ORCID Profile
0000-0003-4909-686X
Current Organisations
University of Tasmania
,
University of Wollongong
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Additive manufacturing | Manufacturing engineering | Microfluidics and nanofluidics | Composite and hybrid materials
Publisher: Springer Science and Business Media LLC
Date: 16-06-2009
Publisher: Springer Science and Business Media LLC
Date: 12-08-2004
Publisher: Wiley
Date: 02-11-2006
Abstract: In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non-infected or non-oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch-cl recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1-deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection- or oxidation compared with wild-type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA lification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1-deficient mice in vivo compared with wild-type animals. In conclusion, induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves autocrine purinoceptor signaling.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2014
DOI: 10.1038/SREP04527
Publisher: Springer Science and Business Media LLC
Date: 05-06-2012
Publisher: Wiley
Date: 22-02-2003
DOI: 10.1016/S0014-5793(03)00172-8
Abstract: P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.
Publisher: PeerJ
Date: 03-2017
DOI: 10.7717/PEERJ.3064
Abstract: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterised by the accumulation of aggregated proteins, microglia activation and motor neuron loss. The mechanisms underlying neurodegeneration and disease progression in ALS are unknown, but the ATP-gated P2X7 receptor channel is implicated in this disease. Therefore, the current study aimed to examine P2X7 in the context of neurodegeneration, and investigate whether the P2X7 antagonist, Brilliant Blue G (BBG), could alter disease progression in a murine model of ALS. Human SOD1 G93A transgenic mice, which normally develop ALS, were injected with BBG or saline, three times per week, from pre-onset of clinical disease (62–64 days of age) until end-stage. During the course of treatment mice were assessed for weight, clinical score and survival, and motor coordination, which was assessed by rotarod performance. Various parameters from end-stage mice were assessed as follows. Motor neuron loss and microgliosis were assessed by immunohistochemistry. Relative amounts of lumbar spinal cord SOD1 and P2X7 were quantified by immunoblotting. Serum monocyte chemoattractant protein-1 was measured by ELISA. Splenic leukocyte populations were assessed by flow cytometry. Relative expression of splenic and hepatic P2X7 mRNA was measured by quantitative real-time PCR. Lumbar spinal cord SOD1 and P2X7 were also quantified by immunoblotting in untreated female SOD1 G93A mice during the course of disease. BBG treatment reduced body weight loss in SOD1 G93A mice of combined sex, but had no effect on clinical score, survival or motor coordination. BBG treatment reduced body weight loss in female, but not male, SOD1 G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1 G93A mice, extending the mean survival time by 4.3% in female mice compared to female mice treated with saline. BBG treatment had no effect on clinical score or motor coordination in either sex. BBG treatment had no major effect on any end-stage parameters. Total amounts of lumbar spinal cord SOD1 and P2X7 in untreated female SOD1 G93A mice did not change over time. Collectively, this data suggests P2X7 may have a partial role in ALS progression in mice, but additional research is required to fully elucidate the contribution of this receptor in this disease.
Publisher: Public Library of Science (PLoS)
Date: 26-03-2014
Publisher: MDPI AG
Date: 03-08-2021
DOI: 10.3390/IJMS22158343
Abstract: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y2 and A2A receptors, and ectoenzymes, CD39 and CD73, in GVHD.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2014
DOI: 10.1038/SREP05060
Publisher: Springer Science and Business Media LLC
Date: 14-05-2016
DOI: 10.1007/S00018-016-2274-2
Abstract: Ectodomain shedding of integral membrane receptors results in the release of soluble molecules and modification of the transmembrane portions to mediate or modulate extracellular and intracellular signalling. Ectodomain shedding is stimulated by a variety of mechanisms, including the activation of P2 receptors by extracellular nucleotides. This review describes in detail the roles of extracellular nucleotides and P2 receptors in the shedding of various cell surface molecules, including amyloid precursor protein, CD23, CD62L, and members of the epidermal growth factor, immunoglobulin and tumour necrosis factor families. This review discusses the mechanisms involved in P2 receptor-mediated shedding, demonstrating central roles for the P2 receptors, P2X7 and P2Y2, and the sheddases, ADAM10 and ADAM17, in this process in a number of cell types.
Publisher: Springer Science and Business Media LLC
Date: 21-02-2019
Publisher: Elsevier BV
Date: 02-2023
Publisher: Portland Press Ltd.
Date: 2020
DOI: 10.1042/CS20191086
Abstract: Background: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. Methods: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. Results: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. Conclusion: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.
Publisher: Oxford University Press (OUP)
Date: 12-2002
Abstract: Dendritic cells (DC) express a number of P2X receptors including the P2X7/P2Z receptor whose activation by extracellular adenosine 5'-triphosphate (ATP) induces the influx of calcium, DC maturation, cytokine release and apoptosis. In B lymphocytes ATP induces the rapid shedding of CD23 and CD62 ligand by activating a membrane metalloprotease. In this study, we examined the expression and early effects of P2X7 receptor activation on monocyte-derived DC, generated from in iduals either wild-type or homozygous for a loss-of-function single nucleotide polymorphism at position 1513 of the P2X7 gene. Labeling with an anti-human P2X7 receptor mAb demonstrated that DC express the P2X7 receptor at a lower level than macrophages. Short-term incubations (5 min) of DC with ATP induced an influx of ethidium+ (314 Da) into wild-type DC, but not into DC homozygous for the loss-of-function polymorphism. In contrast to results with ethidium+, ATP did not induce the influx of the viability dye propidium2+ (415 Da) into DC in short-term incubations. Addition of ATP also induced a rapid loss of CD23 from the surface of wild-type DC (t(1/2 )< 120 s), and this loss was inhibited by oxidized ATP and KN-62 which are known P2X7 receptor antagonists. Moreover, ATP-induced shedding of CD23 was slower from DC homozygous for the loss-of-function polymorphism than from wild-type DC. The data show that monocyte-derived DC express the P2X7 receptor whose activation opens a cation-selective channel, and which leads to rapid and near complete shedding of CD23. Both of these functions of the P2X7 receptor are impaired on DC from subjects who are homozygous for the loss-of-function 1513 polymorphism.
Publisher: Informa UK Limited
Date: 13-10-2023
Publisher: Springer Singapore
Date: 2017
DOI: 10.1007/5584_2017_59
Abstract: The P2X7 receptor is a trimeric ion channel gated by extracellular adenosine 5'-triphosphate. The receptor is present on an increasing number of different cells types including stem, blood, glial, neural, ocular, bone, dental, exocrine, endothelial, muscle, renal and skin cells. The P2X7 receptor induces various downstream events in a cell-specific manner, including inflammatory molecule release, cell proliferation and death, metabolic events, and phagocytosis. As such this receptor plays important roles in heath and disease. Increasing knowledge about the P2X7 receptor has been gained from studies of, but not limited to, protein chemistry including cloning, site-directed mutagenesis, crystal structures and atomic modeling, as well as from studies of primary tissues and transgenic mice. This chapter focuses on the P2X7 receptor itself. This includes the P2RX7 gene and its products including splice and polymorphic variants. This chapter also reviews modulators of P2X7 receptor activation and inhibition, as well as the transcriptional regulation of the P2RX7 gene via its promoter and enhancer regions, and by microRNA and long-coding RNA. Furthermore, this chapter discusses the post-translational modification of the P2X7 receptor by N-linked glycosylation, adenosine 5'-diphosphate ribosylation and palmitoylation. Finally, this chapter reviews interaction partners of the P2X7 receptor, and its cellular localisation and trafficking within cells.
