The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.

The immunosuppressive agents cydosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilin and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cydophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calciumdependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specificay inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.The immunosuppressants cyclosporin A (CsA), FK 506, and rapamycin have proven to be valuable probes for studying T-cell signal transduction (1, 2). While CsA and FK 506 bind to distinct cellular receptors, these agents exhibit essentially identical effects on T-cell activation: both inhibit Ca2+-dependent activation pathways that lead to transcription of lymphokine genes (1-6). Rapamycin, like FK 506, binds to FK 506-binding proteins (FKBPs), but inhibits T-cell activation by interfering with distinct Ca2+-independent signaling pathways (7-9). Rapamycin reverses the action of FK 506, apparently by competitive binding to FKBPs (1, 2, 8, 9). These and other findings have led to the model that an immunosuppressant bound to its cellular receptor (immunophilin) forms an inhibitory complex that interferes with signal transduction (1, 2, 9-11). The complexes of CsA-cyclophilin and FK 506-FKBP are postulated to affect the same signaling component, while rapamycin-FKBP affects a distinct component.Calcineurin, also known as phosphatase 2B, is a Ca2+-and calmodulin-dependent serine/threonine phosphatase consisting of two subunits with predicted molecular masses of 59 kDa and 19 kDa (12). It is expressed ubiquitously in eukaryotic cells, including yeast (13). In mammals, calcineurin is most abundant in brain (12) but also has been detected in T cells (14,15 10%6 (vol/vol) heat-inactivated fetal calf serum (FCS), 100 units of penicillin per ml, 100 j&g of streptomycin per ml, 10 mM Hepes, 2 mM L-glutamine, and 25 juM 2-mercaptoethanol (termed "complete medium") at 370C in humidified air containing 5% CO2. PC12 cells were obtained from K. Wood (Dana-Farber Cancer Institute) and cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS, 5% heat-inactivated horse serum, 100 units of penicillin per ml, 100 jug of streptomycin per ml, and 2 mM L-glutamine at 370C in humified air containing 10% CO2.Immunobl...

Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cisand trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals.Cyclosporin A (CsA) and the structurally unrelated immunosuppressant FK506 have been demonstrated to inhibit T-cell activation and to be effective in organ transplantation (1-3). CsA has been shown to bind to and inhibit the rotamase activity of a cytoplasmic enzyme, cyclophilin (CyP) (4-6). Recently, a cytoplasmic receptor for FK506, FK506 binding protein (FKBP), which is unrelated in sequence to CyP, has also been shown to have similar rotamase activity, suggesting that this common enzymatic property of immunophilin proteins (immunosuppressant binding proteins) may be related to the regulation of T-cell activation (7-10). Rapamycin is a macrolide similar in structure to FK506 and, like FK506,12) MATERIALS AND METHODSReagents, Cell Culture, Interleukin 2 (IL-2) Production, and DNA Fragmentation. The IL-2-producing murine T-cell hybridoma 16.CD2-15.20 (15) was cultured (106 cells per well) with or without a 2% (vol/vol) culture supernatant of the anti-murine CD3 monoclonal antibody (mAb) 145-2C11 (16) in the absence or presence of CsA, FK506, or rapamycin. At 24 hr, culture supernatants were dialyzed and assayed for the presence of IL-2 as described (15). DNA was isolated from the cells and assayed for induction of DNA fragmentation or apoptosis on an ethidium bromide-stained...

The use of social media as a recruitment tool for research with humans is increasing, and likely to continue to grow. Despite this, to date there has been no specific regulatory guidance and little in the bioethics literature to guide investigators and IRBs faced with navigating the ethical issues it raises. We begin to fill this gap by first defending a non-exceptionalist methodology for assessing social media recruitment; second, examining respect for privacy and investigator transparency as key norms governing social media recruitment; and, finally, analyzing three relatively novel aspects of social media recruitment: (i) the ethical significance of compliance with website ‘terms of use’; (ii) the ethics of recruiting from the online networks of research participants; and (iii) the ethical implications of online communication from and between participants. Two checklists aimed at guiding investigators and IRBs through the ethical issues are included as Appendices.