Gene profiling in mucosal biopsies of Eosinophilic Esophagitis patients pre and post glucocorticoid steroid treatment
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Shaolei Lu
PROVIDER: E-GEOD-36725 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
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Project description:Coeliac disease is a small intestinal disorder caused by an abarrent immune response towards dietary gluten due to activation of pro-inflammatory gluten specific CD4+ T cells. Histological evaluation and classification of gluten induced musosal changes is part of the diagnostic work up. of adults. The kinetics of mucosal recovery following commencesment of a gluten free diet (disease remission) and the degree of mucosal changes induced by gluten reintroduction (gluten challenge) varies between patients. Also, patients classified with similar clinical and histological disease remission, can develop different degree of mucosal damage following the same gluten challenge regime. This variation poses a challenge for the interpretation of gluten induced mucosal changes both in a diagnostic and clinical trial settings. In this study, we have analysed material from small intestinal biopsies collected from 19 treated coeliac disease patients before and after completion of a 14-day oral gluten challenge. These patients are part of a previoslpreviouslyu described study where all patients were in clinical and mucosal remission at baseline but only some patients developed histological changes in the mucosa in response to gluten. We have performed shotgun LC-MSMS analysis and label-free quantification of total gut tissue and from laser capture microdissected epithelial cell layer samples. We found that differences in tissue proteome expression could separate patients as responders and non-responders to the gluten challenge. Patients whoich responded strongly to gluten , had signs of gut inflammation already at baseline, supported by presence of low-level blood inflammatory parameters and a slight increase in numbers of gluten-specific CD4+ T cells at baseline. Our proteomics analysis demonstrated baseline differences in gut tissue state between patients that were not evident from routine clinical and histological evaluation. These baseline differences likely explains why some patients respond more strongly to gluten challenge than others.
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Project description:One and four month formalin-fixed paraffin embedded biopsies from 48 kidney transplant recipients (24 AKI donors, 24 non-AKI) underwent global gene expression profiling using DNA microarrays (96 arrays). At one month, there were 898 differentially expressed genes in the AKI group (p-value <0.005; FDR <10%), but by 4-months there were no longer any differences. One and four month formalin-fixed paraffin embedded (FFPE) biopsies from 48 kidney transplant recipients (24 AKI donors, 24 controls; 96 total samples) were profiled using Affymetrix U133PM Plus PM plate arrays on a GeneTitan MC instrument. All samples were selected from the SCD cohort only. AKI donor and controls samples were matched by date of transplantation. Total RNA was extracted from four 20-micron sections using Ambion RecoverAll Total Nucleic Acid Kit and processed for chip hybridization using the new Affymetrix SensationPlus™ FFPE Amplification and 3’ IVT Labeling kit according to the manufacturer’s protocol. Quantile data normalization by RMA and analysis was performed using Partek Genomics Suite for PDR-corrected differential gene expression by ANOVA. Pathway mapping was performed using Ingenuity Pathway Analysis (IPA). This dataset is part of the TransQST collection.
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