, 2006; Lifshitz et al., 2009). The tissue-protective and immunomodulatory functions of Epo on the one hand and erythropoiesis on the other are mediated by different EpoR (Brines et al., 2004; Brines & Cerami, 2008). The hematopoietic receptor is a homodimer of EpoR subunits with a very high affinity to Epo, corresponding to picomolar concentrations Z-VAD-FMK ic50 of circulating Epo. The tissue-protective receptor, in contrast, is a heterodimer consisting of one EpoR subunit disulfide-linked to the β common receptor (CD131). Its affinity for Epo is lower and local concentrations of Epo therefore need to be higher. Efforts have been made to design Epo analogues with confined receptor specificity, allowing tissue-protective, but
not erythropoietic activity (Brines et al., 2008). The pyroglutamate helix B surface peptide (ARA290) is a short peptide of 11 amino acids, designed for specificity to the EpoR–CD131 heterocomplex and without erythropoietic
function (Brines et al., 2008). The tissue-protective and lack of erythropoitetic activity have been reported for ARA290 with in vitro and animal studies. Here, we sought to investigate the influence of ARA290 on two parameters crucial for UTI pathogenesis, early immune response and cellular infection by UPEC, using a cell culture model of E. coli UTI. All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) GSK-3 inhibitor review and maintained in an appropriate medium (Gibco, Carlsbad, CA) at 37 °C in a 5% CO2 and humidified atmosphere. The human bladder cell lines T24 (HTB-4) and 5637 (HTB-9) were cultured in McCoy’s medium and RPMI-1640 medium containing l-glutamine, respectively, supplemented with 10% fetal bovine serum. Primary human bladder epithelium progenitor cells were purchased from CELLnTEC (Bern, Switzerland). Cells were maintained in CnT-58 medium supplemented with antibiotics to final GNAT2 concentrations of 100 U mL−1 penicillin, 100 μg mL−1 streptomycin and 250 ng mL−1
amphotericin B (CELLnTEC) in a 5% CO2 and humidified atmosphere at 35 °C following the instructions of the supplier. For all the experiments, cells reaching confluence were used. The monocytic cell line THP-1 (TIB-202) was maintained in RPMI-1640 medium containing l-glutamine and supplemented with 10% fetal bovine serum, 1 mM HEPES and 0.05 mM 2-mercaptoethanol. In all the experiments, 106 THP-1 cells mL−1 were used. The E. coli cystitis strain NU14 was used for cell stimulation. Bacteria were grown in a static Luria–Bertani broth to enhance the expression of type 1 fimbriae and collected by centrifugation at 3500 g for 10 min. Bacteria were inactivated by the addition of gentamicin to the cell culture medium (40 μg mL−1) to allow longer stimulation without perturbing the viability of epithelial cells. Alternatively, bacteria were heat-inactivated when cells were used for subsequent infection assays. For this purpose, E.