Serum microRNA-223 as a potential biomarker for allergic rhinitis and its correlation to eosinophil-derived neurotoxin.

Allergic rhinitis (AR) is a global health problem. It is an inflammatory condition defined by a malfunction of the immune system's regulatory mechanism. MicroRNA-223 (miRNA-223) has been linked to the modulation of AR in the last few years. The goal of this study was to determine whether miR-223 can be utilized as a potential biomarker for diagnosis of AR, and whether it correlates with the total nasal symptom score (TNSS) along with serum interleukin-17 (IL-17), interleukin-4 levels (IL-4) and eosinophil-derived neurotoxin (EDN). This study included 76 adult participants, consisted of 38 AR patients and 38 apparently healthy controls. Serum levels of miR-223 were assayed using real-time PCR. The levels of EDN, IL-17 and IL-4 in the serum were determined using an enzyme-linked immunosorbent assay. The optimal cutoff value for the analyzed factors to diagnose AR was determined using a receiver operating characteristic curve analysis (ROC). The demographic features (age and gender) of the two study groups were matched. Patients with pollen-induced AR had significantly higher levels of miR-223 in their serum compared to the controls (median = 3.82; median = 1.03, respectively, p < 0.001). In AR cases, a significant positive association was observed between miR-223 expression level and TNSS (r = 0.492, p = 0.002), EDN serum level (r = 0.427, p = 0.008), IL-4 serum level (r = 0.341, p = 0.036) and IL-17 serum level (r = 0.324, p = 0.047). MiR-223, at a cutoff value of 1.18, had a sensitivity and specificity of 94.9 % and 92.5%, respectively. In conclusion, miR-223 expression is significantly greater in blood of AR patients. There is a significant association between miR-223 and clinical severity of AR, each of IL-17 and IL-4 as well as EDN. Therefore, miR-223 may be employed as an effective biomarker for AR diagnosis.

Antiviral effect of high-pressure nasal stimulation.

OBJECTIVE: In a previous study, we reported an increase of nasal nerve growth factor (NGF) in patients treated with high-pressure administration of sterile saline isotonic solution (HPpSIS). Herein we characterized the nasal mucosa in terms of innate immune response and cytokine signature, including antiviral properties. Potential NGF and antiviral benefits of HPpSIS were also discussed. PATIENTS AND METHODS: Twenty (20) patients (11 males, 9 females; age range 30-75 years old) underwent HPpSIS and nasal samples were collected before and after treatment. Nasal scraping was used for morphological (smears and Quick May-Grunwald Giemsa staining, MGG), biochemical (Histamine, Serotonin; ELISA) and molecular (messenger RNA, mRNA) analyses. Amplification of transcripts specific for Toll-like receptor (TLR) 3 (TLR3), TLR7, TLR9, Interleukin-(IL) 18 (IL18), IL13, IL12, eosinophil-derived neurotoxin (EDN), Eosinophil Cationic Protein (ECP), γ Interferon (γIFN), tryptase and serotonin was performed using the 2-step real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR). Clinical and laboratory data were analyzed and compared. RESULTS: The clinical evaluation showed a protective effect of our therapy. Smears showed the presence of leucocytes, eosinophils (EOs) and mast cells (MCs), and increased immunoreactivity for ECP/RNase3 and EDN after HPpSIS. ELISA showed increased levels of Serotonin and EDN associated with unchanged levels of substance P(SP) and histamine. Increased eosinophil-derived neurotoxin eosinophil-derived neurotoxin (EDN) levels were confirmed by in situ fluorescent analysis. HPpSIS induced the upregulation of TLR3, TLR7 and TLR9 transcripts, while no changes were observed for Intercellular Adhesion Molecule 1 (ICAM1), IL18, Interleukin-15 (IL15) and IL12 transcripts nor for Interleukin-6 (IL6) and IL13. No changes were also observed for γIFN and EDN/RNase2 transcripts, while ECP/RNase3 transcripts were significantly upregulated after HPpSIS. Finally, tryptase transcripts were unchanged while serotonin transcripts were significantly increased after HPpSIS. CONCLUSIONS: The clinical and biomolecular changes observed at the nasal mucosa due to HpSS treatment suggest the activation of an innate surveillance, by means of TLR transcription, and a possible anti-viral response due to EDN upregulation. It remains to be verified if NGF, known to be released locally upon HpSIS treatment, might in part be responsible for this local activation.

