Antigen cross-presentation is a crucial step in the assembly of an antitumor immune response leading to activation of naïve CD8 T cells. This process has been extensively used in clinical trials, in which dendritic cells generated in vitro are loaded with tumor antigens and then autotransplanted to the patients. Recently, the use of autologous transplant of dendritic cells fused with dying tumor cells has demonstrated good results in clinical studies. In this work, we generated a similar process in vivo by treating mice with dead tumor cells [cell bodies (CBs)] expressing the fusogenic protein of the infectious salmon anemia virus (ISAV). ISAV fusion protein retains its fusogenic capability when is expressed on mammalian cells in vitro and the CBs expressing it facilitates DCs maturation, antigen transfer by antigen-presenting cells, and increase cross-presentation by DCs in vitro. Additionally, we observed in the melanoma model that CBs with or without ISAV fusion protein reduce tumor growth in prophylactic treatment; however, only ISAV expressing CBs showed an increase CD4 and CD8 cells in spleen. Overall, our results suggest that CBs could be used as a complement with other type of strategies to amplify antitumor immune response.
Piscirickettsia salmonis , an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.
Ethanol (EtOH) enhances glycinergic currents in the central nervous system (CNS). Because evidence for an interaction between the α1 subunit of the glycine receptor (α1GlyR) and the G protein Gβγ subunit exists in vitro and because cAMP levels are known to increase in response to EtOH, we wanted to investigate the interaction between Gβγ and α1GlyR in response to EtOH treatment in HEK293 cells and to explore the possible sites of interaction between EtOH and the Gαs subunit. His pull-down assays in GlyRHis6-transfected HEK293 cells incubated with ethanol or propofol revealed that only EtOH treatment increased the binding of Gβγ heterodimers to α1GlyR. Using molecular modelling (protein structure prediction), was modelled the hGαs protein for the first time and validated this model by site-directed mutagenesis. By molecular docking, we identified some potential regions of interaction between hGαs and EtOH that are located on the SIII and SI regions of the Gαs. Therefore, we conclude that ethanol increases the interaction between α1GlyR and Gβγ in HEK293 cells, an effect that might be attributed to the interaction between EtOH and hGαs, which consequently stimulates hGαs.
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