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1.  Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma 
Blood Cancer Journal  2012;2(8):e82-.
Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK+ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK+ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK+ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2active cells, but not Sox2inactive cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK+ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.
PMCID: PMC3432482  PMID: 22885405
Sox2; transcriptional activity; NPM-ALK; STAT3; tumorigenicity
2.  Anti‐β2GPI‐antibody‐induced endothelial cell gene expression profiling reveals induction of novel pro‐inflammatory genes potentially involved in primary antiphospholipid syndrome 
Annals of the Rheumatic Diseases  2007;66(8):1000-1007.
To determine the effects of primary antiphospholipid syndrome (PAPS)‐derived anti‐β2GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays.
Anti‐β2GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A‐2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real‐time PCR analysis or at the protein level by ELISA.
A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti‐β2GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL‐18 receptor 1, and growth factors CSF2, CSF3 IL‐6, IL1β and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real‐time RT‐PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C).
This study reveals a complex gene expression response in HUVEC to anti‐β2GPI antibodies with multiple chemokines, pro‐inflammatory cytokines, pro‐thrombotic and pro‐adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti‐β2GPI antibody‐regulated genes could contribute to the vasculopathy associated with this disease.
PMCID: PMC1954708  PMID: 17223652
5.  Axillary vein thrombosis in adolescent onset systemic sclerosis. 
Annals of the Rheumatic Diseases  1990;49(7):557-559.
A 16 year old girl with a two year history of systemic sclerosis developed left axillary vein thrombosis. Prolonged euglobulin clot lysis time, anti-endothelial cell antibodies, and raised von Willebrand factor antigen were shown.
PMCID: PMC1004151  PMID: 2383084
6.  Progressive multifocal leukoencephalopathy, myelodysplastic syndrome type II and prostatic carcinoma. 
Postgraduate Medical Journal  1984;60(700):157-158.
A case is described of progressive multifocal leukoencephalopathy (PML) occurring in a man with myelodysplastic syndrome type II (MDS II) and prostatic carcinoma. This is the first case, to our knowledge, of the association of PML with MDS II.
PMCID: PMC2417702  PMID: 6709552
7.  Effect of sulphasalazine on pulmonary inactivation of prostaglandin F2 alpha in the pig. 
British Journal of Pharmacology  1982;76(2):319-326.
1 The metabolism of prostaglandin F2 alpha (PGF2 alpha) 15 nm in 100,000 g supernatant fractions from piglet lung homogenates was inhibited by sulphasalazine with an IC50 value of 25 micrometers. 2 The piglet isolated lung perfused with Krebs solution, containing either albumin or Ficoll 70 to prevent oedema and vascular damage, efficiently metabolized PGF2 alpha given as a bolus injection (1 ng in 0.1 ml; 30 nm). 3 In Krebs solution containing Ficoll 70, sulphasalazine inhibited the pulmonary inactivation of PGF2 alpha in a dose-dependent manner with an IC50 value of 110 micrometers. No inhibition of inactivation by sulphasalazine was found when the perfusion fluid contained albumin, which is known to bind this drug effectively. 4 Analysis of the separated efflux profiles for PGF2 alpha and its metabolites with reference to the dilution curve for an extracellular marker provided evidence that sulphasalazine inhibited PGF2 alpha uptake into lung cells. 5 We conclude that the effect of sulphasalazine on pulmonary prostaglandin inactivation is primarily due to inhibition of prostaglandin transport, and not to inhibition of prostaglandin metabolism.
PMCID: PMC2071779  PMID: 6178459
9.  Regional lung function in ankylosing spondylitis. 
Thorax  1976;31(4):433-437.
Xenon-133 was used to study regional pulmonary function in nine patients with chest cage rigidity due to ankylosing spondylitis. In comparison with normal subjects, the patients showed an overall diminution in lung volume and the proportion of inhaled xenon reaching the lung apices was reduced but the distribution of injected xenon was normal. The possible relationship between these findings and apical lung disease in ankylosing spondylitis is mentioned.
