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‣ Eficácia terapêutica do Metotrexato na Artrite Reumatóide depende da expressão de células T reguladoras CD39+?; Eficácia terapêutica do Metotrexato na Artrite Reumatóide depende da expressão de células T reguladoras CD39+?

Peres, Raphael Sanches
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 10/05/2012 Português
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Introdução: Artrite reumatóide (AR) é uma doença autoimune caracterizada por uma inflamação crônica das articulações. A estratégia terapêutica mais utilizada na AR consiste no uso de doses baixas de Metotrexato (MTX), um antagonista do folato, que promove a manutenção de altos níveis de adenosina (ADO) extracelular. No entanto, uma parte considerável dos pacientes é refratária ao tratamento com MTX e o mecanismo pelo qual este fenômeno ocorre ainda não é estabelecido. Estudos demonstram que células Tregs expressam em suas superfícies as ectonucleotidases CD39/ENTPD1 e CD73/ecto-5 'nucleotidase, enzimas que geram ADO através da degradação de ATP. Estes achados, associados ao fato que a ADO possui potente atividade imunomoduladora, sugere que a atividade antiinflamatória do MTX está relacionada com os efeitos das Tregs. Objetivos: Investigar se os mecanismos de refratariedade ao MTX em pacientes com AR podem estar relacionados com uma deficiência na atividade bioquímica e função supressora de células Tregs, focando principalmente na expressão das ectonucleotidases CD39 e CD73. Pacientes e Métodos: No presente estudo, amostras do sangue periférico de pacientes com AR (n= 89) e doadores saudáveis (n =16) foram coletadas. Por citometria de fluxo...

‣ The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

Marcus, A J; Broekman, M J; Drosopoulos, J H; Islam, N; Alyonycheva, T N; Safier, L B; Hajjar, K A; Posnett, D N; Schoenborn, M A; Schooley, K A; Gayle, R B; Maliszewski, C R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/03/1997 Português
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We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

‣ RanBPM associates with CD39 and modulates ecto-nucleotidase activity

Wu, Yan; Sun, Xiaofeng; Kaczmarek, Elzbieta; Dwyer, Karen M.; Bianchi, Elisabetta; Usheva, Anny; Robson, Simon C.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
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CD39/ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1) is an ecto-nucleotidase that influences P2 receptor activation to regulate vascular and immune cell adhesion and signalling events pivotal in inflammation. Whether CD39 interacts with other membrane or cytoplasmic proteins has not been established to date. Using the yeast two-hybrid system, we note that the N-terminus of CD39 binds to RanBPM (Ran binding protein M; also known as RanBP9), a multi-adaptor scaffolding membrane protein originally characterized as a binding protein for the small GTPase Ran. We confirm formation of complexes between CD39 and RanBPM in transfected mammalian cells by co-immunoprecipitation studies. Endogenous CD39 and RanBPM are also found to be co-expressed and abundant in cell membranes of B-lymphocytes. NTPDase activity of recombinant CD39, but not of N-terminus-deleted-CD39 mutant, is substantially diminished by RanBPM co-expression in COS-7 cells. The conserved SPRY [repeats in splA and RyR (ryanodine receptor)] moiety of RanBPM is insufficient alone for complete physical and functional interactions with CD39. We conclude that CD39 associations with RanBPM have the potential to regulate NTPDase catalytic activity. This intermolecular interaction may have important implications for the regulation of extracellular nucleotide-mediated signalling.

‣ CD39 and control of cellular immune responses

Dwyer, Karen M.; Deaglio, Silvia; Gao, Wenda; Friedman, David; Strom, Terry B.; Robson, Simon C.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
Português
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CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting...

‣ Rat pancreas secretes particulate ecto-nucleotidase CD39

Sørensen, Christiane E; Amstrup, Jan; Rasmussen, Hans N; Ankorina-Stark, Ieva; Novak, Ivana
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
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In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokinin-octapeptide-8 (CCK-8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK-8-stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate...