Publisher: Wiley
Date: 10-03-2020
DOI: 10.1111/BPH.15009
Publisher: Elsevier BV
Date: 06-2019
DOI: 10.1016/J.TRIM.2019.02.003
Abstract: Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4
Publisher: Springer Science and Business Media LLC
Date: 04-01-2022
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6LC00843G
Abstract: By exploiting the Dean-flow-coupled elasto-inertial effects, continuous, sheathless, and high purity plasma extraction under viscoelastic fluid in a straight channel with asymmetrical expansion–contraction cavity arrays (ECCA channel) is demonstrated.
Publisher: American Chemical Society (ACS)
Date: 05-01-2021
Publisher: MDPI AG
Date: 13-11-2020
DOI: 10.3390/IJMS21228572
Abstract: Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.
Publisher: Elsevier BV
Date: 11-2010
Publisher: Wiley
Date: 28-11-2007
Abstract: Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.
Publisher: Springer Science and Business Media LLC
Date: 08-06-2017
Publisher: Institute of Electrical and Electronics Engineers (IEEE)
Date: 12-2017
Publisher: Springer US
Date: 2022
DOI: 10.1007/978-1-0716-2384-8_5
Abstract: The murine anti-human P2X7 receptor monoclonal antibody (mAb) (clone L4) has been used to study the expression and function of the P2X7 receptor on primary leukocytes, keratinocytes, osteoblasts and neuronal cells, as well as various cell lines. This antibody has also been used to characterize polymorphic variants and isoforms of the P2RX7 gene and P2X7 site-directed mutations, and to identify molecules coassociated with P2X7 in the plasma membrane. This chapter describes the maintenance and cryopreservation of the L4 hybridoma cell line, as well as the generation of tissue culture supernatant containing the anti-human P2X7 mAb, and its subsequent purification by Protein A chromatography and conjugation to DyLight™ 488. Moreover, this chapter describes flow cytometric assays to assess the blocking activity and binding of the anti-human P2X7 mAb against P2X7 on human RPMI 8226 multiple myeloma cells.
Publisher: Portland Press Ltd.
Date: 02-2021
DOI: 10.1042/CS20201352
Abstract: Graft-versus-host disease (GVHD) is a severe inflammatory response arising from allogeneic haematopoietic stem cell transplantation. Previous studies revealed that antagonism of the P2X7 receptor with Brilliant Blue G (BBG) reduced liver GVHD but did not alter clinical GVHD in a humanised mouse model. Therefore, the present study aimed to trial a modified injection regime using more frequent dosing of BBG to improve outcomes in this model of GVHD. NOD-scid IL2Rγnull (NSG) mice were injected intraperitoneally (i.p.) with 10 × 106 human peripheral blood mononuclear cells (hPBMCs) (day 0), then daily with BBG (50 mg/kg) or saline (days 0–10). BBG significantly reduced clinical score, mortality and histological GVHD compared with saline treatment (endpoint). BBG significantly increased proportions of human regulatory T cells (Tregs) and human B cells and reduced serum human interferon-γ compared with saline treatment prior to development of clinical GVHD (day 21). To confirm the therapeutic benefit of P2X7 antagonism, NSG mice were injected i.p. with 10 × 106 hPBMCs (day 0), then daily with pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (300 mg/kg) or saline (days 0–10). PPADS increased human Treg proportions compared with saline treatment (day 21), but potential clinical benefits were confounded by increased weight loss with this antagonist. To investigate the role of P2X7 antagonism on Treg survival, hPBMCs were cultured in reduced serum conditions to promote cell death. BBG increased proportions of Tregs (and B cells) compared with saline under these conditions. In conclusion, P2X7 antagonism reduces clinical and histological GVHD in a humanised mouse model corresponding to an increase in human Tregs.
Publisher: Cold Spring Harbor Laboratory
Date: 03-03-2020
DOI: 10.1101/2020.03.02.972240
Abstract: Invasive infections due to Group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates 5448 and animal passaged variant 5448AP. Neutrophil co-incubation with 5448AP allowed proliferation of GAS while it also lowered the production of reactive oxygen species by neutrophils when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 activation and caspase-4 expression in vitro , with caspase-1 activation detected in vivo . Thus, GAS infection of neutrophils corresponds to increased caspase-1 activity and caspase-4 expression, consistent with inflammasome activation and pyroptosis. GAS infections that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the speed and severity of invasive GAS infections.
Publisher: Springer Science and Business Media LLC
Date: 27-09-2013
Publisher: Elsevier BV
Date: 09-2009
DOI: 10.1016/J.IJCARD.2008.05.017
Abstract: We examined if a loss-of-function polymorphism in the P2X(7) receptor (1513C) corresponded with circulating interleukin(IL)-18 concentrations in heart failure (HF) patients. IL-18 values were significantly elevated in HF subjects compared to healthy control subjects. No association was seen between the polymorphism and IL-18 concentrations in HF patients. In HF patients, IL-18 values had an inverse relationship with ejection fraction, mean arterial pressure and body mass index, while high IL-18 concentrations were associated with increased mortality.
Publisher: Portland Press Ltd.
Date: 09-2022
DOI: 10.1042/BSR20211986
Abstract: Graft-versus-host disease (GVHD) is a major complication that occurs following allogeneic haematopoietic stem cell transplantation (HSCT) for the treatment of haematological cancers and other blood-related disorders. GVHD is an inflammatory disorder, where the transplanted donor immune cells can mediate an immune response against the recipient and attack host tissues. Despite over 60 years of research, broad-range immune suppression is still used to prevent or treat GVHD, leading to an increased risk of cancer relapse and infection. Therefore, further insights into the disease mechanisms and development of predictive and prognostic biomarkers are key to improving outcomes and reducing GVHD development following allogeneic HSCT. An important preclinical tool to examine the pathophysiology of GVHD and to understand the key mechanisms that lead to GVHD development are preclinical humanised mouse models. Such models of GVHD are now well-established and can provide valuable insights into disease development. This review will focus on models where human peripheral blood mononuclear cells are injected into immune-deficient non-obese diabetic (NOD)-scid-interleukin-2(IL-2)Rγ mutant (NOD-scid-IL2Rγnull) mice. Humanised mouse models of GVHD can mimic the clinical setting for GVHD development, with disease progression and tissues impacted like that observed in humans. This review will highlight key findings from preclinical humanised mouse models regarding the role of donor human immune cells, the function of cytokines and cell signalling molecules and their impact on specific target tissues and GVHD development. Further, specific therapeutic strategies tested in these preclinical models reveal key molecular pathways important in reducing the burden of GVHD following allogeneic HSCT.