PMCID: PMC470455  PMID: 968800
10.  The estimation of 3,4-dihydroxyphenylacetic acid, homovanillic acid and homo-isovanillic acid in nervous tissue by gas-liquid chromatography and electron capture detection. 
British Journal of Pharmacology  1975;53(1):143-148.
1 A gas chromatographic method using electron capture detection is described for the estimation of three acidic metabolites of dopamine, 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-hydroxy-4-methoxyphenylacetic acid (homo-isovanillic acid, iso-HVA). The method is based on the formation of the trifluoroacetyl-hexafluoroisopropyl derivatives of the three acids. 2 The method has been applied to the estimation of DOPAC, HVA and iso-HVA in tissues from the central and peripheral nervous systems.
PMCID: PMC1666273  PMID: 164966
11.  Relationships between chemical structure and affinity for postganglionic acetylcholine receptors of the guinea-pig ileum 
1 Some phenylacetyl, diphenylacetyl, benziloyl and (±)-cyclohexylphenylglycolloyl esters have been made with 2- and 3-hydroxymethylpyrrolidines, 3-hydroxymethyl-N-methylpiperidine, piperidin-3-ols, piperidin-4-ols, 2,2,6,6-tetramethyl-N-methylpiperidin-4-ol, tropine, pseudotropine and quinuclidin-3-ol, and the affinity of these compounds and of their metho- and etho- derivatives has been measured for postganglionic acetylcholine receptors of the guinea-pig isolated ileum.
2 Some of the compounds were very active indeed; the benziloyl esters of N-methylpiperidin-4-ol methiodide, tropine methiodide, and quinculidin-3-ol, and the (±)-cyclohexylphenylglycolloyl esters of N-methylpiperidin-4-ol and its methiodide had affinity constants greater than 1010.
3 The effects of inserting an additional methylene group onto the nitrogen were extremely variable, ranging from a decrease in log K of 1.64 units to an increase of 0.97 units. The effects of replacing hydrogen by phenyl in the acid portion ranged from an increase of 1.04 units to an increase of 3.06 units and of replacing hydrogen by hydroxyl from a decrease of 0.09 units to an increase of 1.94 units.
4 The extent of the variation in the effects of a particular change in structure on affinity does not appear to be any different in these relatively rigid compounds from that observed with the same changes in open-chain aminoalcohols.
5 Reasons for the variable effects of groups on affinity are discussed. If differences in effects on preferred conformations of these particular compounds in solution are of secondary importance, the effect of a group on affinity will be the net result of what it could contribute to binding, offset by the disturbance it causes to existing binding. The maximum effect observed in a large number of comparisons may indicate the contribution in the absence of disturbance and for groups containing only carbon and hydrogen it appears to be related to size, assessed from the increments in apparent molal volume at infinite dilution. The variation in the effects of these groups also appears to be related to size. Changes involving groups containing oxygen can produce bigger contributions to binding, and a bigger variation in contribution, than would be expected from their size.
6 It is difficult to predict the extent to which groups may fail to produce their maximum effects. Variation is greatest with groups which could produce the biggest changes and so are of the greatest interest.
7 The relevance of the results to the successful prediction of biological activity is discussed.
PMCID: PMC1776805  PMID: 4441797
12.  A comparison of the affinities of antagonists for acetylcholine receptors in the ileum, bronchial muscle and iris of the guinea-pig 
British Journal of Pharmacology  1972;46(2):300-312.
1. Isolated preparations of bronchial strip and of intact iris from the guineapig have been adapted for the measurement of affinity constants of substances which block post-ganglionic acetylcholine receptors.
2. The affinity constants of 28 compounds on bronchial muscle and of 8 compounds on the iris have been compared with values measured on the guinea-pig ileum.
3. Although the compounds differed up to a million-fold in affinity, most of the estimates of log affinity constant for the bronchial muscle and iris differed only slightly from those on the ileum.
4. Some of the differences could be attributed to the actions of hexamethonium, used in the tests on the ileum but not, initially, in the tests with the bronchial strip and iris. Hexamethonium reduced most of the estimates of log K for the receptors in the bronchial strip by a variable but significant amount, which could be due, at least in part, to a weak post-ganglionic blocking (atropine-like) action. On average, hexamethonium had little effect on the estimates made with the ileum, appreciably decreasing estimates with some compounds and increasing those with others.