‣ Central role of Sp1-regulated CD39 in hypoxia/ischemia protection

Eltzschig, Holger K.; Köhler, David; Eckle, Tobias; Kong, Tianqing; Robson, Simon C.; Colgan, Sean P.
Fonte: American Society of Hematology Publicador: American Society of Hematology
Tipo: Artigo de Revista Científica
Publicado em 01/01/2009 Português
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Hypoxia is common to several inflammatory diseases, where multiple cell types release adenine-nucleotides (particularly adenosine triphosphate/adenosine diphosphate). Adenosine triphosphate/adenosine diphosphate is metabolized to adenosine through a 2-step enzymatic reaction initiated by CD39 (ectonucleoside-triphosphate-diphosphohydrolase-1). Thus, extracellular adenosine becomes available to regulate multiple inflammatory endpoints. Here, we hypothesized that hypoxia transcriptionally up-regulates CD39 expression. Initial studies revealed hypoxia-dependent increases in CD39 mRNA and immunoreactivity on endothelia. Examination of the human CD39 gene promoter identified a region important in hypoxia inducibility. Multiple levels of analysis, including site-directed mutagenesis, chromatin immunoprecipitation, and inhibition by antisense, revealed a critical role for transcription-factor Sp1 in hypoxia-induction of CD39. Using a combination of cd39−/− mice and Sp1 small interfering RNA in in vivo cardiac ischemia models revealed Sp1-mediated induction of cardiac CD39 during myocardial ischemia. In summary, these results identify a novel Sp1-dependent regulatory pathway for CD39 and indicate the likelihood that CD39 is central to protective responses to hypoxia/ischemia.

‣ Vascular smooth muscle cell expression of ectonucleotidase CD39 (ENTPD1) is required for neointimal formation in mice

Behdad, Amir; Sun, Xiaofeng; Khalpey, Zain; Enjyoji, Keiichi; Wink, Marcia; Wu, Yan; Usheva, Anny; Robson, Simon C.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
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Vascular smooth muscle cell (VSMC) migration and proliferation are critical steps in the pathogenesis of atherosclerosis, post-angioplasty restenosis, neointimal hyperplasia, and chronic allograft rejection. Extracellular nucleotides are known to influence both migration and proliferation of VSMC. Although it is well established that vascular endothelial Cd39/ENTPD1 regulates blood nucleotide concentrations, whether Cd39 associated with VSMC also impacts vascular wall pathology has not been investigated. The objective of this paper is to determine levels of expression of Cd39 on VSMC and functional consequences of gene deletion in vitro and in vivo. Cd39 is the major ectonucleotidase in VSMC, as shown by substantive decreases in ecto-ATPase and -ADPase activity in Cd39-null cells compared to wild type. Significant decreases in neointimal lesion formation are observed in Cd39-null mice at 21 days post arterial balloon injury. Stimulated Cd39-null VSMC have pronounced proliferative responses in vitro. However, using Transwell systems, we show that Cd39-null VSMC fail to migrate in response to ATP, UTP, and PDGF. Cd39 is the dominant ectonucleotidase expressed by VSMC. Deletion of Cd39 in mice results in decreased neointimal formation after vascular injury and is associated with impaired VSMC migration responses in vitro.

‣ Vascular stasis, intestinal hemorrhage, and heightened vascular permeability complicate acute portal hypertension in cd39-null mice

Sun, Xiaofeng; Cárdenas, Andrés; Wu, Yan; Enjyoji, Keichi; Robson, Simon C.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
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Vasoactive factors that regulate splanchnic hemodynamics include nitric oxide, catecholamines, and possibly extracellular nucleosides/nucleotides (adenosine, ATP). CD39/ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1) is the major vascular ectonucleotidase that hydrolyzes extracellular nucleotides. CD39 activity may be modulated by vascular injury, inflammation, and altered oxygen tension. Altered Cd39 expression by the murine hepatosplanchnic vasculature may impact hemodynamics and portal hypertension (PHT) in vivo. We noted that basal portal pressures (PPs) were comparable in wild-type and Cd39-null mice (n = 9). ATP infusions resulted in increments in PP in wild-type mice, but, in contrast, this significantly decreased in Cd39-null mice (n = 9) post-ATP in a nitric oxide-dependent manner. We then studied Cd39/NTPDase1 deletion in the regulation of portal hemodynamics, vascular integrity, and intestinal permeability in a murine model of PHT. Partial portal vein ligation (PPVL) was performed in Cd39-null (n = 44) and wild-type (n = 23) mice. Sequential measurements obtained after PPVL were indicative of comparable levels of PHT (ranges 14–29 mmHg) in both groups. There was one death in the wild-type group and eight in the Cd39-null group from intestinal bleeding (P = 0.024). Circulatory stasis in the absence of overt portal vein thrombosis...

