aim is to evaluate the diagnostic accuracy of eosinophils granules proteins for the detection of acute appendicitis, as well as for distinguishing between complicated and uncomplicated acute appendicitis. Eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EP) are the most well-known eosinophil granule proteins. From August 2021 to April 2022, we present a prospective single-center study to evaluate the EDN, ECP, and EP concentrations simultaneously in appendicular lavage fluid (ALF) and the serum of 22 patients with acute phlegmonous appendicitis (APA), 24 with acute gangrenous appendicitis (AGA), and 14 normal controls. Concerning EDN, no differences were found between groups. ECP concentrations in ALF and serum were significantly higher in the histologically confirmed acute appendicitis compared to the control groups (p < 0.0001 and p < 0.0001, respectively). In ALF, no differences were found between ECP levels in APA: 38.85 ng/mL (IQR 26.50-51.77) and AGA 51.55 ng/mL (IQR 39.55-70.09) groups (p = 0.176). In the serum, no difference was found between ECP levels at APA: 39 ng/mL (IQR 21.30-56.90) and AGA: 51.30 ng/mL (IQR 20.25-62.59) (p = 0.100). For EP, the concentrations in ALF (p < 0.001) and serum (p < 0.001) were both higher in acute appendicitis compared to the control. In ALF, no difference was found between APA: 240.28 ng/mL (IQR 191.2-341.3) and AGA: 302.5 (IQR 227.7-535.85) (p = 0.236). In the serum, no differences were found between APA: 158.4 ng/mL (IQR 111.09-222.1) and AGA: 235.27 (IQR 192.33-262.51) (p = 0.179). Globally, the ALF concentrations were higher than serum concentrations, reflecting an intense inflammatory local reaction in AA. The optimal ECP cut-off for discriminating between acute appendicitis and the controls was >11.41 ng/mL, with a sensitivity of 93.5%, but with a specificity for identifying appendicitis of 21.4%, good discriminative power (AUC = 0.880). For EP, the optimal cut-off was >93.20 ng/mL, with a sensitivity of 87%, but with a specificity of 14.3% (AUC = 0.901), excellent discriminative power. For the diagnosis of perforated AA, the discriminative power of ECP and EP serum concentrations are weak (AUC = 0.562 and AUC = 0.664, respectively). Concerning the presence of peritonitis, the discriminative power of ECP and EP serum concentrations is acceptable, respectively: AUC = 0.724 and AUC = 0.735. Serum levels of EDN (p = 0.119), ECP (p = 0.586) and EP (p = 0.08) in complicated appendicitis were similar to uncomplicated appendicitis. Serum concentrations of ECP and EP can be added to decision-making AA diagnosis. A Th2-type immune response is present in AA. These data bring forward the role of an allergic reaction in the pathogenesis of acute appendicitis.

Airway Sensory Nerve Density Is Increased in Chronic Cough.

Rationale: Chronic cough is characterized by frequent urges to cough and a heightened sensitivity to inhaled irritants. Airway sensory nerves trigger cough. We hypothesized that sensory nerve density is increased in chronic cough, which may contribute to excessive and persistent coughing.Objectives: To measure airway nerve density (axonal length) and complexity (nerve branching, neuropeptide expression) in humans with and without chronic cough.Methods: Bronchoscopic human airway biopsies were immunolabeled for nerves and the sensory neuropeptide substance P. Eosinophil peroxidase was also quantified given previous reports showing associations between eosinophils and nerve density. Three-dimensional image z-stacks of epithelium and subepithelium were generated using confocal microscopy, and from these z-stacks, total nerve length, the number of nerve branch points, substance P expression, and eosinophil peroxidase were quantified within each airway compartment.Measurements and Main Results: Nerve length and the number of branch points were significantly increased in epithelium, but not subepithelium, in chronic cough compared with healthy airways. Substance P expression was scarce and was similar in chronic cough and healthy airways. Nerve length and branching were not associated with eosinophil peroxidase nor with demographics such as age and sex in either group.Conclusions: Airway epithelial sensory nerve density is increased in chronic cough, suggesting sensory neuroplasticity contributes to cough hypersensitivity.

First report of eosinophil peroxidase in starry flounder (Platichthys stellatus): Gene identification and gene expression profiling.