5. The results indicate that, although there may be differences between the acetylcholine receptors in the three types of tissue, there is no conclusive evidence, because the differences in affinity which we have observed could have arisen from differences in the experimental conditions. This is illustrated by results obtained with the guinea-pig ileum recorded with the same technique as was used for the bronchial strip, which are presented as an appendix.
6. Such differences as may exist between these three types of acetylcholine receptor are likely to be limited to the replacement of one aminoacid in the receptor protein by a homologue.
PMCID: PMC1666339  PMID: 4405611
16.  IgG antiendothelial cell autoantibodies from scleroderma patients induce leukocyte adhesion to human vascular endothelial cells in vitro. Induction of adhesion molecule expression and involvement of endothelium-derived cytokines. 
Journal of Clinical Investigation  1996;97(1):111-119.
IgG autoantibodies that bind human endothelial cells (AECA) were detected by ELISA in 30 of 42 samples of sera from patients with scleroderma. Pretreatment of human umbilical vein endothelial cells with AECA-positive scleroderma sera, or IgG purified from these sera, led to a dose- and time-dependent increase in the ability of the cells to bind human U937 monocytic cells. Threshold-active IgG concentrations were 1-10 micrograms/ml; effects were significant after 3 h and maximal after 6-12 h. IgG from AECA-negative sera or normal sera were without effect. Increased adhesion of U937 cells was accompanied by increased expression of endothelial intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Transfer of endothelial cell-conditioned media after pretreatment with AECA and immunodepletion of IgG demonstrated the presence of transferable activity that mimicked the effects of AECA. Treatment with neutralizing anticytokine antibodies indicated that IL-1, generated by the endothelial cells in response to AECA, was involved in the upregulation of adhesion molecules and U937 cell adhesion. We conclude that AECA can play a pathogenic role in scleroderma by activating endothelial cells, in part due to autocrine or paracrine actions of IL-1.
PMCID: PMC507068  PMID: 8550821
18.  The endothelium: its role in scleroderma. 
Annals of the Rheumatic Diseases  1991;50(Suppl 4):866-871.
PMCID: PMC1033322  PMID: 1750799
19.  Action of calcitonin gene-related peptide upon bovine vascular endothelial and smooth muscle cells grown in isolation and co-culture. 
1. Bovine aortic endothelial cells (BAE) and smooth muscle cells (BASM) were grown separately and in co-culture. 2. Calcitonin gene-related peptide (CGRP) caused dose-dependent activation of adenylate cyclase in each cell type when grown in isolation. The concentration of CGRP causing half-maximal activation in BAE and BASM was 200 nM and 310 nM, respectively. 3. In cells grown in co-culture exposure to bradykinin produced dose-dependent elevations in cyclic GMP content which were maximal 1 min after application of the agonist. 4. CGRP (1 nM-1 microM) did not stimulate a rise in cyclic GMP in co-cultures. 5. Displaceable CGRP binding was identified throughout the wall of the bovine aorta. 6. We conclude that CGRP receptors coupled to adenylate cyclase are present on BAE and BASM, but there is no coupling of these receptors to the release of any agent (such as endothelium-derived relaxing factor) that activates guanylate cyclase.
PMCID: PMC1917523  PMID: 2184911
20.  Purinoceptor mediated stimulation of prostacyclin release in the porcine pulmonary vasculature. 
British Journal of Pharmacology  1984;83(2):457-462.
Prostacyclin (PGI2) release from the piglet isolated perfused lung was measured by radioimmunoassay of 6-keto-PGF1 alpha in the venous effluent. Basal release of PGI2 was transiently stimulated up to 30 fold, in a dose-dependent manner, by bolus injections of ATP (0.03-3 mumol). A continuous infusion of ATP also produced a transient response. Dose-response curves for purinergic stimulation of PGI2 release showed that ADP was equipotent with ATP, while AMP and adenosine were virtually inactive. The non-hydrolyzable ATP analogue, ATP-gamma-S, elicited PGI2 release of similar magnitude and duration to that of ATP, suggesting that pulmonary catabolism of ATP is not required to induce PGI2 release. The results suggest that the porcine pulmonary vasculature possesses P2-purinoceptors through which the synthesis and release of PGI2 can be mediated.