Publisher: Springer Science and Business Media LLC
Date: 05-08-2020
Publisher: Elsevier BV
Date: 07-2004
Publisher: Public Library of Science (PLoS)
Date: 03-03-2021
DOI: 10.1371/JOURNAL.PONE.0247861
Abstract: Over 50% of women with detrusor overactivity (DO), who do not respond to therapy have been shown to have bacteriuria, which may stimulate the release of inflammatory cytokines than can enhance nerve signalling, leading to symptoms of urgency. This study made use of a consecutive series of urine s les collected from women with refractory DO, who participated in a clinical trial of rotating antibiotic therapy. The aim was to determine the effect of bacteriuria and antibiotic treatment on the levels of urinary cytokines, and to correlate the cytokine concentration with patient outcome measures relating to urgency or urge incontinence. The urinary cytokines chosen were IL-1α, IL-1 receptor antagonist, IL-4, IL-6, IL-8, IL-10, CXCL10 (IP-10), MCP-1 and TNF-α. The presence of bacteriuria stimulated a significant increase in the concentrations of IL-1α (P 0.0216), IL-1 receptor antagonist (P 0.0264), IL-6 (P 0.0003), IL-8 (P 0.0043) and CXCL-10 (P 0.009). Antibiotic treatment significantly attenuated the release of IL-1α (P 0.005), IL-6 (P 0.0027), IL-8 (P 0.0001), IL-10 (P 0.049), and CXCL-10 (P 0.042), i.e. the response to the presence of bacteria was less in the antibiotic treated patients. Across the 26 weeks of the trial, antibiotic treatment reduced the concentration of five of the nine cytokines measured (IL-1α, IL-6, IL-8, IL-10 and CXCL-10) this did not reach significance at every time point. In antibiotic treated patients, the urinary concentration of CXCL-10 correlated positively with four of the six measures of urgency. This study has shown that cytokines associated with activation of the innate immune system (e.g. cytokines chemotactic for or activators of macrophages and neutrophils) are reduced by antibiotic therapy in women with refractory DO. Antibiotic therapy is also associated with symptom improvement in these women, therefore the inflammatory response may have a role in the aetiology of refractory DO.
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0DT00319K
Abstract: A new nickel Schiff base complex shows selective binding behaviour towards quadruplex DNA and cytotoxicity against cancer cells.
Publisher: Hindawi Limited
Date: 2016
DOI: 10.1155/2016/2097219
Abstract: Extracellular adenosine 5′-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1 β , from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally erse isatin derivatives on P2X7-mediated IL-1 β release from murine J774 macrophages. ATP-induced IL-1 β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1 β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo- N -( p -methoxybenzyl)isatin (NAI) and 3- { 4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl } propanoic acid (NAI-imine) enhanced P2X7-induced IL-1 β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N -alkyl-substituted isatins enhance P2X7 receptor-induced IL-1 β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment.
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.VETIMM.2012.09.040
Abstract: Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin-Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5'-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2'(3')-O-(4-benzoylbenzoyl)ATP induced ethidium(+) uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.
Publisher: Wiley
Date: 07-2004
Publisher: Hindawi Limited
Date: 2013
DOI: 10.1155/2013/271813
Abstract: The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium + or YO-PRO-1 2+ uptake into both cell lines. ATP induced ethidium + uptake into EOC13 cells in a concentration-dependent manner, with an EC 50 of ~ 130 μ M. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca 2+ influx and K + efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.
Publisher: Springer Science and Business Media LLC
Date: 11-07-2008
Publisher: Frontiers Media SA
Date: 15-02-2016
Publisher: Wiley
Date: 29-04-2019
DOI: 10.1111/IMCB.12251
Abstract: Allogeneic hematopoietic stem cell transplantation is a curative therapy for a number of hematological malignancies, but is limited by the development of graft-versus-host disease (GVHD). CD39 and CD73 form an ectoenzymatic pathway that hydrolyzes extracellular adenosine 5'-triphosphate (ATP) to adenosine, which respectively exacerbate or alleviate disease in allogeneic mouse models of GVHD. The current study aimed to explore the role of the CD39/CD73 pathway and adenosine receptor (AR) blockade in a humanized mouse model of GVHD. Immunodeficient nonobese diabetic-severe combined immunodeficiency-IL-2 receptor γ
Publisher: MDPI AG
Date: 31-08-2023
Publisher: Springer Science and Business Media LLC
Date: 17-07-2023
Publisher: Wiley
Date: 16-05-2023
DOI: 10.1111/IMCB.12652
Abstract: Graft‐ versus ‐host disease (GVHD) is a life‐threatening complication following donor hematopoietic stem cell transplantation, where donor T cells damage host tissues. This study investigated the effect of tocilizumab (TOC) combined with post‐transplant cyclophosphamide (PTCy) on immune cell engraftment and GVHD development in a humanized mouse model. NOD‐ scid ‐IL2Rγ null (NSG) mice were injected intraperitoneally with 2 × 10 7 human (h) peripheral blood mononuclear cells and cyclophosphamide (33 mg kg −1 ) or saline on days 3 and 4, then TOC or control antibody (0.5 mg mouse −1 ) twice weekly for 28 days. Mice were monitored for clinical signs of GVHD for either 28 or 70 days. Spleens and livers were assessed for human leukocyte subsets, and serum cytokines and tissue histology were analyzed. In the short‐term model (day 28), liver and lung damage were reduced in PTCy + TOC compared with control mice. All groups showed similar splenic hCD45 + leukocyte engraftment (55–60%) however, PTCy + TOC mice demonstrated significantly increased (1.5–2‐fold) splenic regulatory T cells. Serum human interferon gamma was significantly reduced in PTCy + TOC compared with control mice. Long‐term (day 70), prolonged survival was similar in PTCy + TOC (median survival time, 70 days) and PTCy mice (median survival time, 56 days). GVHD onset was significantly delayed in PTCy + TOC, compared with TOC or control mice. Notably, natural killer cells were reduced (77.5%) in TOC and PTCy + TOC mice. Overall, combining PTCy with TOC increases regulatory T cells and reduces clinical signs of early GVHD, but does not improve long‐term survival compared with PTCy alone.
Publisher: Wiley
Date: 21-07-2010
DOI: 10.1111/J.1600-0625.2009.01029.X
Abstract: Extracellular ATP via the activation of purinergic P2 receptors has an emerging role in cutaneous biology however, the distribution of these receptors in mouse skin is poorly defined. This study investigated whether murine epidermal cell subpopulations express functional purinergic P2X(7) receptors. P2X(7) expression was examined by immunoblotting and immunofluorescence staining of epidermal cells from C57Bl/6 mice. P2X(7) function was evaluated by nucleotide-induced ethidium(+) uptake measurements in epidermal cells from C57Bl/6 mice, and from P2X(7) deficient mice and wild-type littermate controls. P2X(7) was detected in whole epidermal cell preparations, and specifically on Langerhans cells (LCs) and keratinocytes (KCs). ATP induced ethidium(+) uptake into LCs and KCs, with EC(50) values of 503 and 482 microm, respectively. BzATP, and to a lesser extent ATPgammaS and ADP, also induced ethidium(+) uptake while UTP, alphabeta-meth-ATP and NAD were ineffective. ATP-induced ethidium(+) uptake was impaired by Na(+) and Mg(2+), and the P2X(7) antagonist, A-438079 and was absent in LCs and KCs from P2X(7) deficient mice. These results demonstrate that murine LCs and KCs express functional P2X(7), and support a role for this receptor in cutaneous biology.
