ABSTRACT
Natural killer (NK) cells are a part of the innate immune system and represent the first line of defense against infections and tumors. NK cells can eliminate tumor cells without major histocompatibility restriction and are independent of the expression of tumor-associated antigens. Therefore, they are considered an emerging tool for cancer immunotherapy. However, the general toxicity and biodistribution of NK cells after transplantation remain to be understood. This study was conducted to evaluate the general toxicity and biodistribution of human NK cells after single or repeated intravenous dosing in severely combined immunodeficient (SCID) mice. There were no test item-related toxicological changes in single and repeated administration groups. The no observed adverse effect level of human NK cells was 2 × 107 cells/head for both male and female SCID mice. Results from the biodistribution study showed that human NK cells were mainly distributed in the lungs, and a small number of the cells were detected in the liver, heart, spleen, and kidney of SCID mice, in both the single and repeated dose groups. Additionally, human NK cells were completely eliminated from all organs of the mice in the single dose group on day 7, while the cells persisted in mice in the repeated dose group until day 64. In conclusion, transplantation of human NK cells in SCID mice had no toxic effects. The cells were mainly distributed in the lungs and completely disappeared from the body over time after single or repeated intravenous administration.
ABSTRACT
The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.
Subject(s)
Astrocytes/radiation effects , Gliosis/etiology , Microglia/radiation effects , Animals , Astrocytes/drug effects , Astrocytes/pathology , Cells, Cultured , Coculture Techniques , Culture Media, Conditioned , Cyclooxygenase 2/drug effects , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/metabolism , Dinoprostone/radiation effects , Electrophoretic Mobility Shift Assay , Glial Fibrillary Acidic Protein/metabolism , Gliosis/pathology , Immunoblotting , Immunohistochemistry , Mice , Microglia/drug effects , Microglia/metabolism , Nitrobenzenes/pharmacology , RNA, Messenger/analysis , Radiation, Ionizing , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sulfonamides/pharmacologyABSTRACT
The postsynaptic density (PSD) is a proteinaceous cellular structure that is specialized for postsynaptic signal transduction. Here, we show that eukaryotic translation factor-1A (eEF1A; formerly known as eEF-1alpha) is associated with the excitatory PSD in rat forebrain. Immunoblot analysis showed that eEF1A in the PSD fraction is enriched over homogenate. Salt (1.0M NaCl), but not non-ionic detergents such as Triton X-100 (1.0%) and n-octyl glucoside (1.0%), could dissociate eEF1A from the PSD core. In cultured cortical neurons, eEF1A was colocalized with postsynaptic markers (PSD95 and SynGAPalpha), but not with a presynaptic marker (synaptophysin). These results indicate that eEF1A is present in the PSD of the excitatory synapses.
Subject(s)
Cerebral Cortex/metabolism , Peptide Elongation Factor 1/metabolism , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/ultrastructure , Immunoblotting , Prosencephalon/cytology , Prosencephalon/metabolism , Prosencephalon/ultrastructure , Rats , Signal Transduction , Synapses/metabolismABSTRACT
The current study examined the potential involvement of phosphatidylinositol 3 phosphate kinase (PI3K) in interferon-gamma (IFN-gamma)-stimulated nitric oxide (NO) generation in BV2 murine microglial cells. We found that LY294002, a PI3K inhibitor, markedly reduced IFN-gamma-induced morphological changes, NO production, and cell death. The inhibitory effect of LY294002 on NO generation may be mediated through specific inhibition of signal transducer and activator-1 (STAT1) and NF-kappaB, which are activated by IFN-gamma. Induction of the mRNA for IFN-gamma-mediated interferon response factor (IRF-1) and inducible protein-10 (IP-10) was not significantly affected by LY294002, indicating that suppression of PI3K may not be sufficient for downregulation of these genes. Although it remains unclear how PI3K signaling is involved in IFN-gamma-mediated inflammatory reactions in the brain, our findings provide some insight into the inflammatory mechanisms of IFN-gamma in the brain and suggest that regulators of the PI3K pathway may act as anti-inflammatory agents in microglia.
Subject(s)
Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Interferon-gamma/antagonists & inhibitors , Microglia/drug effects , Morpholines/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Animals , Cell Death/drug effects , Cell Death/physiology , Cell Line , DNA-Binding Proteins/metabolism , Drug Interactions , Enzyme Activation/drug effects , Gene Expression/drug effects , Interferon-gamma/pharmacology , Mice , Microglia/enzymology , Microglia/metabolism , NF-kappa B/metabolism , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Oncogene Protein v-akt , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Retroviridae Proteins, Oncogenic/metabolism , STAT1 Transcription Factor , Signal Transduction/drug effects , Trans-Activators/metabolism , Tyrosine/metabolismABSTRACT
We investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of synaptic proteins in dissociated E18 rat cortical cells. TCDD (0-50 nM) was added to plating media, and cell viability and expression of synaptic proteins were assayed on 4 and 7 days in vitro (DIV), respectively. TCDD had no apparent effect on early neurite outgrowth 12 h after plating. However, on 4 DIV, cell viability was reduced significantly, and neurons often revealed vacuoles in 20 or 50 nM culture and had limited secondary or higher order dendritic processes in 20 or 50 nM culture. Immunoblot analyses of cell homogenates indicated upregulation of NMDA receptor subunits (NR1, NR2A, and NR2B), but downregulation of synaptic organizing proteins (PSD-95, densin-180, and septin6 homologue) and a synapse-enriched enzyme (alphaCaMKII). Changes in the expression of synaptic proteins may be a underlying mechanism for altered synaptic transmission and neuropathy by TCDD.
