Differential response of the murine IL-12 p35 gene to lipopolysaccharide compared with interferon-gamma and CD40 ligation.

Expression of the heterodimeric cytokine interleukin-(IL-)12 is induced by pattern recognition receptors responding to microbial stimuli such as lipopolysaccharide (LPS) and products of the immune system such as interferon-gamma (IFN-gamma) and CD40L. The formation of bioactive IL-12 requires equimolar synthesis of p35 and p40 subunits. However, p35 expression limits the amount of IL-12 formed. Transcription of the gene for the p35 subunit of IL-12 initiates within the first exon, an alternate first exon (exon 1a), or second exon. Here we show that LPS and IFN-gamma/CD40 ligation increase the amount of total p35 mRNA in splenic adherent cells (SAC) to a similar extent. However, the exon 1 transcript was a smaller fraction of total p35 mRNA in IFN-gamma/CD40-stimulated cells than in unstimulated or LPS-stimulated cells. Despite comparable levels of total p35 mRNA, LPS-induced p35 exon 1 transcripts led to significantly more bioactive IL-12 from SAC than IFN-gamma/CD40-induced exon 1a/exon 2 transcripts as measured by ELISA. The data suggest that LPS-inducible p35 synthesis from exon 1 p35 transcripts leads to greater amount of bioactive IL-12 than IFN-gamma/CD40-induced p35 expression from alternate p35 exon 1a/exon 2 transcripts.

Reduction of the cost of testing for antibody to human immunodeficiency virus, without losing sensitivity, by pooling sera.

The sensitivity of testing pooled sera instead of individual sera for antibody against human immunodeficiency virus (HIV) was evaluated using a non-competitive enzyme-linked immunosorbent assay (ELISA). For this purpose, 42 HIV antibody positive sera were titrated and introduced into 42 sets of pools of 2, 4, 8, 16, 32 or 64 sera in such a manner that each pool had one positive sample and the rest, HIV antibody negative sera. When the pools were tested in ELISA, all pools with high titred antibody positive sera were reactive irrespective of pool size, while some of the pools containing medium or low titred sera were non-reactive when pool size exceeded 16. Subsequently the pool size was limited to 16. When 208 previously unscreened samples were tested in 52 pools of 4, 26 pools of 8 or 13 pools of 16 sera, or individually, 6 antibody positive sera were correctly identified. Thus, it was found that the pooling method did not reduce the sensitivity of ELISA test, whereas the cost was reduced to less than half of that of individual testing.