Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1µM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.

Pimecrolimus, a topical calcineurin inhibitor used in the treatment of atopic eczema.

INTRODUCTION: Pimecrolimus , a calcineurin inhibitor, is a non-steroidal treatment option in patients aged ≥ 2 years with mild-to-moderate atopic eczema (AE). It was approved as a viable therapeutic option by the FDA in 2001 and in the European Union a year later in 2002. Calcineurin inhibitors inhibit the synthesis of inflammatory cytokines released from T cells and mast cells. In contrast to corticosteroids, calcineurin inhibitors act specifically on proinflammatory cells. Pimecrolimus shows comparative efficacy to mild topical corticosteroids and a special antipruritic effect. Furthermore, examinations of the systemic absorption of pimecrolimus implicated no systemic immunosuppression. In 2006, the FDA set a black box warning in the packaging materials of pimecrolimus alluding to the risk of skin malignancy or lymphomas due to theoretical consideration. AREAS COVERED: The authors provide a review of pimecrolimus as a treatment for AE. Specifically, the authors present the pharmacokinetic and pharmacodynamic information on pimecrolimus and also review its efficacy. The authors also discuss pimecrolimus' safety and tolerability profile. EXPERT OPINION: Pimecrolimus represents a valuable part of active and proactive therapy in AE. That being said, the long-term safety of topical calcineurin inhibitors remains to be investigated. Given the results from experimental photocarcinogenicity studies, effective sun protection should be employed during the therapy, although an increased risk for skin malignancies and lymphomas was not found in recent studies. Pimecrolimus should be considered as an alternative therapeutic approach in AE treatment management going along with a corticoid-sparing effect.

Immunopotentiation on murine spleen lymphocytes induced by polysaccharide fraction of Panax ginseng via upregulating calcineurin activity.

Calcineurin (CN), a unique Ca2+/calmodulin (CaM)-dependent serine/threonine protein phosphatase, plays a pivotal role in the activation and proliferation of T lymphocytes. Based on the effective molecular screening model established in our laboratory, we found that a part of polysaccharides from the stem and leaves of Panax ginseng, termed PGP-SL, could activate CN activity. Subsequently, we investigated whether PGP-SL also has immunological competence on murine spleen lymphocytes. In the present study, we demonstrated that PGP-SL could significantly promote in vitro spleen lymphocyte proliferation in the absence of either concanavalin A or LPS in a concentration-dependent manner at concentrations ranging from 100 to 500 microg/ml (p<0.001). In addition, the proliferation of cyclosporin A (CsA)-treated spleen lymphocytes was also significantly promoted in the same pattern (p<0.001); the production of IL-2 was elevated and the effect appeared as early as 24 h after PGP-SL treatment. The results of RT-PCR also indicated that the IL-2 mRNA level was markedly enhanced, particularly at PGP-SL concentrations of 300 and 500 microg/ml, and Fura-2/AM fluorescence probe analysis showed that PGP-SL could dramatically increase the intracellular free calcium concentration of spleen lymphocytes, i.e. [Ca2+]i was significantly increased by approximately 181 and 107% at 300 and 500 microg/ml of PGP-SL, respectively. However, this effect could be totally inhibited by verapamil treatment. Taking our results together, we suggest that PGP-SL exhibits immunopotentiation effects on murine spleen lymphocytes by the Ca2+-CN-NFAT-IL-2 signaling pathway.

FK506 and its analogs - therapeutic potential for neurological disorders.

Immunophilin ligands such as FK506 and Cyclosporin A, used in immunosuppression, are well-characterized drugs. In the past, they had been the center of attention as a putative therapeutic strategy for neuroregeneration and neuroprotection. In contrast to Cyclosporin A, FK506 readily crosses the brain-blood-barrier and, thus together with its derivatives, may represent a novel approach to the treatment of neurological disorders. FK506 exerts profound neuroprotective and neuroregenerative effects in vivo and in vitro. The mechanism underlying neuroregeneration is fairly well understood. It is independent of the inhibition of calcineurin, which is responsible for the immunosuppression, but operates via the binding of FKBP52 and the heat shock protein (Hsp) 90. In contrast, the underlying pathways of neuroprotection are far less understood. Protection is apparently independent of calcineurin, as shown by non-calcineurin inhibiting derivatives, such as V-10,367 and GPI-1046, but the intracellular actions remain to be defined. FK506 has been shown to interfere with the apoptotic pathway of neuronal cells, including inhibiting JNK activity, cytochrome c release, caspase 3 activation, and CD95 ligand expression. These effects are in part mediated by the inhibition of calcineurin and may not contribute to protection. Our recent studies suggest that the protective properties of FK506 and its non-calcineurin inhibiting derivatives are realized by a fast induction of heat shock proteins. The induction of the heat shock response by immunophilin ligands might prove to be an interesting target for neuroregeneration and neuroprotection.

[Role of calcineurin-dependent drugs on the immunosuppressive effect induced by the anti-LFA-1 antibody in a fetal intestinal transplantation model in mice]. / Rôle des drogues calcineurine-dépendantes sur l'effet immunosuppresseur induit par l'anticorps anti-LFA-1 dans un modèle de greffe intestinale foetale chez la souris.

STUDY AIM: We have previously demonstrated that anti-LFA-1 monoclonal antibody (mAb) can efficiently protect against rejection of small bowel allograft in a mouse model. The aim of the present work was to determine, in the same model, the optimum conditions for utilisation of anti-LFA-1 mAb and the effects of calcineurin-dependent drugs on the immunosuppression induced by anti-LFA-1 mAb treatment. MATERIALS AND METHODS: Foetal small intestines of C57Bl/6 (H-2b) mice were transplanted into adult C3H/He (H-2k) mice. Recipients were treated with anti-LFA-1 mAb alone (with or without day-1 injection), or combined to cyclosporin (20 mg.kg-1.j-1 for 14 days), or to tacrolimus (1 mg.kg-1.j-1 from day 0 to day 7). Biopsies were performed after engraftment from day 5 to day 30. RESULTS: Administration of anti-LFA-1 mAb alone is sufficient to induce significant prolongation of intestinal allograft survival, provided that the treatment starts one day before engraftment. This tolerogenic effect is reversed by the transitory administration of tacrolimus (p = 0.008). CONCLUSION: Treatment with anti-LFA-1 mAb has to be started before the allogeneic response has begun. Calcineurin-dependent drugs can modulate the tolerogenic effect induced by anti-LFA-1. A transgenic mice model should give precise details about underlying mechanisms of these interactions, before a possible utilisation of anti-LFA-1 mAb in intestinal transplantation in humans.