Publisher: Elsevier BV
Date: 04-2001
Publisher: Springer Science and Business Media LLC
Date: 11-05-2001
Abstract: B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.JIM.2007.06.002
Abstract: The P2X(7) receptor is a ligand-gated cation channel activated by extracellular ATP and highly expressed on monocytes, macrophages and lymphocytes. Activation of this receptor by exposure to extracellular ATP opens a selective cation channel that allows Ca(2+) and Ba(2+) influx, and K(+) efflux. Over the first minute the channel adopts a second and larger permeability state allowing the uptake of ethidium(+), followed by a cascade of intracellular downstream effects. Current methods used to study the P2X(7) receptor function, do not give quantitative measurement in sub-populations of a mixed cell suspension. We describe a quantitative method to determine the P2X(7) receptor function using time-resolved two-colour flow cytometry by assessing ATP-induced ethidium(+) uptake. Practical factors such as ethidium bromide concentration, agonists, temperature and buffers are also studied. Moreover, the ATP-induced ethidium(+) uptake method is compared to ATP induced barium (Ba(2+)) influx with Fura-Red. These two compatible methods can be used to screen the channel ore function of the cell surface P2X(7) receptor among in iduals and the results may be useful to estimate susceptibility of subjects to certain infectious diseases.
Publisher: Wiley
Date: 06-2001
DOI: 10.1002/DDR.1173
Abstract: Extracellular ATP has been shown to induce apoptotic death of many cell types of hematopoietic origin. This action of ATP is mediated via activation of P2X 7 purinergic receptors, which show the unusual property of time‐dependent channel dilation to accept permeants as large as ethidium cation (314 Da). P2X 7 function, measured by area under the ATP‐induced ethidium uptake curve, was 5‐fold greater for monocytes than lymphocytes, while polymorphs and platelets showed no ethidium uptake. Expression of P2X 7 receptor, measured by the binding of a monoclonal antibody, was also 5‐fold greater on monocytes than lymphocytes. However, in some subjects, both normal and with chronic lymphocytic leukemia, the P2X 7 receptor was nonfunctional despite good expression of P2X 7 protein. Three single nucleotide polymorphisms were found in the P2X 7 cDNA coding region, one of which correlated with P2X 7 function. Thus, the homozygous substitution of alanine for glutamic acid at amino acid 496 led to complete loss of function of the P2X 7 receptor, while the heterozygous polymorphism gave function half that of the germline P2X 7 receptor. Drug Dev. Res. 53:72–76, 2001. © 2001 Wiley‐Liss, Inc.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.VETIMM.2013.11.002
Abstract: Binding of extracellular adenosine 5'-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs.
Publisher: MDPI AG
Date: 04-05-2023
DOI: 10.3390/IJMS24098225
Abstract: The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.
Publisher: Springer Science and Business Media LLC
Date: 21-06-2013
Publisher: Oxford University Press (OUP)
Date: 24-07-2017
DOI: 10.1111/CEI.13005
Abstract: Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.
Publisher: Wiley
Date: 27-11-2019
DOI: 10.1111/AGE.12884
Abstract: Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random s le of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within in idual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded.
Publisher: Springer Science and Business Media LLC
Date: 28-04-2022
DOI: 10.1007/S11302-022-09863-5
Abstract: Mutant superoxide dismutase 1 (SOD1) can be constitutively released from motor neurons and transmitted to naïve motor neurons to promote the progression of amyotrophic lateral sclerosis (ALS). However, the biological impacts of this process and the precise mechanisms of SOD1 release remain to be fully resolved. Using biochemical and fluorescent techniques, this study aimed to determine if P2X7 receptor activation could induce mutant SOD1 release from motor neurons and whether this released SOD1 could be transmitted to motor neurons or microglia to mediate effects associated with neurodegeneration in ALS. Aggregated SOD1
Publisher: MDPI AG
Date: 16-09-2022
DOI: 10.3390/BIOM12091309
Abstract: P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.
Publisher: Springer US
Date: 2022
DOI: 10.1007/978-1-0716-2384-8_18
Abstract: Humanized mouse models of graft-versus-host disease (GVHD), where human immune cells are injected into immune deficient mice, are well established and provide opportunities to investigate pathways involved in GVHD development. This chapter provides an overview of human immune cell isolation, injection of these cells into immune deficient mice, monitoring of mice for signs of GVHD, and assessment of human cell engraftment using flow cytometry. Further, this chapter focuses on the P2X7 signaling pathway involved in GVHD, and describes a strategy to block the P2X7 receptor and examine the effect of this on GVHD development.
Publisher: Frontiers Media SA
Date: 28-01-2021
DOI: 10.3389/FCIMB.2020.596023
Abstract: Invasive infections due to group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates 5448 and animal passaged variant 5448AP. Co-incubation of neutrophils with 5448AP resulted in proliferation of GAS and lowered the production of reactive oxygen species when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 and caspase-4 expression in vitro , with inflammatory caspase activation detected in vitro and in vivo . GAS infections involving strains such as 5448AP that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the severity of invasive GAS infections.
Publisher: CSIRO Publishing
Date: 2017
DOI: 10.1071/CH17062
Abstract: The ability of aurothiomalate and auranofin to alter the production of several cellular mediators of inflammation by RAW264.7 macrophages, was compared with each other and that of gold nanoparticles (Au NPs). Addition of auranofin was found to have a pronounced ability to lower the production of reactive nitrogen and oxygen species (RNS and ROS respectively), as well as interleukin-10 (IL-10) and tumour necrosis factor (TNF), by macrophages that were subsequently treated with lipopolysaccharide (LPS) to stimulate production of the mediators. In contrast, prior treatment of the cells with either aurothiomalate or Au NPs had either little or no significant effect on production of RNS and ROS. Treatment of the macrophages with Au NPs had a small effect on production of TNF by cells that were subsequently stimulated with LPS however, the effect was much smaller than that elicited by auranofin. Similarly, aurothiomalate had a small but significant effect on production of IL-10. Varying the size of the Au NPs or the identity of the protective sheath surrounding the nanoparticles did not have a significant effect on the production of RNS or ROS by LPS-stimulated macrophages. The results of some of these investigations are discussed in the light of other studies reported in the literature. In addition, results obtained by scanning electron microscopy and energy-dispersive X-ray spectroscopy are presented that provide evidence for the accumulation of gold within macrophages exposed to Au NPs.
Publisher: MDPI AG
Date: 20-05-2022
DOI: 10.3390/IJMS23105739
Abstract: The adenosine 5′-triphosphate-gated P2X4 receptor channel is a promising target in neuroinflammatory disorders, but the ability to effectively target these receptors in models of neuroinflammation has presented a constant challenge. As such, the exact role of P2X4 receptors and their cell signalling mechanisms in human physiology and pathophysiology still requires further elucidation. To this end, research into the molecular mechanisms of P2X4 receptor activation, modulation, and inhibition has continued to gain momentum in an attempt to further describe the role of P2X4 receptors in neuroinflammation and other disease settings. Here we provide an overview of the current understanding of the P2X4 receptor, including its expression and function in cells involved in neuroinflammatory signalling. We discuss the pharmacology of P2X4 receptors and provide an overview of P2X4-targeting molecules, including agonists, positive allosteric modulators, and antagonists. Finally, we discuss the use of P2X4 receptor modulators and antagonists in models of neuroinflammatory cell signalling and disease.
