No description available.

No description available.

During latent Epstein-Barr virus (EBV) infection, the T helper cell response to the EBV latent membrane protein (LMP)1 is dominated by the production of IL-10, but by IFN- γ during acute EBV infection. The purpose of this thesis was to develop methods for the enumeration and characterisation of the IL-10 producing CD4+ T cells that respond to peptides of the EBV protein LMP1, and to investigate the possible involvement of IL-27 in the development of these cells. It was found that some human donors have very high concentrations of IL-27 within serum, which is not dependent on EBV infection status but demonstrates relatively low heritability. The addition of IL-27 to cultures of human T cells did not induce IL-10 but the production of IL-17 was inhibited. To identify and characterise LMP1 responsive cells I used CD154 as a marker of activated T cells. Having optimised the methodology, 14 donors of known EBV serostatus were tested for activated IL-10 producing cells after culturing peripheral blood mononuclear cells with LMP1 peptides. However in most cases the frequency of CD4+ lymphocytes upregulating CD154 and IL-10 in response to LMP1 peptides was below the assay’s sensitivity. When the CD154+IL-10+ CD4+ cells were stained for T helper cell subset markers they were positive for every marker and isotype control, suggesting that the cells were non-specifically binding labelled antibody. Both single positive CD154+ and single positive IL-10+CD4+ cells were also present in response to LMP1, but again typically at frequencies below the level of sensitivity for this assay. The conclusions of the work are that IL-27 responses are heterogeneous, but unlikely to play an important role in the induction of IL-10+ T cells in EBV infection. The frequency of LMP1 responsive T cells is very low (<0.08%) in most donors.

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

No description available.

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Univ., 2006. / Härtill 4 uppsatser.