Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: P2X 7 R expression is elevated prior to end-stage AD. Representative immunoblots using an antibody against P2X 7 R of total brain homogenates from (a) BA9 prefrontal cortex and (b) BA21 temporal cortex. β-actin was included as a loading control. Bar charts show quantification of P2X 7 R protein relative to β-actin in each sample as percentage of average control (Braak 0-II). Representative immunoblots of the post-synaptic and pre-synaptic markers (c,e) PSD-95 and (d, f) synaptophysin (SYP) in BA9 and BA21 total homogenates. Bar charts show quantification of synaptic proteins relative to neuron-specific enolase (NSE) in each sample as percentage of average control (Braak 0-II). Data is mean ± SEM. Following (a,c,d) d’Agostino and Pearson or (b,e,f) Shapiro-Wilk normality tests, data was analysed using a parametric one-way ANOVA with Dunnett́s multiple comparison test or a non-parametric Kruskal-Wallis test with Dunńs multiple comparison test. (a,c,d) n = 25 (Braak 0-II), 19 (Braak III-IV), 16 (Braak V-VI). (b,e,f) n = 4 (Braak 0-II), n = 5 (Braak III-IV), n = 5 (Braak V-VI). *p < 0.05. g,h) Volcano plots showing changes in expression of P2R transcripts in temporal cortex from (g) AD and (h) PSP brain versus age-matched controls obtained from the Mayo RNA-seq cohort of the AMP-AD consortium. Dotted lines indicate the cut-off p-value of 0.05.

Article Snippet: The following primary antibodies were used: Aβ clone 6E10 (1:200, BioLegend, 803001), Aldh1L1 (1:100, UCDavis/NIH NeuroMab facility, N103/39), β-actin (1:5,000, Abcam, Ab8226), lipocalin-2 (1:500, R&D systems, AF1857), neuronal specific enolase (NSE) (1:10,000, DAKO, M0873), phosphorylated Ser536-NFκB p65 (1:1,000, Cell signalling, 3033), total NFκB p65 (1:1,000, Cell signalling, 6956), P2X 7 R (1:200, Alomone, APR-004), PHF1 (1:1,000, kindly gifted by Peter Davies), PSD-95 (1:1,000, Cell signalling, 3450), synaptophysin (1:1,000, Santa Cruz, Sc-17750), total tau (1:10,000, DAKO, A0034).

Techniques: Expressing, Western Blot, Control, Comparison, RNA Sequencing Assay

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: Microglial P2X 7 R activation induces the formation of ASC specks and release of IL-1β. a) Schematic depicting the stimulation of microglia with 100 ng/mL LPS for 3 h, after which medium was replaced and cells pre-treated with 0.1% DMSO (vehicle) or 1 µM Cp2 for 1 h prior to the addition of 1 mM ATP for 20 min. Untreated and LPS-primed cells were included as additional controls. b) Representative confocal images of microglia immunolabelled using an antibody against ASC (green) under basal conditions (untr.), following stimulation with LPS only, LPS and ATP only, or with LPS and ATP in the presence of vehicle (DMSO) or Cp2. ASC specks are indicated by white arrowheads. Hoechst-33342 was used as to stain nuclei. Insets indicate representative regions of each image displayed at higher magnification. Scale bar: 50 µm. c) Quantification of the number of ASC specks normalised to the number of Hoechst + nuclei per condition, displayed as a percentage relative to vehicle-treated cells exposed to LPS + ATP (n = 4). d) Bar graph shows quantification of the amounts of IL-1β in the supernatant of microglial cultures primed with LPS, followed by pre-treatment with vehicle or Cp2 and stimulated with ATP. Untreated, LPS-primed and LPS + ATP only conditions were also included (n = 3). e) Representative cytokine array of cytosolic fractions isolated from post-mortem BA9 AD and control brain at different Braak stages (0-II, III-IV, V-VI). IL-1β coordinates are indicated in purple. f) Quantification of IL-1β amounts in Braak stage III-IV and V-VI BA9 displayed relative to Braak 0-II tissues. n = 10 per group (Braak 0-II, III-IV, V-VI). Following Shapiro-Wilk normality test, data was analysed using (c,d) one-way ANOVA with Dunnett’s multiple comparison test or (f) Kruskal-Wallis test with Dunńs multiple comparison test. Data is mean ± SEM. *p < 0.05, **p < 0.01,****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: Aβ clone 6E10 (1:200, BioLegend, 803001), Aldh1L1 (1:100, UCDavis/NIH NeuroMab facility, N103/39), β-actin (1:5,000, Abcam, Ab8226), lipocalin-2 (1:500, R&D systems, AF1857), neuronal specific enolase (NSE) (1:10,000, DAKO, M0873), phosphorylated Ser536-NFκB p65 (1:1,000, Cell signalling, 3033), total NFκB p65 (1:1,000, Cell signalling, 6956), P2X 7 R (1:200, Alomone, APR-004), PHF1 (1:1,000, kindly gifted by Peter Davies), PSD-95 (1:1,000, Cell signalling, 3450), synaptophysin (1:1,000, Santa Cruz, Sc-17750), total tau (1:10,000, DAKO, A0034).