PMCID: PMC1987114  PMID: 6091831
21.  Effects of sulphinpyrazone and aspirin on prostaglandin I2 (prostacyclin) synthesis by endothelial cells. 
British Journal of Pharmacology  1978;64(4):481-483.
Synthesis of prostaglandin I2, (PGI2, prostacyclin) by vascular endothelium (assayed by the ability of cultured endothelial cells to inhibit platelet aggregation) was inhibited by aspirin. At 100 mumol/l aspirin completely blocked measurable PGI2 production, but endothelial cells had substantially recovered their ability to synthesize PGI2 24 h after removal of the drug. In contrast, the effect of 1 mmol/l aspirin was still evident 24 h after drug withdrawal. Sulphinpyrazone also inhibited PGI2 synthesis, but was about 100 fold less potent than aspirin, and the effect of the drug was lost within 24 h of its addition, even when endothelial cells were left in contact with the drug during this period.
PMCID: PMC1668445  PMID: 365283
22.  Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells. 
British Journal of Pharmacology  1996;118(7):1761-1771.
1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6. Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7. These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.
PMCID: PMC1909850  PMID: 8842442
23.  Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine. 
British Journal of Pharmacology  1994;112(2):487-492.
1. The kinetics, specificity, pH- and Na(+)-dependency of L-citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)-activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 microgram ml-1) for 24 h. 2. In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mM and Vmax = 32 pmol micrograms-1 protein min-1), pH-insensitive and partially Na(+)-dependent. In contrast to arginine, transport of citrulline was unchanged in LPS-activated (1 microgram ml-1, 24 h) cells. 3. Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mM) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2-0.3 mM), but were poor inhibitors of arginine transport. 4. Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01-10 mM), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 microM NG-nitro-L-arginine methyl ester or NG-iminoethyl-L-ornithine. 5. Intracellular metabolism of L-[14C]-citrulline to L-[14C]-arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon-gamma. 6. We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1910348  PMID: 8075867
24.  Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of L-arginine transport, in activated murine macrophage J774 cells. 
British Journal of Pharmacology  1993;110(4):1401-1406.
1. Effects of dexamethasone on induction of nitric oxide (NO) synthase and L-arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of L-arginine to L-citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production. 2. Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 microgram ml-1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 microgram ml-1) inhibited the time-dependent (1-8 h) induction of NO synthase and arginine transport mediated by LPS. 3. Induction of NO synthase by LPS (1 microgram ml-1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular L-citrulline levels. These changes were further enhanced in the presence of interferon-gamma (IFN-gamma, 100 units ml-1, 24 h). 4. Dexamethasone (1 microM) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN-gamma. In contrast, treatment of cells with dexamethasone (10 microM) had no effect on the LPS-mediated induction of arginine transport or the decrease in intracellular arginine concentration. 5. We conclude that induction of arginine transporter activity in LPS-stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC2175842  PMID: 7508326
25.  Propofol stimulates nitric oxide release from cultured porcine aortic endothelial cells. 
Propofol, an intravenous anaesthetic agent, causes marked vasodilatation in vivo. In the present study the effects of propofol on the release of nitric oxide (NO) from vascular endothelial cells was determined in vitro. Application of propofol to co-cultures of porcine aortic endothelial and smooth muscle cells resulted in a rapid increase in cyclic GMP formation. This increase was significantly inhibited following pretreatment of the cells with either NG-nitro-L-arginine (L-NOARG) or in the presence of haemoglobin. When applied to smooth muscle cells alone, propofol did not result in an increase in cyclic GMP levels. These results demonstrate that propofol stimulates the production and release of NO from cultured endothelial cells and suggest that the vasodilatation and hypotension observed when propofol is given in vivo may be due to NO release.
PMCID: PMC2175593  PMID: 8388302

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