22 results on '"Pizzey AR"'
Search Results
2. Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry
- Author
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Coffin, RS, Thomas, SK, Thomas, NSB, Lilley, CE, Pizzey, AR, Griffiths, CH, Gibb, BJ, Wagstaff, MJD, Inges, SJ, Binks, MH, Chain, BM, Thrasher, AJ, Rutault, K, and Latchman, DS
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- 1998
- Full Text
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3. The 'stomatin' gene and protein in overhydrated hereditary stomatocytosis
- Author
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FRICKE B, ARGENT AC, CHETTY MC, PIZZEY AR, TURNER EJ, HO MM, VON DURING M, STEWART G.W., IOLASCON, ACHILLE, Fricke, B, Argent, Ac, Chetty, Mc, Pizzey, Ar, Turner, Ej, Ho, Mm, Iolascon, Achille, VON DURING, M, and Stewart, G. W.
- Published
- 2003
4. Investigation of systemic inflammatory response in first trimester pregnancy failure.
- Author
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Calleja-Agius J, Jauniaux E, Pizzey AR, and Muttukrishna S
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- 2012
5. Pro- and antiinflammatory cytokines in threatened miscarriages.
- Author
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Calleja-Agius J, Muttukrishna S, Pizzey AR, and Jauniaux E
- Abstract
OBJECTIVE: The purpose of this study was to evaluate circulating and intracellular levels of Th1 and Th2 cytokines in women with threatened miscarriage (TM) and subsequent outcome. STUDY DESIGN: Plasma levels of tumor necrosis factor (TNF)-receptors 1 and 2, TNF[alpha], interferon gamma (IFN[gamma]), and interleukins (IL) -6 and -10 were measured by flow cytometric bead assays in 80 women with TM: 53 women with normal outcome and 27 women who miscarried. Fluorescent antibody labeling was also performed on whole blood in a subgroup of 27 women of TM: 16 women with normal outcome and 11 women who miscarried. RESULTS: Monocyte expression of TNF[alpha] and circulating levels of TNF[alpha], IFN[gamma], IL-10, IL-6, and TNF-R1 were significantly lower, whereas circulating levels of TNF[alpha]/IL-10, IFN[gamma]/IL-10, and TNF[alpha]/IL-6 ratios were significantly higher, in women with TM who subsequently miscarried, compared with the women with normal outcome. CONCLUSION: An increased Th1 type of immune response, which was similar to that observed in preterm delivery, was found in TM cases that were complicated by a subsequent miscarriage. [ABSTRACT FROM AUTHOR]
- Published
- 2011
6. The Presence of Two MyoD Genes in a Subset of Acanthopterygii Fish Is Associated with a Polyserine Insert in MyoD1.
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White LJ, Russell AJ, Pizzey AR, Dasmahapatra KK, and Pownall ME
- Abstract
The MyoD gene was duplicated during the teleost whole genome duplication and, while a second MyoD gene ( MyoD2 ) was subsequently lost from the genomes of some lineages (including zebrafish), many fish lineages (including Alcolapia species) have retained both MyoD paralogues. Here we reveal the expression patterns of the two MyoD genes in Oreochromis ( Alcolapia) alcalica using in situ hybridisation. We report our analysis of MyoD1 and MyoD2 protein sequences from 54 teleost species, and show that O. alcalica , along with some other teleosts, include a polyserine repeat between the amino terminal transactivation domains (TAD) and the cysteine-histidine rich region (H/C) in MyoD1. The evolutionary history of MyoD1 and MyoD2 is compared to the presence of this polyserine region using phylogenetics, and its functional relevance is tested using overexpression in a heterologous system to investigate subcellular localisation, stability, and activity of MyoD proteins that include and do not include the polyserine region.
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- 2023
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7. N1-Src Kinase Is Required for Primary Neurogenesis in Xenopus tropicalis .