Techniques: Activation Assay, Staining, Isolation, Control, Comparison

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: Astrocytic P2X 7 R regulates the expression of Lcn2, activates NF κ B and induces cytokine production. a) mRNA levels of Lcn2 in astrocytes stimulated with BzATP (300 µM, 4 h) expressed as relative abundance to control (n = 4). b) Representative immunoblots of Lcn2 and Aldh1L1 in control or BzATP stimulated astrocytes. Bar graph shows the quantification of Lcn2 levels normalised to Aldh1L1 in BzATP stimulated astrocytes expressed as percentage of control (n = 6). c) mRNA levels of Lcn2 in astrocytes pre-treated with 0.1 % DMSO (vehicle) or the P2X 7 R antagonist Cp2 (1 µM) for 1 h prior to stimulation with BzATP expressed as relative abundance to control (n = 4). d) Representative immunoblots of Lcn2 and Aldh1L1 in vehicle-treated or astrocytes treated with the indicated concentrations of Cp2 for 1 h prior to stimulation with BzATP. Bar graph shows the quantification of Lcn2 normalised to Aldh1L1 expressed relative to vehicle (n = 3–4). e) Representative images of NFκB labelling (green) in control and BzATP stimulated astrocytes (upper panel). Hoechst-33342 was used as a nuclear stain. Merged images are displayed in the lower panel. Scale bar: 50 µm. Bar chart shows quantification of the mean nuclear intensity of NFκB relative to mean total cell intensity per well expressed relative to control (n = 3). f-g) Representative immunoblots of Ser536 phosphorylated (p-)NFκB p65 subunit and total NFκB (p65) in (f) BzATP-stimulated and control astrocytes and (g) unstimulated astrocytes, treated with vehicle or with the indicated concentrations of Cp2 for 1 h prior to stimulation with BzATP. Bar graph displays the quantification of p-NFκB normalised to NFκB relative to (f) untreated (n = 6) or (g) vehicle (n = 3–4). h-j) mRNA levels of (h) ccl2 (n = 5) , (i) cxcl1 (n = 4) (j) il-6 (n = 5) in control astrocytes and astrocytes stimulated with BzATP, treated with vehicle or Cp2 (1 µM) for 1 h prior to stimulation with BzATP. Data is expressed as relative abundance to vehicle. k) Representative cytokine array from BA9 control and AD brain at different Braak stages (0-II, III-IV, V-VI). CCL2 and IL-6 coordinates are indicated in purple. n = 10 per group. l-m) Quantification of (l) CCL2 and (m) IL-6 amounts in Braak III-IV and V-VI AD BA9 relative to Braak 0-II. Following a Shapiro-Wilk normality tests, (a,c,h-j) p-values were obtained from log-transformed values using unequal variance Welch́s t -test. Data is mean ± SD. Data analysed using (b,e,f) unpaired t -test, (d,g) one-way ANOVA with Dunnett́s multiple comparison test or (l, m) Kruskal-Wallis test with Dunńs multiple comparison test . Data is mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: Aβ clone 6E10 (1:200, BioLegend, 803001), Aldh1L1 (1:100, UCDavis/NIH NeuroMab facility, N103/39), β-actin (1:5,000, Abcam, Ab8226), lipocalin-2 (1:500, R&D systems, AF1857), neuronal specific enolase (NSE) (1:10,000, DAKO, M0873), phosphorylated Ser536-NFκB p65 (1:1,000, Cell signalling, 3033), total NFκB p65 (1:1,000, Cell signalling, 6956), P2X 7 R (1:200, Alomone, APR-004), PHF1 (1:1,000, kindly gifted by Peter Davies), PSD-95 (1:1,000, Cell signalling, 3450), synaptophysin (1:1,000, Santa Cruz, Sc-17750), total tau (1:10,000, DAKO, A0034).