Identification of novel genes influencing eosinophil-specific protein levels in asthma families.

BACKGROUND: Eosinophils play a key role in the asthma allergic response by releasing cytotoxic molecules such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) that generate epithelium damages. OBJECTIVE: We sought to identify genetic variants influencing ECP and EDN levels in asthma-ascertained families. METHODS: We performed univariate and bivariate genome-wide association analyses of ECP and EDN levels in 1018 subjects from the EGEA study with follow-up in 153 subjects from the Saguenay-Lac-Saint-Jean study and combined the results of these 2 studies through meta-analysis. We then conducted Bayesian statistical fine mapping together with quantitative trait locus and functional annotation analyses to identify the most likely functional genetic variants and candidate genes. RESULTS: We identified 5 genome-wide significant loci (P &lt; 5 × 10^-8) including 7 distinct signals associated with ECP and/or EDN levels. The genes targeted by our fine mapping and functional search include RNASE2 and RNASE3 (14q11), which encode EDN and ECP, respectively, and 4 other genes that regulate ECP and EDN levels. These 4 genes were JAK1 (1p31), a transcription factor that plays a key role in the immune response and acts as a potential therapeutic target for eosinophilic asthma; ARHGAP25 (2p13), which is involved in leukocyte recruitment to inflammatory sites; NDUFA4 (7p21), which encodes a component of the mitochondrial respiratory chain and is involved in cellular response to stress; and CTSL (9q22), which is involved in immune response, extracellular remodeling, and allergic inflammation. CONCLUSION: Analysis of specific phenotypes produced by eosinophils allows the identification of genes that play a major role in allergic response and inflammation, and offers potential therapeutic targets for asthma.

Cluster of highly expressed interferon-stimulated genes associate more with African ancestry than disease activity in patients with systemic lupus erythematosus. A systematic review of cross-sectional studies.

Type I interferons (IFN) are central players in the pathogenesis of systemic lupus erythematosus (SLE) and the up-regulation of interferon-stimulated genes (ISGs) in SLE patients is subjected to increasing scrutiny as for its use in diagnosis, stratification and monitoring of SLE patients. Determinants of this immunological phenomenon are yet to be fully charted. The purpose of this systematic review was to characterize expressions of ISGs in blood of SLE patients and to analyze if they associated with core demographic and clinical features of SLE. Twenty cross-sectional, case-control studies comprising 1033 SLE patients and 602 study controls could be included. ISG fold-change expression values (SLE vs controls), demographic and clinical data were extracted from the published material and analyzed by hierarchical cluster analysis and generalized linear modelling. ISG expression varied substantially within each study with IFI27, IFI44, IFI44L, IFIT4 and RSAD2, being the top-five upregulated ISGs. Analysis of inter-study variation showed that IFI27, IFI44, IFI44L, IFIT1, PRKR and RSAD2 expression clustered with the fraction of SLE cases having African ancestry or lupus nephritis. Generalized linear models adjusted for prevalence of lupus nephritis and usage of hydroxychloroquine confirmed the observed association between African ancestry and IFI27, IFI44L, IFIT1, PRKR and RSAD2, whereas disease activity was associated with expression of IFI27 and RNASE2. In conclusion, this systematic review revealed that expression of ISGs often used for deriving an IFN signature in SLE patients were influenced by African ancestry rather than disease activity. This underscores the necessity of taking ancestry into account when employing the IFN signature for clinical research in SLE.

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.

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.

Phenotype of ribonuclease 1 deficiency in mice.

