Journal: ACS Central Science

Article Title: A Bioinspired Scaffold with Anti-Inflammatory Magnesium Hydroxide and Decellularized Extracellular Matrix for Renal Tissue Regeneration

doi: 10.1021/acscentsci.8b00812

Figure Lengend Snippet: In vitro cytocompatibility and cell proliferation on PLGA/ECM/Mg(OH) 2 scaffolds. (a) Fluorescence images of HRCEpC labeled with PHK26 around the edges and in the center of scaffolds (S) and collagen gel (G) on the seeding day (0 d) and after 3 days (scale bar = 200 μm). (b) Number of cells in the scaffolds on these days. Values are expressed as mean ± SD ( n = 8). * p < 0.05 and ** p < 0.005.

Article Snippet: Renal epithelial cell growth kit, human renal cortical epithelial cells (HRCEpC), and renal epithelial cell basal medium were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: In Vitro, Fluorescence, Labeling

Journal: Cancers

Article Title: T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity

doi: 10.3390/cancers14081998

Figure Lengend Snippet: Co-expression of PD1-41BB does not change the in vitro safety profile of the lead PRAME-SLL-TCR. ( A ) Functional avidity of CD8+ TCR-Ts, with or without PD1-41BB, following stimulation with PD-L1-transduced T2 cells loaded with graded amounts of PRAME peptide (10 −10 M–10 −5 M). Cells were cultured at an E:T ratio of 1:1. The graph shows nonlinear regression curves of relative IFN-γ release. Peptide concentration needed for half maximal IFN-γ secretion is indicated by the dashed line. The data represent means of duplicates measured at each peptide concentration. ( B ) Left part: The recognition of 191 peptides with high sequence homology with the specific PRAME-SLL peptide by CD8+ TCR-Ts with or without PD1-41BB was analyzed. Peptides were loaded onto T2_PD-L1 cells at a concentration of 10 −6 M. IFN-γ release data for 30 peptides are shown as an example; data for all peptides are shown in . Right part: PRAME-negative, PD-L1-expressing SNB-19 cells were transfected with RNA constructs encoding either a midigene (~400 bp) coding for single peptides (SLL, 1, 26, 66) or minigene (~90 bp per peptide) constructs (MG) coding for up to five variant peptides and co-cultured with effector cells. All constructs included an epitope recognized by a positive control TCR. UT T-cells served as negative control. For read-out, supernatants were harvested after 20 h and analyzed by IFN-γ ELISA. The data represent means of duplicates. ( C ) IFN-γ released by CD8+ TCR-Ts with or without PD1-41BB co-cultured with 36 LCLs covering frequent HLA-A, -B, and -C alleles. UT CD8+ T-cells served as negative control, and PRAME-SLL-peptide-loaded HLA-A*02:01 positive LCLs were used as positive control. ( D ) IFN-γ released by CD8+ TCR-Ts with or without PD1-41BB co-cultured with normal cells derived from critical healthy tissues. As positive controls, cells were loaded with PRAME SLL-peptide. HREpC: human renal epithelial cells, HRCEpC: human renal cortical epithelial cells, RPTEC: renal proximal tubule epithelial cells, NHBE: normal human bronchial epithelial cells, NHLF: normal human lung fibroblasts, HOB: human osteoblasts, Mono: monocytes, iDC: immature dendritic cells, mDC: mature dendritic cells, iCardio: iCell Cardiomyocytes. The data represent means of duplicates. ( E ) Cytotoxicity of CD8+ TCR-Ts with or without PD1-41BB against PRAME-positive PD-L1-negative (red) and PRAME-negative PD-L1-positive (green) 3D tumor cell spheroids. Fresh PRAME-positive Mel624.38 spheroids (red) were added to the co-cultures on days 3 and 6, indicated by black arrows. For days 3 and 6 pictures, before and after adding fresh tumor cell spheroids are shown. Experiments in A–E were repeated at least three times with T-cells derived from different donors; data with cells from one representative donor are shown.

Article Snippet: Primary HLA-A*02:01-positive normal human bronchial epithelial cells (NHBE, Lonza), human renal cortical epithelial cells (HRCEpC, PromoCell, Heidelberg, Germany), human renal epithelial cells (HREpC, PromoCell), renal proximal tubule epithelial cells (RPTEC, Lonza), normal human lung fibroblasts (NHLF, Lonza), human osteoblasts (HOB, Lonza), and induced pluripotent stem cell-derived iCell Cardiomyocytes (iCardio, FujiFilm CDI, Madison, WI, USA) were cultured according to the manufacturers’ instructions.

Techniques: Expressing, In Vitro, Functional Assay, Cell Culture, Concentration Assay, Sequencing, Transfection, Construct, Variant Assay, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Derivative Assay