Publisher: Springer Science and Business Media LLC
Date: 11-10-2022
Publisher: Springer Science and Business Media LLC
Date: 05-2004
Publisher: Wiley
Date: 04-2010
DOI: 10.1096/FJ.09-150862
Abstract: The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.
Publisher: Wiley
Date: 02-03-2023
DOI: 10.1002/JBM.A.37524
Abstract: The current study investigates the therapeutic and optical properties of bismuth oxide (Bi 2 O 3 ) particles for selective melanoma therapy and prevention. The Bi 2 O 3 particles were prepared using a standard precipitation method. The Bi 2 O 3 particles induced apoptosis in human A375 melanoma cells but not human HaCaT keratinocytes or CCD‐1090Sk fibroblast cells. This selective apoptosis appears to be associated with a combination of factors: increased particle internalization (2.29 ± 0.41, 1.16 ± 0.08 and 1.66 ± 0.22‐fold of control) and enhanced production of reactive oxygen species (ROS) (3.4 ± 0.1, 1.1 ± 0.1 and 2.05 ± 0.17‐fold of control) in A375 cells compared to HaCaT and CCD‐1090SK cells, respectively. As a high‐Z element, bismuth is also an excellent contrast agent for computer tomography, which renders Bi 2 O 3 a theranostic material. Moreover, Bi 2 O 3 displays high UV absorption and low photocatalytic activity compared to other semiconducting metal oxides, which opens further potential fields of application as a pigment or as an active ingredient in sunscreens. Overall, this study demonstrates the multifunctional properties of Bi 2 O 3 particles surrounding the treatment and prevention of melanoma.
Publisher: American Thoracic Society
Date: 15-02-2007
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 13-06-2014
Abstract: The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflammatory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described.
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.BBAMEM.2010.07.022
Abstract: The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium(+) uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium(+) uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium(+) uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.
Publisher: Elsevier BV
Date: 10-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0TB00629G
Abstract: Low photocatalytic CeO 2 /TiO 2 nanocomposite particles with high UV attenuation and reduced ROS generation for application in sunscreen products.
Publisher: Wiley
Date: 02-2001
DOI: 10.1046/J.1440-1711.2001.00967.X
Abstract: There is little evidence that cutaneous dendritic cells (DC), including epidermal Langerhans cells (LC), can induce immunity to UV radiation (UVR)-induced skin tumours. Here, it is shown that cells within skin can induce protective antitumour immunity against a UVR-induced fibrosarcoma. Transplantation of the skin overlying subcutaneous tumours onto naïve recipients could induce protective antitumour immunity, probably because the grafting stimulated the tumour Ag-loaded DC to migrate to local lymph nodes. This suggests that cutaneous APC can present tumour Ag to induce protective antitumour immunity. Previously, it has been shown that immunization of mice with MHC class II+ epidermal cells (EC) pulsed with tumour extracts could induce delayed-type hypersensitivity against tumour cells. Here, this same immunization protocol could induce protective immunity against a minimum tumorigenic dose of UVR-induced fibrosarcoma cells, but not higher doses. Epidermal cells obtained from semiallogeneic donors and pulsed with tumour extract could also induce protective immunity. However, presentation of BSA Ag from the culture medium was found to contribute to this result using semiallogeneic EC. The results suggest that LC overlying skin tumours may be able to induce protective immunity to UVR-induced tumours if stimulated to migrate from the skin.
Publisher: Springer Science and Business Media LLC
Date: 07-03-2022
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.VETIMM.2012.05.004
Abstract: P2X7, a damage-associated molecular pattern receptor and adenosine 5'-triphosphate (ATP)-gated cation channel, plays an important role in the activation of the NALP3 inflammasome and subsequent release of interleukin (IL)-1β from human monocytes however its role in monocytes from other species including the dog remains poorly defined. This study investigated the role of P2X7 in canine monocytes, including its role in IL-1β release. A fixed-time flow cytometric assay demonstrated that activation of P2X7 by extracellular ATP induces the uptake of the organic cation, YO-PRO-1(2+), into peripheral blood monocytes from various dog breeds, a process impaired by the specific P2X7 antagonist, A438079. Moreover, in five different breeds, relative P2X7 function in monocytes was about half that of peripheral blood T cells but similar to that of peripheral blood B cells. Reverse transcription-PCR demonstrated the presence of P2X7, NALP3, caspase-1 and IL-1β in LPS-primed canine monocytes. Immunoblotting confirmed the presence of P2X7 in LPS-primed canine monocytes. Finally, extracellular ATP induced YO-PRO-1(2+) uptake into and IL-1β release from these cells, with both processes impaired by A438079. These results demonstrate that P2X7 activation induces the uptake of organic cations into and the release of IL-1β from canine monocytes. These findings indicate that P2X7 may play an important role in IL-1β-dependent processes in dogs.
Publisher: MDPI AG
Date: 24-05-2023
DOI: 10.3390/IJMS24119196
Abstract: Since its inception by the late Geoffrey Burnstock in the early 1970s [...]
Publisher: Springer Science and Business Media LLC
Date: 23-05-2001
DOI: 10.1007/PL00006685
Abstract: In this study we compared the effects of subinflammatory and inflammatory doses of solar-simulated ultraviolet (UV) radiation on enhancement of skin tumor growth, sensitization to haptens and cellular changes within the epidermis of C3H/HeN mice. Tumors transplanted into mice 3 days after exposure to inflammatory, but not subinflammatory, doses of UV radiation had a higher growth rate than those tumors inoculated into unirradiated control mice. Both doses of UV radiation suppressed the induction of contact hypersensitivity and induced tolerance when hapten was painted onto the skin 3 days after irradiation. Skin exposed to the higher, but not the lower, dose of UV radiation contained significantly increased numbers of CD11b+, CD45+ MHC class II- and CD45+ MHC class II(hi) inflammatory cells 3 days post-irradiation. The immunosuppression correlated with a reduction in Langerhans cells and dendritic epidermal T cells. Collectively, this suggests that suppression to contact sensitizers is due to the UV radiation effects on Langerhans cells and dendritic epidermal T cells. While these effects may also suppress the induction of anti-tumor immunity, at higher doses of UV radiation inflammatory cells may enhance tumor growth by a non-immunological mechanism.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C6RA25328H
Abstract: Sheathless particle focusing and separation in viscoelastic fluid is demonstrated using an integrated ECCA (straight channel section with asymmetrical expansion–contraction cavity arrays) straight channel.
