Inhibitors of sialoadhesin for the treatment of diseases caused by enveloped viruses
A technology of enveloped viruses and compositions, applied in the direction of resisting vector-borne diseases, viruses, drug combinations, etc., can solve problems such as unknown precise mechanisms
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[0361] 4. Liposome Preparation
[0362] Large unilamellar vesicles (LUVs) were prepared according to the extrusion method previously described below. See Mayer L, et al., Vesicles Biochim. Biophys. Acta 1986;858:161-168. Lipids and gangliosides are commercially available (Avanti Polar Lipids, Inc., Alabaster, AL, US; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, US). LUV HIV-tRed The lipid composition is: 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphocholine (POPC) 25mol%: 1,2-dipalmitoyl-sn-glyceryl-3-choline Alkaline phosphate (DPPC) 16mol%: brain sphingomyelin (SM) 14mol%: cholesterol (Chol) 45mol% and when Cer, PS or gangliosides were present (4mol%), the amount of SM was reduced to 10mol%. LUV POPC-tRed The lipid composition was 96 mol% POPC with or without 4 mol% Cer, GM3, GM2 or GM1. All LUVs contained 2 mol% of 1,2-dihexadecanoyl-sn-propanetriyl-3-phosphatidylethanolamine (DHPE)-Texas Red (Molecular Probes; Invitrogen Corp., Carslbad, CA , US). Lipids were mixed i...
Embodiment 1
[0383] Gangliosides in the outer lobes of HIV-1 or cyst membranes can act as viral attachment factors generating mDC uptake
[0384] Glycosphingolipids are enriched in raft-like plasma membrane domains from which HIV-1 is thought to bud. Based on this premise, the potential role of glycosphingolipids for HIV-1 capture by mDCs was investigated. GM3 in HIV derived from the T cell line MT-4 NL4.3 The presence of in was confirmed by mass spectrometry. In addition, several other gangliosides of GM1, GM2 and GD1 included in HIV-1 membranes were also detected. see Figure 1A , 1B and 1C.
[0385] To test whether gangliosides in HIV-1 or in the outer lobes of the cyst membrane could act as viral attachment factors to generate mDC uptake, mimicking HIV-1 (LUV HIV-tRed ) size and lipid composition and containing different gangliosides, Texas Red (tRed) labeled large unilamellar vesicles (LUVs) were prepared. see Figure 6 , Lorizate M, et al., J. Biol. Chem. 2009;284:22238-22247....
Embodiment 2
[0388] Ganglioside-containing large unilamellar vesicles and VLPs utilize a common entry structure into mDCs and reach the same compartment within mDCs
[0389] To identify LUVs containing gangliosides HIV-tRed and VLP HIV-Gag-eGFP (and HIV-1) are common entry structures into mDCs, several competition experiments were performed. Mature DCs utilize reduced amounts of GM2-containing LUVs HIV-tRed and a fixed amount of VLP HIV-Gag-eGFP Pulse at 37°C for 4 hours. After extensive washing, the percentage of eGFP- and tRed-positive cells was determined by FACS. LUV containing GM2 HIV-tRed effectively competes for VLP uptake in a dose-dependent manner HIV-Gag-eGFP To mDC (PHIV-tRed , no competition for VLP uptake was observed. Therefore, the LUV containing the GM HIV-tRed and VLP HIV-Gag-eGFP Entry into mDCs uses a common entry structure, which relies on carbohydrate end groups.
[0390] Then, whatever LUV contains the GM HIV-tRed and VLP HIV-Gag-eGFP Whether or not reachi...
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