In this study, we investigated the effects of IL-19 in the pathogenesis of esophageal carcinoma in vitro and in vivo. We also determined the effect of anti-IL-19 monoclonal antibody (mAb) on reducing esophageal carcinoma tumor growth in a mouse model. Materials and MEK162 CAS Methods Patients and Tissue Specimens This retrospective study was done in accordance with the guidelines of the Chi-Mei Medical Center Institutional Review Board (IRB9705-003). De-identified esophageal squamous cell carcinoma (SCC) samples of 60 patients obtained from the Pathology Archive, part of Chi-Mei Medical Center Tumor and Serum Bank between January 2009 and December 2011 were used for immunostaining. Healthy tissue samples were from non-pathological areas distant from tumors in surgical specimens (confirmed by histology examination).
Non-tumor tissue samples with signs of inflammation were excluded [34]. The clinicopathologic variables evaluated are listed in Table 1. Table 1 Associations between IL-19 expression in 60 esophageal SCC tumors with important clinicopathologic variables. Cell Lines and Culture Conditions Esophageal cancer cell line CE81T/VGH was purchased from The Food Industry Research and Development Institute (Hsinchu City, Taiwan) [35]. Cells were grown in Dulbecco��s modified Eagle��s minimal essential medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco BRL) and kept at 37��C in a 5% CO2/95% air atmosphere.
Expression and Purification of hIL-19 and hIL-20R1 Recombinant Protein A cDNA clone coded for the human (h)IL-19 and extracellular domain of hIL-20R1 sequences from was inserted into the expression vector of Pichia pastoris (pPICZ-��; Invitrogen, San Diego, CA, USA). We used affinity chromatography to express and purify hIL-19 and hIL-20R1 from the culture medium of the yeast cells [29]. The biological function was tested by treating IL-19 in peripheral mononuclear blood cells (PBMCs), which induced IL-10 production [36]. Generating anti-hIL-19 and -hIL-20R1 Monoclonal Antibodies (mAb) Monoclonal antibodies against hIL-19 (anti-hIL-19 mAb, 1BB1) and hIL-20R1 (anti-hIL-20R1 mAb, 51D) were generated following the standard protocols [29]. In brief, the hybridoma cells (1��106) were injected intraperitoneally into pristine-pretreated BALB/c mice.
Ascites fluid was collected after 2 weeks, and then 1BB1 or 51D mAb were purified with a Protein-A column (Pharmacia, Uppsala, Sweden). We previously reported [25], [33] that 1BB1 neutralized hIL-19. The 1BB1 mAb Cilengitide specifically recognized IL-19 but not other human IL-10 family cytokines such as IL-10, -20, -22, -24, and -26 [32]. Immunohistochemistry Paraffin-embedded-tissue samples were used for immunohistochemical staining with purified 1BB1 (diluted 150) at 4��C overnight [27], [32], [33].