MOG35-55 specific T-cell responses in mice treated with TFM-C.

<p>A) Popliteal and inguinal lymph node cells from TFM-C-, celecoxib-treated or control mice were incubated in the presence of MOG35-55 for 48h. Proliferative responses were determined by the uptake of [³H] thymidine. B and C). The levels of IFN-γ and IL-17 in culture supernatants were measured by ELISA. The data shown are from a single experiment representative of three similar experiments. Error bars represent + SEM of 3 mice per group. *<i>P</i><0.05 compared with control group, **<i>P</i><0.05 compared with both control and celecoxib-treated groups. </p

Effect of TFM-C on cytokine production in organotypic cerebellar cultures.

<p>A) After 7 <i>DIV</i> organotypic cultures were treated with TFM-C (50µM) for 6h and then stimulated with 15µg/ml LPS for 24h in presence of TFM-C. Panel a) Lay-out of cytokine-specific antibody spots in the 16-plex cytokine Stripwell array (left image) and visualization of cytokine-specific chemiluminescence in culture medium of LPS-treated organotypic cultures in the absence or presence of TFM-C (right images). Cyotkine levels significantly affected by TFM-C are highlighted in grey. Panel b) pg/ml of cytokines were indicated. sd: standard deviation. ND: not determinable. B) Effect of TFM-C on IL-1β, IL-6, IL-10, IL-12p35, IL-23p19, TNF-α and HERP mRNA in organotypic cultures stimulated by LPS for 6h and 24h in presence or absence of 50µM TFM-C. The levels of mRNA are shown as <i>n</i>-fold increase compared with baseline level (-) and normalized to those of the housekeeping gene <i>Hprt1</i>. Asterisks indicate significant differences at *<<i>P</i> 0.05 compared with LPS control by ANOVA test. </p

A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

<div><p>Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1β, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.</p> </div

Effect of TFM-C on ER stress, oxidative stress, demyelination and axonal damage in organotypic cerebellar cultures.

<p>A) Organotypic cultures were stimulated with LPS for 24h or pre-treated with TFM-C (50µM) for 2h and then stimulated with LPS/TFM-C for 24h. Panel a-b: immunostaining for Iba1 (red) and HERP or iNOS (green). Panel c: immunostaining for NFL (red) and MBP (green); Panel d: immunistaining for total neurofilament-heavy (NFH; red) and non-phosphorylated neurofilament (SMI32; green). In the white box inset are shown axonal spheroids and occurrence of axonal transection (end-bulbs). Scale bar 10µm. B) 10µg of total protein were loaded for HERP, CNPase and iNOS Western blot analysis. Quantification of band intensity was calculated in the graphs below after normalization for total protein loaded. Results are expressed as percentage compared to the LPS only (100%). Error bars indicate the standard error. *<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.001 by ANOVA test. C) After 7 <i>DIV</i> organotypic cultures were treated with TFM-C for 2h and then stimulated with 15µg/ml LPS for 6 and 24h. iNOS mRNA expression was analyzed by quantitative PCR. The levels of mRNA are shown as <i>n</i>-fold increase compared with the level of baseline condition (-) and normalized to those of the housekeeping gene <i>Hprt1</i>. All values represent the averages of three independent experiments. Error bars indicate the standard error. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by ANOVA test. D) Percentage of non-phosphorylated neurofilaments with respect to total neurofilaments in cerebellar cultures stimulated for 24h with LPS in presence or absence of TFM-C. Error bars indicate the standard error. *<i>P</i> <0.05 by ANOVA test. </p

The effect of TFM-C on cytokine production from BMDCs.

<p>BMDCs were incubated in TFM-C, celecoxib or vehicle for 16h and subsequently stimulated with LPS (0.1µg/ml) (A) or heat killed H37Ra Mtb (10µg/ml) (B) in the presence of TFM-C, celecoxib or vehicle. Cytokines were detected by ELISA. IL-12, IL-1β or IL-6 were measured 24h after stimulation. IL-23 and TNF-α were measured 6h after stimulation. The data shown are pooled from two similar experiments. Error bars represent + SEM. *<i>P</i><0.05 control versus TFM-C celecoxib-treated group, **<i>P</i><0.05 control versus TFM-C -treated group. </p

The effect of TFM-C on EAE.

<p>Clinical EAE scores of female B6 mice following immunization with MOG35-55. The mice were injected i.p. with 10 µg/kg TFM-C (closed circles), celecoxib (closed triangles) or vehicle (open squares) every other day starting on the day of immunization. *<i>P</i><0.05 TFM-C-treated versus vehicle-treated group. Results shown are the mean + SEM of 14-16 mice per group in TFM-C- or vehicle-treated groups and of 5 mice in the celecoxib-treated group. The data shown are pooled data from three similar experiments. </p

Intracellular localization of TNF-α in BV2 cell line and TNF-α secretion in primary astrocytes.

