Effect of SN from hCMEC/D3 cultures on HIV replication in macrophages. Macrophages were derived from human primary monocytes after 7-day culture and then pretreated with SN from control (Control/SN), LyoVec (LyoVec/SN) or PolyI:C (PolyI:C/SN) -stimulated hCMEC/D3 cultures for 24 hours. Macrophages were then infected with HIV Jago for overnight incubation, and after washing 3 times, cells were maintained in fresh medium for 8 days postinfection. (A) Morphologic observation of HIV-infected macrophages with no pretreatment, LyoVec/SN, or PolyI:C/SN pretreatment under phase contrast microscope (arrows indicate syncytium). (B,C) Intracellular HIV GAG gene expression in macrophages (B) or extracellular HIV reverse transcriptase (RT) activity in macrophage cultures (C) with 10% (volume to volume ratio [v/v]) of indicated SN pretreatments. For PolyI:C/SN, the SNs were collected from 0.1, 0.25, 1, or 2.5 μg/mL PolyI:C-stimulated hCMEC/D3 cultures. (D) HIV GAG gene expression at 8 days postinfection in macrophages pretreated with indicated volumes of 1 μg/mL PolyI:C-stimulated hCMEC/D3 cultures for 24 hours. Representative data from at least 5 donor macrophages were shown and were expressed as mean ± SD from triplicate wells. Asterisks indicate that the differences between indicated groups are statistically significant (*P < .05; **P < .01). GAPDH, glyceraldehyde phosphate dehydrogenase.