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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene Expression Analysis of IgM+CD27+ B Cells in HCV-Infected Individuals with Mixed Cryoglobulinemia

ABSTRACT: Analysis of IgM+CD27+ B cells in individuals with hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC). We have previously shown that HCV+MC+ individuals have clonal expansions of IgM+ memory B cells. This study aims to characterize differentially expressed genes in peripheral IgM+CD27+ B cells in HCV RNA+MC+ individuals, compared to HCV RNA+MC- subjects and HCV RNA- healthy controls. IgM+ B cells were isolated form donor PBMCs by immunomagnetic negative selection, and the CD27+ subset was further purified by positive selection. 2ng RNA was isolated from cells and amplified using the Nugen WT-Ovation Amplification Kit. cDNA was synthesized, biotin labeled, and hybridized to Illumina human v2 microarrays. Data was collected in BeadStudio and exported to GeneSpring for analysis. Raw data were normalized by transforming measurements less than 0.01 to 0.01, normalizing per chip to the 50th percentile, and normalizing per gene to the median. Normalized data were then filtered to remove genes with control signals <170. Genes that were significantly upregulated in HCV RNA+MC+ vs HCV RNA+MC- subjects' IgM+CD27+ B cells were identified using a Welch t-test with p-calue cutoff 0.05 and Benjamini-Hochberg false discovery rate of 0.05. The resulting gene list was then filtered for genes showing >2-fold differential expression. 4 HCV Ab-, 6 HCV AB+/HCV RNA-, 12 HCV RNA+/IgM MC-, and 11 HCV RNA+/IgM MC+ samples analyzed. The supplementary file 'GSE18084_non-normalized_data.txt' contains non-normalized data for Samples GSM452094-GSM452126. This file was obtained from the GeneSpring platform.

ORGANISM(S): Homo sapiens

SUBMITTER: Edgar Charles

PROVIDER: E-GEOD-18084 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene Expression Analysis of IgM+CD27+ B Cells in HCV-Infected Individuals with Mixed Cryoglobulinemia

Project description:Analysis of IgM+CD27+ B cells in individuals with hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC). We have previously shown that HCV+MC+ individuals have clonal expansions of IgM+ memory B cells. This study aims to characterize differentially expressed genes in peripheral IgM+CD27+ B cells in HCV RNA+MC+ individuals, compared to HCV RNA+MC- subjects and HCV RNA- healthy controls. IgM+ B cells were isolated form donor PBMCs by immunomagnetic negative selection, and the CD27+ subset was further purified by positive selection. 2ng RNA was isolated from cells and amplified using the Nugen WT-Ovation Amplification Kit. cDNA was synthesized, biotin labeled, and hybridized to Illumina human v2 microarrays. Data were collected and analyzed with Illumina Genome Studio. Raw data were normalized by transforming measurements less than 0.01 to 0.01, normalizing per chip to the 50th percentile, and normalizing per gene to the median. Normalized data were then filtered to remove genes with control signals <170. Genes that were significantly upregulated in HCV RNA+MC+ vs HCV RNA+MC- subjects' IgM+CD27+ B cells were identified using a Welch t-test with p-calue cutoff 0.05 and Benjamini-Hochberg false discovery rate of 0.05. The resulting gene list was then filtered for genes showing >2-fold differential expression.

2009-10-01 | GSE18084 | GEO

Hepatitis C-like viruses are produced in cells from rabbit and hare DNA

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Project description:This study characterizes the effects of chronic Hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naive HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136 gene signature, including 66 genes elevated in infected individuals. Most of the up-regulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15 and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. Validation of these genes was carried out using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the up-regulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of interferon-stimulated genes. Whole blood was collected from a group of 10 patients who were infected with genotype 1 hepatitis C, before initiation of treatment at the Indiana University School of Medicine. Control blood samples were harvested from healthy student volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of the cohort using centrifugation through a 10-ml Ficoll-Hypaque gradient (Amersham/Pharmacia, Piscataway, NJ), and RNA was isolated within a few hours. RNA was then processed according to the protocols recommended by Affymetrix (Santa Clara, CA), and run on an Affymextrix Human Genome U133 array.

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Effect of HCV in B cells and changes in B cell lymphoma (BCL)

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2014-02-09 | E-GEOD-54722 | biostudies-arrayexpress

Homo sapiens

Project description:Gene Expression Analysis of IgM+CD27+ B Cells in HCV-Infected Individuals with Mixed Cryoglobulinemia

| PRJNA119359 | ENA

The blood transcriptional signature of chronic HCV

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Project description:Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein-interactions, we performed an affinity purification mass spectrometry screen (AP-MS) of HCV infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p)7 or nonstructural protein 4B (NS4B), as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Detected were five ApoE binding proteins, 12 p7 binders, seven primary E2-interactors and 24 proteins interacting with NS4B. Cell-derived PREB, STT3B, SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.

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