Virtual Touch (TM) Quantification to Diagnose and Monitor Liver Fibrosis in Hepatitis B and Hepatitis C: A NICE Medical Technology Guidance

King’s Technology Evaluation Centre is funded by NICE to act as an External Assessment Centre for the Medical Technologies Evaluation Programme. The NHS has a financial interest in the guidance issued by NICE as a result of this work and two authors are NHS employees (Prof. Keevil and Dr. Lewis

The neuropeptide PACAP and the gliopeptide ODN prevent 6-hydroxydopamine-induced apoptosis of cerebellar granule neurons

Poster prizeInternational audienc

Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

International audienceAstroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS and inhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase

Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis

International audienceOxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H(2)O(2)-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H(2)O(2) (300 μM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H(2)O(2)-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H(2)O(2). Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation

The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a PKA/MAPK-dependent mechanism.

Astrocytes synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN) an endogenous ligand of both central-type benzodiazepine (CBR) and metabotropic receptors. We have recently shown that ODN exerts a protective effect against hydrogen peroxide (H(2)O(2))-induced oxidative stress in astrocytes. The purpose of the present study was to determine the type of receptor and the transduction pathways involved in the protective effect of ODN in cultured rat astrocytes. We have first observed a protective activity of ODN at very low concentrations that was abrogated by the metabotropic ODN receptor antagonist cyclo(1-8)[DLeu(5)]OP, but not by the CBR antagonist flumazenil. We have also found that the metabotropic ODN receptor is positively coupled to adenylyl cyclase in astrocytes and that the glioprotective action of ODN upon H(2)O(2)-induced astrocyte death is PKA- and MEK-dependent, but PLC/PKC-independent. Downstream of PKA, ODN induced ERK phosphorylation, which in turn activated the expression of the anti-apoptotic gene Bcl-2 and blocked the stimulation by H(2)O(2) of the pro-apoptotic gene Bax. The effect of ODN on the Bax/Bcl-2 balance contributed to abolish the deleterious action of H(2)O(2) on mitochondrial membrane integrity and caspase-3 activation. Finally, the inhibitory effect of ODN on caspase-3 activity was shown to be PKA and MEK-dependent. In conclusion, the present results demonstrate that the potent glioprotective action of ODN against oxidative stress involves the metabotropic ODN receptor coupled to the PKA/ERK-kinase pathway to inhibit caspase-3 activation

Schematic representation of the signaling pathways likely involved in the protective effect of ODN against H₂O₂-induced astroglial cell apoptosis.

<p>ODN activates both adenylyl cyclase (AC) and phospholipase C (PLC) in astrocytes, however only the cAMP-dependent protein kinase A (PKA) pathway is involved in the effect of ODN on cell survival. ODN stimulates phosphorylation of extracellular regulated kinase (ERK) in a PKA-dependent manner. Downstream, ODN activates the expression of the anti-apoptotic gene Bcl-2, stimulates glutathione (GSH) formation and abolishes H₂O₂-induced decrease of mitochondrial potential (Ψm) via the formation of highly reactive oxygen species (ROS), the inhibition of Bcl-2 and the stimulation of the pro-apoptotic gene Bax. Finally, ODN prevents H₂O₂-evoked activation of caspase-3 leading to astrocyte death. Cyt c, cytochrome c; DAG, diacylglycerol; IP₃, inositol trisphosphate; H89, protein kinase A inhibitor; MEK, mitogen-activated protein kinase kinase; PKC, protein kinase C; U0126, MAP kinase kinase inhibitor. +, activation; –, inhibition.</p

Protective effect of ODN on astroglial cell death induced by H₂O₂.

<p>(A) Cultured astrocytes were pre-incubated for 10 min in the absence or presence of graded concentrations of ODN (1 fM–1 nM) and then incubated for 1 h with medium alone (□) or with 300 µM H₂O₂ without (•) or with ODN (▪). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test: <i>** p</i><0.01; <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^#<i>p</i><0.05; ^###<i>p</i><0.01; ns, not statistically different <i>vs.</i> H₂O₂-treated cells. (B) Typical images illustrating the protective effect of ODN on H₂O₂-induced cell death. Cells were pre-incubated for 10 min in the absence (a, c) or presence of 0.1 nM ODN (b, d), and then incubated for 1 h with medium alone (a), ODN (b) or with 300 µM H₂O₂ without (c) or with ODN (d). Living astrocytes were labeled with calcein-AM (green fluorescence staining). Scale bar = 50 µm. (C) Time-course effect of ODN on H₂O₂-induced cell death. Cells were incubated for the indicated times with medium alone (□) or 300 µM H₂O₂ without (•) or with ODN (0.1 nM; ▪). After 30, 60, 90, 120 or 150 min of incubation, a refill of 0.1 nM ODN (arrows; ▴) was added in the culture medium. Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. ^##<i>p</i><0.01; ^###<i>p</i><0.001 <i>vs.</i> H₂O₂-treated cells.</p

Effect of ODN on ERK phosphorylation in astroglial cells.

<p>(A–D) Cultured astrocytes were incubated with medium alone, with graded concentration of ODN (1 fM–1 nM; A) for 1 h, with 0.1 nM ODN for the times indicated (B), or with ODN (0.1 nM) in the absence or presence of flumazenil (1 µM; C), cyclo_1–8 [DLeu⁵] OP (c[DLeu⁵] OP;1 µM; C), H89 (20 µM; D) or U0126 (20 µM; D) for 1 h. Active ERK1 and ERK2 were detected by Western blotting using antibodies against phosphorylated ERK and quantified by using total ERK and actin as internal controls. The results are expressed as percentages of control. Each value represents the mean (± SEM) of 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. ** <i>p</i><0.01; *** <i>p</i><0.001; NS, not statistically different <i>vs.</i> control.</p

Pharmacological characterization of the receptor involved in the protective effect of ODN on astroglial cells.

<p>(A) Cultured astrocytes were pre-incubated for 30 min in the absence or presence of ODN (0.1 nM), the metabotropic receptor agonist OP (0.1 nM), the inactive ODN analog [Ala¹⁵]ODN (0.1 nM) or the specific CBR agonist clonazepam (Clona; 10 nM), and then incubated for 1 h with medium alone or with 300 µM H₂O₂ without or with receptor ligands. (B) Cells were pre-incubated for 30 min in the absence or presence of the metabotropic receptor antagonist cyclo_1–8 [DLeu⁵] OP (c[DLeu⁵] OP; 1 µM) or the CBR antagonist flumazenil (1 µM), and then incubated for 1 h with medium alone or with 300 µM H₂O₂ without or with ODN (0.1 nM). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least 12 different wells from three independent cultures. ANOVA followed by the Bonferroni's test. <i>*** p</i><0.001; NS, not statistically different <i>vs.</i> control. ^###<i>p</i><0.001; ns, not statistically different <i>vs.</i> H₂O₂-treated cells.</p