Representative high-magnification photomicrographs showing double immunofluorescence for NAAA, CD19, CD3 and CD14 in order to characterize the immune cells in the mucosa infiltrate of UC patients.

<p>NAAA immunofluorescence was observed in CD19+ B lymphocytes (A–C), CD3+ T lymphocytes (D–F) and CD14+ macrophages (G–I).</p

Primers sequences used for RT-PCR.

<p>Primers sequences used for RT-PCR.</p

Representative photomicrographs and densitometrical quantification of PPARα (A–D), NAAA (E–H), NAPE-PLD (I–L) and FAAH (M–P) immunoreactivity in human healthy (control; A, E, I, M), active UC (B, F, J, N) and quiescent UC (C, G, K, O) colonic epithelium depending on treatment.

<p>Active UC at disease onset showed a decrease in PPARα and NAPE-PLD immunoreactivity, but an increase in NAAA immunoreactivity. PPARα immunoreactivity showed significant differences depending on treatment. Treatment with 5-ASA in UC patients restored completely PPARα and NAPE-PLD protein levels to control ones. However, PPARα expression dropped again in UC patients treated with 5-ASA and glucocorticoids, and 5-ASA, glucocorticoids and immunomodulators compared to control group. In contrasts, treatment with 5-ASA and glucocorticoids increased NAPE-PLD immunoreactivity. No changes were observed in FAAH immunoreactivity in the epithelium of control, active (untreated) UC and quiescent (treated) UC patients. Mann-Whitney U and Wilcoxon tests (N = 22–24): *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus control group; ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>versus</i> UC group; ^&<i>P</i><0.05 <i>versus</i> 5-ASA-treated quiescent UC group.</p

Relative quantification of PPARα (A), PPARγ (B), NAAA (C), iNOS (D), NAPE-PLD (E) and FAAH (F) gene expression in the colonic mucosa of active UC patients compared to human healthy colonic tissue (control).

<p>Absolute values were normalized with regard to the housekeeping gene SP1. Active UC at disease onset showed lower PPARα, PPARγ and NAAA gene expression, but higher iNOS and FAAH gene expression compared to control. No change was detected for NAPE-PLD gene expression. Student <i>t</i> test (N = 8): *<i>P</i><0.05, **<i>P</i><0.01 <i>versus</i> control group.</p

Clinical characteristics of control group.

*<p>BMI: body mass index Kg/m²; Medical treatment at the time to take samples of colon mucosa.</p

Representative high-magnification photomicrographs showing double immunofluorescence for FAAH, NAPE-PLD, CD38 and CD3 in order to characterize the immune cells in the mucosa infiltrate of UC patients.

<p>Nearly all FAAH immunofluorescent cells are plasma cell-specific CD38 (A–C). NAPE-PLD immunofluorescence was observed in both CD38+ plasma cells (D–F) and CD3+ T lymphocytes (G–I).</p

Schematic drawings that hypothesize the normal pattern of NAE-PPARα activity (A), and the pro- and anti-inflammatory NAE-PPARα signaling that may occur in the colonic epithelial cells of active UC at disease onset (B) and after a putative treatment with 5-ASA and/or glucocorticoids (C).

<p>Schematic drawings that hypothesize the normal pattern of NAE-PPARα activity (A), and the pro- and anti-inflammatory NAE-PPARα signaling that may occur in the colonic epithelial cells of active UC at disease onset (B) and after a putative treatment with 5-ASA and/or glucocorticoids (C).</p

Analysis of the number of FAAH-ir cells per area (µm²) in the lamina propria of acute and quiescent (5-ASA and corticoid-treated) UC patients compared to control ones.

<p>A–F: Representative high-magnification photomicrographs showing FAAH immunostaining in the lamina propria. G: Acute UC at disease onset was associated with a dramatic increase in the number of FAAH-ir cells in the infiltrate of the lamina propria. The number of FAAH-ir cells was significantly dropped after treatment, but do not reach control levels. Mann-Whitney U and Wilcoxon tests (N = 15–22): ***<i>P</i><0.001 <i>versus</i> control group; ^###<i>P</i><0.001 <i>versus</i> acute UC group.</p

Characteristics of UC patients.

$<p>C-reactive protein at diagnosis (mg/L);</p>*<p>Ulcerative colitis extension by Montreal criteria;</p>&<p>Modified Truelove–Witts Severity index (MTWSI): Mild 11–15 points, Moderate 16–20 points, Severe 21–27 points.</p

Analysis of the number of NAPE-PLD-ir cells per area (µm²) in the lamina propria of acute and quiescent (5-ASA and corticoid-treated) UC patients compared to control ones.

<p>A–D: Representative high-magnification photomicrographs showing NAPE-PLD immunostaining in the lamina propria. E: No significant change in the number of NAPE-PLD-ir cells was observed in the infiltrate of the lamina propria of acute UC patients and after treatment (5-ASA and corticoids). Mann-Whitney U and Wilcoxon tests (N = 15–22).</p