Techniques: Expressing, Control, Western Blot, Staining, Transformation Assay, Comparison

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: P2X 7 R inhibition reduces tau aggregate levels in BSCs expressing P301L/S320F human tau. a) Volcano plots showing changes in expression of P2r transcripts in cortex from rTg4510 mice versus non-transgenic controls at 2.5, 4.5 and 6 months, obtained from the AD-AMP Knowledge Portal ( https://doi.org/10.7303/syn3157182) . Dotted lines indicate the cut-off p-value of 0.05. b) Schematic represents the preparation of organotypic brain slice cultures (BSCs) from CD1 mice at P7-P8. BSCs were transduced with 1x10 11 vg/mL rAAV2/8 expressing EGFP-tagged 0N4R WT or (P301L/S320F) human tau (hTau) under the hybrid cytomegalovirus enhancer chicken β-actin (hCBA) promoter at 0 DIV. Region-matched slices were treated with 10 µM Cp2 at 14 DIV and every 2–3 days thereafter with each media change until 28 DIV. Control slices were treated identically with vehicle (0.1 % DMSO). Representative confocal images of BSCs transduced with EGFP-tagged WT or P301L/S320F-hTau at 28 DIV. Scale bar: 50 µm. Representative immunoblots of low-speed supernatant (LSS), high-speed supernatant (HSS) and sarkosyl-insoluble (SI) fractions probed with antibodies against (c) total tau (DAKO), β-actin and (d) tau phosphorylated at Ser396/404 (PHF1). Bar graphs display the quantification of (c) insoluble tau, determined as the proportion of total tau in the SI fraction relative to total tau in LSS from the same sample and (d) phosphorylated insoluble tau, calculated as the amount of PHF1 relative to total tau in the SI fraction, shown relative to vehicle-treated slices expressing WT-hTau. Representative immunoblots of synaptoneurosome (SNS) and cytosolic (cyt) fractions of BSCs immunoblotted with antibodies against (e) total tau, PSD-95 and (f) PHF1. Bar charts display the ratio of (e) total tau and (f) PHF1-immunoreactive tau normalised to total tau in SNS relative to the cyt compartment, expressed as percentage relative to BSCs expressing WT-hTau and treated with DMSO. Following Shapiro-Wilk normality test, data was analysed using two-way ANOVAs with Sidaḱs multiple comparison test. n = 3. Data is mean ± SEM. *p < 0.05, **p < 0.01,***p < 0.001.

Article Snippet: The following primary antibodies were used: Aβ clone 6E10 (1:200, BioLegend, 803001), Aldh1L1 (1:100, UCDavis/NIH NeuroMab facility, N103/39), β-actin (1:5,000, Abcam, Ab8226), lipocalin-2 (1:500, R&D systems, AF1857), neuronal specific enolase (NSE) (1:10,000, DAKO, M0873), phosphorylated Ser536-NFκB p65 (1:1,000, Cell signalling, 3033), total NFκB p65 (1:1,000, Cell signalling, 6956), P2X 7 R (1:200, Alomone, APR-004), PHF1 (1:1,000, kindly gifted by Peter Davies), PSD-95 (1:1,000, Cell signalling, 3450), synaptophysin (1:1,000, Santa Cruz, Sc-17750), total tau (1:10,000, DAKO, A0034).

