Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Significance of the rapid increase in GSH levels in the protective response to cadmium exposure through phosphorylated Nrf2 signaling in Jurkat T-cells.

Although cadmium (Cd) is a redox system disruptor, the systematic defensive responses to Cd-induced oxidative stress remain unclear. In this study, we initially determined that when human T-cell-derived Jurkat cells were exposed to a low concentration of Cd, the glutathione (GSH) concentration rapidly increased via the transient nuclear accumulation of the transcription factor Nrf2. Therefore, we hypothesized that this increase in the GSH levels was a significant event that occurred in response to the Cd toxicity in the Jurkat T-cells. To test this hypothesis, the expression of Nrf2 in the cells was silenced using siRNA transfection. These restricted expression conditions demonstrated that the sensitivity of the Jurkat T-cells to Cd toxicity was significantly higher in the knockdown cells. Whereas we could not find differences in the metallothionein (MT) expression responses, accumulation of Nrf2 in the nuclei and the GSH increase after Cd exposure were clearly suppressed in the Nrf2 knockdown cells. These findings strongly suggest that the Cd-induced activation of GSH synthesis is initiated as an acute response for Cd detoxification. Furthermore, the Cd remaining in the Jurkat T-cells did not cause a significant inhibition of cell growth after the rapid and transient increase in the GSH concentration returned to its basal level. Additionally, we found that MT expression induced by Cd occurred much later, with the expression seen at least 12h or more after the Nrf2-dependent immediate responses were almost completed. These results indicate that the rapid increase in GSH is an essential defensive response, with the subsequent induction of MT potentially chelating the Cd retained in the cell, thereby leading to continued suppression of Cd toxicity.