Olfactory cleft mucus eosinophil-derived neurotoxin better reflects olfactory loss than blood eosinophil counts in patients with chronic rhinosinusitis.

BACKGROUND: Eosinophils are associated with olfactory dysfunction in chronic rhinosinusitis (CRS) and eosinophil-derived neurotoxin (EDN) is a sensitive marker of intense eosinophil activation. This study aimed to analyze olfactory cleft mucus and olfactory mucosa EDN levels and their association with olfactory dysfunction in CRS. METHODS: We prospectively recruited 150 patients with CRS electing endoscopic sinus surgery and 25 healthy controls. Both superior turbinate biopsy specimens and olfactory cleft mucus were collected to analyze EDN levels. Sniffin' Sticks test scores, olfactory cleft computed tomography (CT) scores, and olfactory cleft endoscopy scale (OCES) were obtained. Multivariable logistic regression analysis was applied to analyze the predictability of EDN levels for olfactory dysfunction in CRS. RESULTS: Chronic rhinosinusitis with olfactory dysfunction presented significantly higher olfactory mucosa (p = 0.016) and olfactory cleft mucus (p < 0.001) EDN levels than CRS without olfactory dysfunction. Mucus EDN levels were positively correlated with blood eosinophils (r = 0.625, p = 0.002), olfactory cleft CT scores (r = 0.738, p < 0.001), and OCES (r = 0.605, p = 0.004) in CRS. Furthermore, mucus EDN levels were significantly negatively correlated with threshold, discrimination, and identification (TDI) (r = -0.688), olfactory threshold (r = -0.606), olfactory discrimination (r = -0.608), and olfactory identification (r = -0.697) scores. After adjusting for patient demographics and comorbidities, mucus EDN levels were significantly associated with olfactory dysfunction in CRS (odds ratio = 2.162; p = 0.027). Mucus EDN levels showed a significantly better performance for predicting olfactory dysfunction than blood eosinophil counts (area under the curve, 0.873 vs. 0.764, p = 0.024). CONCLUSION: Olfactory cleft mucus EDN level may be a better biomarker for predicting olfactory dysfunction in CRS than blood eosinophil counts.

The Role of Eosinophil-Derived Neurotoxin and Vascular Endothelial Growth Factor in the Pathogenesis of Eosinophilic Asthma.

Asthma is a chronic complex pulmonary disease characterized by airway inflammation, remodeling, and hyperresponsiveness. Vascular endothelial growth factor (VEGF) and eosinophil-derived neurotoxin (EDN) are two significant mediators involved in the pathophysiology of asthma. In asthma, VEGF and EDN levels are elevated and correlate with disease severity and airway hyperresponsiveness. Diversity in VEGF polymorphisms results in the variability of responses to glucocorticosteroids and leukotriene antagonist treatment. Targeting VEGF and eosinophils is a promising therapeutic approach for asthma. We identified lichochalcone A, bevacizumab, azithromycin (AZT), vitamin D, diosmetin, epigallocatechin gallate, IGFBP-3, Neovastat (AE-941), endostatin, PEDF, and melatonin as putative add-on drugs in asthma with anti-VEGF properties. Further studies and clinical trials are needed to evaluate the efficacy of those drugs. AZT reduces the exacerbation rate and may be considered in adults with persistent symptomatic asthma. However, the long-term effects of AZT on community microbial resistance require further investigation. Vitamin D supplementation may enhance corticosteroid responsiveness. Herein, anti-eosinophil drugs are reviewed. Among them are, e.g., anti-IL-5 (mepolizumab, reslizumab, and benralizumab), anti-IL-13 (lebrikizumab and tralokinumab), anti-IL-4 and anti-IL-13 (dupilumab), and anti-IgE (omalizumab) drugs. EDN over peripheral blood eosinophil count is recommended to monitor the asthma control status and to assess the efficacy of anti-IL-5 therapy in asthma.

Urine eosinophil-derived neurotoxin: A potential marker of activity in select eosinophilic disorders.

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.

Serum Levels of Vascular Endothelial Growth Factor, Platelet Activating Factor and Eosinophil-Derived Neurotoxin in Chronic Spontaneous Urticaria-A Pilot Study in Adult Patients.

