Graham P Cook

University of Leeds, Leeds, ENG, United Kingdom

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Publications (30)133.65 Total impact

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    ABSTRACT: Deregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated.
    BMC Cancer 10/2014; 14(1):754. · 3.33 Impact Factor
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    ABSTRACT: Rheumatoid arthritis (RA) is a polygenic disorder usually arising from combined genetic predisposition and environmental influences with associated dysfunctional immune responses. The X-box binding protein 1 (XBP1) transcription factor is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes including RA. XBP1 can also be activated in direct response to Toll-like Receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Our aim was to investigate the relevance of interactions between UPR and TLR signalling pathways in the serum and synovial fibroblasts (SFs) of patients with RA, using samples from healthy individuals and patients with osteoarthritis (OA) as controls Peripheral blood mononuclear cells (PBMC) were obtained from control groups: healthy individuals (n = 24) and RA patients, comprising active disease (n = 47) and remission (n = 12). SFs from RA and OA patients were isolated by digestion of synovial biopsies. Gene expression profiling was performed using qPCR for the detection of sXBP1 and enzyme-linked immunosorbent assays (ELISA) to quantify levels of pro-inflammatory cytokines, IL-6 and TNF. siRNA targeting of XBP1 was used for knockdown experiments in SFs. We investigated the expression of ER stress response genes in patients with active RA and patients in remission. We show that TLR-dependent XBP1 activation is operative in the SFs of patients with active RA. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission (p = 0.01). Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SFs by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent IL-6 and TNF production. We also show that TNF itself induces sXBP1 in SFs, thus generating a potential feedback loop for sustained activation of these cells. sXBP1 plays a central role in two different cellular processes that may first appear unconnected. However, linking inflammatory pathways with ER stress provides SFs with a timely, coordinated and protective response. XBP1 activation may therefore be a suitable target in the treatment of RA, since it forms a cornerstone of two different molecular processes implicated in the pathogenesis of RA.
    Annals of the rheumatic diseases 03/2014; 73 Suppl 1:A22. · 8.11 Impact Factor
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    ABSTRACT: Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.
    Molecular Immunology 12/2013; 58(2):160-168. · 2.65 Impact Factor
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    ABSTRACT: In paroxysmal nocturnal hemoglobinuria (PNH), hematopoietic cells lacking glycosylphosphatidylinositol (GPI)-linked proteins on their surface (GPI(neg)) exist alongside normal (GPI+) cells. Analysis of NK cell subsets in forty-seven PNH patients revealed that the ratio of CD56(bright):CD56(dim) NK cells differed in the GPI+ and GPI(neg) populations, with GPI(neg)CD56(bright) NK cells significantly more abundant in peripheral blood than their normal GPI+ counterparts. Indeed, GPI+CD56(bright) NK cells were not detected in the peripheral blood of some patients, suggesting their trafficking to a niche unavailable to the GPI(neg)CD56(bright) NK cell population. Defective cellular trafficking in this disease was supported by findings showing differential chemokine receptor expression between GPI+ and GPI(neg) NK cells and impaired SDF-1 induced chemotaxis of GPI(neg) NK cells. Our results indicate a role for GPI-linked proteins in NK cell subset homeostasis and suggest that differential chemokine responses might contribute to the balance of GPI+ and GPI(neg) populations in this disease.
    Blood 07/2013; · 9.78 Impact Factor
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    ABSTRACT: BACKGROUND: The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). METHODS: Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. RESULTS: At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. CONCLUSIONS: This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.
    Annals of the rheumatic diseases 05/2013; · 8.11 Impact Factor
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    ABSTRACT: The rodent papillomaviruses (PVs) have great potential to be utilized as a model for PV infection and oncogenesis since mice and rats have well-studied genetics and similar physiology to humans, as well as the ready availability of many reagents for the study of host cell biology and immunology. Rodent PVs have been isolated and their genomes have been sequenced, however the role of the rodent PV oncoproteins in cell transformation is still relatively unknown. We have cloned and expressed the E6 and E7 open reading frames (ORFs) of Micromys minutus papillomavirus 1 (MmiPV1), introducing epitope tags to enable the identification of expressed proteins. Initial results showed that both E6 and E7 were located in the nucleus and the cytoplasm of transiently-transfected established mouse BALB/c 3T3 cells and A549 cells. In contrast, E6 and E7 of HPV16 were localized predominantly in the nucleus. Preliminary observations on the transforming ability of individual MmiPV1 E6 and E7 ORFs suggested that neither protein was able to morphologically or functionally transform established BALB/c 3T3 or primary baby rat kidney (BRK) cells. Current investigations are focused on possible cooperation of both proteins and the involvement of cellular oncogenes (e.g. activated ras) in transformation of established and primary cells.
    Faculty of Biological Sciences Postgraduate Symposium 2013; 04/2013
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    ABSTRACT: X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.
    Journal of Autoimmunity 01/2013; · 8.15 Impact Factor
