BACKGROUND: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective antitumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific CD8+ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. METHODS: Bone marrow-derived DC (BM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of beta-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using beta-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. RESULTS: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. CONCLUSION: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I-restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.
Animals ; Antigen Presentation ; Antigen-Presenting Cells ; beta-Galactosidase ; Brefeldin A ; Dendritic Cells* ; Enzyme Assays ; Immune System ; Immunization ; Immunotherapy ; Liposomes ; Mice ; Ovum ; T-Lymphocytes ; Vaccination

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.
Animals ; Brefeldin A ; pharmacology ; Cell Line, Tumor ; Cytosol ; drug effects ; metabolism ; Humans ; Intracellular Space ; drug effects ; metabolism ; Membrane Proteins ; metabolism ; Protein Transport ; drug effects ; Rats ; Vesicular Transport Proteins ; metabolism ; trans-Golgi Network ; drug effects ; metabolism

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6alpha, and the phosphorylation of eukaryotic initiation factor-2alpha and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.
Animals ; *Apoptosis ; Brefeldin A/pharmacology ; Cell Line ; Cytoprotection ; DNA-Binding Proteins/metabolism ; Endoplasmic Reticulum/drug effects/*physiology ; Flavanones/*pharmacology ; Heat-Shock Proteins/biosynthesis ; Hippocampus/cytology ; Membrane Potential, Mitochondrial/drug effects ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; Neurons/*drug effects/physiology ; Reactive Oxygen Species/*metabolism ; Signal Transduction/drug effects ; Thapsigargin/pharmacology ; Transcription Factor CHOP/*biosynthesis ; Transcription Factors/metabolism ; Unfolded Protein Response/drug effects