Chronic spontaneous urticaria (CSU) is a skin disease characterized by the presence of wheals, angioedema, or both for at least 6 weeks. Although, CSU is often regarded as autoimmune in nature, its etiology is not fully explained and interactions between various small molecules are still taken under account. The aim of this research was to investigate the mean serum concentration of vascular endothelial growth factor (VEGF), platelet activating factor (PAF), and eosinophil-derived neurotoxin (EDN) in relation to the disease activity and pruritus intensity in adult patients with CSU. Fifteen patients with CSU and 15 healthy subjects participated in this pilot study. Blood samples were taken to examine the mean serum levels of VEGF, PAF, and EDN by the enzyme-linked immunosorbent assay test (ELISA). The Urticaria Activity Score (UAS7) and The Visual Analogue Scale (VAS) were used to assess the disease activity and the pruritus intensity, respectively. Obtained results revealed that VEGF, PAF, and EDN concentrations were higher in patients with CSU compared with those of the control group, but only for VEGF it was statistically significant (p = 0.008). However, levels of all investigated cytokines were not significantly correlated neither with the disease activity nor with the pruritus intensity. Our results showed higher serum levels of VEGF, PAF, and EDN among CSU patients which may highlight a functional role of these cytokines in the disease's pathogenesis. In contrast, VEGF, PAF, or EDN might not be useful to reflect the severity of symptoms.

Single-cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti-Inflammatory Macrophage Phenotype under Myeloid-TLR4 Dependent Regulation in Anti-Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN).

Previously, this study demonstrates the critical role of myeloid specific TLR4 in macrophage-mediated progressive renal injury in anti-glomerular basement membrane (anti-GBM) crescentic glomerulonephritis (cGN); however, the underlying mechanism remains largely unknown. In this study, single-cell RNA sequencing (scRNA-seq), pseudotime trajectories reconstruction, and motif enrichment analysis are used, and macrophage diversity in anti-GBM cGN under tight regulation of myeloid-TLR4 is uncovered. Most significantly, a myeloid-TLR4 deletion-induced novel reparative macrophage phenotype (Nr4a1+ Ear2+) with significant upregulated anti-inflammatory and tissue repair-related signaling is discovered, thereby suppressing the M1 proinflammatory responses in anti-GBM cGN. This is further demonstrated in vitro that deletion of TLR4 from bone marrow-derived macrophages (BMDMs) induces the Nr4a1/Ear2-expressing anti-inflammatory macrophages while blocking LPS-stimulated M1 proinflammatory responses. Mechanistically, activation of the Nr4a1/Ear2-axis is recognized as a key mechanism through which deletion of myeloid-TLR4 promotes the anti-inflammatory macrophage differentiation in vivo and in vitro. This is confirmed by specifically silencing macrophage Nr4a1 or Ear2 to reverse the anti-inflammatory effects on TLR4 deficient BMDMs upon LPS stimulation. In conclusion, the findings decode a previously unidentified role for a myeloid-TLR4 dependent Nr4a1/Ear2 negative feedback mechanism in macrophage-mediated progressive renal injury, implying that activation of Nr4a1-Ear2 axis can be a novel and effective immunotherapy for anti-GBM cGN.

Blood eosinophil cationic protein and eosinophil-derived neurotoxin are associated with different asthma expression and evolution in adults.

BACKGROUND: Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are proteins released by activated eosinophils whose role in adult asthma remains unclear. OBJECTIVE: To study associations between ECP, EDN and various asthma characteristics in adults from the Epidemiological Study on the Genetics and Environment of Asthma (EGEA). METHODS: Plasma ECP and EDN levels were measured by ELISA. Cross-sectional analyses were performed in 941 adults (43±16 years old, 39% with asthma) at EGEA2 (2003-2007). Longitudinal analyses investigated the associations between EDN level at EGEA2 and changes in asthma characteristics between EGEA2 and EGEA3 (2011-2013, n=817). We used generalised estimated equations adjusted for age, sex, smoking status and body mass index to take into account familial dependence. RESULTS: At EGEA2, both high ECP and EDN levels were associated with current asthma (adjusted OR (aOR) (95% CI): 1.69 (1.35-2.12) and 2.12 (1.76-2.57)). Among asthmatics, high EDN level was associated with asthma attacks (aOR: 1.50 (1.13-1.99)), wheezing and breathlessness (aOR: 1.38 (1.05-1.80)), use of asthma treatments (aOR: 1.91 (1.37-2.68)) and bronchial hyper-responsiveness (aOR: 2.03 (1.38-2.97)), even after further adjustment on ECP. High ECP level was associated with high neutrophil count and tended to be associated with chronic bronchitis. High EDN level at EGEA2 was associated with persistent asthma (aOR: 1.62 (1.04-2.52)), nocturnal symptoms (aOR from 2.19 to 3.57), worsening wheezing and breathlessness (aOR: 1.97 (1.36-2.85)) and nocturnal shortness of breath (aOR: 1.44 (1.04-1.98)) between EGEA2 and EGEA3. CONCLUSIONS: EDN and ECP were associated with different asthma expression in adults. EDN could be a potential biomarker to monitor asthma evolution in adults.

