Simultaneous determination of d- and l-thyroxine in human serum by liquid chromatography with electrochemical detection

A method for the determination of d- and l-thyroxine in human serum is described. The method involves extraction of thyroxine from serum and the separation of thyroxine enantiomers on a reversed-phase, high-performance liquid chromatographic column by use of a chiral eluent containing l-proline and cupric sulfate. Satisfactory resolution of the enantiomers of thyroxine, triiodothyronine, and reverse triiodothyronine can be achieved in 12 min and, employing amperometric detection to monitor the separation, the detection limit for serum thyroxine is in the range of 1–3 ng per injected sample.     

Cytokine-Independent Proliferation of IL-2-Nonproducing CTL Clones in Association with High TCR-Signal Transduction Responses

Alloreactive CTL clone D2-23 proliferated in response to antigenic cells without IL-2 production. Among subclones of D2-23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fc receptors. Cyclosporin A, which totally blocks IL-2 production of Th1 cells, barely or only partially inhibited PMA- or aCD3-induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL-2 plus antigenic cells, or even IL-2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR-linked mechanism for CTL proliferation that is independent of costimulatory cell- or cytokine-mediated signaling, but is originally prepared by prior stimulation with IL-2.     

TCR-Independent Induction of Low Responsiveness by Chemically Fixed Cells in Alloreactive CTL Clones and Its Prevention through Cognate Cell-Cell Interaction

We established BALB/c-derived CD8⁺ CTL clones D2-22 (V_β 6⁺), D2-23 (V_β 8⁺) and D2-24 (V_β 8⁺) specific for B10.D2 minor H antigen. D2-22 and D2-23 proliferated without producing IL-2 in response to X-ray-irradiated antigenic cells, Con A, aCD3, PMA and IL-2. Paraformaldehyde-fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL-2-producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen-specific unresponsiveness in these IL-2-nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen-nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2-23 cells. Close contact of D2-23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell-cell interaction could be replaced by exogenously added IL-2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2-23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL-2-nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.