Dendritic cells mediate the anti-inflammatory action of omega-3 long-chain polyunsaturated fatty acids in experimental autoimmune uveitis.

We previously showed that dietary omega (ω)-3 long-chain polyunsaturated fatty acids (LCPUFAs) suppress inflammation in mice with experimental autoimmune uveitis (EAU). We have now investigated the role of antigen presenting cells (APCs) in this action of ω-3 LCPUFAs. C57BL/6 mice were fed a diet supplemented with ω-3 or ω-6 LCPUFAs for 2 weeks, after which splenocytes were isolated from the mice and cocultured with CD4+ T cells isolated from mice with EAU induced by injection of a human interphotoreceptor retinoid-binding protein peptide together with complete Freund's adjuvant. The proliferation of and production of interferon-γ and interleukin-17 by T cells from EAU mice in vitro were attenuated in the presence of splenocytes from ω-3 LCPUFA-fed mice as compared with those from mice fed ω-6 LCPUFAs. Splenocyte fractionation by magnetic-activated cell sorting revealed that, among APCs, dendritic cells (DCs) were the target of ω-3 LCPUFAs. Adoptive transfer of DCs from mice fed ω-3 LCPUFAs attenuated disease progression in EAU mice as well as the production of pro-inflammatory cytokines by T cells isolated from these latter animals. The proliferation of T cells from control Balb/c mice was also attenuated in the presence of DCs from ω-3 LCPUFA-fed mice as compared with those from ω-6 LCPUFA-fed mice. Furthermore, T cell proliferation in such a mixed lymphocyte reaction was inhibited by prior exposure of DCs from mice fed an ω-6 LCPUFA diet to ω-3 LCPUFAs in vitro. Our results thus suggest that DCs mediate the anti-inflammatory action of dietary ω-3 LCPUFAs in EAU.

Novel vitamin D3 antipsoriatic antedrugs: 16-En-22-oxa-1alpha,25-(OH)2D3 analogs.

A series of 16-en-22-oxa-derivatives of vitamin D3 based on the structure of maxacalcitol (2) were prepared. Maxacalcitol is currently used topically for the treatment of psoriasis and is recognized as the most successful antedrug of natural vitamin D(3) because it retains the original antiproliferative activity of calcitriol without increased calcemic activity. We introduced 16-olefinic functionality to accelerate the oxidative metabolism of the drug in liver, presumed to be essential for the reduction of calcemic activity, and modified the side-chain moiety by placing the 22-oxygen on the more labile allylic carbon center. Novel 22-oxa analogs (7a-i), carrying either the 24-alkynyl bond or 24-hydroxy functionality in addition to the 16-double bond were synthesized and their pharmacokinetics were evaluated.

Overexpression of ROCK in human breast cancer cells: evidence that ROCK activity mediates intracellular membrane traffic of lysosomes.

Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/ transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.

In vivo time-course of receptor binding in the parathyroid gland of the vitamin D analogue [(3)H]1,25-dihydroxy-22-oxavitamin D(3) compared with [(3)H]1,25-dihydroxyvitamin D(3), determined by micro-autoradiography.

1,25-Dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT), is a new synthetic analogue of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol), to be used in the treatment of secondary hyperparathyroidism. This study used receptor micro-autoradiography in the parathyroid gland to determine and compare the time-course of receptor binding between OCT and 1,25(OH)(2)D(3). Mice were injected with 4 microg/kg of [26-(3)H]OCT or [26,27-methyl-(3)H]1,25(OH)(2)D(3), and killed at 5, 15, 30 min, 1, 2, 4, 8, 12, and 24 h afterwards. Thyroid-parathyroid tissue was excised and autoradiograms were prepared. Under identical conditions of dose and adjusted specific radioactivity between [(3)H]OCT and [(3)H]1,25(OH)(2)D(3), the plasma concentration of [(3)H]OCT was much lower than that of [(3)H]1,25(OH)(2)D(3). In the parathyroid at all time points, chief cell nuclei were labelled with varying degrees while connective tissue cells remained unlabelled. Nuclear receptor binding of [(3)H]OCT appeared equal to or higher than that of [(3)H]1,25(OH)(2)D(3). Nuclear uptake of [(3)H]OCT was maximal at 15 min and higher than that of [(3)H]1,25(OH)(2)D(3), which was maximal at 1 h after injection. Low levels of nuclear retention of the two compounds were still similarly detectable at 12 h. The results indicate the high affinity of OCT to parathyroid cells, and suggest that OCT has a higher therapeutic potential than 1,25(OH)(2)D(3), especially under clinical conditions, at which OCT with its lower calcaemic effect would allow treatment with a dose several times higher than 1,25(OH)(2)D(3).