- Author
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Lewis PA, Bradley IC, Pizzey AR, Isaacs HV, and Evans GJO
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- Animals, Cell Differentiation physiology, Enzyme Activation, Male, Protein Isoforms, Xenopus anatomy & histology, src-Family Kinases genetics, Neural Stem Cells cytology, Neural Stem Cells enzymology, Neurogenesis physiology, Neurons cytology, Neurons enzymology, Xenopus physiology, src-Family Kinases metabolism
- Abstract
The presence of the neuronal-specific N1-Src splice variant of the C-Src tyrosine kinase is conserved through vertebrate evolution, suggesting an important role in complex nervous systems. Alternative splicing involving an N1-Src -specific microexon leads to a 5 or 6 aa insertion into the SH3 domain of Src. A prevailing model suggests that N1-Src regulates neuronal differentiation via cytoskeletal dynamics in the growth cone. Here we investigated the role of n1-src in the early development of the amphibian Xenopus tropicalis , and found that n1-src expression is regulated in embryogenesis, with highest levels detected during the phases of primary and secondary neurogenesis. In situ hybridization analysis, using locked nucleic acid oligo probes complementary to the n1-src microexon, indicates that n1-src expression is highly enriched in the open neural plate during neurula stages and in the neural tissue of adult frogs. Given the n1-src expression pattern, we investigated a possible role for n1-src in neurogenesis. Using splice site-specific antisense morpholino oligos, we inhibited n1-src splicing, while preserving c-src expression. Differentiation of neurons in the primary nervous system is reduced in n1-src -knockdown embryos, accompanied by a severely impaired touch response in later development. These data reveal an essential role for n1-src in amphibian neural development and suggest that alternative splicing of C-Src in the developing vertebrate nervous system evolved to regulate neurogenesis. SIGNIFICANCE STATEMENT The Src family of nonreceptor tyrosine kinases acts in signaling pathways that regulate cell migration, cell adhesion, and proliferation. Srcs are also enriched in the brain, where they play key roles in neuronal development and neurotransmission. Vertebrates have evolved a neuron-specific splice variant of C-Src, N1-Src, which differs from C-Src by just 5 or 6 aa. N1-Src is poorly understood and its high similarity to C-Src has made it difficult to delineate its function. Using antisense knockdown of the n1-src microexon, we have studied neuronal development in the Xenopus embryo in the absence of n1-src , while preserving c-src Loss of n1-src causes a striking absence of primary neurogenesis, implicating n1-src in the specification of neurons early in neural development., (Copyright © 2017 Lewis, Bradley et al.)
- Published
- 2017
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8. Successful outcomes achieved in assisted reproduction cycles using sperm with high levels of high DNA stainability.
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Speyer BE, Pizzey AR, Abramov B, Saab W, Doshi A, Sarna U, Harper JC, and Serhal P
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- Adult, Female, Humans, Male, Pregnancy, Pregnancy Outcome, DNA analysis, Sperm Injections, Intracytoplasmic statistics & numerical data, Spermatozoa
- Abstract
The sperm chromatin structure assay (SCSA) has been proposed as a useful addition to the battery of tests routinely used to explore semen quality and hence to give an indication of the likelihood of a successful pregnancy. As usually performed at present, the assay yields two main sperm variables, the DNA fragmentation index (DFI) and the high DNA stainability (HDS). In the present study 275 patients undergoing 215 in vitro fertilization (IVF) and 215 intracytoplasmic sperm injection (ICSI) cycles were studied with the purpose of defining the clinical significance of HDS in IVF and ICSI cycles. Using the Spearman correlation test there were no significant statistical relationships between %HDS and fertilization rate, rate of embryo growth, blastocyst rate, implantation rate, or live birth rate. Rate of pregnancy loss showed a negative relationship significant at the 0.05 level which is unexplained. It is not known whether the normal practice of using processed sperm for fertilization plays any part in this lack of a negative effect of HDS level upon the stages of the cycle. A total of 16 patients with HDS levels >28% had an average live birth rate of 47.8% and an average pregnancy loss of 8.7%, which compared favourably with the group of patients as a whole.
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- 2015
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9. Fall in implantation rates following ICSI with sperm with high DNA fragmentation.
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Speyer BE, Pizzey AR, Ranieri M, Joshi R, Delhanty JD, and Serhal P
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- Adult, Female, Humans, Male, Pregnancy, Pregnancy Outcome, Pregnancy Rate, Spermatozoa ultrastructure, DNA Fragmentation, Embryo Implantation genetics, Sperm Injections, Intracytoplasmic, Spermatozoa physiology
- Abstract
Background: There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy., Methods: The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm., Results: The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients., Conclusions: These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.
