Galectin-4 and its domains distinctively reduce secretion of pro-inflammatory cytokines.

<p>(A) Cytokine release was tested in PBT stimulated with anti-CD3/CD28 for 72 hours. Data represent mean±SEM of six individual experiments. *p≤0.01 for decrease vs. baseline. (B) IL-17 secretion of LPT and PBT was determined by an IL-17 specific ELISA. LPT and PBT were activated by anti-CD2 or -CD3/CD28 mAb, respectively and cultured in the presence or absence of 100 µg/ml Gal-4. Data represent mean±SEM of three individual experiments.</p

Galectin-4 induces T cell apoptosis caspase-independently via calpain-mediated pathways.

<p>(A) PBT and LPT were stimulated with anti-CD3/CD28 or -CD2, respectively, in the presence of 0, 50, 100 or 200 µg/ml Gal-4 and apoptosis was determined by annexin-V staining. Data represent mean±SEM of eight individual experiments. *p≤0.05 for increase vs. baseline. (B) PBT were stimulated in the presence of 0, 25, 50, 100 or 200 µg/ml Gal-4. Apoptosis was detected by annexin V/PI staining, necrosis by PI staining followed by flow cytometry. Data represent mean±SEM of six individual experiments. *p≤0.05 for increase vs. baseline. (C) PBT were transfected with Gal-4 siRNA or scrambled control, activated by anti-CD3/CD28 and cultured in the presence of the TNF-α blocker adalimumab for three days. Apoptosis was detected by annexin V/PI staining followed by flow cytometry. Data are representative for three individual experiments. (D) Caspase-3, -8, and -9 activity in anti-CD3/CD28 stimulated T cells cultured in the presence or absence of 100 µg/ml Gal-4. Data are representative for three individual experiments. (E) PBT were activated by anti-CD3/CD28 and cultured for 24 h in the presence or absence of 100 µg/ml Gal-4 and 50 mM Calpain inhibitor z-LLY-fmk, 4 mM EGTA or 30 mM BAPTA-AM. Data are representative for three individual experiments. (F) Disruption of the mitochondrial membrane potential was detected by rhodamine123 staining followed by flow cytometric analysis. T cells were stimulated with anti-CD3/CD28 and incubated for 3 h in the presence or absence of 100 µg/ml Gal-4. Afterwards cells were analysed by flow cytometry. All data are representative for three individual experiments. (G) Disruption of the mitochondrial membrane potential was detected after 12 h by rhodamine123 staining followed by flow cytometric analysis. All data are representative for three individual experiments.</p

Galectin-4 binds to stimulated, but not resting T cells.

<p>(A) Flow cytometric analysis of Gal-4 binding to resting and anti-CD3/CD28 stimulated T cells using Gal-4 detected by an anti-Gal-4 Ab and PE-labeled secondary Ab. Carbohydrate-dependence of the binding was analysed by addition of 50 mM lactose as a pan-galectin inhibitor and 50 mM sucrose as control. Data are representative of three individual experiments. (B) Immunoprecipitation of Gal-4 binding complexes. BSA served as negative, Gal-1 as positive control. Data are representative for four individual experiments.</p

Identification of Galectin-4 expression.

<p>(A) Gal-4 expression was detected in cryosections of the GI tract of healthy volunteers. The results are representative for four individuals (original magnifications: ×100). (B) PCR analysis of Gal-4 mRNA expression in resting and anti-CD3/CD28 stimulated PBT. (C) Flow cytometric analysis of intra- and extracellular Gal-4 content in resting and anti-CD3/CD28 stimulated PBT. Data are representative of three individual experiments.</p

Galectin-4 ameliorates experimental colitis by inducing apoptosis and reduction of pro-inflammatory cytokine secretion.

<p>(A) Disease activity index in acute DSS-induced colitis, treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. *p≤0.01 for change vs. control. (B) Detection of apoptotic cells by TUNEL staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (C) Apoptotic cells after TUNEL staining were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.05 for change vs. control. (D) Detection of proliferating cells by BrdU staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (E) BrdU positive cells were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.01 for change vs. control. (F) Cytokine secretion was determined by CBA analysis from colonic cultures from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. All data represent mean±SEM of ten individual experiments. *p≤0.05 for change vs. control.</p

Cost-utility of biological treatment sequences for luminal Crohn’s disease in Europe

<p><b>Background</b>: This study aims to compare the cost-effectiveness of treatment sequences with available biologics, including adalimumab (ADA), biosimilar infliximab (bsIFX), originator infliximab (IFX) and vedolizumab (VEDO) for luminal Crohn’s disease in nine European countries.</p> <p><b>Methods</b>: A Markov-model was constructed to simulate five-year medical costs and quality-adjusted life years (QALYs). Data on clinical efficacy were obtained from randomised controlled trials. Country-specific unit costs, discount rates and a third-party payer perspective were applied.</p> <p><b>Results</b>: The bsIFX versus conventional therapy resulted in the most favourable incremental cost-utility ratios (ICURs) ranging from €34,580 (Hungary) to €77,062/QALY (Sweden). Compared to bsIFX, the bsIFX-ADA sequence was more cost-effective than the bsIFX-VEDO sequence with ICURs varying between €70,277 (France) and €162,069/QALY (Germany). The ICURs of the bsIFX-ADA-VEDO sequence versus the bsIFX-ADA strategy were between €206,266 (The Netherlands) and €363,232/QALY (Spain).</p> <p><b>Conclusion</b>: We are the first to compare cost-effectiveness of multiple biological sequences for luminal Crohn’s disease. Based on our findings, bsIFX can be recommended as a first-line treatment in patients unresponsive to conventional treatments. While biological sequences only slightly differ in their associated health gains, their costs vary greatly. The bsIFX-ADA-VEDO seems to be the most cost-effective sequence of the available biologics across Europe.</p