Activation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1B release from canine monocytes

P2X7, a damage-associated molecular pattern receptor and adenosine 5\u27-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-12+, 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 LPSprimed canine monocytes. Immunoblotting confirmed the presence of P2X7 in LPS-primed canine monocytes. Finally, extracellular ATP induced YO-PRO-12+ 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

R270C polymorphism leads to loss of function of the canine P2X7 receptor

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 sample 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-clamp 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 individual 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

Pharmacological and genetic characterisation of the canine P2X4 receptor

Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca 2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key Results: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2′(3′)-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans

Mapping past human land use using archaeological data: A new classification for global land use synthesis and data harmonization.

In the 12,000 years preceding the Industrial Revolution, human activities led to significant changes in land cover, plant and animal distributions, surface hydrology, and biochemical cycles. Earth system models suggest that this anthropogenic land cover change influenced regional and global climate. However, the representation of past land use in earth system models is currently oversimplified. As a result, there are large uncertainties in the current understanding of the past and current state of the earth system. In order to improve representation of the variety and scale of impacts that past land use had on the earth system, a global effort is underway to aggregate and synthesize archaeological and historical evidence of land use systems. Here we present a simple, hierarchical classification of land use systems designed to be used with archaeological and historical data at a global scale and a schema of codes that identify land use practices common to a range of systems, both implemented in a geospatial database. The classification scheme and database resulted from an extensive process of consultation with researchers worldwide. Our scheme is designed to deliver consistent, empirically robust data for the improvement of land use models, while simultaneously allowing for a comparative, detailed mapping of land use relevant to the needs of historical scholars. To illustrate the benefits of the classification scheme and methods for mapping historical land use, we apply it to Mesopotamia and Arabia at 6 kya (c. 4000 BCE). The scheme will be used to describe land use by the Past Global Changes (PAGES) LandCover6k working group, an international project comprised of archaeologists, historians, geographers, paleoecologists, and modelers. Beyond this, the scheme has a wide utility for creating a common language between research and policy communities, linking archaeologists with climate modelers, biodiversity conservation workers and initiatives

Mapping past human land use using archaeological data: A new classification for global land use synthesis and data harmonization

In the 12,000 years preceding the Industrial Revolution, human activities led to significant changes in land cover, plant and animal distributions, surface hydrology, and biochemical cycles. Earth system models suggest that this anthropogenic land cover change influenced regional and global climate. However, the representation of past land use in earth system models is currently oversimplified. As a result, there are large uncertainties in the current understanding of the past and current state of the earth system. In order to improve representation of the variety and scale of impacts that past land use had on the earth system, a global effort is underway to aggregate and synthesize archaeological and historical evidence of land use systems. Here we present a simple, hierarchical classification of land use systems designed to be used with archaeological and historical data at a global scale and a schema of codes that identify land use practices common to a range of systems, both implemented in a geospatial database. The classification scheme and database resulted from an extensive process of consultation with researchers worldwide. Our scheme is designed to deliver consistent, empirically robust data for the improvement of land use models, while simultaneously allowing for a comparative, detailed mapping of land use relevant to the needs of historical scholars. To illustrate the benefits of the classification scheme and methods for mapping historical land use, we apply it to Mesopotamia and Arabia at 6 kya (c. 4000 BCE). The scheme will be used to describe land use by the Past Global Changes (PAGES) LandCover6k working group, an international project comprised of archaeologists, historians, geographers, paleoecologists, and modelers. Beyond this, the scheme has a wide utility for creating a common language between research and policy communities, linking archaeologists with climate modelers, biodiversity conservation workers and initiatives

Mapping past human land use using archaeological data: A new classification for global land use synthesis and data harmonization

In the 12,000 years preceding the Industrial Revolution, human activities led to significant changes in land cover, plant and animal distributions, surface hydrology, and biochemical cycles. Earth system models suggest that this anthropogenic land cover change influenced regional and global climate. However, the representation of past land use in earth system models is currently oversimplified. As a result, there are large uncertainties in the current understanding of the past and current state of the earth system. In order to improve repre- sentation of the variety and scale of impacts that past land use had on the earth system, a global effort is underway to aggregate and synthesize archaeological and historical evi- dence of land use systems. Here we present a simple, hierarchical classification of land use systems designed to be used with archaeological and historical data at a global scale and a schema of codes that identify land use practices common to a range of systems, both imple- mented in a geospatial database. The classification scheme and database resulted from an extensive process of consultation with researchers worldwide. Our scheme is designed to deliver consistent, empirically robust data for the improvement of land use models, while simultaneously allowing for a comparative, detailed mapping of land use relevant to the needs of historical scholars. To illustrate the benefits of the classification scheme and meth- ods for mapping historical land use, we apply it to Mesopotamia and Arabia at 6 kya (c. 4000 BCE). The scheme will be used to describe land use by the Past Global Changes (PAGES) LandCover6k working group, an international project comprised of archaeologists, historians, geographers, paleoecologists, and modelers. Beyond this, the scheme has a wide utility for creating a common language between research and policy communities, link- ing archaeologists with climate modelers, biodiversity conservation workers and initiatives.publishedVersio

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells

P2X7 antagonism using Brilliant Blue G reduces body weight loss and prolongs survival in female SOD1G93A amyotrophic lateral sclerosis mice

Background 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. Methods Human SOD1G93A 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 SOD1G93A mice during the course of disease. Results BBG treatment reduced body weight loss in SOD1G93A 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, SOD1G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1G93A 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 SOD1G93A mice did not change over time. Discussion 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