). A band with a molecular weight of about 46 000 with a faint band underneath appeared, which was greatly enhanced when B cells were activated with CD40L + IL-4 (Fig. 1c). AID and A3G mRNA expression were then evaluated by real-time PCR. All results were expressed as mean (± SEM) relative to unstimulated cells, which were accorded an arbitrary value of 100. CD40L induced an increase in AID mRNA from 100 to 258 (± 131) and to a lesser extent in A3G mRNA to 128 (± 13), these failed to reach the 5% level of significance (Table 1). However, IL-4 significantly R788 mw up-regulated AID (P = 0·037) but not A3G (P = 0·29). The combined CD40L + IL-4 B-cell agonists up-regulated significantly both

AID and A3G mRNA (P < 0·05), and this was much greater for AID than A3G mRNA (Table 1). CD40L + HLA class II antibodies (177 ± 25) was more effective for A3G mRNA (P = 0·027) than that for AID mRNA (295 ± 128, P = 0·11). As with immunofluorescence the other B-cell agonists were not pursued further. The results suggest that CD40L + IL-4 www.selleckchem.com/GSK-3.html yielded the most consistent and significant increases in both mRNA and protein of AID and A3G. We have evaluated a major function of AID in B cells by demonstrating a significant

increase in the cell-surface expressions of IgG (P < 0·001) and IgA (P < 0·0001) (Fig. 2b,c) when stimulated with CD40L + HLA-II mAb and to a lesser extent with CD40L + IL-4 (P < 0·03). The IgM also increased but to a greater extent with CD40L + IL-4 than CD40L + HLA-II mAb (Fig. 2a). These studies were then extended to the culture supernatants of the B-cell-agonist-stimulated cells. Using the Luminex bead technology confirmed the increase in IgA antibodies in the 4-day culture supernatants

of CD40L + IL-4-stimulated B cells (Fig. 3). The 6·4-fold higher concentration of IgA compared with IgG1 was surprising as the reverse is normally found Ureohydrolase in serum. This might be related to the shorter half-life of IgA (about 9 days) compared with that of IgG (about 21 days). After 7 days of culture, the supernatants showed a significant increase in IgG4 by stimulation with CD40L + IL-4 (P = 0·01), though the total concentration was moderate (12·6 ± 5·4 ng/ml) (Fig. 3b). A functional effect of up-regulation of A3G by stimulating primary B cells with the selected agonists was studied in HIV-1 (BaL) infectivity of autologous CD4+ T cells. Isolated B cells were stimulated with CD40L + IL-4 or HLA-II mAb for 3 days, followed by co-culturing the B cells with autologous CD4+ T cells (activated with phytohaemagglutinin and IL-2 for 3 days) and infected with serial dilution HIV-1 (BaL) for 9 days. The results showed dose-dependent inhibition of HIV-1 replication with the B cells pre-treated with either of the B-cell agonists, compared with the untreated B cells (Fig. 4a,b).