Publisher: Informa UK Limited
Date: 07-02-2019
DOI: 10.1080/10408444.2019.1579780
Abstract: Nanotechnology has the potential to bring about revolutionary changes in manufacturing products, including sunscreens. However, a knowledge gap between benefits and detriments of engineered nano-materials used in sunscreens exists, which gives rise to safety concerns. This article is concerned with the protection of consumers without impairing the embellishment of this promising technology. It is widely argued that the harm associated with nano-sunscreens may only occur under certain conditions related mainly to users skin vulnerability, which can be avoided by informed and careful use of such a product. We thus recognize the need for fostering the growth of nanotech simultaneously with preventing potential harm. We revisit the Australian sunscreens regulatory policies, which embrace a "wait and see" approach, through the lens of regulatory policies in the European Union (EU) that are influenced by a "precautionary principle." We highlight the importance of informing consumers about the sunscreen they are using and recommend that product labels should disclose the presence of nano-ingredients in line with the EU disclosure requirements. This will allow users to carefully apply the product in order to avoid any potential harm and to protect manufacturers from possible costly litigation in future. This can be achieved through a combined collaborative effort of regulators, supply chain entities, and end users.
Publisher: The American Association of Immunologists
Date: 15-11-2003
DOI: 10.4049/JIMMUNOL.171.10.5442
Abstract: Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X7 pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-primed, mycobacteria-infected macrophages from wild-type in iduals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from in iduals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X7-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.
Publisher: Wiley
Date: 03-1998
DOI: 10.1046/J.1365-2222.1998.00223.X
Abstract: Both genetic and environmental factors are thought to contribute to specific IgE responses, however, the relative contribution of each in the responses to in idual ryegrass pollen allergens is largely unknown even though some responses to allergens have been linked to certain HLA complexes. Using a large group of monozygotic and dizygotic twins, this study was designed to investigate the IgE binding profiles of in idual ryegrass pollen (Lolium perenne) components to assess the relative contribution of genetic and environmental factors in determining IgE responses to specific allergens. Ryegrass pollen proteins were separated by electrophoresis and immunoblotted with sera from 191 pairs of twins where at least one sibling had a SPT > 2 mm to perennial ryegrass. Concordance levels for in idual ryegrass pollen components were compared between monozygotic and dizygotic twins in a subset group where both twins had SPT > 3 mm to perennial ryegrass. Immunoblotting revealed 23 in idual IgE-binding components from ryegrass pollen. Although there was a significantly greater proportion of monozygotic twins with SPT wheals greater than 3 mm when compared with the dizygotic twins, the mean case-wise concordance for the immunoblot components was similar for both groups of twins. The mean case-wise concordance when at least four pairs of sera were involved was 44% for the MZ twins (n=11 components) and 45% for the DZ twins (n=12 components). We found no significant differences in concordance levels for any of the 23 in idual components including allergens previously associated with HLA. Evidence for genetic control of allergen-specific IgE responses in a large population s le of twins to in idual ryegrass allergens is limited, indicating that the IgE responses to specific ryegrass pollen allergens are determined largely by environmental factors.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3EY00239J
Publisher: Elsevier BV
Date: 2006
Publisher: Wiley
Date: 30-03-2015
DOI: 10.1111/TRF.13101
Abstract: Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5'-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl- fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks. The propensity of RBCs to undergo P2X7-mediated PS exposure does not alter during in vivo and ex vivo aging. Thus, P2X7 activation is unlikely to be involved in the removal of senescent RBCs or stored RBCs after transfusion.
Publisher: Elsevier BV
Date: 03-2013
DOI: 10.1016/J.BBRC.2013.01.134
Abstract: Activation of the purinergic P2X7 receptor by extracellular ATP induces the shedding of cell-surface molecules including the low-affinity IgE receptor, CD23 from leukocytes. CD23 is a known substrate of a disintegrin and metalloprotease (ADAM)10. The aim of the current study was to determine if P2X7 activation induced the shedding of the chemokine CXCL16, an ADAM10 substrate. Using immunolabelling and flow cytometry we demonstrate that human RPMI 8226 multiple myeloma B cells, which have been previously shown to express P2X7, also express CXCL16. Flow cytometric and ELISA measurements of ATP-induced loss of cell-surface CXCL16 showed that ATP treatment of RPMI 8226 cells induced the rapid shedding of CXCL16. Treatment of RPMI 8226 cells with the specific P2X7 antagonists, AZ10606120 and KN-62 impaired ATP-induced CXCL16 shedding by ~86% and ~90% respectively. RT-PCR demonstrated that ADAM10 is expressed in these cells and treatment of cells with the ADAM10 inhibitor, GI254023X, impaired ATP-induced CXCL16 shedding by ~87%. GI254023X also impaired P2X7-induced CD23 shedding by ∼57%. This data indicates that human P2X7 activation induces the rapid shedding of CXCL16 and that this process involves ADAM10.
Publisher: Oxford University Press (OUP)
Date: 07-2005
DOI: 10.1086/430622
Abstract: Stimulation of the P2X7 purinergic receptor (P2X7) in bacille Calmette-Guerin (BCG)-infected human macrophages with extracellular adenosine triphosphate (ATP) leads to pore formation and killing of mycobacteria. We examined the effect of polymorphisms in the P2X7 gene (P2X7) on the capacity of macrophages to kill mycobacteria. Polymorphisms and mutations in P2X7 were identified by both DNA sequence analysis and determination of uptake of ethidium by time-resolved flow cytometry. Macrophages from affected subjects were infected with Mycobacterium bovis BCG. Apoptosis was determined by use of Annexin V staining, and BCG growth was determined by use of quantitative mycobacterial cultures. Three new mutations were identified. Macrophages from subjects heterozygous for a polymorphism in P2X7 had a 50% reduction in uptake of ethidium and a 75% reduction in the number of apoptotic cells, compared with macrophages from wild-type (wt) subjects, after stimulation with interferon (IFN)- gamma and ATP. Furthermore, after stimulation with IFN- gamma and ATP, there was a reduction in BCG growth of up to approximately 0.5 log10 in macrophages from single-heterozygous subjects, compared with a reduction of 1.0 log10 in macrophages from wt subjects. Interestingly, BCG-infected macrophages from compound-heterozygous subjects, for different combinations of polymorphisms in P2X7, had no uptake of ethidium, failed to undergo apoptosis, and were unable to kill mycobacteria after stimulation with IFN- gamma and ATP. Various polymorphisms in P2X7 abrogate IFN- gamma /ATP-induced killing of mycobacteria by human macrophages and, thus, may contribute to variability in susceptibility to mycobacterial infections.
Publisher: Elsevier BV
Date: 10-2004
Publisher: Elsevier BV
Date: 04-1998
DOI: 10.1016/S0091-6749(98)70356-2
Abstract: Although some studies have shown genetic control of specific IgE responses to purified grass allergens, studies with other allergens have not supported this. The extent of such control for house dust mite (HDM) (Dermatophagoides pteronyssinus) allergens is unclear. We sought to determine the extent to which genetic factors control the specificity of IgE responses to in idual HDM allergens by comparing the immunoblot patterns of IgE binding of serum from monozygotic and dizygotic members of a large cohort of Australian twins. HDM proteins separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis were immunoblotted with sera from 317 twin pairs in which at least one twin had at least a weak HDM skin test response. Concordance levels for IgE binding to the in idual HDM components were compared in the subset of 142 pairs of twins in which both twins were allergic to HDMs (skin prick test wheal diameter, > 3 mm). Over all 36 blotted bands, the mean case-wise concordance was 41% for monozygotic twins and 17% for dizygotic twins. Of the components detected, only those of molecular weights 23 kd and 16 kd were significantly different between the groups (p < 0.01). Differences observed between the monozygotic and dizygotic twins could be partly explained by overall IgE hyperresponsiveness. Evidence for genetic control of IgE responses to 36 IgE-binding HDM components from a large s le of twins showed significant differences in concordance for two components and nonsignificant differences for several others. In the monozygotic twins, concordance never exceeded 67% for any band, and most monozygotic in iduals recognized components their co-twin did not. Genetic control of overall atopy in monozygotic twins is far stronger than that controlling specific sensitization to HDM allergens.
