This randomised, open label, phase I, immunotherapeutic study investigated the effects of interle... more This randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. Twelve HIV-1(+) patients on suppressive cART with baseline CD4 T-cell counts >400 cells/mm(3) blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n=3); (2) vaccine alone (n=4); or (3) IL-2, GM-CSF and rhGH (n=5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. Median ba...
T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell ... more T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell proliferation, cytokine production and differentiation into effector cells. These cascades comprise an array of protein-tyrosine kinases, phosphatases, GTP-binding proteins and adaptor proteins that regulate generic and specialised functions. The integration of these signals is essential for the normal development, homeostasis and function of T cells. Defects in a single mediator can produce T cells that are unable to participate fully in an immune response and/or that mount an inappropriate response, which leads to immunodeficiency, autoimmunity or leukaemia/lymphomas. This review highlights some of the key players in T-cell signalling and their involvement in the development of various clinical disease states. Some of these immune-specific signalling proteins are attractive potential targets in the development of therapies to augment T-cell responses to antigen or tumours, and to treat immune cell disorders.
Successful antiretroviral therapy (ART) suppresses plasma HIV-1 RNA below detection limits, reduc... more Successful antiretroviral therapy (ART) suppresses plasma HIV-1 RNA below detection limits, reducing the chronic insult to the immune systems of infected individuals and supporting a degree of immunological recovery. However, the surface phenotypic profile of T cells in ART-treated patients does not resemble that of healthy, uninfected individuals, but rather shows upregulation of proteins associated with an exhausted immune system. We sought to address whether aviraemic HIV-1 infection, therefore, contributed to long-term alterations in intracellular signalling events within the T cells of infected individuals that contributed to the exhausted phenotype. A flow cytometric approach was employed to assess levels of phosphorylation within T-cell signalling proteins in ART-treated HIV-1-positive patients and HIV-negative individuals. The relative phosphorylation levels of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38, zeta-chain-associated protein kinase 70 (ZAP70), linker of activated T cells, SLP76, nuclear factor kappaB were measured within resting and stimulated CD4(+) and CD8(+) T cells from aviraemic HIV-1-positive and healthy individuals by intracellular staining and flow cytometric analysis. Basal levels of phospho-ZAP70, phospho-ERK and phospho-JNK were two-fold to three-fold higher in HIV-1-positive individuals compared with healthy controls, with phospho-p38 also showing a tendency to increase in HIV-1-positive individuals. Interestingly, in contrast to healthy controls, peripheral blood mononuclear cells from aviraemic, infected individuals were refractory to stimulation with IL-2 and CD3/CD28 showing no enhancement of phosphorylation. CD4(+) and CD8(+) T cells from HIV-1-positive individuals are poorly responsive to direct stimulation through the T-cell receptor due to chronically raised basal activation levels of intracellular signalling molecules.
... Full Text. HIV Therapy. January 2010, Vol. 4, No. 1, Pages 83-99 , DOI 10.2217/hiv.09.51. (do... more ... Full Text. HIV Therapy. January 2010, Vol. 4, No. 1, Pages 83-99 , DOI 10.2217/hiv.09.51. (doi:10.2217/hiv.09.51). T-cell dysfunction in HIV-1 infection: targeting the inhibitors. Jocelyn S Downey & Nesrina Imami Author for correspondence. Sections: ...
CTLA-4 is a co-receptor that plays a pivotal role in regulating the threshold for T-cell activati... more CTLA-4 is a co-receptor that plays a pivotal role in regulating the threshold for T-cell activation. We recently reported that CTLA-4 ligation can over-ride the stop-signal induced by anti-CD3 ligation [Schneider H, Downey J, Smith A, Zinselmeyer BH, Rush C, Brewer JM, et al. Reversal of the TCR stop-signal by CTLA-4. Science 2006;313:1972]. While these studies compared CTLA-4 positive and negative T-cells from normal mice, little is known regarding the behaviour of T-cells from diseased Ctla4 deficient mice with auto-proliferative disease. In this study, we show that while activated wild-type and Ctla-4−/− T-cells have similar rates of motility, Ctla-4−/− T-cells show a marked resistance to the induction of a stop-signal by anti-CD3 ligation. By contrast, T-cells from normal mice and CD28 deficient mice underwent a normal slowing of motility in response to anti-CD3 ligation. Our findings identify a fundamental difference between normal versus CTLA-4−/− T-cells from diseased mice in the regulation of motility by anti-CD3 ligation. This dysregulation of motility may contribute to the tissue infiltration and the autoimmune disorder observed in Ctla-4−/− mice.