Biological roles for extracellular RNA (eRNA) have become apparent. For example, eRNA can induce contact activation in blood via activation of the plasma proteases factor XII (FXII) and factor XI (FXI). We sought to reveal the biological role of the secretory enzyme ribonuclease 1 (RNase 1) in an organismal context by generating and analyzing RNase 1 knockout (Rnase1-/-) mice. We found that these mice are viable, healthy, and fertile, though larger than Rnase1+/+ mice. Rnase1-/- plasma contains more RNA than does the plasma of Rnase1+/+ mice. Moreover, the plasma of Rnase1-/- mice clots more rapidly than does wild-type plasma. This phenotype appeared to be due to increased levels of the active form of FXII (FXIIa) in the plasma of Rnase1-/- mice compared to Rnase1+/+ mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of Rnase1-/- mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1-/- mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo.

Integrative approach identifies corticosteroid response variant in diverse populations with asthma.

BACKGROUND: Although inhaled corticosteroid (ICS) medication is considered the cornerstone treatment for patients with persistent asthma, few ICS pharmacogenomic studies have involved nonwhite populations. OBJECTIVE: We sought to identify genetic predictors of ICS response in multiple population groups with asthma. METHODS: The discovery group comprised African American participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity (SAPPHIRE) who underwent 6 weeks of monitored ICS therapy (n = 244). A genome-wide scan was performed to identify single nucleotide polymorphism (SNP) variants jointly associated (ie, the combined effect of the SNP and SNP × ICS treatment interaction) with changes in asthma control. Top associations were validated by assessing the joint association with asthma exacerbations in 3 additional groups: African Americans (n = 803 and n = 563) and Latinos (n = 1461). RNA sequencing data from 408 asthmatic patients and 405 control subjects were used to examine whether genotype was associated with gene expression. RESULTS: One variant, rs3827907, was significantly associated with ICS-mediated changes in asthma control in the discovery set (P = 7.79 × 10-8) and was jointly associated with asthma exacerbations in 3 validation cohorts (P = .023, P = .029, and P = .041). RNA sequencing analysis found the rs3827907 C-allele to be associated with lower RNASE2 expression (P = 6.10 × 10-4). RNASE2 encodes eosinophil-derived neurotoxin, and the rs3827907 C-allele appeared to particularly influence ICS treatment response in the presence of eosinophilic inflammation (ie, high pretreatment eosinophil-derived neurotoxin levels or blood eosinophil counts). CONCLUSION: We identified a variant, rs3827907, that appears to influence response to ICS treatment in multiple population groups and likely mediates its effect through eosinophils.

Ribonucleotide incorporation enables repair of chromosome breaks by nonhomologous end joining.

The nonhomologous end-joining (NHEJ) pathway preserves genome stability by ligating the ends of broken chromosomes together. It employs end-processing enzymes, including polymerases, to prepare ends for ligation. We show that two such polymerases incorporate primarily ribonucleotides during NHEJ-an exception to the central dogma of molecular biology-both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. Moreover, additions of ribonucleotides but not deoxynucleotides effectively promote ligation. Repair kinetics suggest that ribonucleotide-dependent first-strand ligation is followed by complementary strand repair with deoxynucleotides, then by replacement of ribonucleotides embedded in the first strand with deoxynucleotides. Our results indicate that as much as 65% of cellular NHEJ products have transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.

The genetically determined production of the alarmin eosinophil-derived neurotoxin is reduced in visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Recent findings indicate that dendritic cells have a key role in the defense against the Leishmania parasite and that the activity of this cell may be modified by the eosinophil secretory protein eosinophil-derived neurotoxin (EDN). We hypothesized that the interactions between dendritic cells and EDN might be of importance in the disease development. Cellular content of EDN was analyzed by ELISA. The single-nucleotide polymorphisms at positions 405, 416, and 1122 in the EDN gene were analyzed by real-time PCR with TaqMan® reagents. The study cohorts comprised 239 Sudanese subjects (65 healthy controls and 174 with VL) and 300 healthy Swedish controls. The eosinophil content of EDN was lower in VL as compared with controls (p < 0.0001). The EDN405 (G>C) genotype distribution was similar among Swedish and Sudanese controls, whereas VL subjects had a higher prevalence of the EDN405-GG genotype (p < 0.0001). The content of EDN in the eosinophils was closely linked to the EDN405 polymorphism (p = 0.0002). Our findings suggest that the predisposition to acquire VL is related to the genetic polymorphism of the EDN gene and the reduced production by the eosinophil of this gene product.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome.