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- 2010
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10. Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia.
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Gale RE, Hills R, Pizzey AR, Kottaridis PD, Swirsky D, Gilkes AF, Nugent E, Mills KI, Wheatley K, Solomon E, Burnett AK, Linch DC, and Grimwade D
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- Adolescent, Adult, Child, Child, Preschool, Female, Furans, Gene Expression Profiling, Humans, Infant, Leukemia, Promyelocytic, Acute mortality, Male, Middle Aged, Mutation, Oligonucleotide Array Sequence Analysis, Prognosis, Survival Analysis, Tretinoin pharmacology, Antineoplastic Agents pharmacology, Carbazoles pharmacology, Indoles pharmacology, Leukemia, Promyelocytic, Acute drug therapy, Leukemia, Promyelocytic, Acute genetics, fms-Like Tyrosine Kinase 3 genetics
- Abstract
The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD+D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 x 10(9)/L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P = .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P = .04, but no significant difference in relapse risk (28% vs 23%; P = .5) or overall survival (59% vs 67%; P = .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n = 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD+ cells, but this inhibition was reduced in the presence of ATRA. Furthermore, in the presence of CEP-701, ATRA-induced differentiation was reduced in FLT3/ITD+ cells. These data carry implications for the use of FLT3 inhibitors as frontline therapy for APL.
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- 2005
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11. Generation of potent antitumor CTL from patients with multiple myeloma directed against HM1.24.
- Author
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Rew SB, Peggs K, Sanjuan I, Pizzey AR, Koishihara Y, Kawai S, Kosaka M, Ozaki S, Chain B, and Yong KL
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- Antigens, CD, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Cell Line, Tumor, Cytotoxicity Tests, Immunologic, Cytotoxicity, Immunologic immunology, Dendritic Cells immunology, Dendritic Cells metabolism, Flow Cytometry methods, GPI-Linked Proteins, Humans, Immunophenotyping, Interferon-gamma metabolism, Leukocyte Common Antigens immunology, Membrane Glycoproteins genetics, Membrane Glycoproteins physiology, Multiple Myeloma pathology, Perforin, Plasma Cells immunology, Pore Forming Cytotoxic Proteins, Signal Transduction immunology, T-Lymphocytes immunology, T-Lymphocytes metabolism, Transfection, Membrane Glycoproteins immunology, Multiple Myeloma immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Purpose: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma., Experimental Design: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays., Results: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-gamma ELISPOT assay (median, 342; range, 198-495 IFN-gamma-producing cells/10(5) cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti-MHC class I but not anti-MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid-transfected dendritic cells had no effect on lysis of autologous MPC., Conclusion: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.
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- 2005
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12. Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model.
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Ahmed F, Ings SJ, Pizzey AR, Blundell MP, Thrasher AJ, Ye HT, Fahey A, Linch DC, and Yong KL
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- Animals, Antigens, CD34 analysis, Bone Marrow Cells chemistry, Cell Cycle immunology, Cell Movement drug effects, Cells, Cultured, Chemokine CXCL12, Chemokines, CXC pharmacology, Culture Media pharmacology, Cytokines physiology, Fas Ligand Protein, Flow Cytometry, Hematopoietic Stem Cells chemistry, Humans, Membrane Glycoproteins physiology, Mice, Mice, Inbred NOD, Mice, SCID, Bone Marrow Cells cytology, Cell Movement immunology, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Severe Combined Immunodeficiency pathology
- Abstract
The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.
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- 2004
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13. The upregulation of CC chemokine receptor 7 and the increased migration of maturing dendritic cells to macrophage inflammatory protein 3beta and secondary lymphoid chemokine is mediated by the p38 stress-activated protein kinase pathway.
