Tim Hewson - Academia.edu (original) (raw)

Papers by Tim Hewson

American Journal of Respiratory and Critical Care Medicine, 2000

The Journal of Immunology, 2001

Immunology and Cell Biology, 1999

Human immunodeficiency virus (HIV) infects about 40 million people worldwide. HIV is the causativ... more Human immunodeficiency virus (HIV) infects about 40 million people worldwide. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). AIDS is characterised by a progressive decline in protective immunity that leads to opportunistic infection and eventually death. Although HIV-1 causes a decline in CD4' T-cell number and this undoubtedly contributes to the general immune deficit of AIDS, CD4+ T-cell loss does not completely explain the pathogenesis of AIDS. Death and anergy of uninfected T-cells is observed in AIDS, as are deficits in innate and specific immunity. Antigen presenting cells (APCs, including macrophages and dendritic cells, DCs, which are infectable by M-tropic strains of HIV-1 via the CCR-5 chemokine receptor) play a key role in orchestrating innate and adaptive immune responses and controlling T-cell activities including activation, anergy, deletion, tolerisation and memory by the provision of appropriate signals. APC dysregulation results in deficits of innate and adaptive immune responses. It is known that HIV-1 can cause APC dysregulation; this thesis examines some mechanisms by which this might occur. The HIV-1 envelope glycoprotein gpl20 mediates HIV-1 infection by binding to target cells via CD4 and CCR-5 and is focussed on throughout this work. Because gpl20 is found on the surface of HIV-1 and dissolved in the serum of HIV-1 infected patients, it has the ability to disrupt the function of both infected and uninfected APCs. Data in this thesis demonstrate that gp 120 causes a decline of cell-surface CD4 from human macrophages in vitro. A mechanism for this loss is proposed based on observations that it is significantly more substantial when CCR-5-binding gpl20, derived from M-tropic HIV-1 is used as opposed to CXCR-4-binding gpl20. CD4 loss is absent from macrophages that fail to express surface CCR-5 due to homozygosity for the naturally occurring ccr5A32 mutation. It appears that CD4 loss by this novel CCR-5-dependent mechanism requires cross-linking of CCR-5, CD4 and gp 120 at the cell surface leading to receptor-mediated endocytosis of this protein complex. Confocal microscopy was used to visualise these endocytosed proteins inside macrophages and RT-PCR was used to investigate transcriptional regulation of CD4 and CCR-5 recovery. Endocytosis of the protein complex may change antigen presentation efficiencies. Possible implications for protective-and auto¬ immunity are discussed. This thesis also presents evidence that pre-treatment with gp!20 leads to reduction in an APC's ability to stimulate antigen-specific proliferation of a T-cell line. Because this effect is not dependent on the tropism of the HIV-1 strain from which the gpl20 is derived, an alternative mechanism to CD4-loss was sought. The hypothesis that APC dysfunction is due to HIV-1 subversion of physiological mechanisms involving prostaglandin and the Notch signalling pathway, leading to inappropriate tolerance induction, was examined. Treatment of macrophages and DCs with gpl20 caused the transcriptional up-regulation of genes involved in the Notch pathway including Notch ligands, the presence of which on an APC has previously been shown to abrogate T-cell activation by the induction of an anergic phenotype. Preventing HIV-1 infection of APCs and the subsequent dysregulation of immune responses is a therapeutic goal. Branched, synthetic peptides based on discontinuous epitopes of gpl20 and previously demonstrated to disrupt binding to CD4 and CCR-5 are shown to protect macrophages from infection with M-tropic HIV-1Bal-Possible refinements to peptide structure and their utility as anti-HIV-1 therapeutics or vaccines are discussed. 2 DECLARATION I hereby declare that the work presented in this thesis is my own, except where stated in the text. The work has not been submitted in any previous application for a degree.

American Journal of Respiratory and Critical Care Medicine, 2000

The Journal of Immunology, 2001

Immunology and Cell Biology, 1999

Human immunodeficiency virus (HIV) infects about 40 million people worldwide. HIV is the causativ... more Human immunodeficiency virus (HIV) infects about 40 million people worldwide. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). AIDS is characterised by a progressive decline in protective immunity that leads to opportunistic infection and eventually death. Although HIV-1 causes a decline in CD4' T-cell number and this undoubtedly contributes to the general immune deficit of AIDS, CD4+ T-cell loss does not completely explain the pathogenesis of AIDS. Death and anergy of uninfected T-cells is observed in AIDS, as are deficits in innate and specific immunity. Antigen presenting cells (APCs, including macrophages and dendritic cells, DCs, which are infectable by M-tropic strains of HIV-1 via the CCR-5 chemokine receptor) play a key role in orchestrating innate and adaptive immune responses and controlling T-cell activities including activation, anergy, deletion, tolerisation and memory by the provision of appropriate signals. APC dysregulation results in deficits of innate and adaptive immune responses. It is known that HIV-1 can cause APC dysregulation; this thesis examines some mechanisms by which this might occur. The HIV-1 envelope glycoprotein gpl20 mediates HIV-1 infection by binding to target cells via CD4 and CCR-5 and is focussed on throughout this work. Because gpl20 is found on the surface of HIV-1 and dissolved in the serum of HIV-1 infected patients, it has the ability to disrupt the function of both infected and uninfected APCs. Data in this thesis demonstrate that gp 120 causes a decline of cell-surface CD4 from human macrophages in vitro. A mechanism for this loss is proposed based on observations that it is significantly more substantial when CCR-5-binding gpl20, derived from M-tropic HIV-1 is used as opposed to CXCR-4-binding gpl20. CD4 loss is absent from macrophages that fail to express surface CCR-5 due to homozygosity for the naturally occurring ccr5A32 mutation. It appears that CD4 loss by this novel CCR-5-dependent mechanism requires cross-linking of CCR-5, CD4 and gp 120 at the cell surface leading to receptor-mediated endocytosis of this protein complex. Confocal microscopy was used to visualise these endocytosed proteins inside macrophages and RT-PCR was used to investigate transcriptional regulation of CD4 and CCR-5 recovery. Endocytosis of the protein complex may change antigen presentation efficiencies. Possible implications for protective-and auto¬ immunity are discussed. This thesis also presents evidence that pre-treatment with gp!20 leads to reduction in an APC's ability to stimulate antigen-specific proliferation of a T-cell line. Because this effect is not dependent on the tropism of the HIV-1 strain from which the gpl20 is derived, an alternative mechanism to CD4-loss was sought. The hypothesis that APC dysfunction is due to HIV-1 subversion of physiological mechanisms involving prostaglandin and the Notch signalling pathway, leading to inappropriate tolerance induction, was examined. Treatment of macrophages and DCs with gpl20 caused the transcriptional up-regulation of genes involved in the Notch pathway including Notch ligands, the presence of which on an APC has previously been shown to abrogate T-cell activation by the induction of an anergic phenotype. Preventing HIV-1 infection of APCs and the subsequent dysregulation of immune responses is a therapeutic goal. Branched, synthetic peptides based on discontinuous epitopes of gpl20 and previously demonstrated to disrupt binding to CD4 and CCR-5 are shown to protect macrophages from infection with M-tropic HIV-1Bal-Possible refinements to peptide structure and their utility as anti-HIV-1 therapeutics or vaccines are discussed. 2 DECLARATION I hereby declare that the work presented in this thesis is my own, except where stated in the text. The work has not been submitted in any previous application for a degree.