Cytokine levels in PBMCs from AD patients with follow-up at 12 and 24 months after inclusion.

<p>IL1-β, TNF-α and IL-6 levels measured by the 3-plex Luminex xMAP^® assay in cell lysates (INTRA) and in culture media (EXTRA) at baseline (day 0 inclusion), 12 months (M12) and 24 months (M24) after pre-treatment of PBMCs with 1 μM C16 <i>versus</i> its vehicle (DMSO). Cytokine levels were expressed in pg/mg protein for INTRA and in pg/mL for EXTRA samples. Results are mean ± SEM. Intra-individual comparisons between DMSO and C16 conditions at each time</p><p>****p<0.0001 by Wilcoxon matched-pairs signed rank test, Intra-individual comparisons between D0, M12 and M24 in DMSO or C16 conditions</p><p>^†p<0.05 compared to DMSO-treated PBMCs at M12</p><p>^‡‡p<0.01 compared to C16-treated PBMCs at Day 0 by ANOVA for repeated measures and post-hoc Sheffe’s test. Friedman test: p = 0.0190 and p = 0.0087 for TNF-α in INTRA and EXTRA with DMSO treatment, respectively and p = 0.0052 for IL-6 INTRA with C16 treatment.</p><p>Cytokine levels in PBMCs from AD patients with follow-up at 12 and 24 months after inclusion.</p

Cytokine levels in PBMCs from healthy patients in three experimental conditions.

<p>IL1-β, TNF-α and IL-6 levels measured by the 3-plex Luminex xMAP^® assay in cell lysates (INTRA) and in culture media (EXTRA) after pre-treatment of PBMCs with 1 μM C16 <i>versus</i> its vehicle (DMSO) in three experimental conditions during 48h: serum medium, serum-free medium and serum-free medium with 20 μM Aβ42. Cytokine levels were expressed in pg/mg protein for INTRA and in pg/mL for EXTRA samples. Results are mean ± SEM. Intra-individual comparisons between DMSO and C16 conditions at each time</p><p>**p<0.01 by Wilcoxon matched-pairs signed rank test. Intra-individual comparisons between serum medium, free-serum medium, free serum medium with 20μM Aβ42 in DMSO or C16</p><p>^†††p<0.001 compared to DMSO-treated PBMCs in serum medium</p><p>^‡‡p<0.01 compared to C16-treated PBMCs in serum medium</p><p>^#p<0.05 compared to C16-treated PBMCs in serum-free medium by ANOVA for repeated measures and post-hoc Sheffe’s test.</p><p>Cytokine levels in PBMCs from healthy patients in three experimental conditions.</p

Expression of autophagic markers in PBMCs from healthy patients.

<p>Representative immunoblots showed the immunoreactivity of Beclin-1 (panel A), p62 (panel B), LC3 I and LC3 II (panel C) in PBMCs of healthy patients cultured in three experimental culture conditions (serum medium, serum free medium and 20μM Aβ42 in serum free medium) with or without 1 μM C16 or its vehicle (0.8% DMSO) for 48hrs as described in method section. Semi-quantitative analysis of immunoblot was performed using Gene Tools software (Syngene, Ozyme France). The immunoreactivity of protein was normalized to β-actin immunoreactivity. The line on each graph represents the mean. *p < 0.05 compared to DMSO-treated PBMCs in serum medium, ^$p < 0.05 compared to Aβ42-treated PBMCs in serum free medium with DMSO by Wilcoxon matched-pairs signed rank test.</p

Correlation between inflammation and autophagic markers in PBMCs from AD patients.

<p>Spearman correlations were performed between Beclin-1, p62, LC3 I and LC3 II levels and IL1-β, TNF-α and IL-6 levels measured in intra- and extracellular media (INTRA and EXTRA) of vehicle-treated PBMCs at D0, M12 or M24. No correlation was observed for p62 (data not shown). Some signals were not analyzed on the blot due to a very low or absent signal, therefore the number of patients was indicated in each row. In table, rho and p values were indicated. The level of significance was p < 0.05 and significant correlations were shown in bold.</p><p>Correlation between inflammation and autophagic markers in PBMCs from AD patients.</p

MMSE and ADAScog scores from AD patients at baseline, 12 and 24 months follow-up.

<p>AD patients naive of treatment for AD and have a CRP < 10 mg/L were included in this ancillary clinical study (part of Cytocogma program). Cognitive evaluation included MMSE and ADAScog scale through a 2-year long follow-up. Mean ± SD of MMSE and ADAScog scores was indicated in the table. For intra-individual comparison between D0 and M24</p><p>**p<0.001</p><p>***p<0.0001 and between M12 and M24</p><p>^†p<0.05</p><p>^††p<0.01 using ANOVA repeated measures and post-hoc Sheffe’s test.</p><p>MMSE and ADAScog scores from AD patients at baseline, 12 and 24 months follow-up.</p

Longitudinal monitoring of Beclin-1 expression in PBMCs from AD patients.

<p>Representative immunoblots (panel A) showed the immunoreactivity of Beclin-1 in PBMCs of AD patients at the Day of inclusion (D0, panel B) and after a follow-up at 12 months (M12, panel C) and 24 months (M24, panel D). PBMCs were isolated from blood and cultured either with 1 μM C16 or its vehicle (0.8% DMSO) for 48hr as described in method section. Semi-quantitative analysis of immunoblot was performed using Gene Tools software (Syngene, Ozyme France). The immunoreactivity of protein was normalized to β-actin immunoreactivity. The line on the graphs (B to D) represents the mean of 36, 33 and 27 patients at D0, M12 and M24, respectively. The mean expression of Beclin-1 in PBMCs at D0, M12 and M24 are shown in the panels E and F, without or with the C16 treatment, respectively. *p < 0.05 compared to vehicle-treated PBMCs at M12 by Wilcoxon matched-pairs signed rank test; ^<b>$</b>p < 0.05 compared to PBMCs at M12 by ANOVA for repeated measures and post-hoc Sheffe’s test. Friedman test: p = 0.0019 for Beclin-1 in vehicle condition and p = 0.0002 in C16 condition.</p