Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca²⁺-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.     

Production of IL2 and induction of IL2 receptor: two independent and dissociable events during allogenic stimulation in vitro in man

Donors from HLA genotyped families are extremely useful for immunogenetic studies. Analysis of the in vitro allogenic response of lymphocytes from HLA recombinant siblings can dissect the respective role of HLA class I and class II antigens for the lymphocyte activation. Using lymphocytes from such donors, we have demonstrated that IL2 production is triggered by HLA class II disparities (but not by HLA class I), HLA class I disparities can prime the cytotoxic T lymphocytes precursors inducing the IL2 receptor on the specific clones which will proliferate in presence of IL2 either from endogenous origin (in the case of HLA class II disparities) or from experimentally added IL2 in the culture.     

Dendritic cell recovery post-lymphodepletion: a potential mechanism for anti-cancer adoptive T cell therapy and vaccination

Adoptive transfer of autologous tumor-reactive T cells holds promise as a cancer immunotherapy. In this approach, T cells are harvested from a tumor-bearing host, expanded in vitro and infused back to the same host. Conditioning of the recipient host with a lymphodepletion regimen of chemotherapy or radiotherapy before adoptive T cell transfer has been shown to substantially improve survival and anti-tumor responses of the transferred cells. These effects are further enhanced when the adoptive T cell transfer is followed by vaccination with tumor antigens in combination with a potent immune adjuvant. Although significant progress has been made toward an understanding of the reasons underlying the beneficial effects of lymphodepletion to T cell adoptive therapy, the precise mechanisms remain poorly understood. Recent studies, including ours, would indicate a more central role for antigen presenting cells, in particular dendritic cells. Unraveling the exact role of these important cells in mediation of the beneficial effects of lymphodepletion could provide novel pathways toward the rational design of more effective anti-cancer immunotherapy. This article focuses on how the frequency, phenotype, and functions of dendritic cells are altered during the lymphopenic and recovery phases post-induction of lymphodepletion, and how they affect the anti-tumor responses of adoptively transferred T cells.     

Effect of Temporal Expression of Integral Membrane Proteins by Baculovirus Expression Vector System

Integral membrane proteins (IMPs) are popular target for drugs, but their resolved structures have been overlooked when compared with cytosolic proteins. The main reason is that IMPs usually need intensive post-translational modifications and they are bound to membranes, which increase the complexity of purifying or crystalizing them. Although different expression systems are used to express IMPs, baculovirus is considered one of the most successful expression systems for those proteins. Despite that, there are always unknown discrepancies in the level of IMPs expression in the baculovirus expression system. Retrospective studies have shown that expression of an immunoglobulin (anti-Chymase mouse monoclonal IgG1) driven by vp39 promoter was more efficient compared to its expression under polyhedrin (polh) promoter; however, this conclusion was not tested on different IMPs to generalize such a conclusion. In this study, the expression of eight different IMPs has been compared under vp39 and polh promoters of Autographa californica nucleopolyhedrovirus. Although different IMPs have shown different patterns of expression, the expression driven by vp39 promoter was found to be generally more efficient than the polh promoter.