... MAP kinase pathways Wei Zhu, Jocelyn S. Downey, Yingqiu Li, Jiahuai Han Department of Immunol... more ... MAP kinase pathways Wei Zhu, Jocelyn S. Downey, Yingqiu Li, Jiahuai Han Department of Immunology, The Scripps Research Institute, La Jolla, California, USA ... Nature 1994; 372: 739. 13. Hambleton J, McMahon M, DeFranco AL. ...
Biochemical and Biophysical Research Communications, Jan 1, 1998
The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC... more The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC rats, which develop a spontaneous form of hepatitis at 3-4 months of age, and compared to trace metal concentrations in the LEA rat, its asymptomatic congenic strain. Consistent with results found by other groups, copper was found to accumulate within the liver of LEC rats to levels more than 50 times those measured in LEA rats. In addition, liver selenium concentration in LEC rats was found to be around 50% of that in LEA rats. The enzyme activity, and RNA for the selenium dependant enzyme, glutathione peroxidase, was also found to be reduced in LEC rat liver. These results indicate that hepatic selenium in the LEC rat is depleted and that, as a result of this, the capacity to protect cells from copper-induced free-radical damage is reduced.
Septic shock is an increasingly important clinical condition, characterized by systemic hypotensi... more Septic shock is an increasingly important clinical condition, characterized by systemic hypotension, ischemia, and ultimately organ failure. In Gram negative infection, the bacterial cell wall component, lipopolysaccharide (endotoxin, LPS), has been strongly linked to the pathophysiological responses that result in septic shock. LPS is bound in plasma to a protein called LPS-binding protein (LBP), which facilitates the binding of LPS to a cell surface receptor, CD14. Binding to CD14 stimulates cell signaling mechanisms that result in the production of inflammatory cytokines. However, the events which follow LPS binding to CD14 and which lead to the production of cytokines remain unclear. It has recently become evident that a number of phosphorylation cascades including MAP kinase pathways and NF-kappaB activation pathway are initiated by exposure of cells to LPS. These cascades act at both the transcriptional and translational levels to regulate cytokine production. This review will focus on the signaling pathways that are initiated by LPS and the cellular effects of the signaling pathways.
Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor n... more Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.
This randomised, open label, phase I, immunotherapeutic study investigated the effects of interle... more This randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. Twelve HIV-1(+) patients on suppressive cART with baseline CD4 T-cell counts >400 cells/mm(3) blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n=3); (2) vaccine alone (n=4); or (3) IL-2, GM-CSF and rhGH (n=5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. Median ba...
T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell ... more T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell proliferation, cytokine production and differentiation into effector cells. These cascades comprise an array of protein-tyrosine kinases, phosphatases, GTP-binding proteins and adaptor proteins that regulate generic and specialised functions. The integration of these signals is essential for the normal development, homeostasis and function of T cells. Defects in a single mediator can produce T cells that are unable to participate fully in an immune response and/or that mount an inappropriate response, which leads to immunodeficiency, autoimmunity or leukaemia/lymphomas. This review highlights some of the key players in T-cell signalling and their involvement in the development of various clinical disease states. Some of these immune-specific signalling proteins are attractive potential targets in the development of therapies to augment T-cell responses to antigen or tumours, and to treat immune cell disorders.
Successful antiretroviral therapy (ART) suppresses plasma HIV-1 RNA below detection limits, reduc... more Successful antiretroviral therapy (ART) suppresses plasma HIV-1 RNA below detection limits, reducing the chronic insult to the immune systems of infected individuals and supporting a degree of immunological recovery. However, the surface phenotypic profile of T cells in ART-treated patients does not resemble that of healthy, uninfected individuals, but rather shows upregulation of proteins associated with an exhausted immune system. We sought to address whether aviraemic HIV-1 infection, therefore, contributed to long-term alterations in intracellular signalling events within the T cells of infected individuals that contributed to the exhausted phenotype. A flow cytometric approach was employed to assess levels of phosphorylation within T-cell signalling proteins in ART-treated HIV-1-positive patients and HIV-negative individuals. The relative phosphorylation levels of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38, zeta-chain-associated protein kinase 70 (ZAP70), linker of activated T cells, SLP76, nuclear factor kappaB were measured within resting and stimulated CD4(+) and CD8(+) T cells from aviraemic HIV-1-positive and healthy individuals by intracellular staining and flow cytometric analysis. Basal levels of phospho-ZAP70, phospho-ERK and phospho-JNK were two-fold to three-fold higher in HIV-1-positive individuals compared with healthy controls, with phospho-p38 also showing a tendency to increase in HIV-1-positive individuals. Interestingly, in contrast to healthy controls, peripheral blood mononuclear cells from aviraemic, infected individuals were refractory to stimulation with IL-2 and CD3/CD28 showing no enhancement of phosphorylation. CD4(+) and CD8(+) T cells from HIV-1-positive individuals are poorly responsive to direct stimulation through the T-cell receptor due to chronically raised basal activation levels of intracellular signalling molecules.
