8A, even 5 μl of HA/ml did not stimulate reactivation of HIV-1 in

8A, even 5 μl of HA/ml did not stimulate reactivation of HIV-1 in ACH-2 cells, as characterized by western blot analysis see more of the p24 Ag, while a 48 h treatment led to a comparable increase in expression of p24 Ag in cells stimulated with PMA only as well as with PMA and HA. Stimulation of the cells with 10 U/ml of TNF-α led to an even higher expression of p24 Ag, while 1 U/ml induced a relatively smaller expression of p24 Ag. On the other hand, any concentration of phytohemagglutinin A tested (PHA; 0.5, 2.5; 5 μg/ml) alone or in combination with 1 μM ionomycine did not yield a positive signal of p24 Ag in western blot analysis (Fig. 8A and data not

shown). ELISA analysis of culture supernatants revealed similar changes in levels of the p24 antigen as the western blot analysis (Fig. 8B). However, it is obvious that the overall release of p24 by ACH-2 cells stimulated with PMA for 48 h was stronger than by ACH-2 cells stimulated with PMA and HA for the same time period. This effect is possibly due to the death

of the learn more PMA- and HA-stimulated cells or to the inhibitory effects of CO and bilirubin on HIV-1 reactivation as discussed below. The same stimulatory agents were also used for treatment of A2 and H12 cells for 48 h. As shown in Fig. 8C, expression of EGFP was stimulated with HA alone weakly in both cells, very strongly with PMA and even more strongly with PMA and HA. The stimulation with 10 U/ml of TNF-α or 0.5–1 μg/ml PHA was comparable to the effect of PMA, while the stimulation with 1 U/ml TNF-α induced a relatively weaker expression of EGFP. It can be observed that the effect of 1 U/ml TNF-α was comparable to the effect of HA (2.5 μl/ml)

in H12 cells, while it was stronger in A2 cells. The stimulatory effects of individual agents on the expression of EGFP were also studied using flow cytometry (Fig. 8D, Supplementary data Table S3). Again, these results reveal similar tendencies as western blot analysis, but as mentioned above, H12 cells reveal a higher background expression of EGFP in untreated cells than A2 cells, and in general respond with a smaller fold-increase than A2 cells. Based on various criteria used in this analysis, it can be concluded that A2 cells are more responsive to TNF-α than H12 from cells. When analyzing the cell viability, neither PMA nor TNF-α alone or in combination with HA were found to decrease it. On the other hand, PHA reduced cell viability relatively strongly. In addition to the previous studies, we have explored the ability of T-cells to get activated by PMA in the presence of HA. The A3.01 cells were stimulated with PMA and expression of CD69 on the cell surface was determined. In these assays, HA revealed no negative effects on the T-cell activation characterized by this activation marker at any concentration of PMA tested (1 and 10 ng/ml; data not shown), especially not even at the lowest concentration used throughout the experiments (0.5 ng/ml; Fig. 9).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>