Tomofumi Miyamoto

Identification of Distinct Ligands for the C-type Lectin Receptors Mincle and Dectin-2 in the Pathogenic Fungus Malassezia

Various C-type lectin receptors (CLRs), including Mincle and Dectin-2, function as pattern recognition receptors and play a central role in immunity to fungal pathogens. However, the precise structures of the CLR ligands in various pathogenic fungi have yet to be completely defined. Here we report that Malassezia, an opportunistic skin fungal pathogen, is cooperatively recognized by Mincle and Dectin-2 through distinct ligands. Solvent-based fractionation revealed that Mincle and Dectin-2 recognize lipophilic and hydrophilic components of Malassezia, respectively. Mass spectrometry and nuclear magnetic resonance (NMR) revealed glyceroglycolipid and unique mannosyl fatty acids linked to mannitol as two Mincle ligands. An O-linked mannobiose-rich glycoprotein was identified as a Malassezia ligand for Dectin-2. Cytokine production in response to the Mincle ligands and the Dectin-2 ligand was abrogated in Mincle(-/-) and Dectin-2(-/-) dendritic cells, respectively. These results demonstrate that Mincle and Dectin-2 recognize distinct ligands in Malassezia to induce host immune responses.

Role of macrophages in inflammatory lymphangiogenesis: Enhanced production of vascular endothelial growth factor C and D through NF-κB activation ☆

The close association of inflammation, angiogenesis and cancer progression is now highlighted, and in this study we especially focused on a close association of inflammation and lymphangiogenesis. We found that proinflammatory cytokine, interleukin-1beta (IL-1beta), could induce lymphangiogenesis in mouse cornea through enhanced production of potent lymphangiogenic factors, VEGF-A, VEGF-C and VEGF-D. IL-1beta-induced lymphangiogenesis, but not angiogenesis, was inhibited by administration of a selective anti-VEGF receptor-3 (VEGFR-3) neutralizing antibody. And in mouse cornea we observed recruitment of monocyte/macrophages and neutrophils by IL-1beta implanted cornea. Depletion of macrophages by a bisphosphonate encapsulated in liposomes inhibited this IL-1beta-induced lymphangiogenesis and also up-regulation of VEGF-A, VEGF-C, and VEGF-D. Furthermore, IL-1beta-induced lymphangiogenesis and angiogenesis were suppressed by NF-kappaB inhibition with marked suppression of VEGF-A, VEGF-C, and VEGF-D expression.

Costunolide, a sesquiterpene lactone from Saussurea lappa, inhibits the VEGFR KDR/Flk-1 signaling pathway

Costunolide (CT), a sesquiterpene lactone constituent isolated from Saussurea lappa (Compositae), exerted an antiangiogenic effect. CT selectively inhibited the endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Further, CT was also found to inhibit the VEGF-induced chemotaxis of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. From these results, we hypothesized that CT might inhibit angiogenesis by blocking the angiogenic factor signaling pathway. VEGF interacts with its cognate receptors, KDR/Flk-1 and Flt-1, and exerts its angiogenic effect. CT inhibited the autophosphorylation of KDR/Flk-1 without affecting that of Flt-1. Moreover, administration of CT reduced VEGF-induced neovascularization in a mouse corneal micropocket assay. These results suggest that CT may prove useful for the development of a novel angiogenesis inhibitor.

Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses

Background: Cholesterol crystals are involved in various chronic inflammatory diseases; however, direct pattern recognition receptors (PRRs) recognizing cholesterol crystals have not been identified. Results: We purified endogenous components interacting with human Mincle and identified cholesterol. Conclusion: Direct interaction of human Mincle with cholesterol crystals activates innate immune responses. Significance: Human Mincle could be a therapeutic target of chronic inflammation. C-type lectin receptors (CLRs) are an emerging family of pattern recognition receptors that recognizes pathogens or damaged tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. MS analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells expressed a set of inflammatory genes in response to cholesterol crystals, and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, whereas other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and that they activate innate immune responses.

Cytotoxic spirostane-type saponins from the roots of Chlorophytum borivilianum.