Noninvasive biomarkers identify eosinophilic esophagitis: A prospective longitudinal study in children.

BACKGROUND: Esophageal histology is critical for diagnosis and surveillance of disease activity in eosinophilic esophagitis (EoE). A validated noninvasive biomarker has not been identified. We aimed to determine the utility of blood and urine eosinophil-associated proteins to diagnose EoE and predict esophageal eosinophilia. METHODS: Blood and urine were collected from children undergoing endoscopy with biopsy. Absolute eosinophil count (AEC), plasma eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), major basic protein-1 (MBP-1), galectin-10 (CLC/GAL-10), Eotaxin-2 and Eotaxin-3, and urine osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) were determined. Differences were assessed between EoE and control, and with treatment response. The capacity to predict EoE diagnosis and esophageal eosinophil counts was assessed. RESULTS: Of 183 specimens were collected from 56 EoE patients and 15 non-EoE controls with symptoms of esophageal dysfunction; 33 EoE patients had paired pre- and post-treatment specimens. Plasma (CLC/GAL-10, ECP, EDN, Eotaxin-3, MBP-1) and urine (OPN) biomarkers were increased in EoE compared to control. A panel comprising CLC/GAL-10, Eotaxin-3, ECP, EDN, MBP-1, and AEC was superior to AEC alone in distinguishing EoE from control. AEC, CLC/GAL-10, ECP, and MBP-1 were significantly decreased in patients with esophageal eosinophil counts <15/hpf in response to treatment. AEC, CLC/GAL-10, ECP, EDN, OPN, and MBP-1 each predicted esophageal eosinophil counts utilizing mixed models controlled for age, gender, treatment, and atopy; AEC combined with MBP-1 best predicted the counts. CONCLUSIONS: We identified novel panels of eosinophil-associated proteins that along with AEC are superior to AEC alone in distinguishing EoE from controls and predicting esophageal eosinophil counts.

Eosinophil-derived neurotoxin: A biologically and analytically attractive asthma biomarker.

There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a biomarker in asthma, including association with eosinophilic airway inflammation, assessment of disease severity and potential for predicting pathogenic risks, including exacerbations. However, to interpret any biomarker data with confidence, it is first important to understand the preanalytical factors and biological variation that may affect its reliable measurement and results interpretation. In this study we defined the healthy serum EDN reference range for men and women as 1.98 to 26.10 ng/mL, with no significant gender differences. Smoking did not impact the mean EDN levels and no circadian rhythm was identified for EDN, unlike blood eosinophils (EOS) where levels peaked at 00:00h. EDN expression in different cell types was investigated and shown to occur primarily in eosinophils, indicating they are likely to be the main cellular repository for EDN. We also confirm that the quantification of serum EDN is not influenced by the type of storage tube used, and it is stable at ambient temperature or when refrigerated for at least 7 days and for up to one year when frozen at -20°C or -80°C. In summary, EDN is a stable biomarker that may prove useful in precision medicine approaches by enabling the identification of a subpopulation of asthma patients with activated eosinophils and a more severe form of the disease.

Experimental Modeling of Eosinophil-Associated Diseases.