Publisher: Elsevier BV
Date: 2015
DOI: 10.1016/J.JINORGBIO.2014.09.013
Abstract: Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.
Publisher: Wiley
Date: 16-04-2020
DOI: 10.1111/IMCB.12328
Publisher: Wiley
Date: 26-08-2015
DOI: 10.1038/ICB.2014.69
Abstract: Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.
Publisher: Elsevier BV
Date: 09-2023
Publisher: Elsevier BV
Date: 07-2019
DOI: 10.1016/J.INTIMP.2019.04.037
Abstract: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A
Publisher: Frontiers Media SA
Date: 29-03-2022
Publisher: Frontiers Media SA
Date: 28-10-2015
Publisher: Medknow
Date: 2017
Publisher: American Chemical Society (ACS)
Date: 09-06-2021
Publisher: Frontiers Media SA
Date: 02-10-2020
Publisher: Wiley
Date: 11-10-2011
DOI: 10.1111/J.1399-0039.2011.01780.X
Abstract: The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel ore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.
Publisher: Springer Science and Business Media LLC
Date: 03-2020
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.CELLIMM.2018.12.001
Abstract: Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.
Publisher: Bentham Science Publishers Ltd.
Date: 04-2011
DOI: 10.2174/187221511794839219
Abstract: The human P2X7 receptor is a trimeric ligand-gated cation channel coded by the P2XR7 gene located at chromosome position 12q24. P2X7 is expressed in a wide variety of normal and disease-associated cell types. Activation of this receptor by extracellular adenosine 5'-triphosphate results in numerous downstream events including the release of pro-inflammatory mediators, cell proliferation or death, and killing of intracellular pathogens. As a result, P2X7 plays important roles in inflammation, immunity, bone homeostasis, neurological function and neoplasia. The P2XR7 gene encodes a P2X7 subunit 595 amino acids in length, however splice isoforms that can alter receptor expression and function, and modify the signaling properties downstream of receptor activation also exist. Moreover, the relative amount of P2X7 function varies between human in iduals due to numerous single nucleotide polymorphisms resulting in either loss- or gain-of-function. Combinations of these polymorphisms give rise to various haplotypes that can also modify P2X7 function. Collectively, P2X7, and its splice and polymorphic variants are attracting considerable interest in relation to human health and disease, including the development and publication of a number of patents.
Publisher: Medknow
Date: 2023
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.BBRC.2007.11.008
Abstract: A panel of 18 protein tyrosine kinase antagonists were tested for their inhibitory effect on human P2X(7) receptor-mediated (86)Rb(+) (K(+)) efflux. The most potent compound (compound P), a phthalazinamine derivative and an inhibitor of vascular endothelial growth factor receptor kinase, blocked ATP-induced (86)Rb(+)-efflux in human B-lymphocytes and erythrocytes by 76% and 66%, respectively. This inhibition was dose-dependent in both cell types with an IC(50) of approximately 5muM. Kinetic analysis showed compound P was a non-competitive inhibitor of P2X(7). This compound also inhibited ATP-induced ethidium(+) influx into B-lymphocytes and P2X(7)-transfected-HEK-293 cells, as well as ATP-induced (86)Rb(+)-efflux from canine erythrocytes. Externally, but not internally, applied compound P impaired ATP-induced inward currents in P2X(7)-transfected-HEK-293 cells. This study demonstrates that a novel protein tyrosine kinase antagonist directly impairs native and recombinant human P2X(7) receptors. The data suggests that antagonists which target ATP-binding sites of kinases may potentially block the P2X(7) receptor.
Publisher: Wiley
Date: 02-1996
DOI: 10.1111/J.1600-0625.1996.TB00090.X
Abstract: This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one ex le of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.
Publisher: American Physiological Society
Date: 15-07-2014
DOI: 10.1152/PHYSIOLGENOMICS.00195.2013
Abstract: The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random s le of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-cl measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between in idual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.BBRC.2007.01.124
Abstract: Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X(7) receptor, an ATP-gated cation channel. The two most potent P2X(7) agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP>ATP>2MeSATP>ATPgammaS however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC(50) of approximately 75 microM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X(7) receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca(2+). These results show that P2X(7) activation by extracellular ATP can induce PS exposure in erythrocytes.
Publisher: Wiley
Date: 13-06-2021
DOI: 10.1111/IMM.13374
Abstract: Graft‐versus‐host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) that develops when donor T cells in the graft become reactive against the host. Post‐transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo‐HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NOD‐ scid ‐IL2Rγ null mice were injected intraperitoneally (i.p.) with 20 × 10 6 human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg −1 ) (PTCy‐mice) or saline (saline‐mice) (days 3 and 4). Mice were assessed for T‐cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSE low ) human (h) CD3 + T cells in PTCy‐mice compared with saline‐mice. Over 10 weeks, PTCy‐mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline‐mice. PTCy‐mice also demonstrated increased splenic hCD4 + :hCD8 + T‐cell ratios and reduced splenic Tregs (hCD4 + hCD25 + hCD127 lo ) compared with saline‐mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.BBAGEN.2010.07.001
Abstract: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC₅₀ of ~116 μM, and this uptake was reduced in the presence of extracellular Ca²+ and Mg²+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.
Publisher: Wiley
Date: 14-04-2005
DOI: 10.1016/J.FEBSLET.2005.03.091
Abstract: The P2X(7) gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X(7) function. A splice site mutation (g-->t) was found at position +1 of the first intron of the P2X(7) gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A-->C polymorphism of the P2X(7) gene. RT-PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X(7) null allele in 1-2% of the Caucasian population.
Publisher: Elsevier BV
Date: 05-2003
Publisher: Elsevier BV
Date: 09-2005
Publisher: Baishideng Publishing Group Inc.
Date: 2016
DOI: 10.5315/WJH.V5.I4.88
Publisher: MDPI AG
Date: 08-08-2023
Abstract: The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV+ patients contained increased proportions of CD39−CD73−CD4+ T cells and reduced proportions of CD39−/+CD73+CD4+ T cells compared to the equivalent cells from HPV− patients. Notably, the pretreatment CD4+:CD8+ T cell ratios and CD39+CD73+CD19+ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.
Publisher: Baishideng Publishing Group Inc.