‣ Isolated CD39 Expression on CD4+ T Cells Denotes both Regulatory and Memory Populations

Zhou, Q.; Yan, J.; Putheti, P.; Wu, Y.; Sun, X.; Toxavidis, V.; Tigges, J.; Kassam, N.; Enjyoji, K.; Robson, S. C.; Strom, T. B.; Gao, W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Foxp3+ regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4+CD39+ T-cell pool contains two roughly equal size Foxp3+ and Foxp3− populations. While Foxp3+CD39+ cells are CD73bright and are the bone fide Tregs, Foxp3−CD39+ cells do not have suppressive activity and are CD44+CD62L−CD25−CD73dim/−, exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3−CD39− naïve T cells, Foxp3−CD39+ cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-γ (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3−CD39+ cells inhibits TGF-β induction of Foxp3 in Foxp3−CD39− cells. Furthermore, when transferred in vivo, Foxp3−CD39+ cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3−CD39− cells. Thus...

‣ The Expression Level of Ecto-NTP Diphosphohydrolase1/CD39 Modulates Exocytotic and Ischemic Release of Neurotransmitters in a Cellular Model of Sympathetic Neurons

Corti, Federico; Olson, Kim E.; Marcus, Aaron J.; Levi, Roberto
Fonte: The American Society for Pharmacology and Experimental Therapeutics Publicador: The American Society for Pharmacology and Experimental Therapeutics
Tipo: Artigo de Revista Científica
Publicado em /05/2011 Português
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Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-differentiated PC12 cells, a cellular model of sympathetic neuron, in which dopamine is the predominant catecholamine. We report that E-NTPDase1/CD39 deletion markedly increases depolarization-induced exocytosis of ATP and dopamine and increases ATP-induced dopamine release. Moreover, overexpression of E-NTPDase1/CD39 resulted in enhanced removal of exogenous ATP, a marked decrease in exocytosis of ATP and dopamine, and a large decrease in ATP-induced dopamine release. Administration of a recombinant form of E-NTPDase1/CD39 reproduced the effects of E-NTPDase1/CD39 overexpression. Exposure of PC12 cells to simulated ischemia elicited a release of ATP and dopamine that was markedly increased in E-NTPDase1/CD39-silenced cells and decreased in E-NTPDase1/CD39-overexpressing cells. Therefore...

‣ Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Künzli, Beat M.; Bernlochner, Maria-Isabell; Rath, Stephan; Käser, Samuel; Csizmadia, Eva; Enjyoji, Keiichi; Cowan, Peter; d’Apice, Anthony; Dwyer, Karen; Rosenberg, Robert; Perren, Aurel; Friess, Helmut; Maurer, Christoph A.; Robson, Simon C.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
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Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples...

‣ Liver damage and systemic inflammatory responses are exacerbated by the genetic deletion of CD39 in total hepatic ischemia