Eosinophils are an important subtype of leukocytes derived from bone marrow multipotent hematopoietic stem cells and represent about 1% of leukocytes in circulating blood. In homeostatic conditions, eosinophils reside in the intestine to maintain the balance of immune responses by communicating with gut microbes without causing inflammation. However, under the stressed or diseased condition, eosinophils degranulate, releasing their granule-derived cytotoxic proteins that are involved in inflammatory responses. Various eosinophil-associated inflammatory diseases are eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EG), and eosinophilic colitis (EC), together called EGID, asthma, hypereosinophilic syndrome, and eosinophilic pneumonia (EP). Eosinophil degranulation results in the release of their four toxic proteins [major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN)] which promote disease pathogenesis. Pancreatitis is the inflammatory disease of the pancreas that arises due to blockage of the pancreatic duct, trypsinogen mutation, alcohol consumption, and repeated occurrence of pancreatitis leading to chronic pancreatitis (CP); subsequently some CP patients may also develop pancreatic cancer. The presence of eosinophils is now shown in various case reports with acute, recurrent acute, and chronic pancreatitis and pancreatic cancer indicating the role of eosinophils in the pathogenesis of various pancreatic inflammatory disorders. However, the details of eosinophil accumulation during pancreatic diseases are not well explored and need further attention. Overall, the chapter provides the current understanding of reported eosinophils associated with inflammatory diseases like EGID diseases, asthma, and pancreatic disorders, i.e., acute, chronic pancreatitis, and pancreatic cancer. This knowledge will be helpful for future studies to develop novel treatment options for the eosinophils associated diseases. Therefore, more efforts are needed to perform preclinical and clinical studies in this field for the successful development of eosinophil-targeting treatments for a variety of eosinophil-associated diseases.

Identification of a novel immune prognostic model in gastric cancer.

PURPOSE: The tumor immune microenvironment (TIME) is now considered as an important factor during gastric cancer (GC) development. This study identified a novel immune-related risk model for predicting prognosis and assessing the immune status of GC patients. METHODS: Transcriptomic data were obtained from the TCGA database. The differential expressed immune-related genes (IRGs) were identified through the ImmPort portal. Enrichment analysis was performed to explore the potential molecular mechanism of these IRGs. By the Cox regression analysis, we constructed the immune prognostic model. Then, the association between the model and the immune microenvironment was estimated. The model was validated in the GSE84433 dataset. RESULTS: Totally, we identified 222 differentially expressed IRGs. These IRGs were closely correlated with immune response and immune signaling pathways. Through the Cox regression analysis, we developed the immune prognostic model based on the expression of seven IRGs (CXCL3, NOX4, PROC, FAM19A4, RNASE2, IGHD2-15, CGB5). Patients were stratified into two groups according to immune-related risk scores. Survival analysis indicated that the prognosis of high-risk patients was poorer than low-risk patients. Moreover, the immune-related risk score was an independent prognostic biomarker. More importantly, we found that the infiltration level of immunosuppressive cells and the expression of inhibitory immune checkpoints were higher in high-risk patients. The immune microenvironment tended to be a suppressive status in patients with high-risk scores. CONCLUSION: This study demonstrated that our model had predictive value for prognosis and the TIME in GC. It might be a robust tool to improve personalized patient management.

Using Eosinophil Biomarkers From Rectal Epithelial Samples to Diagnose Food Protein-induced Proctocolitis: A Pilot Study.

OBJECTIVES: The gold standard diagnostic procedure for food protein-induced proctocolitis (FPIP) requires flexible sigmoidoscopy (FS). To date there is no validated, noninvasive test to confirm FPIP diagnosis. Eosinophil-derived neurotoxin (EDN), a product of eosinophil (EOS) degranulation, has been shown to correlate with eosinophil infiltration in other tissues. Our objective was to compare EDN concentrations in rectal epithelial samples from infants with FPIP with those from a control population. METHODS: Children who underwent routine FS at Arnold Palmer Hospital for Children were enrolled in an IRB-approved, prospective, open-label pilot study between July 2017 and May 2019. We obtained rectal epithelial samples via: rectal swab, cytology brushing through FS, and rectal biopsy through FS. We then measured EDN levels in the samples and compared levels found in infants with FPIP against levels found in the control group. FPIP was defined as more than 60 EOS per 10 high-power fields (HPF) in rectal epithelial tissue obtained via rectosigmoid biopsy. RESULTS: Twenty-four patients were enrolled. The control group (n = 13) included patients with normal histopathology (84% boys, mean age 19 months, SD 6 months) and the FPIP group (n = 11) included patients with FPIP confirmed via biopsy (45% boys, mean age 6.9 months, SD 9 months). EDN concentration was significantly higher in the FPIP group than in the control group, for 2 sampling methods: rectal biopsy (183.6 ±â€Š114.6 vs 76.6 ±â€Š71.0 µg/mL; P = 0.010) and rectal swab (66.2 ±â€Š64.8 vs 20.4 ±â€Š22.2 µg/mL; P = 0.025). CONCLUSIONS: EDN concentrations measured from rectal swab and rectal biopsy samples is elevated and may be a useful tool to screen for FPIP in children.

