A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian ... more A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian species is described. This expedites the generation of recombinant proteins and cell lines that can provide alternatives to animal experiments. This was achieved by the collection of RNA from a comprehensive range of tissues from a variety of species, and the optimisation of cDNA synthesis. This "zooplate" has been successfully used for the simultaneous amplification and cloning of drug targets from multiple species. These products have subsequently been used to develop in vitro assays that support efficacy and safety studies in new drug discovery programmes. Within the framework of the Three Rs, these reagents can reduce the number of animals required to provide material for ex vivo assays and can refine the in vivo studies that are still necessary.
During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcope... more During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis. Satellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56(+) cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised. Large-scale isolation of CD56(+) cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers. We have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.
Objective: Interleukin-1b (IL-1b) plays a key role in the pathogenesis of chronic joint diseases,... more Objective: Interleukin-1b (IL-1b) plays a key role in the pathogenesis of chronic joint diseases, including osteoarthritis (OA), and drives a cascade of inflammatory and destructive responses within the synovial joint. Animal models of arthritis support the role of IL-1b in joint pathology, however, the molecular changes downstream of IL-1b are poorly understood in vivo. This study aimed to evaluate the intra-articular (i.a.) injection of IL-1b in the rat joint as an acute model of joint disease and associate gene and mediator expression with clinical endpoints and pathological changes.
The polymerase chain reaction was used to screen human peripheral blood mononuclear cells (PBMC) ... more The polymerase chain reaction was used to screen human peripheral blood mononuclear cells (PBMC) and Jurkat cells for the presence of GABAA receptor subunit mRNAs. Positive signals were detected for the alpha1, alpha3, beta2, beta3, delta and epsilon subunit mRNAs in both cell populations, with the Jurkat cells giving a positive signal for some additional species. Real-time PCR was used to confirm that PBMC, lymphocytes and monocytes contained significant levels of the alpha1 subunit mRNA and that PBMC and lymphocytes contained low levels of beta2 mRNA. The alpha1 subunit was detected in PBMC and fractionated T-cell populations, as well as Jurkat and HL-60 cell lines, by Western blotting and immunofluorescence using a specific antibody. The application of 1mM GABA reduced the specific increase in intracellular PBMC Ca2+ levels produced by addition of 1 nM fMLP: this effect was mimicked by muscimol, but not glycine, and was blocked by bicuculline. The inhibitory effect of GABA was limited to a subset of PBMC. We conclude that cells within the human PBMC population, including lymphocytes, express functional GABAA receptors and these receptors may modulate immune responses.
Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary... more Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). In this setting they are routinely exposed to cigarette smoke and a range of pathogens including bacteria and viruses. The gene expression changes that result from these challenges may contribute to the initiation and progression of the disease. Understanding such changes is therefore of great interest and could aid the discovery of novel therapeutics. To study this, we stimulated monocyte-derived macrophages (MDM) from smokers and non-smokers with either cigarette smoke extract (CSE) or bacterially derived lipopolysaccharide (LPS) and profiled global transcriptional changes using Affymetrix arrays. LPS and CSE stimulation elicited markedly different transcriptome profiles with the former agent producing a larger number of significant changes. The CSE evoked changes showed some overlap with those observed when comparing habitual smokers with non-smokers, although the latter changes were generally of a more subtle nature. Detailed pathway analyses indicated that a number of genes involved in host defence were regulated following CSE stimulation and in MDM from smokers. In particular the interferon gamma (IFNgamma)-signalling pathway was significantly down-regulated following CSE stimulation, a finding that was confirmed by RT-PCR analysis. Furthermore, these changes were associated with suppressed release of the IFNgamma-induced chemokines, CXCL10 and CXCL9 from CSE treated MDM. In summary, our data provides evidence that smoking alters key mechanisms of host defence in macrophages. Such changes may explain the increased susceptibility of COPD patients to the lung infections that are associated with exacerbations of this disease.
Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully ... more Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully with fumaric acid esters (FAE). Beneficial effects of this medication coincided with decreased production of IFN-+ . Since dendritic cells (DC) regulate the differentiation of T helper (Th) cells, this study focussed on effects of monomethylfumarate (MMF, bioactive metabolite of FAE) on polarization of monocyte-derived DC. MMF-incubated, lipopolysaccharide-stimulated DC (MMF-DC) produced dramatically (p X 0.05) reduced levels of IL-12p70 and IL-10 (8±4% and 20±4%, respectively) compared to control DC. MMF-DC were mature. MMF affected polarization of DC irrespective of polarization factor(s) and ligands for the various Toll-like receptors used. Coculture of MMF-DC with naive and primed allogenous Th cells resulted in lymphocytes producing less IFN-+ , i.e. 59% and 54% of that by the respective Th cells cocultured with control DC. IL-4 production by primed, but not naive Th cells cocultured with MMF-DC was decreased as compared to cocultures with control DC. IL-10 production by naive and primed Th cells cocultured with MMF-DC and control DC did not differ. In addition, MMF inhibited LPS-induced NF-‹ B activation in DC. Together, beneficial effects of FAE in psoriasis involve modulation of DC polarization by MMF such that these cells down-regulate IFN-+ production by Th cells.
Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selecti... more Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selective agonists of human peroxisome proliferator-activated receptor δ (PPARδ. Compound 18 demonstrated efficacy in a biomarker for increased fatty acid oxidation, with upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) in human primary myotubes.
Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivativ... more Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB(VIN), and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI(50) values of 0.19-0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC(50) 1.4-1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.
Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages... more Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs. MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients. In MDMs, PGE(2) potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-alpha release (p[A](50) 8.51+/-0.11, maximum inhibition 95.9+/-4.8%). In human AMs, PGE(2) also inhibited TNF-alpha release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE(2) curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP(4)-receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE(2) in both AMs and MDMs were mediated by activation of EP(4) and EP(2) receptors. These data indicate that both E-prostanoid(4) and E-prostanoid(2) selective agonists may have anti-inflammatory properties in lung diseases where macrophages play a role.
A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian ... more A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian species is described. This expedites the generation of recombinant proteins and cell lines that can provide alternatives to animal experiments. This was achieved by the collection of RNA from a comprehensive range of tissues from a variety of species, and the optimisation of cDNA synthesis. This "zooplate" has been successfully used for the simultaneous amplification and cloning of drug targets from multiple species. These products have subsequently been used to develop in vitro assays that support efficacy and safety studies in new drug discovery programmes. Within the framework of the Three Rs, these reagents can reduce the number of animals required to provide material for ex vivo assays and can refine the in vivo studies that are still necessary.
During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcope... more During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis. Satellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56(+) cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised. Large-scale isolation of CD56(+) cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers. We have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.
Objective: Interleukin-1b (IL-1b) plays a key role in the pathogenesis of chronic joint diseases,... more Objective: Interleukin-1b (IL-1b) plays a key role in the pathogenesis of chronic joint diseases, including osteoarthritis (OA), and drives a cascade of inflammatory and destructive responses within the synovial joint. Animal models of arthritis support the role of IL-1b in joint pathology, however, the molecular changes downstream of IL-1b are poorly understood in vivo. This study aimed to evaluate the intra-articular (i.a.) injection of IL-1b in the rat joint as an acute model of joint disease and associate gene and mediator expression with clinical endpoints and pathological changes.
The polymerase chain reaction was used to screen human peripheral blood mononuclear cells (PBMC) ... more The polymerase chain reaction was used to screen human peripheral blood mononuclear cells (PBMC) and Jurkat cells for the presence of GABAA receptor subunit mRNAs. Positive signals were detected for the alpha1, alpha3, beta2, beta3, delta and epsilon subunit mRNAs in both cell populations, with the Jurkat cells giving a positive signal for some additional species. Real-time PCR was used to confirm that PBMC, lymphocytes and monocytes contained significant levels of the alpha1 subunit mRNA and that PBMC and lymphocytes contained low levels of beta2 mRNA. The alpha1 subunit was detected in PBMC and fractionated T-cell populations, as well as Jurkat and HL-60 cell lines, by Western blotting and immunofluorescence using a specific antibody. The application of 1mM GABA reduced the specific increase in intracellular PBMC Ca2+ levels produced by addition of 1 nM fMLP: this effect was mimicked by muscimol, but not glycine, and was blocked by bicuculline. The inhibitory effect of GABA was limited to a subset of PBMC. We conclude that cells within the human PBMC population, including lymphocytes, express functional GABAA receptors and these receptors may modulate immune responses.
Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary... more Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). In this setting they are routinely exposed to cigarette smoke and a range of pathogens including bacteria and viruses. The gene expression changes that result from these challenges may contribute to the initiation and progression of the disease. Understanding such changes is therefore of great interest and could aid the discovery of novel therapeutics. To study this, we stimulated monocyte-derived macrophages (MDM) from smokers and non-smokers with either cigarette smoke extract (CSE) or bacterially derived lipopolysaccharide (LPS) and profiled global transcriptional changes using Affymetrix arrays. LPS and CSE stimulation elicited markedly different transcriptome profiles with the former agent producing a larger number of significant changes. The CSE evoked changes showed some overlap with those observed when comparing habitual smokers with non-smokers, although the latter changes were generally of a more subtle nature. Detailed pathway analyses indicated that a number of genes involved in host defence were regulated following CSE stimulation and in MDM from smokers. In particular the interferon gamma (IFNgamma)-signalling pathway was significantly down-regulated following CSE stimulation, a finding that was confirmed by RT-PCR analysis. Furthermore, these changes were associated with suppressed release of the IFNgamma-induced chemokines, CXCL10 and CXCL9 from CSE treated MDM. In summary, our data provides evidence that smoking alters key mechanisms of host defence in macrophages. Such changes may explain the increased susceptibility of COPD patients to the lung infections that are associated with exacerbations of this disease.
Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully ... more Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully with fumaric acid esters (FAE). Beneficial effects of this medication coincided with decreased production of IFN-+ . Since dendritic cells (DC) regulate the differentiation of T helper (Th) cells, this study focussed on effects of monomethylfumarate (MMF, bioactive metabolite of FAE) on polarization of monocyte-derived DC. MMF-incubated, lipopolysaccharide-stimulated DC (MMF-DC) produced dramatically (p X 0.05) reduced levels of IL-12p70 and IL-10 (8±4% and 20±4%, respectively) compared to control DC. MMF-DC were mature. MMF affected polarization of DC irrespective of polarization factor(s) and ligands for the various Toll-like receptors used. Coculture of MMF-DC with naive and primed allogenous Th cells resulted in lymphocytes producing less IFN-+ , i.e. 59% and 54% of that by the respective Th cells cocultured with control DC. IL-4 production by primed, but not naive Th cells cocultured with MMF-DC was decreased as compared to cocultures with control DC. IL-10 production by naive and primed Th cells cocultured with MMF-DC and control DC did not differ. In addition, MMF inhibited LPS-induced NF-‹ B activation in DC. Together, beneficial effects of FAE in psoriasis involve modulation of DC polarization by MMF such that these cells down-regulate IFN-+ production by Th cells.
Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selecti... more Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selective agonists of human peroxisome proliferator-activated receptor δ (PPARδ. Compound 18 demonstrated efficacy in a biomarker for increased fatty acid oxidation, with upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) in human primary myotubes.
Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivativ... more Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB(VIN), and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI(50) values of 0.19-0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC(50) 1.4-1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.
Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages... more Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs. MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients. In MDMs, PGE(2) potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-alpha release (p[A](50) 8.51+/-0.11, maximum inhibition 95.9+/-4.8%). In human AMs, PGE(2) also inhibited TNF-alpha release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE(2) curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP(4)-receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE(2) in both AMs and MDMs were mediated by activation of EP(4) and EP(2) receptors. These data indicate that both E-prostanoid(4) and E-prostanoid(2) selective agonists may have anti-inflammatory properties in lung diseases where macrophages play a role.
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Papers by Andrew Walding