Sun, Xiaofeng; Imai, Masato; Nowak-Machen, Martina; Guckelberger, Olaf; Enjyoji, Keiichi; Wu, Yan; Khalpey, Zain; Berberat, Pascal; Munasinghe, Jeeva; Robson, Simon Christopher
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
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Liver ischemia reperfusion injury is associated with both local damage to the hepatic vasculature and systemic inflammatory responses. CD39 is the dominant vascular endothelial cell ectonucleotidase and rapidly hydrolyses both adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate. These biochemical properties, in tandem with 5′-nucleotidases, generate adenosine and potentially illicit inflammatory vascular responses and thrombosis. We have evaluated the role of CD39 in total hepatic ischemia reperfusion injury (IRI). Wildtype mice, Cd39-hemizygous mice (+/−) and matched Cd39-null mice (−/−); (n = 25 per group) underwent 45 min of total warm ischemia with full inflow occlusion necessitating partial hepatectomy. Soluble nucleoside triphosphate diphosphohydrolase (NTPDases) or adenosine/amrinone were administered to wildtype (n = 6) and Cd39-null mice (n = 6) in order to study protective effects in vivo. Parameters of liver injury, systemic inflammation, hepatic ATP determinations by P31-NMR and parameters of lung injury were obtained. All wildtype mice survived up to 7 days with minimal biochemical disturbances and minor evidence for injury. In contrast, 64% of Cd39+/− and 84% of Cd39-null mice required euthanasia or died within 4 h post-reperfusion with liver damage and systemic inflammation associated with hypercytokinemia. Hepatic ATP depletion was pronounced in Cd39-null mice posthepatic IRI. Soluble NTPDase or adenosine administration protected Cd39-deficient mice from acute reperfusion injury. We conclude that CD39 is protective in hepatic IRI preventing local injury and systemic inflammation in an adenosine dependent manner. Our data indicate that vascular CD39 expression has an essential protective role in hepatic IRI.

‣ Segregated Regulatory CD39+ CD4+ T Cell Function: TGF-β-Producing FoxP3− and IL-10-Producing FoxP3+ Cells Are Interdependent for Protection Against Collagen-Induced Arthritis1

Kochetkova, Irina; Thornburg, Theresa; Callis, Gayle; Pascual, David W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic E. coli colonization factor antigen I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-β. To identify the responsible regulatory CD4+ T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39+CD4+ T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39+CD4+ T cells into CIA mice conferred complete protection, while CD39−CD4+ T cells did not. Subsequent analysis of vaccinated FoxP3-GFP mice revealed the CD39+ T cells were composed of FoxP3-GFP− and FoxP3-GFP+ subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced FoxP3− and FoxP3+CD39+CD4+ T cells could protect against CIA, each subset was not as efficacious as total CD39+CD4+ T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed FoxP3− CD39+CD4+ T cells produced TGF-β, and FoxP3+CD39+CD4+ T cells produced IL-10, showing a segregation of function. Moreover, donor FoxP3-GFP− CD4+ T cells converted to FoxP3-GFP+ CD39+CD4+ T cells in the recipients, showing plasticity of these regulatory T cells. TGF-β was found to be essential for protection since in vivo TGF-β neutralization reversed activation of cAMP-response element-binding protein (CREB) and reduced the development of CD39+CD4+ T cells. Thus...

‣ Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

Leal, Fabio E.; Ndhlovu, Lishomwa C.; Hasenkrug, Aaron M.; Bruno, Fernanda R.; Carvalho, Karina I.; Wynn-Williams, Harry; Neto, Walter K.; Sanabani, Sabri S.; Segurado, Aluisio C.; Nixon, Douglas F.; Kallas, Esper G.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 07/02/2013 Português
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HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4+ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4+ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39+CD25+) and effector (CD39+CD25−) function. Here, we investigated the expression of CD39 on CD4+ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39+ CD4+ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39+CD25− CD4+ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39+CD25+ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39−CD25+ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4+ T cells compared to uninfected controls. Taken together...

‣ Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)