Comparison of 18F-FDG PET-MR and fecal biomarkers in the assessment of disease activity in patients with ulcerative colitis.

OBJECTIVE: To compare the diagnostic performance of fecal biomarkers and 18F-fludeoxyglucose (18F-FDG) positron emmision tomography-MR (PET-MR) in the assessment of disease activity in patients with ulcerative colitis. METHODS: This study was conducted under the framework of a single-center clinical trial (clinicaltrials.gov [NCT03781284]). N = 50 participants were enrolled. Fecal samples were collected before bowel preparation. All patients underwent whole-body 18F-FDG PET-MR followed by ileocolonoscopy within 24 h. Diagnostic performance of five fecal biomarkers (calprotectin, lactoferrin, polymorphonuclear leukocyte elastase, S100A12 and eosinophil-derived neurotoxin), MR morphological parameters (MRmorph), diffusion-weighted imaging and PET in detecting active disease determined by Rachmilewitz endoscopic activity index (EAI) were evaluated and compared with each other. Correlations between fecal biomarkers, PET and endoscopy were calculated. RESULTS: According to EAI, n = 38 patients presented with endoscopically active disease (16 mild, 19 moderate and 3 severe). All five biomarkers, PET and MRmorph could differentiate endoscopically active disease from endoscopic remission without significant difference regarding their operating characteristics (accuracies between 0.673 for calprotectin and 0.898 for lactoferrin). In predicting endoscopically moderate to severe disease, PET showed the highest diagnostic performance (accuracy = 0.857) compared to calprotectin and lactoferrin (accuracy = 0.633 and 0.735). PET had also the strongest correlation with endoscopy (ρ = 0.685, p < 0.001), while within fecal biomarkers the levels of lactoferrin and eosinophil-derived neurotoxin correlated significantly with EAI (ρ = 0.423 and 0.528, both p < 0.05). CONCLUSION: Both fecal biomarkers and PET-MR were excellent non-invasive diagnostic tools in the assessment of disease activity in ulcerative colitis. ADVANCES IN KNOWLEDGE: Both fecal biomarkers and PET-MR parameters are able to predict endoscopically active disease with comparable diagnostic performance. PET had the highest correlation with endoscopy and outperformed fecal biomarkers in differentiating moderate to severe from mild disease.

Galectins, Eosinophiles, and Macrophages May Contribute to Schistosoma japonicum Egg-Induced Immunopathology in a Mouse Model.

Schistosomiasis is a severe public health problem, which can cause tissue fibrosis and can even be fatal. Previous studies have proven that galectins and different kinds of cells involve in the regulation of tissue fibrosis process. In this study, outbred Kunming mice were infected with Schistosoma japonicum (S. japonicum). Our results showed that compared with uninfected mice, there were severe egg granulomatous inflammation and tissue fibrosis in the livers, spleens, and large intestines of S. japonicum-infected mice at 8 weeks post-infection (p.i.), and the number of eosinophils by hematoxylin and eosin staining and CD68 macrophage-positive area by immunohistochemical staining were significantly increased. Detected by using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), at 8 weeks after S. japonicum infection, the mRNA expression levels of galectin (Gal)-1, Gal-3, CD69, eosinophil protein X (EPX), and chitinase 3-like protein 3 (Ym1) were significantly increased in liver, spleen, and large intestine; eotaxin-1 (CCL11) and eosinophil cationic protein were significantly increased in both liver and spleen; eotaxin-2 (CCL24) and Arginase1 (Arg1) were significantly increased in both spleen and large intestine; and CD200R was significantly increased in both liver and large intestine. However, interleukin (IL)-1ß and inducible nitric oxide synthase (iNOS) were only significantly increased in liver. The M2/M1 ratio of CD200R/CD86 genes was significantly increased in liver, and ratios of Ym1/IL-1ß and Ym1/iNOS were significantly increased in liver, spleen, and large intestine of S. japonicum-infected mice. Ex vivo study further confirmed that the levels of Gal-1, Gal-3, CD200R, Arg1, and Ym1 were significantly increased, and the ratios of CD200R/CD86 and Ym1/IL-1ß were significantly increased in peritoneal macrophages isolated from S. japonicum-infected mice at 8 weeks p.i. In addition, correlation analysis showed that significant positive correlations existed between mRNA levels of Gal-1/Gal-3 and EPX in liver, between Gal-3 and Ym1 in both liver and large intestine, and between Gal-3 and CD200R in peritoneal macrophages of S. japonicum-infected mice. Our data suggested that Gal-1, Gal-3, eosinophils, and macrophages are likely involved in the development of egg granulomatous response and fibrosis induced by S. japonicum infection.

