Nanostring nCounter human miRNA assay (v1) of eosinophilic esophagitis
ABSTRACT: Nanostring nCounter Human miRNA assay (v1) of esophageal mucosal biopsies from children with eosinophilic esophagitis and controls Individual esophageal mucosal biopsies from children with eosinoniphilic esophagitis and controls were analysed for detection of microRNA
Project description:3 eosinophilic esophagitis biopsies, cultured and stimulated with IL-13 : each of them was either left unstimulated or stimulated (100ng for 48h) We used microarray to uncover the IL-13-induced genes in esophageal epithelial cells of the esophagus Keywords: treated vs non treated Overall design: 3 biopsies from EE patients were obtained and primary epithelial cell were cultured and either left unstimulated or stimulated with IL-13 followed by RNA extraction and hybridization on Affymetrix microarrays.
Project description:3 eosinophilic esophagitis biopsies, cultured and stimulated with IL-13 : each of them was either left unstimulated or stimulated (100ng for 48h); We used microarray to uncover the IL-13-induced genes in esophageal epithelial cells of the esophagus Experiment Overall Design: 3 biopsies from EE patients were obtained and primary epithelial cell were cultured and either left unstimulated or stimulated with IL-13 followed by RNA extraction and hybridization on Affymetrix microarrays.
Project description:Serum from children with active and inactive treated eosinophilic esophagitis was analyzed for detection of microRNA Individual serum samples from children with eosinophilic esophagitis were analyzed for detection of microRNA. (n=5 for active EoE and n=5 for inactive EoE)
Project description:Eosinophlic esophagitis (EoE) is increasely recognized as an antigen-drived disorder. The goal of this study is to reveal the gene expression changes in EoE before and after a successful glucocorticoid steroid treatment. We used microarrays to identify distinct genes involving the pathophysiology of EoE. Esophageal mRNA from the epithelial layers of 5 paired paraffin-embedded biopsies before and after treatment with glucocorticosteroids were harvested and profiled using Affymetrix Human Gene 1.0 ST array to generate differentially regulated mRNA transcripts.
Project description:We utilized RNA sequencing to expand and better define the molecular entities involved in the transcriptional programming within the eosphagus in eosinophilic esophagitis (EoE) Examination of differentially expressed genes from RNA sequencing data performed on esophageal biopsy specimen from 6 healthy controls and 10 patients with active EoE
Project description:Eosinophlic esophagitis (EoE) is increasely recognized as an antigen-drived disorder. The goal of this study is to reveal the miRNA expression changes in EoE before and after a successful glucocorticoid steroid treatment. Total RNA was extracted from the esophageal epithelial layers of 5 paired paraffin-embedded biopsies before and after treatment with glucocorticosteroids using RecoverAll Total Nucleic Acid Extraction Kit for FFPE tissues (Ambion, Austin, TX). Five nanograms of total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit and the Megaplex RT primer Human Pool A (Applied Biosystems). The reverse-transcribed cDNA was then pre-amplified in 12 cycles of PCR using Taqman PreAmp Master Mix and the Megaplex PreAmp primers, Human Pool A (Applied Biosystems). The cDNA’s were then diluted and loaded on to a Taqman Human miRNA Array card A (Platform GPL9731 ; Applied Biosystems), which contains probes for 377 distinct miRNAs. The Array cards were run on an ABI HT7900 qPCR instrument. Ct values were obtained for all miRNAs represented on the cards and fold changes in expression were calculated using the delta delta Ct (ddCt) method.
Project description:Esophageal involvement in patients with systemic sclerosis (SSc) is common, but tissue-specific pathological mechanisms are poorly understood. Fibrosis in the esophagus is thought to disrupt smooth muscle function and lead to esophageal dilatation, but autopsy studies demonstrate esophageal smooth muscle atrophy and the absence of fibrosis in the majority of SSc cases. Molecular characterization of SSc esophageal pathology is lacking. Herein, we perform a detailed characterization of SSc esophageal histopathology and molecular signatures at the level of gene expression. Esophageal biopsies were prospectively obtained during esophagogastroduodenoscopy in 16 consecutive SSc patients and 7 subjects without SSc. Upper and lower esophageal biopsies were evaluated for histopathology and gene expression. Individual patient’s upper and lower esophageal biopsies showed nearly identical patterns of gene expression. Similar to skin, inflammatory and proliferative gene expression signatures were identified suggesting that molecular subsets are a universal feature of SSc end-target organ pathology. The inflammatory signature was present in biopsies without high numbers of infiltrating lymphocytes. Molecular classification of esophageal biopsies was independent of SSc skin subtype, serum autoantibodies and esophagitis. Proliferative and inflammatory molecular gene expression subsets in tissues from patients with SSc may be a conserved, reproducible component of SSc pathogenesis. The inflammatory signature is observed in biopsies that lack large inflammatory infiltrates suggesting that immune activation is a major driver of SSc esophageal pathogenesis. Overall design: Gene expression was measured in upper and lower esophageal biopsies from 16 patients with and 7 subjects without SSc.
Project description:There is no useful biomarker for reflux esophagitis. The aim of this study is to establish novel diagnosis marker for RE by using miRNA. Overall design: In this study, acid reflux esophagitis was induced in rats. Rats were killed day 21 (chronic phase) after induction of RE. Shapm operation rats were used as control. Exosomes were extracted from serum, and the expression patterns of serum miRNAs during the RE chronic phase were analyzed via microarray.
Project description:Background & Aims: Because the pathophysiology of GERD is not fully understood, presently used drug target only one or more of the known underlying mechanisms but are not fully effective in all patients. 1Identifying novel central targets may pave the way to develop more effective agents. Methods: A surgical model of sub-chronic reflux esophagitis was developed. Wistar rats were pretreated for 7days with omeprazole (standard proton pump inhibitor) or STW5 (herbal preparation of established efficacy in gastro-intestinal disorders). Treatment was continued for 10days after surgery, rats were sacrificed and esophagi excised. Histological, proteomic and transcriptomic methods were applied to identify reflux induced changes and treatment responses. Results: Protection against reflux induced inflammation was achieved by both test drugs. Both reduced macroscopic and microscopic lesions of the esophagi as well as most measured pro-inflammatory cytokines without significantly affecting NF-kB activity. Proteomic and transcriptomic analysis identified CINC1-3, MIP-1/3α, MIG, RANTES and IL-1β as highly relevant mediators in GERD. Other highly regulated genes were those of IL-6, CCL3, CCL7 and LOX-1. Many affected cyto-/chemokines were involved in the TREM-1 signaling pathway. The fatty acid receptor GPR84 was highly up-regulated in esophagitis but down-regulated by both drugs. This was confirmed by Western blot and immune-histochemical staining, showing for the first time expression of this receptor in esophageal tissue and its possible involvement in GERD. Conclusion: STW5 and omeprazole target a broad spectrum of molecules involved in immunological and inflammatory processes, of which IL-8 (CINC1-3), TREM-1 pathway and GPR84 are proposed to be most promising novel targets for the treatment of GERD. Refluxesophagitis was surgically induced. Wistar rats were pretreated for 7 days with omeprazole or STW5. Treatment was continued for 10days after surgery, rats were sacrificed and esophagi exiced. The study had 5 groups. Group 1: sham operated, Group 2: esophagitis group, untreated, Group3: esophagitis treated with STW5 0.5ml/kg. Group 4: esophagitis treated with STW5 2ml/kg. Group 5: esophagitis treated with Omeprazole (30mg/kg). 4 microarrays from esophageal tissue and blood from 4 animals of each group were performed.