- Author
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Ardeshna KM, Pizzey AR, Walker SJ, Devereux S, and Khwaja A
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- Butadienes pharmacology, Cellular Senescence drug effects, Chemokine CCL20, Chemokine CCL21, Chemotaxis, Leukocyte drug effects, Chromones pharmacology, Enzyme Inhibitors pharmacology, Humans, Imidazoles pharmacology, Lipopolysaccharides pharmacology, Monocytes physiology, Morpholines pharmacology, Nitriles pharmacology, Pyridines pharmacology, Receptors, CCR6, Receptors, CCR7, Signal Transduction, Up-Regulation, p38 Mitogen-Activated Protein Kinases, Angiogenesis Inhibitors physiology, Chemokines, CC physiology, Dendritic Cells physiology, Macrophage Inflammatory Proteins physiology, Mitogen-Activated Protein Kinases antagonists & inhibitors, Receptors, Chemokine metabolism
- Abstract
Using the p38 stress-activated protein kinase (p38SAPK) inhibitor, SB203580, increased responsiveness of monocyte-derived dendritic cells (MoDCs) to secondary lymphoid chemokine (SLC) and macrophage inflammatory protein 3beta (MIP3beta), following lipopolysaccharide-induced MoDC maturation, was shown to be mediated by the p38SAPK pathway. This was due to the complete abrogation of upregulation of CC chemokine receptor 7, the receptor for MIP3beta/SLC. Once mature, MoDCs utilized both the p38SAPK and phosphoinositide-3 kinase pathways to migrate in response to SLC or MIP3beta. These findings have implications for the mechanism of action of p38SAPK inhibitors, currently in use in clinical trials for patients with autoimmune diseases.
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- 2002
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14. The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells.
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Grimwade D, Outram SV, Flora R, Ings SJ, Pizzey AR, Morilla R, Craddock CF, Linch DC, and Solomon E
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- Cell Lineage, Chromatin chemistry, Chromatin genetics, Deoxyribonuclease I metabolism, Humans, Immunophenotyping, Jurkat Cells, Leukemia, Promyelocytic, Acute pathology, Polymerase Chain Reaction methods, T-Lymphocytes cytology, CD2 Antigens genetics, Chromatin physiology, Leukemia, Promyelocytic, Acute genetics, T-Lymphocytes physiology
- Abstract
The nature of hemopoietic progenitors subject to leukemic transformation in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and downstream flanking regions of CD2 were found to be hypersensitive to DNase I in primary APL blasts, with an identical pattern of hypersensitive sites to those detected in cells of T-lineage. All of the sites were confirmed to be inaccessible to DNase I in B-lineage leukemia cells. The demonstration of T-cell-associated chromatin features in primary APL blasts has implications for the origin of APL that may arise in more primitive progenitors than previously considered to be the case.
- Published
- 2002
15. The PI3 kinase, p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells.
- Author
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Ardeshna KM, Pizzey AR, Devereux S, and Khwaja A
- Subjects
- Cell Differentiation drug effects, Cell Survival, Cells, Cultured, Dendritic Cells drug effects, Humans, Lipopolysaccharides pharmacology, Monocytes cytology, Monocytes drug effects, p38 Mitogen-Activated Protein Kinases, Dendritic Cells cytology, Dendritic Cells physiology, Mitogen-Activated Protein Kinases physiology, NF-kappa B physiology, Phosphatidylinositol 3-Kinases physiology, Signal Transduction
- Abstract
As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal-regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-kappaB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-kappaB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-kappaB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.
- Published
- 2000
16. Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.
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Ardeshna KM, Pizzey AR, Thomas NS, Orr S, Linch DC, and Devereux S
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- Antigens, CD34, Cell Differentiation, Cell Division, Flow Cytometry, Genetic Vectors, Humans, Leukocyte Count, Moloney murine leukemia virus genetics, Stem Cells cytology, Transfection, Dendritic Cells cytology, Leukocytes, Mononuclear cytology
- Abstract
Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 +/- 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.
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- 2000
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17. Preferential induction of apoptosis of leukaemic cells by farnesol.
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Rioja A, Pizzey AR, Marson CM, and Thomas NS
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- Cell Division drug effects, Cell Line, Transformed, Humans, Jurkat Cells, Apoptosis drug effects, Farnesol pharmacology, Leukemia, Myeloid, Acute blood, Monocytes drug effects
- Abstract
Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.
- Published
- 2000
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18. Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.