Comment on: Variables associated subclinical atherosclerosis among rheumatoid arthritis patients of Gulf Cooperative Council countries.

[No Abstract Available].

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis.

BACKGROUND: The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE: We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS: Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS: CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS: CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.

Poor Correlation of Oral Swabs with Esophageal Eosinophil Counts.

Eosinophilic esophagitis (EoE) is a chronic condition that requires repeated endoscopies/biopsies to track the disease and treatment response. This invasive procedure involves risk to the patient and has significant costs. We studied whether the detection of specific proteins (cytokines and eosinophil degranulation products) from oral swabs could serve as a minimally invasive test for EoE. Swabs of the oral cavity (buccal and oropharyngeal) were obtained prior to endoscopy/biopsies in patients with EoE, possible EoE, and non-EoE patients in addition to obtaining additional esophageal biopsy tissue. ELISAs measuring the levels of cytokines IL-5, IL-8, IL-13, and eosinophil degranulation products including major basic protein (MBP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO) were performed on the samples. Comparisons were made to peak esophageal eosinophil counts. Tolerability of the swabs was evaluated. 43 patients, 4-17 years old, participated in the study. Swabs were well tolerated and all showed measurable protein. 26 patients had EoE [14 active (> 15 eosinophils/high power field), 12 non-active], 17 patients did not have EoE. Results obtained from oral swabs showed poor correlation with those from esophageal tissue. Only measurement of eosinophil degranulation products EDN and EPO from esophageal tissues showed strong correlations with eosinophil counts. In this study, the levels of cytokines and eosinophil degranulation products detected from oral swabs did not correlate with esophageal eosinophilia, and their detection would be insufficient to displace endoscopy/biopsies.

Systemic biomarkers of eosinophilic chronic rhinosinusitis.

PURPOSE OF REVIEW: The current understanding of eosinophilic chronic rhinosinusitis (CRS) has developed rapidly over the past decades. Classification of CRS based on the inflammatory endotype more accurately reflects the underlying pathophysiology and better directs treatment. Corticosteroids and more recently biologic agents, target the eosinophil inflammatory that drives this subtype of CRS. Tissue sampling is not always accessible or available and surrogate markers are sought to define this endotype of CRS. The purpose of this review is to assess current systemic predictors of eosinophilic CRS (eCRS) diagnosis. RECENT FINDINGS: Blood eosinophils are a moderate surrogate predictor of eCRS. A blood eosinophil count of more than 0.24 × 10/l predicts eCRS with tissue eosinophilia of more than 10 eosinophils per high-power field. It has been further shown that a blood eosinophil count more than 0.45 × 10/l is associated with need for long-term systemic therapy following endoscopic sinus surgery. Other biomarkers reviewed include IgE, eosinophilic cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, IL-5, periostin, eotaxin-3 and IL-16. SUMMARY: There remains limited data surrounding the prognostic use of biomarkers in eCRS. However, peripheral eosinophilia best predicts the eosinophilic density that best predicts the eCRS phenotype. In addition, it is also prognostic of need for more intensive therapy. Simple haematoxylin and eosin stained sinus mucosa still remains the most reliable tissue for assessment and is more accessible than bronchial biopsies.

Fixed airflow obstruction relates to eosinophil activation in asthmatics.