<p>A) BV2 cells were treated with TFM-C (50µM) for 2h and stimulated with LPS (1µg/ml) for 3, 6 and 24h in presence or absence of TFM-C. Panel a) Staining for TNF-α in permeabilized cells. Scale bar 5µm. Panel b) Intracellular localization of TNF-α by flow cytometry. Results are expressed as fold increase compared to the control at the same time point. B) TNF-α release in astrocyte cultures. Astrocytes were treated with TMF-C (30 or 50µM) for 24h or pre-treated with TMF-C for 2h and then stimulated with LPS (1µg/ml) in presence or absence of TFM-C for 24h and then analyzed by ELISA. Error bars indicate the standard deviation. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by ANOVA test.</p

Effect of TFM-C on HERP and cytokine mRNA and protein production in BV-2 cells.

<p>BV2 cells were treated with TFM-C (30 or 50µM) for 2h and then stimulated with LPS (1µg/ml) for different times in presence or absence of TFM-C. A) Quantification of the kinetics of mRNA production and cytokine secretion (IL-1β, IL-6 and TNF-α). All values represent the averages of three independent experiments. Lower graphs represent cytokine-specific mRNA quantified by QPCR, while the upper graphs represents amount of secreted cytokine quantified using specific ELISA kits. Asterisks indicate significant differences at *<i>P</i>< 0.05, **<i>P</i><0.01, ***<i>P</i><0.001 between TFM-C-treated and LPS-treated cells at each time point using ANOVA test. B) Effect of TFM-C (30 and 50µM) on IL-23p19 and HERP mRNAs in BV2 cells stimulated with LPS. The levels of mRNA levels are shown as fold increase. Asterisks indicate significant differences at *<i>P</i> < 0.05 and **<i>P</i><0.01 compared with LPS only using ANOVA test. C) Effect of TFM-C on HERP protein expression. BV2 cells were treated with TFM-C (50µM) or pretreated with TFM-C for 2h (30 and 50µM) and then stimulated with LPS in presence of TFM-C. Panel a) Immunofluorescence for HERP (green), Iba1 (red) and DAPI (blue) at 12h of LPS/TFM-C or TFM-C treatment. Scale bar 5µm. Panel b-c) 10µg of total protein were loaded for HERP Western blot analysis. Results were expressed as arbitrary units respect to the control at same time point. Error bars indicate the standard error. *<i>P</i><0.05 by ANOVA test. D) Effect of TFM-C treatment (50µM) on the viability of BV2 cells. Apoptotic cells were measured by double propidium iodite (PI) and DAPI staining, and the percentage of damaged DNA and condensed chromatin was calculated at 24h of LPS/TFM-C treatment.</p

Autophagic Marker <i>MAP1LC3B</i> Expression Levels Are Associated with Carotid Atherosclerosis Symptomatology

<div><p>Objectives</p><p>The mechanism by which atheroma plaque becomes unstable is not completely understood to date but analysis of differentially expressed genes in stable versus unstable plaques may provide clues. This will be crucial toward disclosing the mechanistic basis of plaque instability, and may help to identify prognostic biomarkers for ischaemic events. The objective of our study was to identify differences in expression levels of 59 selected genes between symptomatic patients (unstable plaques) and asymptomatic patients (stable plaques).</p><p>Methods</p><p>80 carotid plaques obtained by carotid endarterectomy and classified as symptomatic (>70% stenosis) or asymptomatic (>80% stenosis) were used in this study. The expression levels of 59 genes were quantified by qPCR on RNA extracted from the carotid plaques obtained by endarterectomy and analyzed by means of various bioinformatic tools.</p><p>Results</p><p>Several genes associated with autophagy pathways displayed differential expression levels between asymptomatic and symptomatic (i.e. <i>MAP1LC3B</i>, <i>RAB24</i>, <i>EVA1A</i>). In particular, mRNA levels of <i>MAP1LC3B</i>, an autophagic marker, showed a 5−fold decrease in symptomatic samples, which was confirmed in protein blots. Immune system−related factors and endoplasmic reticulum-associated markers (i.e. <i>ERP27</i>, <i>ITPR1</i>, <i>ERO1LB, TIMP1, IL12B</i>) emerged as differently expressed genes between asymptomatic and symptomatic patients.</p><p>Conclusions</p><p>Carotid atherosclerotic plaques in which <i>MAP1LC3B</i> is underexpressed would not be able to benefit from <i>MAP1LC3B</i>−associated autophagy. This may lead to accumulation of dead cells at lesion site with subsequent plaque destabilization leading to cerebrovascular events. Identified biomarkers and network interactions may represent novel targets for development of treatments against plaque destabilization and thus for the prevention of cerebrovascular events.</p></div

Ranking of gene expression markers according to highest fold change in asymptomatic (A) compared with symptomatic (S) samples quantified by Real Time RT−PCR.

<p>The statistical significance was analyzed with the non-parametrical statistical test Mann-Whitney U test (* P≤0.05 and ** P≤0.0001).</p><p>Ranking of gene expression markers according to highest fold change in asymptomatic (A) compared with symptomatic (S) samples quantified by Real Time RT−PCR.</p