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Common Marker Genes Identified from Various Sample Types for Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex auto-immune disease. Gene expression studies have been conducted to identify SLE-related genes in various types of samples. It is unknown whether there are common marker genes significant for SLE but independent of sample types, which may have potentials for follow-up translational research. The aim of this study is to identify common marker genes across various sample types for SLE. METHODS: Based on four public microarray gene expression datasets for SLE covering three representative types of blood-born samples (monocyte; peripheral blood mononuclear cell, PBMC; whole blood), we utilized three statistics (fold-change, FC; t-test p value; false discovery rate adjusted p value) to scrutinize genes simultaneously regulated with SLE across various sample types. For common marker genes, we conducted the Gene Ontology enrichment analysis and Protein-Protein Interaction analysis to gain insights into their functions. RESULTS: We identified 10 common marker genes associated with SLE (IFI6, IFI27, IFI44L, OAS1, OAS2, EIF2AK2, PLSCR1, STAT1, RNASE2, and GSTO1). Significant up-regulation of IFI6, IFI27, and IFI44L with SLE was observed in all the studied sample types, though the FC was most striking in monocyte, compared with PBMC and whole blood (8.82-251.66 vs. 3.73-74.05 vs. 1.19-1.87). Eight of the above 10 genes, except RNASE2 and GSTO1, interact with each other and with known SLE susceptibility genes, participate in immune response, RNA and protein catabolism, and cell death. CONCLUSION: Our data suggest that there exist common marker genes across various sample types for SLE. The 10 common marker genes, identified herein, deserve follow-up studies to dissert their potentials as diagnostic or therapeutic markers to predict SLE or treatment response.

Eosinophil-Derived Neurotoxin (EDN/RNase 2) and the Mouse Eosinophil-Associated RNases (mEars): Expanding Roles in Promoting Host Defense.

The eosinophil-derived neurotoxin (EDN/RNase2) and its divergent orthologs, the mouse eosinophil-associated RNases (mEars), are prominent secretory proteins of eosinophilic leukocytes and are all members of the larger family of RNase A-type ribonucleases. While EDN has broad antiviral activity, targeting RNA viruses via mechanisms that may require enzymatic activity, more recent studies have elucidated how these RNases may generate host defense via roles in promoting leukocyte activation, maturation, and chemotaxis. This review provides an update on recent discoveries, and highlights the versatility of this family in promoting innate immunity.

Relationship between SNPs and expression level for candidate genes in rheumatoid arthritis.

OBJECTIVES: The study of polymorphisms of genes differentially expressed may lead to the identification of putative causal genetic variants in multifactorial diseases such as rheumatoid arthritis (RA). Based on preceding transcriptomic results, we genotyped 10 single nucleotide polymorphisms (SNPs) belonging to six genes (S100A8, RNASE2, PGLYRP1, RUNX3, IL2RB, and LY96) showing the highest fold change (> 1.9) when level of expression was compared between RA patients and controls. These SNPs were then analysed to evaluate their role in RA. METHOD: The relationship between gene expression and genotypes of SNPs was first investigated by Kruskal-Wallis and Mann-Whitney tests in RA patients and controls. The genetic association of these SNPs with RA were then analysed using family-based association tests in trio families. RESULTS: We found that RNASE2 gene expression was related to rs2013109 genotypes in 14 RA patients (p = 0.030). The association study in a discovery sample of 200 French trio families revealed a significant association with RA for one SNP, PGLYRP1-rs2041992 (p = 0.019); this association was stronger in trios where RA patients carried the HLA-DRB1 shared epitope (SE) (p = 0.003). However, this association was not found in a replication sample of 240 European trio families (p = 0.6). CONCLUSIONS: Family-based association tests did not reveal an association between RA and any SNP of the candidate genes tested. However, RNASE2 gene expression was differentially expressed in RA patients considering a sequence polymorphism. This result led us to highlight the potential disease-specific regulation for this candidate gene in RA.