Techniques: Inhibition, Expressing, Transgenic Assay, Slice Preparation, Transduction, Control, Western Blot, Comparison

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: P2X 7 R expression is elevated prior to end-stage AD. Representative immunoblots using an antibody against P2X 7 R of total brain homogenates from (a) BA9 prefrontal cortex and (b) BA21 temporal cortex. β-actin was included as a loading control. Bar charts show quantification of P2X 7 R protein relative to β-actin in each sample as percentage of average control (Braak 0-II). Representative immunoblots of the post-synaptic and pre-synaptic markers (c,e) PSD-95 and (d, f) synaptophysin (SYP) in BA9 and BA21 total homogenates. Bar charts show quantification of synaptic proteins relative to neuron-specific enolase (NSE) in each sample as percentage of average control (Braak 0-II). Data is mean ± SEM. Following (a,c,d) d’Agostino and Pearson or (b,e,f) Shapiro-Wilk normality tests, data was analysed using a parametric one-way ANOVA with Dunnett́s multiple comparison test or a non-parametric Kruskal-Wallis test with Dunńs multiple comparison test. (a,c,d) n = 25 (Braak 0-II), 19 (Braak III-IV), 16 (Braak V-VI). (b,e,f) n = 4 (Braak 0-II), n = 5 (Braak III-IV), n = 5 (Braak V-VI). *p < 0.05. g,h) Volcano plots showing changes in expression of P2R transcripts in temporal cortex from (g) AD and (h) PSP brain versus age-matched controls obtained from the Mayo RNA-seq cohort of the AMP-AD consortium. Dotted lines indicate the cut-off p-value of 0.05.

Article Snippet: We therefore selected the P2X 7 R antibody manufactured by Alomone for western blotting of total homogenates from BA9 and BA21.

Techniques: Expressing, Western Blot, Control, Comparison, RNA Sequencing Assay

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: Microglial P2X 7 R activation induces the formation of ASC specks and release of IL-1β. a) Schematic depicting the stimulation of microglia with 100 ng/mL LPS for 3 h, after which medium was replaced and cells pre-treated with 0.1% DMSO (vehicle) or 1 µM Cp2 for 1 h prior to the addition of 1 mM ATP for 20 min. Untreated and LPS-primed cells were included as additional controls. b) Representative confocal images of microglia immunolabelled using an antibody against ASC (green) under basal conditions (untr.), following stimulation with LPS only, LPS and ATP only, or with LPS and ATP in the presence of vehicle (DMSO) or Cp2. ASC specks are indicated by white arrowheads. Hoechst-33342 was used as to stain nuclei. Insets indicate representative regions of each image displayed at higher magnification. Scale bar: 50 µm. c) Quantification of the number of ASC specks normalised to the number of Hoechst + nuclei per condition, displayed as a percentage relative to vehicle-treated cells exposed to LPS + ATP (n = 4). d) Bar graph shows quantification of the amounts of IL-1β in the supernatant of microglial cultures primed with LPS, followed by pre-treatment with vehicle or Cp2 and stimulated with ATP. Untreated, LPS-primed and LPS + ATP only conditions were also included (n = 3). e) Representative cytokine array of cytosolic fractions isolated from post-mortem BA9 AD and control brain at different Braak stages (0-II, III-IV, V-VI). IL-1β coordinates are indicated in purple. f) Quantification of IL-1β amounts in Braak stage III-IV and V-VI BA9 displayed relative to Braak 0-II tissues. n = 10 per group (Braak 0-II, III-IV, V-VI). Following Shapiro-Wilk normality test, data was analysed using (c,d) one-way ANOVA with Dunnett’s multiple comparison test or (f) Kruskal-Wallis test with Dunńs multiple comparison test. Data is mean ± SEM. *p < 0.05, **p < 0.01,****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We therefore selected the P2X 7 R antibody manufactured by Alomone for western blotting of total homogenates from BA9 and BA21.

