Hyperglycemic conditions and HIV-1 Nef significantly enhance the production of complement factor C3 in vitro and in vivo. (A) To mimic hyperglycemic conditions close to the blood glucose levels of 180, 270 and 360 mg/dl, U87-MG human astroglioma cells were cultured with 10,15, 20 and 25 mM glucose containing medium for 12 hours. Astrocytes with 5 mM glucose treatment were used as control. The cells were washed and transduced with HIV-1 Nef expressing virus. 48 hours later, the astrocytes and cellular supernatants were collected and subjected to ELISA using manufacturer's protocol to quantify the complement factor 3 (Assay Designs, Ann Arbor, MI) (B). Hyperglycemic conditions and expression of HIV-1 Nef significantly enhanced the production of complement factor 3 in mice brain. Diabetes was induced in C57/BL6 mice by a subcutaneous injection of a single dose of 40 mg/kg body weight streptozotocin (Sigma Chemicals, St.Louis, MO), which has been freshly dissolved in 0.1 mol/L citrate buffer at pH of 4.5. Upon confirmation of diabetes induction (325-425 mg/dl glucose) by a glucometer in these mice, 1 × 107 viral particles generated through HIV-1 based vectors or SNV-based vectors were injected into the mice brain via the mid ventricle, cortex, or the cerebellum as described previously. Age-matched non-diabetic mice injected with an equal volume of citrate buffer were served as control. After eight weeks the mice were sacrificed and the brain and other organs were harvested. ELISA was performed on brain tissue extracts to determine the release of C3 into the brain. The results are the mean values for triplicate samples ± standard errors of the means. The data presented are averages of three independent experiments.