TGF-b isoforms expression revealed that while TGF-b1 and TGF-b2 mRNAs were readily detectable in PBECs, TGF-b3 was close to the limit of detection of our RT-qPCR assay. However, when we examined protein expression, only TGF-b2 was detectable and this was present at significantly higher levels in culture supernatants of PBECs from asthmatic donors. Our failure to detect protein expression of TGF-b1 even though its mRNA was detectable is consistent with the findings of others. Although we did not investigate the reason for the higher levels of TGF-b2 expression by PBECs from asthmatic donors, it has been reported that there are polymorphisms in the TGFB2 gene promoter that are associated with childhood asthma. In this study, one of the asthma-associated promoter variants, 2109 RACAA ins, was a common variant and was shown to increase TGFB2 promoter reporter activity in the BEAS2B bronchial epithelial cell line. It would therefore be of interest to investigate the TGFB2 genotype of the donors used in the present study. TGF-b isoforms are secreted from cells as latent complexes, consisting of mature dimeric growth factor, the latency-associated propeptide, and latent TGF-b binding protein. Latent TGF-b complexes are normally activated by a MK-0683 diverse group of mechanisms including proteases, thrombospodin-1, integrins such as avb6 and avb6, reactive oxygen species, and low pH. While latent TGF-b2 was detectable by ELISA in culture supernatants, we could not detect active TGF-b2 in these samples using the same method; we also had limited success in detection of active TGF-b in a sensitive bioassay using transformed mink lung cells. However, there was robust TGFb activity detected in epithelial cells by phospho-Smad2 immunoblots. Based on our observation that exogenous TGF-b2 promoted viral replication, whereas pan-TGF-b neutralizing antibodies markedly suppressed viral replication, and the predominance of TGF-b2 at both message and protein level, our data would suggest that active TGF-b2 is the predominant isoform promoting viral replication even though it could not be detected by ELISA. The failure to release free active TGF-b into cell supernatants is well described for TGF-b1 that requires direct cell-cell contact for its activation. Active TGF-b is also known to have a substantially shorter half-life than the latent form in plasma and several binding proteins such as a2-macroglobulin allow scavenging of active TGF-b from the extracellular space to keep the TGFb signal local. Furthermore, studies using gene knock out mice have highlighted the roles of the LTBPs in targeting the secreted complex to specific locations in the extracellular matrix. This ability to target the latent complex in a specific manner may explain why our studies with exogenous TGF-b2 required high concentrations of the active TGF-b2 to elicit an effect, since appropriate targeting of the growth factor was missing. However, in view of the presence of TGF-b1 mRNA expression in the PBECs, we cannot exclude the possibility that low levels of cellassociated active TGF-b1 may have been produced that were not detectable as free growth factor in the medium. However, the demonstration of a functional effect of TGF-b2 in the absence of free active TGF-b2 in cell media raises the intriguing possibility that TGF-b2 may be activated by an RGD independent change in its conformational structure. The pleiotropic effects of TGF-b in vivo and in vitro provides it with various roles in growth and development, inflammation and repair and host immunity.