Relative clock gene expression over time after serum shocked primary fibroblasts.

<p>Fitted sin graphs of the expression and mean ± SD of the mean from three individual patients per group are shown. The time of serum removal is 0 in the graphs and the relative expression is normalized to the expression before serum (at time = −2). Δb indicates the general difference in the expression i.e., the baseline of the expression. Phase differences are marked as red solid lines and the difference is indicated in hours.</p

The oscillation of <i>IL-1β</i>, <i>IL-6</i> and <i>TNF-α</i> in RA and OA cells after serum shock.

<p>Relative gene expression over time is displayed. Fibroblasts derived from RA synovium display weaker rhythmic expression of <i>IL-1β</i>, <i>IL-6</i> and <i>TNF-α</i> after clock resetting than cells from OA synovium. Fitted sin graphs of the expression and mean ± SD of the mean from three individual patients per group is shown. The time of serum removal is 0 in the graphs and the relative expression is normalized to the expression before serum (at time = −2). Δb indicates the general difference in the expression i.e., the baseline of the expression. ΔA indicates the differences in the peak amplitude. Phase differences are marked as red solid lines and the difference is indicated in hours.</p

The expression of circadian genes in RA and OA patients.

<p>A. Synovial membrane samples were collected from RA and OA patients (n = 10 for each) and analyzed for circadian gene expression. The relative expression of each gene is expressed as mean ± SD. B. The expression of <i>BMAL1</i> and <i>PER1</i> should be in anti-phase. Pearson correlation and the linear fit for the expression of <i>BMAL1</i> and <i>PER1</i> in the synovial membrane of RA patients reveals that high expression of <i>BMAL1</i> correlates with high <i>PER1</i> expression and <i>vice versa</i>, low <i>BMAL1</i> expression correlates with low <i>PER1</i> expression. In contrast to synovium of RA patients, the high expression of <i>BMAL1</i> was associated with low <i>PER1</i> expression and conversely, low <i>BMAL1</i> expression was coupled with high <i>PER1</i> expression in the synovial membrane of OA patients. Similar findings were observed with <i>NPAS2</i> and <i>PER1</i> as well as with <i>ARNTL2</i> and <i>CRY2</i>. Each dot indicates the Ct value of <i>BMAL1</i>, <i>NPAS2</i> or <i>ARNTL2</i> in the x-axis and <i>PER1</i> or <i>CRY2</i> in the y-axis in the synovial membrane sample of an individual patient. *p<0.05.</p

Serum levels of bone turnover markers, bone metabolism proteins and cytokines.

<p>CTX-I levels are decreased in patients at 6 months follow-up when compared to healthy donors (p = 0.0268). DKK-1, IL-1 β, IL-6, IL-17A, IL-12p70, IL-23, TNF, MCP-1 and TGF-β are increased in patients at baseline when compared to healthy donors. After 6 months of therapy, follow-up patients had decreased levels of IL-17A, IL-23 and TGF-β when compared to their baseline. We have also observed that after therapy the levels of IL-1 β, IL-6, IL-12p70 and TNF were still significantly higher than healthy donors levels’. Each dot represents a sample. Line represents median. * vs Baseline, § vs Follow-up. DKK—dickkopf-related protein, CTX—carboxy-terminal collagen crosslinks, IL—interleukin, TNF—tumor necrosis factor, MCP—monocyte chemmotractant protein, TGF—transforming growth factor.</p

Gene expression profile of stimulated cells in culture for 21 days.

<p>All genes except CTSK are significantly decreased at day 1 in patients at follow-up when compared to healthy donors. At day 1 RANK expression in patients at baseline is also significantly reduced when compared to healthy donors (p = 0.0268). We found no differences between groups throughout the culture time except for Atp6v0d2 expression at day 21 in patients at baseline which is significantly reduced when compared both to healthy donors (p = 0.039) and patients at follow-up (p = 0.0234). Relative gene expression shown in Log scale. Dots in graphs represent median gene expression for each group at each time-point. d—day; CSF1R—gene encoding macrophage-colony stimulating factor (c-fms), RANK—gene encoding for receptor activator of nuclear factor-κβ, NFATc1—gene encoding for nuclear factor of activated T-cells, Atp6v0d2—gene encoding ATPase, H⁺ transporting, lysosomal V0 subunit D2, CTSK—gene encoding cathepsin K. p<0.05 is considered significant.</p

Osteoclast number is reduced in baseline patients, but bone resorption activity is increased after TNF-blocker exposure.

<p>A) Representative images of culture day 21 of cells stimulated with M-CSF, RANKL, dexamethasone and TGF-β and stained for TRAP. OC number increased throughout time and at culture day 21 baseline patients have significantly less osteoclasts than healthy donors (p = 0.0038). No differences were found in the number of nuclei per OC in any studied time of culture. B) Representative images of pit assay at culture day 21. Patients at follow-up had significantly higher number of pits and resorption area at culture day 21 when compared to their baseline (p = 0.0469 for both resorption pit number and percentage of resorbed area). Dots represent median counts for each group at each time-point and bars represent interquartile range [10–90]. d—day; OC—osteoclast; Scale bars 100μm, red arrows—osteoclasts, black arrows—resorption pits.</p