Eosinophils are granular leukocytes that are evolutionarily preserved in the innate immune system of some invertebrates and vertebrates, and these cells can directly remove invading microorganisms and secrete various cytokines, and are also involved in homeostasis. These eosinophils are made up of specific granular proteins that can be differentiated from other cells, and eosinophil peroxidase (EPX) is a peroxidase released only from eosinophils that plays an important role in maintaining the main function and homeostasis of eosinophils. We obtained the sequence information of EPX for the first time from the starry flounder (Platichthys stellatus), and predicted it by amino acid sequencing to confirm sequence alignment and phylogenetic characteristics with other species. Based on analysis of the expression characteristics of PsEPX mRNA in healthy P. stellatus, it was expressed at the highest level in peripheral blood lymphocytes (PBLs) and was also expressed at a relatively high level in the head kidney and intestine, which are immune-related tissues. After artificial infection with Streptococcus parauberis and viral haemorrhagic septicaemia virus, which are the causes of major pathogenic diseases, the expression level of PsEPX was significantly regulated, which showed specific characteristics of pathogens or tissues. These results suggest that PsEPX is an important component of the immune system of P. stellatus and is considered a basic research case for the study of the immunological function of eosinophils in fish.

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.

Inhibition of Myeloperoxidase.

Myeloperoxidase participates in innate immune defense mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. This has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, based on the profound knowledge of structure and function of MPO and its biochemical and biophysical differences with the other homologous human peroxidases, various rational and high-throughput screening attempts were performed in developing specific irreversible and reversible inhibitors. The most prominent candidates as well as MPO inhibitors already studied in clinical trials are introduced and discussed.

Reactions of kynurenic acid with hypobromous acid and hypochlorous acid.

Kynurenic acid, a tryptophan metabolite, acts as antagonist or agonist of several receptors. Hypobromous acid (HOBr) and hypochlorous acid (HOCl) are generated by eosinophils and neutrophils. At inflammation sites, kynurenic acid may encounter HOBr and HOCl to generate products. When kynurenic acid was incubated with HOBr under neutral conditions, kynurenic acid generated a single product almost exclusively. This was identified as 3-bromokynurenic acid. Kynurenic acid reacted with HOCl, generating two products. The major product was identified as 3-chlorokynurenic acid with its oxidative decarboxylation product, 3-chloro-4-hydroxy-2(1H)-quinolinone as a by-product. Free amino acids suppressed the reactions of kynurenic acid with HOBr and HOCl. Taurine suppressed the HOCl reaction but not the HOBr reaction. An eosinophil peroxidase system containing H2O2, NaCl, and NaBr reacted with kynurenic acid, generating 3-bromokynurenic acid under mildly acidic conditions. Although a myeloperoxidase system containing H2O2 and NaCl reacted with kynurenic acid to generate 3-chlorokynurenic acid under mildly acidic conditions, the product was altered to 3-bromokynurenic acid by addition of NaBr to the system. These results suggest that 3-bromokynurenic acid and 3-chlorokynurenic acid may be generated from kynurenic acid at inflammation sites in humans, although their formation will be suppressed by coexistent amino acids.

Dexamethasone and lidocaine suppress eosinophilopoiesis from umbilical cord blood cells.

BACKGROUND: Eosinophils play an important role in allergic inflammation. Glucocorticosteroids have been used as an anti-inflammatory medication for inflammatory diseases involving eosinophil infiltration. Some effect of nebulized lidocaine has been reported when treating certain patients with asthma, which is also an inflammatory disease. The goal of this study was to examine the effects of dexamethasone and lidocaine on eosinophil proliferation and differentiation using a model of human umbilical cord blood mononuclear cells (UCMC) cultured with IL-5. METHODS: UCMC were cultured with IL-5 (5 ng/mL) for 4 weeks. The effects of dexamethasone and lidocaine on the number and morphology of eosinophilic cells were visualized with Wright-Giemsa and cyanide-resistant peroxidase stains. Moreover, the effect on eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX) contents in cultured cells were evaluated using radioimmunoassay. RESULTS: The number of eosinophilic cells and EDN and EPX content in cultured cells increased in a time-dependent manner in the presence of IL-5. Dexamethasone treatment slightly decreased the number of eosinophilic cells in one week, but this effect was lost in 2-4 weeks. Macrophages in cultured UCMC treated with dexamethasone contained more eosinophil granule proteins. Both EDN and EPX content in cultured cells were reduced by dexamethasone. Lidocaine decreased the number of eosinophilic cells and reduced both EDN and EPX contents in cultured cells. CONCLUSIONS: Dexamethasone suppressed the production of eosinophil granule proteins and may also induce apoptosis of eosinophils, while lidocaine suppresses eosinophilopoiesis.

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.

Enzymatic Degradation of Graphene Quantum Dots by Human Peroxidases.

Carbon-based nanomaterials have demonstrated to be potent candidates for biomedical applications. Recently, graphene quantum dots (GQDs) have emerged as an attractive tool for bioimaging, biosensing, and therapy. Hence, studying their biodegradability in living systems is essential to speed up the translation toward real clinical innovations. Here, the enzymatic degradation of GQDs using human myeloperoxidase and eosinophil peroxidase is investigated. Transmission electron microscopy, fluorescence, and Raman spectroscopy are used to evaluate the biodegradation of GQDs. Signs of degradation by both enzymes are observed already after a few hours of incubation with each enzyme, being more evident after a couple of days of treatment. Molecular dynamics simulations show intimate interactions between the enzymes and the GQDs. The conformation of both peroxidases is slightly altered to favor the interactions, while the GQD sheets distort a little to adapt to the surface of the enzymes. The biodegradability of the GQDs ensures their real potential in the practical biomedical applications.