Techniques: Activation Assay, Staining, Isolation, Control, Comparison

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: Astrocytic P2X 7 R regulates the expression of Lcn2, activates NF κ B and induces cytokine production. a) mRNA levels of Lcn2 in astrocytes stimulated with BzATP (300 µM, 4 h) expressed as relative abundance to control (n = 4). b) Representative immunoblots of Lcn2 and Aldh1L1 in control or BzATP stimulated astrocytes. Bar graph shows the quantification of Lcn2 levels normalised to Aldh1L1 in BzATP stimulated astrocytes expressed as percentage of control (n = 6). c) mRNA levels of Lcn2 in astrocytes pre-treated with 0.1 % DMSO (vehicle) or the P2X 7 R antagonist Cp2 (1 µM) for 1 h prior to stimulation with BzATP expressed as relative abundance to control (n = 4). d) Representative immunoblots of Lcn2 and Aldh1L1 in vehicle-treated or astrocytes treated with the indicated concentrations of Cp2 for 1 h prior to stimulation with BzATP. Bar graph shows the quantification of Lcn2 normalised to Aldh1L1 expressed relative to vehicle (n = 3–4). e) Representative images of NFκB labelling (green) in control and BzATP stimulated astrocytes (upper panel). Hoechst-33342 was used as a nuclear stain. Merged images are displayed in the lower panel. Scale bar: 50 µm. Bar chart shows quantification of the mean nuclear intensity of NFκB relative to mean total cell intensity per well expressed relative to control (n = 3). f-g) Representative immunoblots of Ser536 phosphorylated (p-)NFκB p65 subunit and total NFκB (p65) in (f) BzATP-stimulated and control astrocytes and (g) unstimulated astrocytes, treated with vehicle or with the indicated concentrations of Cp2 for 1 h prior to stimulation with BzATP. Bar graph displays the quantification of p-NFκB normalised to NFκB relative to (f) untreated (n = 6) or (g) vehicle (n = 3–4). h-j) mRNA levels of (h) ccl2 (n = 5) , (i) cxcl1 (n = 4) (j) il-6 (n = 5) in control astrocytes and astrocytes stimulated with BzATP, treated with vehicle or Cp2 (1 µM) for 1 h prior to stimulation with BzATP. Data is expressed as relative abundance to vehicle. k) Representative cytokine array from BA9 control and AD brain at different Braak stages (0-II, III-IV, V-VI). CCL2 and IL-6 coordinates are indicated in purple. n = 10 per group. l-m) Quantification of (l) CCL2 and (m) IL-6 amounts in Braak III-IV and V-VI AD BA9 relative to Braak 0-II. Following a Shapiro-Wilk normality tests, (a,c,h-j) p-values were obtained from log-transformed values using unequal variance Welch́s t -test. Data is mean ± SD. Data analysed using (b,e,f) unpaired t -test, (d,g) one-way ANOVA with Dunnett́s multiple comparison test or (l, m) Kruskal-Wallis test with Dunńs multiple comparison test . Data is mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We therefore selected the P2X 7 R antibody manufactured by Alomone for western blotting of total homogenates from BA9 and BA21.

Techniques: Expressing, Control, Western Blot, Staining, Transformation Assay, Comparison

Journal: Brain, Behavior, and Immunity

Article Title: P2X 7 R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

doi: 10.1016/j.bbi.2023.09.011

Figure Lengend Snippet: P2X 7 R inhibition reduces tau aggregate levels in BSCs expressing P301L/S320F human tau. a) Volcano plots showing changes in expression of P2r transcripts in cortex from rTg4510 mice versus non-transgenic controls at 2.5, 4.5 and 6 months, obtained from the AD-AMP Knowledge Portal ( https://doi.org/10.7303/syn3157182) . Dotted lines indicate the cut-off p-value of 0.05. b) Schematic represents the preparation of organotypic brain slice cultures (BSCs) from CD1 mice at P7-P8. BSCs were transduced with 1x10 11 vg/mL rAAV2/8 expressing EGFP-tagged 0N4R WT or (P301L/S320F) human tau (hTau) under the hybrid cytomegalovirus enhancer chicken β-actin (hCBA) promoter at 0 DIV. Region-matched slices were treated with 10 µM Cp2 at 14 DIV and every 2–3 days thereafter with each media change until 28 DIV. Control slices were treated identically with vehicle (0.1 % DMSO). Representative confocal images of BSCs transduced with EGFP-tagged WT or P301L/S320F-hTau at 28 DIV. Scale bar: 50 µm. Representative immunoblots of low-speed supernatant (LSS), high-speed supernatant (HSS) and sarkosyl-insoluble (SI) fractions probed with antibodies against (c) total tau (DAKO), β-actin and (d) tau phosphorylated at Ser396/404 (PHF1). Bar graphs display the quantification of (c) insoluble tau, determined as the proportion of total tau in the SI fraction relative to total tau in LSS from the same sample and (d) phosphorylated insoluble tau, calculated as the amount of PHF1 relative to total tau in the SI fraction, shown relative to vehicle-treated slices expressing WT-hTau. Representative immunoblots of synaptoneurosome (SNS) and cytosolic (cyt) fractions of BSCs immunoblotted with antibodies against (e) total tau, PSD-95 and (f) PHF1. Bar charts display the ratio of (e) total tau and (f) PHF1-immunoreactive tau normalised to total tau in SNS relative to the cyt compartment, expressed as percentage relative to BSCs expressing WT-hTau and treated with DMSO. Following Shapiro-Wilk normality test, data was analysed using two-way ANOVAs with Sidaḱs multiple comparison test. n = 3. Data is mean ± SEM. *p < 0.05, **p < 0.01,***p < 0.001.

