Sphingomyelin

Immunogens (selected peptide – Peptide 5 or LiAg) and PBS (as control) were encapsulated in liposomes, which were used as vaccine carrier. Sphingomyelin/Cholesterol multilamellar liposomes (ratio of 2:1) were prepared by dissolving 25 mg of Sphingomyelin (Sigma-Aldrich) and 6.5 mg Cholesterol (Sigma-Aldrich) in 20 ml chloroform together with traces of methanol. The solution was kept in a 1,000-ml round-bottom flask and the solvent was removed by flash evaporation on a rotary evaporator at 37°C. After drying under reduced pressure for 80 min, the aqueous phase containing 2.1 mg of immunogens in PBS, pH 7.4 was added to the flask. The lipid film was dislodged from the glass by the use of a vortex mixer. The liposomes were retrieved using a Pasteur pipette and then treated with ultrasonic vibration three times during 20s each. The liposome suspension was centrifuged at 8,000 g for 10 min at 4°C to remove non-encapsulated immunogens and the supernatant protein concentration was estimated by spectrophotometry. The encapsulation efficiency for the formulations was 85%. The pelleted liposomes were resuspended and washed three more times with PBS by centrifugation and 0.5 mg Aluminum Hydroxide was added (200 μg/animal). The compound was stored in PBS at 4°C.

SK-N-SH neuronal cells were obtained from the ATCC (Manassas, VA). Primary human fetal brain cells were a kind gift from Prof Guillemin and were prepared as described previously^{33 (link)–35 (link)}, and the study was approved by the University of New South Wales Human Research Ethics Committee (approval number: HREC 03187). Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 1% Glutamax, 0.5% glucose, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in humidified air containing 5% CO₂. SK-N-SH cells were transfected with ABCA5 cDNA or empty vector (control) using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s protocol. After 48 hours the cells were harvested and total RNA prepared for gene expression studies. In the sphingomyelin study, cells were cultured in 12-well plates and treated with 40 µM sphingomyelin (Sigma-Aldrich #85615), 50 µM D609 inhibitor or vehicle (control) for 24 hours. In the regulation study, cells were cultured in 12-well plates and treated with 10 µM T0901317, 10 µM 9-cis retinoic acid, 10 µM pioglitazone (Sigma-Aldrich) or vehicle (control) for 24 h. The cells were harvested and total RNA prepared for gene expression analysis.

Tissue samples were pulverized in aluminum mortar precooled with liquid nitrogen and transferred to tubes containing a solution of methanol and antioxidant (0.01% butylated hydroxytoluene). Lipid fractions were then isolated by the method described by Bligh and Dyer (1959). sphingomyelin was extracted using thin‐lyer chromatography (TLC). The gel bands were scraped off from the plates after their reference to the distance traveled by standard (sphingomyelin, Sigma–Aldrich) and moved into tubes containing internal standard (pentadecanoic acid). After transmethylation the sphingomyelin fatty acids were analyzed using gas‐liquid chromatography (GLC). Hewlett–Packard 5890 Series II system equipped with a double flame‐ionization detector and Agilent CP‐Sil 88 capillary column was utilized. Total sphingomyelin concentration is shown as the sum of individual fatty acid species (expressed in nM/g of the tissue).