Cell membrane-bound CD200 signals both via an extracellular domain and following nuclear translocation of a cytoplasmic fragment.

In previous studies we had reported that the immunosuppressive cell membrane bound molecule CD200 is released from the cell following cleavage by matrix metalloproteases, with the released soluble CD200 acting as an immunosuppressant following binding to, and signaling through, its cognate receptor CD200R expressed on target cells. We now show that although the intracellular cytoplasmic tail (CD200C-tail) of CD200 has no consensus sites for adapter molecules which might signal the CD200+ cell directly, cleavage of the CD200C-tail from the membrane region of CD200 by a consensus γ-secretase, leads to nuclear translocation and DNA binding (identified by chromatin immunoprecipitation followed by sequencing, Chip-sequencing) of the CD200C-tail. Subsequently there occurs an altered expression of a limited number of genes, many of which are transcription factors (TFs) known to be associated with regulation of cell proliferation. Altered expression of these TFs was also prominent following transfection of CD200+ B cell lines and fresh patient CLL cells with a vector construct containing the CD200C-tail. Artificial transfection of non CD200+ Hek293 cells with this CD200C-tail construct resulted in altered expression of most of these same genes. Introduction of a siRNA for one of these TFs, POTEA, reversed CD200C-tail regulation of altered cell proliferation.

Importance of CD200 expression by tumor or host cells to regulation of immunotherapy in a mouse breast cancer model.

Cell-surface CD200 expression by mouse EMT6 breast tumor cells increased primary tumor growth and metastasis to the draining lymph nodes (DLN) in normal (WT) BALB/c female recipients, while lack of CD200R1 expression in a CD200R1-/- host negated this effect. Silencing CD200 expression in EMT6siCD200 tumor cells also reduced their ability to grow and metastasize in WT animals. The cellular mechanisms responsible for these effects have not been studied in detail. We report characterization of tumor infiltrating (TILs) and draining lymph node (DLN) cells in WT and CD200-/- BALB/c mice, receiving WT tumor cells, or EMT6 lacking CD200 expression (EMT6siCD200 cells). Our data show an important correlation with augmented CD8+ cytotoxic T cells and resistance to tumor growth in mice lacking exposure (on either host cells or tumor) to the immunoregulatory molecule CD200. Confirmation of the importance of such CD8+ cells came from monitoring tumor growth and characterization of the TILs and DLN cells in WT mice challenged with EMT6 and EMT6siCD200 tumors and treated with CD8 and CD4 depleting antibodies. Finally, we have assessed the mechanisms(s) whereby addition of metformin as an augmenting chemotherapeutic agent in CD200-/- animals given EMT6 tumors and treated with a previously established immunotherapy regime can increase host resistance. Our data support the hypothesis that increased autophagy in the presence of metformin increases CD8+ responses and tumor resistance, an effect attenuated by the autophagy inhibitor verteporfin.

Ent-kaurane glycosides and sesquiterpene lactones of the hirsutinolide type from Vernonia triflosculosa.

Investigation of the aerial parts of Vernonia triflosculosa afforded three hirsutinolides of which 8alpha-(4alpha-hydroxymethacryloyloxy)-10alpha-hydroxy-1,13-dimethoxy-hirsutinolide is new, three ent-kaurane diterpenes, among which the 19-[alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucopyranosyl] esters of 16beta-hydroxy-ent-kauran-19-oic acid and of 16beta,17-hydroxy-ent-kauran-19-oic acid are also new. Diterpenes are reported here for the first time in the large genus Vernonia. Their structures were elucidated using 1D and 2D NMR measurement as well as ESI, CIMS, and HRMS analysis. Two hirsutinolides were studied for their NF-kappaB DNA binding activity in HaCaT cells (a human cell line similar to keratinocytes) and for their inhibition on IL-8 production in HeLa cells.

New sesquiterpene lactones from Arnica tincture prepared from fresh flowerheads of Arnica montana.

Investigation of an ethanolic extract prepared from fresh Arnica montana flowers afforded three new 1,5- trans-guaianolides, of which 11alpha,13-dihydro-2-O-tigloylflorilenalin and the respective 2-O-isovaleryl derivative are reported for the first time. Additionally, three new and one known 2beta-ethoxy-2,3-dihydrohelenalin esters were isolated. GC/MS studies of the extract after a two year storage at 4 degrees C demonstrated that the latter were artefacts that had been formed by addition of ethanol to the cyclopentenone structure of helenalin. Formation of these adducts gave compounds possessing an inhibitory activity comparable to that of 11alpha,13-dihydrohelenalin derivatives in the NF-kappaB EMSA and the IL-8 ELISA in vitro assays as well as in the in vivo croton oil-induced mouse ear edema test for one adduct, namely 2beta-ethoxy-6-O-acetyl-2,3-dihydrohelenalin. As expected, 6-O-(2-methylbutyryl)- and 6-O-methacryloyl-helenalin exhibited a stronger activity in the NF-kappaB EMSA and IL-8 ELISA. Sesquiterpene lactones seem to be the most important NF-kappaB inhibiting compounds in the Arnica extract. Bioguided fractionation using the luciferase reporter gene assay resulted in the isolation of only moderately active compounds, such as 6-acetoxy-2,2-dimethylchroman-4-one and 10-acetoxy-8,9-epoxythymol isobutyrate.

Comparison of three chiral stationary phases with respect to their enantio- and diastereoselectivity for cyclic beta-substituted alpha-amino acids.

Three chiral stationary phases were examined for the enantio- and diastereoseparation of cycloaliphatic beta-substituted alpha-quaternary alpha-amino acids. Resolution of diastereomeric analytes is feasible with a chiral crown ether based column, whereas the separation of enantiomers, except for one pair of amino acids, could not be achieved. The two chiral stationary phases with the glycopeptide antibiotic teicoplanin and with the copper(II)-D-penicillamine complex, respectively, are, however, both very potent in the separation of the enantiomeric, as well as of the diastereomeric amino acids. A baseline separation of all four stereoisomeric forms in one chromatographic run was possible with the exception of one type of amino acid. The results of the method development are presented in this paper.