Four new spirostane-type saponins named borivilianosides E-H (1-4) were isolated from an ethanol extract of the roots of Chlorophytum borivilianum together with two known steroid saponins (5 and 6). The structures of 1-4 were elucidated using mainly 2D NMR spectroscopic techniques and mass spectrometry. The cytotoxicity of borivilianosides F (2), G (3), and H (4) and three known compounds was evaluated using two human colon cancer cell lines (HT-29 and HCT 116).

Dorisenones, cytotoxic spongian diterpenoids, from the nudibranch Chromodoris obsoleta

Abstract Seven new cytotoxic spongian diterpenoids, dorisenones A ( 1 ), B ( 2 ), C ( 3 ), D ( 4 ), 7α-hydroxyspongian-16-one ( 5 ), 15α, 16α-diacetoxy-11, 12β-epoxyspongian ( 6 ) and 7α-acetoxydendrillol-3 ( 7 ) were isolated from a Japanese marine mollusk Chromodoris obsoleta (Chromodorididae), together with four known spongian diterpenoids. Their structures have been elucidated by spectroscopic evidence and single crystal X-ray analysis.

Acylated triterpene saponins from the roots of Securidaca longepedunculata

Four triterpene saponins, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-d-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-[(6-O-acetyl)-beta-D-glucopyranosyl-(1-->3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-galactopyranosyl-(1-->3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, and 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-[alpha-L-arabinopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, were isolated from the roots of Securidaca longepedunculata, together with three known compounds. Their structures were established mainly by 2D NMR techniques and mass spectrometry.

ISOLATION AND STRUCTURE OF THREE NEW CERAMIDES FROM THE STARFISH ACANTHASTER PLANCI

Three new phytosphingosine-type ceramides, AC-1-6, AC-1-10 and AC-1-11, were isolated from the ceramide molecular species AC-1, obtained from the less polar fraction of the CHCl3-MeOH extract of the starfish Acanthasterplanci. The structures of these ceramides were determined on the basis of chemical and spectroscopic evidence as (2S,2′R,3S,4R,9Z)-2-(2-hydroxyhexadecanoylamino)-9-docosene-1,3,4-triol (AC-1-6), (2S,2′R,3S,4R,)-2-(2-hydroxytricosanoylamino)hexadecane-1,3,4-triol (AC-1-10) and (2S,2′R,3S,4R,)-2(2-hydroxytetracosanoylamino)hexadecane-1,3,4-triol (AC-1-11). Positive ion FABMS/MS of each ceramide gave important information for their structure elucidation. Mass spectrometry of the dimethyl disulfide derivatives of ceramide was also useful for the determination of the double bond position in the long-chain base.

Cytotoxic acacic acid glycosides from the roots of Albizia coriaria.

Two new oleanane-type saponins, coriariosides A (1) and B (2), along with a known saponin, gummiferaoside C (3), were isolated from the roots of Albizia coriaria. Their structures were established by extensive analysis of 1D and 2D NMR experiments (COSY, ROESY, TOCSY, HSQC, and HMBC) and mass spectrometry. Compounds 1 and 3 when tested for cytotoxicity against two colorectal human cancer cells showed activity against the HCT 116 (IC50 4.2 microM for 1 and 2.7 microM for 3) and HT-29 (IC50 6.7 microM for 1 and 7.9 microM for 3) cell lines.

Isolation and Structure of Biologically Active Glycosphingolipids from the Sea CucumberCucumaria echinata

Five cerebrosides, CE-1-1, CE-1-2, CE-1-3, CE-3-1, and CE-3-2, and a ganglioside molecular species CG-1 have been obtained from the less polar and polar fractions, respectively, of the chloroform/methanol extract of the sea cucumber Cucumaria echinata. The structures of these glycosphingolipids have been determined on the basis of chemical and spectroscopic evidence. The cerebrosides show lethality toward brine shrimps. On the other hand, the ganglioside CG-1 exhibits neuritogenic activity toward the rat pheochromocytoma cell line, PC-12 cells.