Ear2 deletion causes early memory and learning deficits in APP/PS1 mice.

To assess the consequences of locus ceruleus (LC) degeneration and subsequent noradrenaline (NA) deficiency in early Alzheimer's disease (AD), mice overexpressing mutant amyloid precursor protein and presenilin-1 (APP/PS1) were crossed with Ear2(-/-) mice that have a severe loss of LC neurons projecting to the hippocampus and neocortex. Testing spatial memory and hippocampal long-term potentiation revealed an impairment in APP/PS1 Ear2(-/-) mice, whereas APP/PS1 or Ear2(-/-) mice showed only minor changes. These deficits were associated with distinct synaptic changes including reduced expression of the NMDA 2A subunit and increased levels of NMDA receptor 2B in APP/PS1 Ear2(-/-) mice. Acute pharmacological replacement of NA by L-threo-DOPS partially restored phosphorylation of ß-CaMKII and spatial memory performance in APP/PS1 Ear2(-/-) mice. These changes were not accompanied by altered APP processing or amyloid ß peptide (Aß) deposition. Thus, early LC degeneration and subsequent NA reduction may contribute to cognitive deficits via CaMKII and NMDA receptor dysfunction independent of Aß and suggests that NA supplementation could be beneficial in treating AD.

The production of the eosinophil proteins ECP and EPX/EDN are regulated in a reciprocal manner.

Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single-nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene. In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil-derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5'nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non-allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non-allergic and allergic subjects.The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.

Basic amino acid residues of human eosinophil derived neurotoxin essential for glycosaminoglycan binding.

Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.

Eosinophil associated genes in the inflammatory bowel disease 4 region: correlation to inflammatory bowel disease revealed.

AIM: To study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP). METHODS: DNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohn's disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5'-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fisher's exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant. RESULTS: The genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subject's genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in µg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes. CONCLUSION: Polymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity.

The human eosinophil granule ribonuclease, eosinophil-derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus-B (RSV-B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine-residue insertion in its carboxy-terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN.

Locus coeruleus damage and noradrenaline reductions in multiple sclerosis and experimental autoimmune encephalomyelitis.

The endogenous neurotransmitter noradrenaline exerts anti-inflammatory and neuroprotective effects in vitro and in vivo. Several studies report that noradrenaline levels are altered in the central nervous system of patients with multiple sclerosis and rodents with experimental autoimmune encephalomyelitis, which could contribute to pathology. Since the major source of noradrenaline are neurons in the locus coeruleus, we hypothesized that alterations in noradrenaline levels are a consequence of stress or damage to locus coeruleus neurons. In C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to develop chronic disease, cortical and spinal cord levels of noradrenaline were significantly reduced versus control mice. Immunohistochemical staining revealed increased astrocyte activation in the ventral portion of the locus coeruleus in immunized mice. The immunized mice showed neuronal damage in the locus coeruleus detected by a reduction of average cell size of tyrosine hydroxylase stained neurons. Analysis of the locus coeruleus of multiple sclerosis and control brains showed a significant increase in astrocyte activation, a reduction in noradrenaline levels, and neuronal stress indicated by hypertrophy of tyrosine hydroxylase stained cell bodies. However, the magnitude of these changes was not correlated with extent of demyelination or of cellular infiltrates. Together these findings demonstrate the presence of inflammation and neuronal stress in multiple sclerosis as well as in experimental autoimmune encephalomyelitis. Since reduced noradrenaline levels could be permissive for increased inflammation and neuronal damage, these results suggest that methods to raise noradrenaline levels or increase locus coeruleus function may be of benefit in treating multiple sclerosis.