... Full Text. HIV Therapy. January 2010, Vol. 4, No. 1, Pages 83-99 , DOI 10.2217/hiv.09.51. (do... more ... Full Text. HIV Therapy. January 2010, Vol. 4, No. 1, Pages 83-99 , DOI 10.2217/hiv.09.51. (doi:10.2217/hiv.09.51). T-cell dysfunction in HIV-1 infection: targeting the inhibitors. Jocelyn S Downey & Nesrina Imami Author for correspondence. Sections: ...
CTLA-4 is a co-receptor that plays a pivotal role in regulating the threshold for T-cell activati... more CTLA-4 is a co-receptor that plays a pivotal role in regulating the threshold for T-cell activation. We recently reported that CTLA-4 ligation can over-ride the stop-signal induced by anti-CD3 ligation [Schneider H, Downey J, Smith A, Zinselmeyer BH, Rush C, Brewer JM, et al. Reversal of the TCR stop-signal by CTLA-4. Science 2006;313:1972]. While these studies compared CTLA-4 positive and negative T-cells from normal mice, little is known regarding the behaviour of T-cells from diseased Ctla4 deficient mice with auto-proliferative disease. In this study, we show that while activated wild-type and Ctla-4−/− T-cells have similar rates of motility, Ctla-4−/− T-cells show a marked resistance to the induction of a stop-signal by anti-CD3 ligation. By contrast, T-cells from normal mice and CD28 deficient mice underwent a normal slowing of motility in response to anti-CD3 ligation. Our findings identify a fundamental difference between normal versus CTLA-4−/− T-cells from diseased mice in the regulation of motility by anti-CD3 ligation. This dysregulation of motility may contribute to the tissue infiltration and the autoimmune disorder observed in Ctla-4−/− mice.
... MAP kinase pathways Wei Zhu, Jocelyn S. Downey, Yingqiu Li, Jiahuai Han Department of Immunol... more ... MAP kinase pathways Wei Zhu, Jocelyn S. Downey, Yingqiu Li, Jiahuai Han Department of Immunology, The Scripps Research Institute, La Jolla, California, USA ... Nature 1994; 372: 739. 13. Hambleton J, McMahon M, DeFranco AL. ...
Biochemical and Biophysical Research Communications, Jan 1, 1998
The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC... more The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC rats, which develop a spontaneous form of hepatitis at 3-4 months of age, and compared to trace metal concentrations in the LEA rat, its asymptomatic congenic strain. Consistent with results found by other groups, copper was found to accumulate within the liver of LEC rats to levels more than 50 times those measured in LEA rats. In addition, liver selenium concentration in LEC rats was found to be around 50% of that in LEA rats. The enzyme activity, and RNA for the selenium dependant enzyme, glutathione peroxidase, was also found to be reduced in LEC rat liver. These results indicate that hepatic selenium in the LEC rat is depleted and that, as a result of this, the capacity to protect cells from copper-induced free-radical damage is reduced.
Septic shock is an increasingly important clinical condition, characterized by systemic hypotensi... more Septic shock is an increasingly important clinical condition, characterized by systemic hypotension, ischemia, and ultimately organ failure. In Gram negative infection, the bacterial cell wall component, lipopolysaccharide (endotoxin, LPS), has been strongly linked to the pathophysiological responses that result in septic shock. LPS is bound in plasma to a protein called LPS-binding protein (LBP), which facilitates the binding of LPS to a cell surface receptor, CD14. Binding to CD14 stimulates cell signaling mechanisms that result in the production of inflammatory cytokines. However, the events which follow LPS binding to CD14 and which lead to the production of cytokines remain unclear. It has recently become evident that a number of phosphorylation cascades including MAP kinase pathways and NF-kappaB activation pathway are initiated by exposure of cells to LPS. These cascades act at both the transcriptional and translational levels to regulate cytokine production. This review will focus on the signaling pathways that are initiated by LPS and the cellular effects of the signaling pathways.
Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor n... more Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.
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