Gene-flow patterns in Atlantic and Mediterranean populations of the Lusitanian sea star Asterina gibbosa.

In this study, the population structure of the Lusitanian sea star Asterina gibbosa was assessed using amplified fragment length polymorphism (AFLP). One hundred and twenty-two AFLP loci were analysed in 159 individuals from eight populations from across the species' range and revealed high levels of genetic diversity, with all individuals but two harbouring a unique banding pattern. As reported for other marine invertebrates, we found high levels of genetic differentiation between the Atlantic and Mediterranean basins, suggesting that the Strait of Gibraltar represents a major barrier to dispersal for this sea star. Our assignment studies suggest that, in the Atlantic, a measurable degree of gene flow occurs between populations, which could result in the isolation-by-distance pattern of differentiation observed in this basin. In contrast, no evidence of contemporary gene flow was found in the Mediterranean, suggesting contrasting patterns of dispersal of Asterina gibbosa in the Atlantic and Mediterranean basins.

A novel aspect of the anti-inflammatory actions of glucocorticoids: inhibition of proximal steps of signaling cascades in lymphocytes.

Glucocorticoid hormones are effective in inhibiting inflammatory responses, but the mechanisms that confer this action have not been completely elucidated. The prevailing view is that these compounds inhibit novel gene transcription regulated by the nuclear factor kappa B and/or activator protein-1 transcription factors. In the last few years, several reports have shown that glucocorticoids can also block signal transduction in lymphocytes at an early, postreceptor step, suggesting novel molecular targets for these hormones. These data will be briefly reviewed and the possible in vivo relevance of these findings discussed, with particular emphasis on T cell development.

Dexamethasone inhibits invasion of murine T cells through cultured fibroblastic monolayers.

Despite the wide clinical use of glucocorticoids in the chemotherapy of leukaemia and lymphoma, there have been limited efforts at understanding the effects of these hormones on metastasis formation. The purpose of this study was to investigate the effects of glucocorticoids on the tissue-infiltrating capability of lymphoid cells. Using an in vitro invasion assay, we found that dexamethasone, a synthetic glucocorticoid analogue, inhibited the invasion of a murine T-cell hybridoma through a monolayer of fibroblast-like cells. Even low doses of dexamethasone were effective at inhibiting cellular transmigration (EC50 = 0.4 nM). A maximal decrease was observed after an overnight culture in the presence of dexamethasone. The effect persisted for at least 24 h after removal of the drug and required the binding of the hormone to its intracellular glucocorticoid receptor. Our results suggest that the decreased invasiveness of dexamethasone-treated cells is not the consequence of reduced motility or deficient production of an autocrine factor required for cell migration. This in vitro study suggests that glucocorticoids may act to reduce dissemination of lymphoma cells in vivo.

Glucocorticoids attenuate T cell receptor signaling.

Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen-major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the zeta chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.

Dexamethasone increases intracellular cyclic AMP concentration in murine T lymphocyte cell lines.

The effects of the synthetic glucocorticoid dexamethasone on the cAMP content of murine T lymphocyte cell lines has been investigated. Incubation of the 3B4.15 T cell hybrids with dexamethasone results in an average 5-fold increase in intracellular cyclic AMP levels after 6 h of treatment. This phenomenon is abolished in the presence of RU486 and of cycloheximide, indicating that it requires binding of the drug to the intracellular glucocorticoids receptor and de novo protein synthesis. Dexamethasone-induced elevation of intracellular cyclic AMP correlates with both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity in T cell hybrids. This modulation of cyclic AMP metabolism is independent of serum-derived factors, suggesting that it is not secondary to transmembrane receptor stimulation by an extracellular ligand. We propose that glucocorticoids interfere with the homeostatic control of intracellular cAMP concentration, leading to a sustained increase in the content of this important second messenger in murine T lymphocyte cell lines. This study suggests that elevation of cAMP levels may represent one way by which glucocorticoids modulate the immune response.

Activation of the liver glycogen phosphorylase by Ca(2+)oscillations: a theoretical study.