Date: 2016
DOI: 10.5314/WJD.V5.I2.72
Publisher: American Chemical Society (ACS)
Date: 17-08-2017
DOI: 10.1021/ACS.ANALCHEM.7B02671
Abstract: This work investigates the on-chip washing process of microparticles and cells using coflow configuration of viscoelastic fluid and Newtonian fluid in a straight microchannel. By adding a small amount of biocompatible polymers into the particle medium or cell culture medium, the induced viscoelasticity can push particles and cells laterally from their original medium to the coflow Newtonian medium. This behavior can be used for particle or cell washing. First, we demonstrated on-chip particle washing by the size-dependent migration speed using coflow of viscoelastic fluid and Newtonian fluid. The critical particle size for efficient particle washing was determined. Second, we demonstrated continuous on-chip washing of Jurkat cells using coflow of viscoelastic fluid and Newtonian fluid. The lateral migration process of Jurkat cells along the channel length was investigated. In addition, the cell washing quality was verified by hemocytometry and flow cytometry with a recovery rate as high as 92.8%. Scanning spectrophotometric measurements of the media from the two inlets and the two outlets demonstrated that diffusion of the coflow was negligible, indicating efficient cell washing from culture medium to phosphate-buffered saline medium. This technique may be a safer, simpler, cheaper, and more efficient alternative to the tedious conventional centrifugation methods and may open up a wide range of biomedical applications.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2008
DOI: 10.1007/S00134-008-1156-Y
Abstract: Critical illness is associated with tissue damage, inflammation and the development of immune dysfunction. Leukocyte reprogramming occurs leading to insufficient production of pro-inflammatory cytokines upon subsequent stimulation. Cellular nucleotides released during tissue damage act via purinergic receptors to modulate immune function. We hypothesized that extracellular nucleotides in concentrations similar to those found near injured and ischemic tissues will modulate cytokine secretion. Bench study in 28 healthy human volunteers using standardized lipopolysaccharide (LPS) stimulated ex vivo whole blood cultures (ILCS). The Nepean Hospital Laboratories, University of Sydney. Nucleotides ATP, ADP and other P2 purinergic receptor agonists ATPgammaS, BzATP, UTP and P1 agonist CV1808 were injected into the ILCS, and cultured for 6, 12 and 24 h as indicated. ATP (100 muM) reduced the LPS stimulated secretion of TNFalpha at 6 and 12 h, as well as IL-12(p70) and IFNgamma at 24 h of incubation. ADP, ATPgammaS, BzATP, and CV1808, but not UTP displayed IL-12(p70) and IFNgamma reducing effect similar to ATP. Higher ATP concentration (500 muM) had even more pronounced immunosuppressive effect. Nucleotides had variable effect on stimulated IL-10 secretion. ATP and ADP at high-micromolar concentrations reduce secretion of the main Th1 cytokines TNFalpha, IL-12(p70) and IFNgamma in LPS stimulated human blood. As immune dysfunction associated with critical illness is characterized by decreased TNFalpha, IL-12 and IFNgamma production by leukocytes, extracellular nucleotides might contribute to its pathogenesis [corrected]
Publisher: Wiley
Date: 30-04-2004
Publisher: Springer Science and Business Media LLC
Date: 12-12-2018
Publisher: American Physiological Society
Date: 06-2020
DOI: 10.1152/AJPCELL.00408.2019
Abstract: The ubiquitous calpains, calpain-1 and -2, play important roles in Ca 2+ -dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca 2+ -dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 ( CAPNS1 −/− ) virtually ablates Ca 2+ -dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 ( CAPN1 −/− ) or -2 ( CAPN2 −/− ) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 ( CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca 2+ -signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.
Publisher: Wiley
Date: 10-05-2023
DOI: 10.1002/CTD2.197
Publisher: LIDSEN Publishing Inc
Date: 06-06-2022
DOI: 10.21926/OBM.TRANSPLANT.2203166
Abstract: Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematological malignancies, but its efficacy is limited by graft-versus-host disease (GVHD). This life-threatening disorder develops when donor (graft) immune cells cause inflammatory damage to recipient (host) tissues. The immune cell receptor channel P2X7 and its ligand adenosine 5’-triphosphate (ATP) have been implicated in GVHD pathogenesis. Therefore, this signaling axis represents a potential therapeutic target. This study aimed to investigate if the specific P2X7 antagonist AZ10606120 (AZ10) could prevent GVHD development in a preclinical, humanized mouse model, in which NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are injected with human peripheral blood mononuclear cells (hPBMCs). Flow cytometric measurements of ATP-induced cation dye uptake revealed that AZ10 blocked P2X7 activity in human RPMI 8226 multiple myeloma cells (IC50 of 1 ± 1 nM) and murine RAW 264.7 macrophages (IC50 of 3 ± 1 nM), as well as primary donor CD4+ and CD8+ T cells. However, AZ10 (2 mg/kg), injected intraperitonealy (i.p.) daily for the first 10 days post-hPBMC injection, did not reduce clinical or histological GVHD development in mice. AZ10 did not impact engraftment of human leukocytes, predominantly CD4+ and CD8+ T cells. However, AZ10 increased serum human interferon gamma (hIFN-γ) concentrations, with CD8+ T cells being the main hIFN-γ producing T cell subset. In conclusion, this study suggests a role for P2X7 activation in impairing hIFN-γ production during GVHD pathogenesis, with the use of P2X7 blockade as a therapeutic strategy warranting further investigation.
Publisher: Elsevier BV
Date: 03-2002
Publisher: Informa UK Limited
Date: 02-12-2017
DOI: 10.1080/15257770.2017.1391395
Abstract: The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate (ATP). Patch cl measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium
Publisher: Portland Press Ltd.
Date: 02-2021
DOI: 10.1042/BSR20203827
Abstract: We sought to determine the effect of time and temperature of blood s le storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood s les from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved s les. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.
Publisher: Springer Science and Business Media LLC
Date: 18-04-2019
Publisher: American Physiological Society
Date: 11-2007
DOI: 10.1152/AJPREGU.00166.2007
Abstract: Over three decades ago, Parker and Snow ( Am J Physiol 223: 888–893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel ore-forming P2X 7 receptor on canine erythrocytes. P2X 7 receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced 86 Rb + (K + ) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X 7 agonist 2′(3′)- O-(4-benzoylbenzoyl)adenosine 5′-trisphosphate (BzATP) was more potent than ATP, and both stimulated 86 Rb + efflux from erythrocytes in a dose-dependent fashion with EC 50 values of ∼7 and ∼309 μM, respectively. 2-Methylthioadenosine 5′-triphosphate and adenosine 5′- O-(3-thiotriphosphate) induced a smaller 86 Rb + efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced 86 Rb + efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X 7 antagonists. ATP also induced uptake of choline + into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in % of cells and caused up to 20% hemolysis. In contrast, % of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium + uptake showed that P2X 7 function was three times lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X 7 receptor channel ore.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2023
Publisher: Elsevier BV
Date: 02-2023
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.BBAMEM.2010.06.002
Abstract: Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium+ uptake into MEL cells in a concentration-dependent fashion and with an EC(50) of approximately 154 microM. The most potent P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium+ uptake. ATP-induced ethidium+ and YO-PRO-1(2+) uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Springer Science and Business Media LLC
Date: 17-07-2019
Start Date: 2023
End Date: 12-2023
Amount: $731,584.00
Funder: Australian Research Council
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