Sputum autoantibodies in patients with severe eosinophilic asthma.

BACKGROUND: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. OBJECTIVES: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. METHODS: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. RESULTS: We report a "polyclonal" autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. CONCLUSION: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

Lung Pathologies in a Chronic Inflammation Mouse Model Are Independent of Eosinophil Degranulation.

RATIONALE: The release of eosinophil granule proteins in the lungs of patients with asthma has been dogmatically linked with lung remodeling and airway hyperresponsiveness. However, the demonstrated inability of established mouse models to display the eosinophil degranulation occurring in human subjects has prevented a definitive in vivo test of this hypothesis. OBJECTIVES: To demonstrate in vivo causative links between induced pulmonary histopathologies/lung dysfunction and eosinophil degranulation. METHODS: A transgenic mouse model of chronic T-helper cell type 2-driven inflammation overexpressing IL-5 from T cells and human eotaxin 2 in the lung (I5/hE2) was used to test the hypothesis that chronic histopathologies and the development of airway hyperresponsiveness occur as a consequence of extensive eosinophil degranulation in the lung parenchyma. MEASUREMENT AND MAIN RESULTS: Studies targeting specific inflammatory pathways in I5/hE2 mice surprisingly showed that eosinophil-dependent immunoregulative events and not the release of individual secondary granule proteins are the central contributors to T-helper cell type 2-induced pulmonary remodeling and lung dysfunction. Specifically, our studies highlighted a significant role for eosinophil-dependent IL-13 expression. In contrast, extensive degranulation leading to the release of major basic protein-1 or eosinophil peroxidase was not causatively linked to many of the induced pulmonary histopathologies. However, these studies did define a previously unappreciated link between the release of eosinophil peroxidase (but not major basic protein-1) and observed levels of induced airway mucin. CONCLUSIONS: These data suggest that improvements observed in patients with asthma responding to therapeutic strategies ablating eosinophils may occur as a consequence of targeting immunoregulatory mechanisms and not by simply eliminating the destructive activities of these purportedly end-stage effector cells.

Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent eosinophil peroxidase (EPO) inhibitors.

There is an increasing interest in developing novel eosinophil peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3 D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC50 values down to 10 nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against eosinophilic disorders.

Dansylglycine, a fluorescent probe for specific determination of halogenating activity of myeloperoxidase and eosinophil peroxidase.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl-) and bromide (Br-), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H2O2) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H2O2. The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3',5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr.

Epicutaneous immunization with ovalbumin and CpG induces TH1/TH17 cytokines, which regulate IgE and IgG2a production.

BACKGROUND: Subcutaneous allergen-specific immunotherapy is a standard route for the immunotherapy of allergic diseases. It modulates the course of allergy and can generate long-term remission. However, subcutaneous allergen-specific immunotherapy can also induce anaphylaxis in some patients, and therefore additional routes of administration should be investigated to improve the safety and tolerability of immunotherapy. OBJECTIVE: We sought to determine whether epicutaneous treatment with antigen in the presence of a Toll-like receptor 9 agonist can suppress TH2-mediated responses in an antigen-specific manner. METHODS: Epicutaneous immunization was performed by applying a skin patch soaked with ovalbumin (OVA) plus CpG, and its suppressor activity was determined by using the mouse model of atopic dermatitis. Finally, adoptive cell transfers were implemented to characterize the regulatory cells that are induced by epicutaneous immunization. RESULTS: Epicutaneous immunization with OVA and CpG reduces the production of OVA-specific IgE and increases the synthesis of OVA-specific IgG2a antibodies in an antigen-specific manner. Moreover, eosinophil peroxidase activity in the skin and production of IL-4, IL-5, IL-10, and IL-13 are suppressed. The observed reduction of IgE synthesis is transferable with T-cell receptor (TCR) αß(+)CD4(+)CD25(-) cells, whereas IgG2a production is dependent on both TCRαß(+) and TCRγδ(+) T cells. Further experiments show that the described phenomenon is myeloid differentiation primary response 88, IFN-γ, and IL-17A dependent. Finally, the results suggest that epicutaneous immunization with OVA and CpG decreases the synthesis of OVA-specific IgE and skin eosinophil peroxidase activity in mice with ongoing skin allergy. CONCLUSION: Epicutaneous application of protein antigen in the presence of adjuvant could be an attractive needle-free and self-administered immunotherapy for allergic diseases.