Cytosolic calcium plays a crucial role as a second messenger in cellular signalling. Various cell types, including hepatocytes, display Ca(2+)oscillations when stimulated by an extracellular signal. However, the biological relevance of this temporal organization remains unclear. In this paper, we investigate theoretically the effect of Ca(2+)oscillations on a particular example of cell regulation: the phosphorylation-dephosphorylation cycle controlling the activation of glycogen phosphorylase in hepatocytes. By modelling periodic sinusoidal variations in the intracellular Ca(2+)concentration, we show that Ca(2+)oscillations reduce the threshold for the activation of the enzyme. Furthermore, as the activation of a given enzyme depends on the kinetics of its phosphorylation-dephosphorylation cycle, specificity can be encoded by the oscillation frequency. Finally, using a model for signal-induced Ca(2+)oscillations based on Ca(2+)-induced Ca(2+)release, we show that realistic Ca(2+)oscillations can potentiate the response to a hormonal stimulation. These results indicate that Ca(2+)oscillations in hepatocytes could contribute to increase the efficiency and specificity of cellular signalling, as shown experimentally for gene expression in lymphocytes (Dolmetsch et al., 1998).

Multidisciplinary discharge planning: developing a process.

In today's health care environment, Frankford Health Care System identified the need for a discharge planning process. A multidisciplinary task force developed and implemented a system that decreased length of stay and duplication of services, improved timeliness of intervention and fostered communication and respect among team members. The process included assessment of patient/family needs and coordination of care, services and referrals for all patients.

Dexamethasone inhibits the early steps of antigen receptor signaling in activated T lymphocytes.

Glucocorticoids are very effective in inhibiting inflammatory and immune responses. In particular, the synthetic analogue dexamethasone has been shown to inhibit T cell proliferation and IL-2 production by interfering with the transcriptional activation of the IL-2 gene. The experiments described in this report were performed to determine whether dexamethasone treatment affects the early steps of TCR signal transduction in T cell hybrids. Incubation of murine T cell hybrids in the presence of dexamethasone prevents the intracellular calcium increase that normally follows TCR/CD3 aggregation. Accordingly, dexamethasone treatment decreases inositol phosphates production and phospholipase-Cgamma1 tyrosine phosphorylation induced after TCR/CD3 stimulation. Dexamethasone has no effect on cell surface expression of TCR-associated structures nor does it inhibit calcium responses induced by a heterologous G protein-coupled muscarinic receptor, suggesting that this hormone analogue specifically inhibits the TCR signaling pathway at a postreceptor stage. We also show that inhibition of membrane proximal events by dexamethasone requires binding to the intracellular glucocorticoid receptor and de novo protein synthesis. When splenic T cells were assayed, only activated but not resting T cells were found to be sensitive to this new immunomodulatory effect of dexamethasone. These findings indicate that in addition to their previously described inhibitory effects on cytokine gene transcription, glucocorticoids block IL-2 production in activated T cells by interfering with an early step of the signal transduction cascade initiated by TCR/CD3 cross-linking.

Induction of T cell unresponsiveness by anti-CD3 antibodies occurs independently of co-stimulatory functions.

Antibodies to the T cell receptor (TcR)-associated CD3 molecules represent potent immunosuppressive agents in vivo in both human and animals models, in spite of their well-characterized mitogenic properties. We demonstrate in this report that antibodies to the B7.2 molecule inhibit IL-2 production in vivo caused by anti-CD3 administration, suggesting that anti-CD3 monoclonal antibodies (mAb) stimulate naive T cells in vivo in a co-stimulation-dependent fashion. To characterize better the mechanisms by which antibodies to CD3 induce antigen unresponsiveness in naive T cells, we developed a model of activation-induced T cell unresponsiveness in vitro. Our data indicate that following interaction with mitogenic anti-CD3 mAb in vitro, naive purified CD4+ T cells become refractory to a further stimulus. This unresponsive state develops independently of co-stimulatory functions, as neither B7-expressing antigen-presenting cells nor anti-CD28 mAb are able to prevent anergy induction in this model. We therefore conclude that induction of unresponsiveness in naive T cells by anti-CD3 mAb is not a consequence of co-stimulus-deficient stimulation, but may develop following a productive response both in vivo and in vitro. Unresponsive T cells display a defective calcium mobilization upon TcR triggering, suggesting that anergy is maintained in these cells through receptor desensitization. The potential role of co-stimulation-independent TcR desensitization in the down-regulation of immune responses in vivo is briefly discussed.

Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor.

A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.