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Lam EW, Glassford J, van der Sman J, Banerji L, Pizzey AR, Shaun N, Thomas B, and Klaus GG
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- Animals, B-Lymphocytes metabolism, Cell Cycle Proteins analysis, Cell Cycle Proteins biosynthesis, Cell Cycle Proteins metabolism, Cells, Cultured, DNA-Binding Proteins analysis, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins metabolism, E2F Transcription Factors, E2F1 Transcription Factor, E2F3 Transcription Factor, E2F4 Transcription Factor, Macromolecular Substances, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Phosphorylation, Retinoblastoma-Binding Protein 1, Transcription Factor DP1, Transcription Factors analysis, Transcription Factors biosynthesis, B-Lymphocytes immunology, CD40 Antigens metabolism, Carrier Proteins, Immunoglobulin M metabolism, Lymphocyte Activation, Receptors, Antigen, B-Cell physiology, Receptors, Fc physiology, Transcription Factors metabolism
- Abstract
Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein/E2F complexes in mouse B lymphocytes following stimulation by anti-IgM, anti-CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two "free" E2F complexes consisting of E2F4/DP1 and E2F1-3/DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB/E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.
- Published
- 1999
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19. p130, p107, and pRb are differentially regulated in proliferating cells and during cell cycle arrest by alpha-interferon.
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Thomas NS, Pizzey AR, Tiwari S, Williams CD, and Yang J
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- B-Lymphocytes cytology, B-Lymphocytes drug effects, Base Sequence, Binding Sites, Cell Cycle drug effects, Cell Division, Cells, Cultured, DNA-Binding Proteins metabolism, E2F Transcription Factors, E2F1 Transcription Factor, E2F4 Transcription Factor, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells drug effects, Humans, Interferon-alpha physiology, Oligodeoxyribonucleotides chemistry, Oligodeoxyribonucleotides metabolism, Phosphorylation, Retinoblastoma-Binding Protein 1, Retinoblastoma-Like Protein p107, Retinoblastoma-Like Protein p130, T-Lymphocytes cytology, T-Lymphocytes drug effects, Transcription Factor DP1, Transcription Factors metabolism, Tretinoin pharmacology, Tumor Cells, Cultured, B-Lymphocytes metabolism, Carrier Proteins, Cell Cycle physiology, Cell Cycle Proteins, Hematopoietic Stem Cells metabolism, Interferon-alpha pharmacology, Nuclear Proteins metabolism, Phosphoproteins metabolism, Proteins, Retinoblastoma Protein metabolism, T-Lymphocytes metabolism
- Abstract
We have determined how the phosphorylation of the retinoblastoma family (pRb, p107, and p130) is governed in individual cell cycle phases of Daudi B-cells during cell cycle exit triggered by alpha-interferon (alpha-IFN). alpha-IFN causes dephosphorylation of pRb and loss of p130 phosphorylated Form 3. However, the change in p130 phosphorylation in response to alpha-IFN occurs before dephosphorylation of pRb is complete because loss of p130 Form 3 occurs throughout the cell cycle prior to complete arrest in G1, whereas pRb is dephosphorylated only in G1. In contrast, p107 is dephosphorylated and is then depleted from cells as they exit the cell cycle. p130, predominantly in Form 1, and hypophosphorylated pRb bind an E2F DNA binding site; p130 complexes E2F-4, whereas pRb binds both E2F-4 and E2F-1. The phosphorylated forms of E2F-4 that bind to the E2F DNA site are different from hyperphosphorylated E2F-4, which predominates in primary hemopoietic cells in G0. We conclude that although cell cycle arrest induced by alpha-IFN may be mediated in part by formation of a complex containing p130 and E2F-4, alpha-IFN does not induce hyperphosphorylation of E2F-4, which characterizes primary hemopoietic cells in G0.
- Published
- 1998
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20. The effects of interleukin-8 on neutrophil fMetLeuPhe receptors, CD11b expression and metabolic activity, in comparison and combination with other cytokines.
