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Two polymorphisms in the epithelial cell-derived neutrophil-activating peptide (ENA-78) gene.

ABSTRACT: Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78) has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing. The polymorphism at position +398 (A/G from the first ATG codon) in exon 2 results in a synonymous substitution not resulting in an amino acid change. The promoter polymorphism was found at position -156 (C/G from the first ATG codon). An assay was designed for the detection of the polymorphisms using SNapshot ddNTP primer extension, followed by capillary electrophoresis (ABI 3100). Allele and genotype frequencies for the promoter -156 polymorphism are presented for 107 healthy Spanish and 54 UK Caucasians. Frequencies for the exon 2 polymorphism are also presented for 63 UK Caucasians.

SUBMITTER: Amoli MM

PROVIDER: S-EPMC3850584 | biostudies-other | 2005

REPOSITORIES: biostudies-other

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Two polymorphisms in the epithelial cell-derived neutrophil-activating peptide (ENA-78) gene.

Amoli Mahsa M MM Larijani Bagher B Thomson Wendy W Ollier William E R WE Gonzalez-Gay Miguel A MA

Disease markers 20050101 2

Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78) has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing. The polymorphism at position +398 (A/G from the first ATG codon) in exon 2 results in a synonymous substitution not ...[more]

PMID: 15920294

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Project description:Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.

Project description:BackgroundInnovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells.MethodsThe immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions.ResultsNeutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions.ConclusionThe MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.

Dataset Information

Two polymorphisms in the epithelial cell-derived neutrophil-activating peptide (ENA-78) gene.

Publications

Two polymorphisms in the epithelial cell-derived neutrophil-activating peptide (ENA-78) gene.

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