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    ABSTRACT: Reovirus is a promising oncolytic virus, acting by both direct and immune-mediated mechanisms, although its potential may be limited by inactivation following systemic delivery. This study addressed whether systemically delivered reovirus might be shielded from neutralising antibodies by cell carriage and whether virus-loaded blood or hepatic innate immune effector cells become activated to kill colorectal cancer cells metastatic to the liver in human systems. We found that reovirus was directly cytotoxic against tumour cells but not against fresh hepatocytes. Whilst direct tumour cell killing by neat virus was significantly reduced in the presence of neutralising serum, reovirus was protected when loaded onto peripheral blood mononuclear cells, which may carry virus following intravenous administration in patients. As well as handing off virus for direct oncolytic killing, natural killer cells within reovirus-treated blood mononuclear cells were stimulated to kill tumour targets, but not normal hepatocytes, in a type I interferon-dependent manner. Similarly, natural killer cells within liver mononuclear cells became selectively cytotoxic towards tumour cells when activated by reovirus. Hence, intravenous reovirus may evade neutralisation by serum via binding to circulating mononuclear cells and this blood cell carriage has the potential to instigate both direct and innate immune-mediated therapy against human colorectal or other cancers metastatic to the liver. © 2012 Wiley Periodicals, Inc.
    International Journal of Cancer 11/2012; · 6.20 Impact Factor
  • 51st Annual Scientific Meeting of the British-Society-for-Haematology; 04/2011
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    ABSTRACT: NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.
    The Journal of Immunology 02/2011; 186(3):1538-45. · 5.52 Impact Factor
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    ABSTRACT: Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the interleukin (IL)-15 induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. Interleukin-15 also promotes NK cell survival and IL-15 excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-15 mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity.
    PLoS ONE 01/2011; 6(9):e22842. · 3.53 Impact Factor
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    ABSTRACT: Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin-mediated delivery of pro-apoptotic serine proteases known as granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. A simple bioinformatics strategy identified a candidate granzyme B cleavage site in the widely expressed BNIP-2 (BCL2/adenovirus E1B-19K protein-interacting protein 2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK (natural killer) cell-mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full-length BNIP-2 or a truncated molecule mimicking the granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID (BH3-interacting domain death agonist) expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximizes the chances of immune-mediated clearance of infected cells or tumour cells.
    Biochemical Journal 11/2010; 431(3):423-31. · 4.65 Impact Factor
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    ABSTRACT: A role for NLRP3 inflammasome in recurrent and chronic inflammation was initially described in a group of rare autoinflammatory conditions, termed cryopyrin-associated periodic syndrome. Subsequently, inflammasomes have been implicated in the pathology of many common diseases, including cancer, gout and diabetes. Despite diverse pathologies, the central role of the inflammasome in innate defences and tumour elimination suggests common therapeutic approaches to reduce inflammation where appropriate.
    European Journal of Immunology 03/2010; 40(3):631-4. · 4.97 Impact Factor
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    ABSTRACT: Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.
    Mobile DNA. 03/2010; 1(1):9.
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    ABSTRACT: NK cells induce apoptosis in target cells via the perforin-mediated delivery of granzyme molecules. Cytotoxic human NK cells can be generated by IL-15-mediated differentiation of CD34(+) cells in vitro and these cultures have been used extensively to analyze the development of the NK cell surface phenotype. We have used NK cell differentiation in vitro together with protease-deficient human NK cells to analyze the acquisition of the cytotoxic phenotype. Granzymes are synthesized as inactive zymogens and are proteolytically activated by the cysteine protease cathepsin C. Cathepsin C is also synthesized as a zymogen and activated by proteolysis. We show that human NK cells generated in vitro undergo granule exocytosis and induce the caspase cascade in target cells. IL-15 and stem cell factor (IL-15 plus SCF) induced the expression of the granzyme B and perforin genes and the activation of cathepsin C and granzyme B zymogens. Perforin activation is also mediated by a cysteine protease and IL-15 plus SCF-mediated differentiation was accompanied by perforin processing. However, cathepsin C-deficient human NK cells revealed that perforin processing could occur in the absence of cathepsin C activity. The combination of IL-15 plus SCF is therefore sufficient to coordinate the development of the NK cell surface phenotype with the expression and proteolytic activation of the cytotoxic machinery, reflecting the central role of IL-15 in NK cell development.
    The Journal of Immunology 08/2009; 183(2):803-13. · 5.52 Impact Factor
  • Graham P Cook
    Dermatology 07/2009; 219(2):187-8. · 2.02 Impact Factor
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    G E Blair, G P Cook
    Oncogene 11/2008; 27(45):5868. · 8.56 Impact Factor
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    ABSTRACT: We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.
    Bone marrow transplantation 11/2008; 43(8):627-35. · 3.00 Impact Factor

Publication Stats

407 Citations
133.65 Total Impact Points

Institutions

  • 2005–2013
    • University of Leeds
      • • Division of Rheumatic and Musculoskeletal Disease
      • • Leeds Institute of Molecular Medicine (LIMM)
      • • School of Biochemistry and Microbiology
      Leeds, ENG, United Kingdom
  • 2010
    • Institute of Genetics and Molecular Medicine
      Edinburgh, Scotland, United Kingdom