BACKGROUND: Some asthmatics develop irreversible chronic airflow obstruction, for example, fixed airflow obstruction (fixed-AO). This is probably a consequence of airway remodelling, but neither its relation to inflammation nor which asthma biomarkers can be clinically useful are elucidated. We hypothesized that the presence of type 2 inflammation relates to fixed-AO. OBJECTIVES: To evaluate the presence of four markers for type 2 inflammation in fixed airflow obstruction among asthmatics. METHODS: This was a cross-sectional study of 403 participants with asthma, aged 17-75 years, from three Swedish centres. Fixed airflow obstruction was defined as forced expiratory volume during the first second (FEV1 ) over forced vital capacity (FVC) being below the lower limit of normal (LLN). The following type 2 inflammation markers were assessed: exhaled nitric oxide (FeNO), serum periostin, serum eosinophil cationic protein (S-ECP), and urinary eosinophil-derived neurotoxin (U-EDN). RESULTS: Elevated U-EDN (values in the highest tertile, ≥65.95 mg/mol creatinine) was more common in subjects with fixed-AO vs. subjects without fixed-AO: 55% vs. 29%, P < 0.001. Elevated U-EDN related to increased likelihood of having fixed-AO in both all subjects and never-smoking subjects, with adjusted (adjusted for sex, age group, use of inhaled corticosteroids last week, atopy, early-onset asthma, smoking history, and packyears) odds ratios (aOR) of 2.38 (1.28-4.41) and 2.51 (1.04-6.07), respectively. In a separate analysis, having both elevated S-ECP (>20 µg/L) and U-EDN was related to having the highest likelihood of fixed-AO (aOR (95% CI) 6.06 (2.32-15.75)). Elevated serum periostin or FeNO did not relate to fixed-AO. CONCLUSIONS AND CLINICAL RELEVANCE: These findings support that type 2 inflammation, and in particular eosinophil inflammation, is found in asthma with fixed-AO. This could indicate a benefit from eosinophil-directed therapies. Further longitudinal studies are warranted to investigate causality and relation to lung function decline.

Eosinophil-derived neurotoxin enhances airway remodeling in eosinophilic chronic rhinosinusitis and correlates with disease severity.

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) that is characterized by intractable nasal polyp formation. Eosinophil-derived neurotoxin (EDN) is an eosinophil granule protein that is closely related to allergic inflammation, but the pathological implications of EDN in ECRS remain unknown. In this study, we evaluated the function of EDN in ECRS pathogenesis and assessed its potential as a disease activity marker. Serum EDN levels were significantly higher in patients with ECRS than in those with other nasal and paranasal diseases, and were positively correlated with clinical disease activity. Production of EDN from isolated human eosinophils was induced by stimulation with IL-5 in vitro. Human nasal epithelial cells were stimulated with EDN, and the resultant changes in gene expression were detected by RNA sequencing. Pathway analysis revealed that the major canonical pathway affected by EDN stimulation was 'regulation of the epithelial-mesenchymal transition pathway'; the only gene in this pathway to be up-regulated was matrix metalloproteinase 9 (MMP-9). Consistent with this, immunostaining analysis revealed intense staining of both EDN and MMP-9 in nasal polyps from patients with ECRS. In conclusion, our data demonstrate that serum EDN level is a useful marker for the evaluation of ECRS severity. Furthermore, EDN induces production of MMP-9 from the nasal epithelium, which may be involved in the pathogenesis of ECRS.

Blind esophageal brushing offers a safe and accurate method to monitor inflammation in children and young adults with eosinophilic esophagitis.

Patients with eosinophilic esophagitis (EoE) require frequent evaluation of mucosal inflammation via endoscopy. Instead of endoscopy, mucosal evaluation in adults with esophageal cancer and candidiasis is achieved using a cytology brush inserted through a nasogastric tube (NGT). We conducted a prospective cross-sectional study in children and young adults scheduled for routine esophagogastroduodenoscopy (EGD) where in Phase 1, we performed esophageal brushing through the endoscope under direct visualization and in Phase 2, we inserted the brush through a Cortrak® NGT prior to endoscopy. Eosinophil-derived neurotoxin (EDN) measured by ELISA in the samples extracted from brushes was validated as the sensitive biomarker. We collected 209 esophageal brushing samples from 94 patients and we found that EDN in brushing samples collected via EGD or NGT was significantly higher in patients having active EoE (n = 81, mean EDN 381 mcg/mL) compared with patients having gastroesophageal reflux disease (n = 31, mean EDN 1.9 mcg/mL, P = 0.003), EoE in remission (n = 47, mean EDN 3.7 mcg/mL, P = 0.003), or no disease (n = 50, mean EDN 1.1 mcg/mL, P = 0.003). EDN at a concentration of ≥10 mcg/mL of brushing sample was found to accurately detect active EoE. NGT brushing did not cause any significant adverse effects. We concluded that blind esophageal brushing using an NGT is a fast, less invasive, safe, and well-tolerated technique compared with EGD to detect and monitor EoE inflammation using EDN as the sensitive biomarker.