Article Snippet: We therefore selected the P2X 7 R antibody manufactured by Alomone for western blotting of total homogenates from BA9 and BA21.

Techniques: Inhibition, Expressing, Transgenic Assay, Slice Preparation, Transduction, Control, Western Blot, Comparison

Journal: PLoS ONE

Article Title: Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

doi: 10.1371/journal.pone.0015299

Figure Lengend Snippet: (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

Article Snippet: The protein was identified by incubating the membrane with anti-GLT-1 antibody (1∶1000; Millipore) or anti- purinergic P2X 7 R antibody (1∶1000; Alomone Labs, Israel) overnight at 4°C, followed by HRP-conjugated secondary antibody (1∶2000) and ECL solution.

Techniques: Cell Culture, Western Blot, Infection, Fluorescence

Journal: Journal of Inflammation (London, England)

Article Title: The role of the purinergic P2X 7 receptor in inflammation

doi: 10.1186/1476-9255-4-5

Figure Lengend Snippet: Summary of the production of active IL-1β . This process can be divided into 3 stages. Stage 1: LPS stimulates monocytes/macrophages (M∅) to produce pro-ICE and pro-IL-1β. Stage 2: ATP stimulates the P2X 7 R expressed on M∅ to cause a fall in intracellular K + concentration ([K + ] i ) which in turn converts pro-ICE to ICE. Stage 3: LPS-primed M∅ following ATP stimulation results in activated ICE which converts inactive pro-IL-β to active IL-1β. It should be noted that this process is intracellular and the figure is for illustrative purposes only (see text for references).

Article Snippet: Since the P2X 7 R is important in the production of both TNF-α and IL-1β and as inhibitors of both are in clinical use for the treatment of rheumatoid arthritis [ ] and other inflammatory conditions, such observations possibly underlie why AstraZeneca, Pfizer and Abbot amongst others are currently developing P2X 7 R antagonists.

Techniques: Concentration Assay

Journal: Journal of Inflammation (London, England)

Article Title: The role of the purinergic P2X 7 receptor in inflammation

doi: 10.1186/1476-9255-4-5

Figure Lengend Snippet: Possible outcomes of an inflammatory response . Tissue damage (inflammation initiation) can lead to cell death by apoptosis or necrosis. The balance between these two types of cell death can determine the outcome of the inflammatory response e.g. propagation (leading to chronic inflammation) or resolution. Resolution is more common when cell death is predominantly apoptotic, however, the phagocytosis of apoptotic or necrotic cells is also an important determinant of the outcome of inflammation. As can be seen, the P2X 7 R may be critical to determining the outcome of an inflammatory response.

Article Snippet: Since the P2X 7 R is important in the production of both TNF-α and IL-1β and as inhibitors of both are in clinical use for the treatment of rheumatoid arthritis [ ] and other inflammatory conditions, such observations possibly underlie why AstraZeneca, Pfizer and Abbot amongst others are currently developing P2X 7 R antagonists.

Techniques:

Journal: Journal of Inflammation (London, England)

Article Title: The role of the purinergic P2X 7 receptor in inflammation

doi: 10.1186/1476-9255-4-5

Figure Lengend Snippet: Diagrammatic representation of the interplay between inflammatory mediators and cells . Tissue damage or inflammatory stimuli results in ATP release which activates the P2X 7 R causing eosinophils to release IL-8 which amplifies the initial inflammatory response.

Article Snippet: Since the P2X 7 R is important in the production of both TNF-α and IL-1β and as inhibitors of both are in clinical use for the treatment of rheumatoid arthritis [ ] and other inflammatory conditions, such observations possibly underlie why AstraZeneca, Pfizer and Abbot amongst others are currently developing P2X 7 R antagonists.

Techniques: