selleckchem The Inhibitors,Modulators,Libraries concentrations of IL 1B and IL 8 were sig nificantly higher in wild type infected compared with Vpr infected cul ture at infection phase. The increase and or decrease in protein levels were directly correlated with the RNA transcript levels. Figure 2B represents one of six individual experiments demonstrating the correl ation. For instance, IL 1B and Inhibitors,Modulators,Libraries TNF showed a decrease on day 8 both at the protein and transcript levels fol lowed by a reduction on day 12 compared to HIV 1wt infected culture. Absence of Vpr reduces activation of p38 and SAPK JNK and not ERK1 2 in MDMs MAPK signaling cascade is known to play a role in the production of cytokine Inhibitors,Modulators,Libraries chemokine by macrophages microglia and astrocytes and hence activate immune re sponse in the host cells.

Therefore, next we examined whether deletion of Vpr could modulate phos phorylation of the three most important members of MAPK family including ERK1 2, p38 and SAPK JNK that are known to regulate proinflammatory Inhibitors,Modulators,Libraries cytokine production. Phosphorylation of ERK1 2, p38 and SAPK JNK was assessed by western blot from 6 hours to 20 days post infection after activating the MDMs with LPS and the bands were quantified by densitometry. Results indicate an increase in phosphor ylation of p38, SAPK JNK and ERK1 2 at 6 hours post infection in both HIV 1wt and HIV 1Vpr infected MDMs compared to control. This in crease was followed by a decrease at 12, 24 and 36 hours. Phosphorylation of MAPKs at 6 hours may be the consequence of gp120 binding and Vpr may not have specific effect at this stage.

The difference of phosphoryl ation levels of p38 and SAPK JNK Inhibitors,Modulators,Libraries between HIV 1wt and HIV 1Vpr infected MDMs was first observed at 48 hours post infection and was maintained up to day 8. Phosphorylation of SAPK JNK was most pronounced at day 8. However, ERK1 2 showed no change in phosphor ylation between HIV 1wt and HIV 1Vpr infected MDMs over a period of 20 days except the initial expos ure. To confirm the involvement of p38 and SAPK JNK signaling molecules and to cross check the effect of ERK1 2 on IL 1B, IL 8 and TNF upregulation, MDMs were pretreated with SB203580, SP600125 and PD98059 prior to infection with HIV 1wt or HIV 1Vpr or mock. Supernatants were collected 48 hours post infection and the concentrations of TNF, IL 1B and IL 8 by ELISA were measured in multiple donors.

MAPK inhibitors Pazopanib GW786034 reduced the levels of all tested proin flammatory factors in all infected MDM supernatants compared to control MDMs. Blocking of ERK1 2 path way with PD98059 showed reduction in IL 1B, IL 8 and TNF levels in both infected and uninfected MDM supernatants. However, SB203580, that blocks p38 path way, significantly suppressed the production of IL 1B as well as IL 8 in HIV 1wt infected MDMs compared to HIV 1Vpr infected culture suggesting that activation of p38 pathway is involved in production of IL 1B and IL 8 in MDMs.