Baek, Amy E.; Kanthi, Yogendra; Sutton, Nadia R.; Liao, Hui; Pinsky, David J.
Fonte: Federation of American Societies for Experimental Biology Publicador: Federation of American Societies for Experimental Biology
Tipo: Artigo de Revista Científica
Publicado em /11/2013 Português
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The ectoenzyme CD39 suppresses thrombosis and inflammation by suppressing ATP and ADP to AMP. However, mechanisms of CD39 transcriptional and post-translational regulation are not well known. Here we show that CD39 levels are modulated by inhibition of phosphodiesterase 3 (PDE3). RAW macrophages and human umbilical vein endothelial cells (HUVECs) were treated with the PDE3 inhibitors cilostazol and milrinone, then analyzed using qRT-PCR, immunoprecipitation/Western blot, immunofluorescent staining, radio-thin-layer chromatography, a malachite green assay, and ELISA. HUVECs expressed elevated CD39 protein (2-fold [P<0.05] for cilostazol and 2.5-fold [P<0.01] for milrinone), while macrophage CD39 mRNA and protein were both elevated after PDE3 inhibition. HUVEC ATPase activity increased by 25% with cilostazol and milrinone treatment (P<0.05 and P<0.01, respectively), as did ADPase activity (47% and 61%, P<0.001). There was also a dose-dependent elevation of soluble CD39 after treatment with 8-Br-cAMP, with maximal elevation of 60% more CD39 present compared to controls (1 mM, P<0.001). Protein harvested after 8-Br-cAMP treatment showed that ubiquitination of CD39 was decreased by 43% compared to controls. A DMSO or PBS vehicle control was included for each experiment based on solubility of cilostazol...

‣ CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia-reperfusion injury

Yoshida, Osamu; Kimura, Shoko; Jackson, Edwin K.; Robson, Simon C.; Geller, David A.; Murase, Noriko; Thomson, Angus W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Hepatic innate immune cells, in particular interstitial dendritic cells (DC), regulate inflammatory responses and may promote inherent liver tolerogenicity. Following tissue injury, adenosine triphosphate (ATP) is released and acts as a damage-associated molecular pattern that activates innate immune cells via pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DC might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid (m) DC were hyporesponsive to ATP compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDC. Human liver mDC expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDC correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39-/- mouse liver mDC exhibited a more mature phenotype, greater responsiveness to Toll-like receptor 4 ligation, and stronger pro-inflammatory and immunostimulatory activity than wild-type (WT) liver mDC. To investigate the role of CD39 on liver mDC in vivo...

‣ Bilayer Mechanical Properties Regulate Transmembrane Helix Mobility and Enzymatic State of CD39

Guidotti, Guido; Grinthal, Alison Elizabeth
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
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CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In the present study we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone shaped or inverse cone shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state as does removing or solubilizing the transmembrane domains. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix...

‣ CD39 and Control of Cellular Immune Responses

Dwyer, Karen M.; Deaglio, Silvia; Gao, Wenda; Friedman, David J.; Strom, Terry Barton; Robson, Simon Christopher
Fonte: Springer Verlag Publicador: Springer Verlag
Tipo: Artigo de Revista Científica
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CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5'–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg...

‣ CD39 deletion exacerbates experimental murine colitis and human polymorphisms increase susceptibility to inflammatory bowel disease

Friedman, David J.; Künzli, Beat M.; A-Rahim, Yousif I.; Sevigny, Jean; Berberat, Pascal O.; Enjyoji, Keiichi; Csizmadia, Eva; Friess, Helmut; Robson, Simon C.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 29/09/2009 Português
Relevância na Pesquisa
27.931047%
CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to generate adenosine. As purinergic mediators have been implicated in intestinal inflammation, we hypothesized that CD39 might protect against inflammatory bowel disease. We studied these possibilities in a mouse model of colitis using mice with global CD39 deletion. We then tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. We induced colitis in mice using Dextran Sodium Sulfate (DSS). Readouts included disease activity scores, histological evidence of injury, and markers of inflammatory activity. We used HapMap cell lines to find SNPs that tag for CD39 expression, and then compared the frequency of subjects with high vs. low CD39-expression genotypes in a case-control cohort for Crohn's disease. Mice null for CD39 were highly susceptible to DSS injury, with heterozygote mice showing an intermediate phenotype compared to wild type (WT). We identified a common SNP that tags CD39 mRNA expression levels in man. The SNP tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case-control cohort comprised of 1,748 Crohn's patients and 2,936 controls (P = 0.005–0.0006). Our data indicate that CD39 deficiency exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans.

‣ cAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression*

Liao, Hui; Hyman, Matthew C.; Baek, Amy E.; Fukase, Keigo; Pinsky, David J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.931047%
CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser133) and ATF2 (Thr71) in a PKA-...