AimsCongenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models.Methods and resultsSkin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes IKr potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patient's mother), multi-electrode array and patch-clamp electrophysiology of LQT2–hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2–hiPSC cardiomyocytes were exposed to E4031 (an IKr blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2–hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by β-blockers, propranolol, and nadolol, the latter being used for the patient's therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol.ConclusionsFindings from this paper demonstrate that patient LQT2–hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.
Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.
Functional cardiomyocytes can now be derived routinely from hPSCs (human pluripotent stem cells), which collectively include embryonic and induced pluripotent stem cells. This technology presents new opportunities to develop pharmacologically relevant in vitro screens to detect cardiotoxicity, with a view to improving patient safety while reducing the economic burden to industry arising from high drug attrition rates. In the present article, we consider the need for human cardiomyocytes in drug-screening campaigns and review the strategies used to differentiate hPSCs towards the cardiac lineage. During early stages of differentiation, hPSC-cardiomyocytes display gene expression profiles, ultra-structures, ion channel functionality and pharmacological responses reminiscent of an embryonic phenotype, but maturation during extended time in culture has been demonstrated convincingly. Notably, hPSC-cardiomyocytes have been shown to respond in a highly predictable manner to over 40 compounds that have a known pharmacological effect on the human heart. This suggests that further development and validation of the hPSC-cardiomyocyte model as a tool for assessing cardiotoxicity is warranted.
Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1 (IL-1) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1 and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1 dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1 and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1 and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1 is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.
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