Unknown,Transcriptomics,Genomics,Proteomics

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Metabolic stress is a barrier to Epstein-Barr virus mediated B-cell immortalization

ABSTRACT: Early after infection, Epstein-Barr Virus (EBV) induces a transient period of hyper-proliferation that is suppressed by the activation of the DNA damage response and a G1/S phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation. We used microarray analysis to uncover changes in gene expression that could give us a better understanding of the pathways that attenuate immortalization. Human PBMCs were labeled with CellTrace Violet and infected with the B95-8 strain of EBV. At 4 days post-infection, the cells were labeled with a second proliferation dye, CFSE. Cells that proliferated are called PP and those that initially proliferated and then arrested are called PA. Total mRNA was isolated from sorted PA and PP cells using an RNeasy kit . The RNA was processed using an Ambion MessageAmp Premier Package and hybridized to a Human Genome U133 Plus. 2.0 Chip by the Duke Center for Genomic and Computational Biology Microarray Core. The resultant CEL files were RMA normalized (Partek) and the data was analyzed with GenePattern and GSEA v2.

ORGANISM(S): Homo sapiens

SUBMITTER: Karyn Mcfadden

PROVIDER: E-GEOD-76137 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments, the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Experiment Overall Design: Six healthy adult volunteers (5 females, 1 male, 36 Â± 13 yrs, mean age Â± SD) who had been infected with H. ducreyi twice were inoculated a third time. Each volunteer was inoculated at 3 sites on the upper arm with live H. ducreyi 35000HP (a human passaged isolate of strain 35000) and at 1 site with sterile PBS. Forty-eight hours after inoculation, lesion size was measured and RNA was isolated from the infected site that had the largest diameter. RNA was also obtained from the PBS control site (uninfected site). We obtained peripheral blood 6 to 12 months after inoculation of the 6th subject and derived myeloid DC in vitro. In brief, CD14+ peripheral blood monocytes were isolated from PBMC by positive selection magnetic beads (Miltenyi Biotec, Auburn, CA) and grown in the presence of recombinant human (rh) IL-4 (1 ng/ml) and rhGM-CSF (0.2 ng/ml) (R&D Systems, Minneapolis, MN). The cells were HLA-DR+, CD86+ CD40+, CD3-, CD14-, and CD19- by flow cytometry. The experiment was done in pairs so that DC from 1 RR subject were exposed to the same inoculum as DC from 1 PP subject. DC were incubated with nonopsonized H. ducreyi for 90 minutes at an MOI of 30:1. The DC were washed to remove non-associated bacteria, and incubated an additional 22.5 hours. Cells were collected and used for microarray analysis. Supernatants were collected and analyzed for cytokines using the Human Th1/Th2 II Cytometric Bead Array Kit per manufacturerâs instructions (BD Biosciences).

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Metabolic stress is a barrier to Epstein-Barr virus mediated B-cell immortalization

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