- Author
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Roberts PJ, Pizzey AR, Khwaja A, Carver JE, Mire-Sluis AR, and Linch DC
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- Antigens, CD analysis, Arachidonic Acid blood, CD11 Antigens, Cells, Cultured, Cytokines pharmacology, Humans, Interleukin-8 physiology, Neutrophils immunology, Neutrophils metabolism, Receptors, Formyl Peptide, Receptors, Immunologic metabolism, Receptors, Peptide metabolism, Respiratory Burst drug effects, Superoxides metabolism, Antigens, CD drug effects, Interleukin-8 pharmacology, Neutrophils drug effects, Receptors, Immunologic drug effects, Receptors, Peptide drug effects
- Abstract
The effect of the chemotactic cytokine, IL-8, on neutrophil function was compared with that of of other cytokines, GM-CSF, G-CSF TNF alpha and IFN-gamma. IL-8 rapidly stimulated a three-fold enhancement of the fMLP-stimulated respiratory burst, but this priming effect was transient compared with the slower and sustained effects of GM-CSF and IFN gamma. Apart from G-CSF, IL-8 was the weakest priming agent and was weaker than GM-CSF in priming arachidonic acid metabolism stimulated by calcium ionophore. When incubated in combination, IL-8 and TNF alpha were highly synergistic in their effects on respiratory burst priming, whereas IL-8 and GM-CSF showed little synergy. In contrast, IL-8 was as potent as GM-CSF at increasing the expression of neutrophil chemotactic peptide receptors and the beta 2 integrin, CD11b. The latter was maximally upregulated within 5 min of stimulation with IL-8, whereas the effect of GM-CSF was much slower. The kinetics of neutrophil respiratory burst priming by IL-8 were the same when measured in whole blood samples and in purified cell suspensions, and IL-8 dose-response curves were similar, showing that the low affinity IL-8 receptors on erythrocytes do not rapidly sequester circulating IL-8. The data suggest that IL-8 plays a minor role in priming neutrophil function and that a more major activity is the regulation of neutrophil adhesion and migration.
- Published
- 1993
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21. The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor.
- Author
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Roberts PJ, Pizzey AR, and Linch DC
- Abstract
The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 +/- 10% (mean +/- S.E.M., n = 13) and 182 +/- 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b beta(2)-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.
- Published
- 1993
- Full Text
- View/download PDF
22. Pentoxifylline at clinically achievable levels inhibits FMLP-induced neutrophil responses, but not priming, upregulation of cell-adhesion molecules, or migration induced by GM-CSF.
- Author
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Roberts PJ, Yong KL, Khwaja A, Johnson BV, Pizzey AR, Carver JE, Addison IE, and Linch DC
- Subjects
- Antigens, CD metabolism, CD11 Antigens, Cell Adhesion Molecules metabolism, Chemotaxis, Leukocyte drug effects, Cyclic AMP metabolism, Humans, Pentoxifylline administration & dosage, Pentoxifylline blood, Respiratory Burst drug effects, Sepharose, Tumor Necrosis Factor-alpha pharmacology, Granulocyte-Macrophage Colony-Stimulating Factor pharmacology, N-Formylmethionine Leucyl-Phenylalanine pharmacology, Neutrophils drug effects, Neutrophils physiology, Pentoxifylline pharmacology
- Abstract
Pentoxifylline (PTX) administered after bone-marrow transplantation reduces procedure-related organ damage mediated by TNF alpha. GM-CSF is also given post-transplant to stimulate earlier neutrophil recovery. Because PTX has been shown to inhibit neutrophil function, we sought to determine whether it also inhibited the effects of GM-CSF on neutrophil activity. The study confirmed that PTX at clinically achievable concentration (5-10 mumol/l) attenuated the responses of human neutrophils to chemotactic peptide, whereas it did not inhibit the effect of GM-CSF on neutrophil function even at high concentrations. In experiments with human neutrophils, neither the direct effects of GM-CSF such as stimulation of migration and increased expression of CD11b, nor the priming effects of GM-CSF on the respiratory burst, were inhibited by PTX. In experiments with monkeys, intravenous administration of PTX did not block subsequent GM-CSF-induced neutrophil CD11b upregulation or phagocyte margination, even when near millimolar plasma levels of pentoxifylline were obtained. The retention of cytokine-stimulated activities suggests that PTX will not compromise the response of neutrophils to stimuli from infectious foci.
- Published
- 1993
- Full Text
- View/download PDF
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