Relative improvement of experimental colitis with roflumilast 5 mg/kg/d and pumafentrine 5 mg/kg/d.

<p>Data is depicted as mean ± standard error * p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001, DSS: Dextrane sodium sulfate; TNFα: tumor necrosis factor-α.</p><p>Data are depicted as % improvement of the dextrane sulfate sodium (DSS) treatment groups versus DSS methocel groups.</p

Effect of pumafentrine on clinical score, colon length and histologic score. A. Efficacy of pumafentrine in DSS-induced colitis.

<p>Mice were exposed to 3.5% DSS in drinking water for eleven days. Either 1.5 mg/kg/d pumafentrine (n = 8), 5 mg/kg/d pumafentrine (n = 16) or 4% methocel (n = 16) were administered orally once daily for eleven days. Non-DSS mice received either 20 mg/kg/d pumafentrine or 4% methocel (n = 8). The degree of colitis was quantified by the clinical score assessing weight loss, stool consistency and rectal bleeding (range from 0 =  healthy to 4 =  maximal disease activity). The scores are depicted as mean ± SEM; *p<0.05, **p<0.01 versus DSS+methocel. <b>B. Reduction of colon length shortening in DSS-induced colitis by pumafentrine.</b> Mice were exposed to 3.5% DSS in drinking water for an eleven day period. Pumafentrine treatment (either 1.5 mg/kg/d (n = 8) or 5 mg/kg/d (n = 16) orally, once daily for 11 days) or 4% methocel (n = 12) were started the same day as DSS administration. Non-DSS mice received 20 mg/kg/d pumafentrine or 4% methocel (n = 8), respectively. Values are depicted as mean ± SEM. **p<0.01 versus DSS+methocel. <b>C. Effect on histologic signs of colonic inflammation by pumafentrine.</b> Mice were exposed to 3.5% DSS in drinking water for eleven days and were treated with pumafentrine (either 1.5 mg/kg/d (n = 8) or 5 mg/kg/d (n = 16) orally once daily for 11 days) or 4% methocel (n = 12). Non-DSS mice received 20 mg/kg/d pumafentrine or 4% methocel (n = 8). At day 11 mice were euthanized, colon rings were stained and the histologic score (degree of inflammation: 0 =  no changes to 6 =  extensive cell infiltration and tissue damage) was determined in a blinded fashion as described in detail in the <i>Material and Methods</i>. Scores are depicted as means ± SEM.</p

Reduction of colonic mucosa TNFα content by roflumilast.

<p>Mice were exposed to 3.5% DSS in drinking water for eleven days and were treated with roflumilast (either 1 or 5 mg/kg/d orally once daily for 11 days, n = 8) or 4% methocel (n = 5). At day 11 the colon was removed, weighed, vortexed in PBS and centrifuged. TNFα was quantified in the eluate by ELISA. Values represent mean ± SEM; *p<0.05.</p

Effect of pumafentrine on colonic mucosa TNFα concentration.

<p>Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine (either 1.5 mg/kg/d (n = 8) or 5 mg/kg/d (n = 16) orally once daily for 11 days) or 4% methocel (n = 12). Non-DSS mice received 20 mg/kg/d pumafentrine or 4% methocel (n = 8). At day 11, the end of experiment, the colon was removed, weighed, vortexed in PBS and centrifuged. TNFα was quantified in the eluate by ELISA. Values represent mean ± SEM; *p<0.05 versus DSS+methocel.</p

Systemic effects of pumafentrine. A Effect of <i>in vivo</i> administered pumafentrine on PMA/ionomycine-induced IFNγ synthesis by splenocytes from DSS-exposed mice.

<p>Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated. After a 20 h incubation period with PMA 25 ng/ml and ionomycine 500 ng/ml, the experiment was stopped by three freeze-thaw cycles and total IFNγ was measured by ELISA. Results are depicted as mean ± SEM. *p<0.05 versus DSS+methocel. <b>B. Effect of </b><b><i>in vivo</i></b><b> administered pumafentrine on PMA/ionomycine-induced CD69 expression by splenocytes from DSS-exposed mice.</b> Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated and stimulated with PMA 25 ng/ml and ionomycine 500 ng/ml. After 20 h incubation period the CD69-expression was determined by flow cytometry. Bars represent the mean ± SEM of CD69 positive splenocytes. *p<0.05 versus DSS+methocel.</p

Effect of roflumilast on clinical score and colon length. A. Mitigation of DSS-induced colitis by roflumilast.

<p>Mice were exposed to 3.5% DSS in drinking water for 11 days. Either 1 mg/kg/d roflumilast, 5 mg/kg/d roflumilast or 4% methocel were administered orally once daily for 11 days (n = 8). Non-DSS-treated mice received 5 mg/kg/d roflumilast (n = 8) or 4% methocel (n = 5). The degree of colitis was quantified by the clinical score assessing weight loss, stool consistency and rectal bleeding (range from 0 =  healthy to 4 =  maximal disease activity). Scores are depicted as mean ± SEM; *p<0.05, **p<0.01 versus DSS+methocel. <b>B. Effect of roflumilast on colon length shortening in DSS-induced colitis.</b> Mice were exposed to 3.5% DSS in drinking water for an 11 day period. Roflumilast treatment (either 1 mg/kg/d or 5 mg/kg/d orally, once daily for eleven days) or 4% methocel were started on the same day as DSS administration (n = 8). Non-DSS mice received 5 mg/kg/d roflumilast (n = 8) or 4% methocel (n = 5). Values are depicted as mean ± SEM. **p<0.01, ***p<0.001 versus DSS+methocel.</p

Murine models of inflammation-driven carcinogenesis.

<p>(A) Endoscopic images of inflamed mucosa and tumor of AOM/DSS-colitis as well as the IL10^−/− model. Tumorigenesis was assessed by high resolution endoscopy <i>in vivo</i>. Methylene blue was used to enhance dysplasia detection as shown exemplarily for AOM/DSS colitis (B) Tumor size as rated by the tumor size score described in the methods section, IFNγ -levels, and inflammation score as assessed in H.E. staining for untreated controls, AOM/DSS-mice, and IL10^−/− mice. Bars represent means, whishkers represent standard error of the mean (SEM). Colonic mucosa samples of 10 healthy untreated C57BL/6J-mice were applied to assess histologic parameters and IFNγ -expression.</p

Ploidy assessment according to Auer's classification, p53-, beta-catenin-, and Ki67-immunohistochemistry for murine tissue analyzed.

<p>AOM-CA_n: carcinomas of AOM/DSS-colitis. IL10CA_n: carcinomas of IL10^−/−-mice. CNTRL_n: premalignant tissue.</p><p>DNA-ploidy was assessed according to Auer (please refer to the text). p53 was assessed semiquantitatively, 0: no expression, 1 1–20% positive mucosa cells, 2: 21–50%, 3>50%</p><p>Beta-catenin: 1: membranous, 2: membranous-cytoplasmatic, 3: cytoplasmatic, 4: cytoplasmatic-nulcear, to 5, nuclear.</p

Immunohistochemistry for beta-catenin, p53 and Ki67 of murine premalignant tissue and CACs, details of 100x magnification.

<p>Immunohistochemistry for beta-catenin, p53 and Ki67 of murine premalignant tissue and CACs, details of 100x magnification.</p

Overview SWS.

<p>Plot of SWS determined with all three methods in all patients. Additionally, the reference value for stage F2 fibrosis is shown for ARFI quantification and 2D-SWE.</p