PCR-based simultaneous analysis of the interferon-alpha family reveals distinct kinetics for early interferons.

Here we describe a PCR-based analysis system that allows the simple simultaneous assessment of murine interferons (IFN)-alpha and IFN-beta induction in a single reaction. In this analysis, the so-called early IFN-alpha4 can be distinguished from the so-called late IFN-nonalpha4 by employing a primer mixture that amplifies a part of the IFN-alpha genes in which IFN-alpha4 exhibits a deletion of 15 nucleotides compared to IFN-nonalpha4. By including a final denaturation and a slow cooling step at the end of the PCR procedure, hybrid formation was avoided that regularly occurred when standard protocols were used. Separation of the amplification products on 4.5% agarose gels allowed the comparative assessment of the classical type I IFNs. Using this analysis system, we could show that in immortalized adult fibroblasts, IFN-beta is induced first and the two types of IFN-alpha are induced later and simultaneously. When similar fibroblasts derived from mice that lack IFN-beta were tested, the IFN response was delayed. However, now IFN-alpha4 appeared first and apparently induced the cascade of IFN-nonalpha4. This confirms the role of IFN-beta as master regulator of the normal IFN response.

Survival of lethal poxvirus infection in mice depends on TLR9, and therapeutic vaccination provides protection.

Poxviruses such as the causative agent of smallpox have developed multiple strategies to suppress immune responses, including the suppression of DC activation. Since poxviruses are large DNA viruses, we hypothesized that their detection by DCs may involve the endosomal DNA recognition receptor TLR9. Indeed, we have shown here that DC recognition of ectromelia virus (ECTV), the causative agent of mousepox, completely depended on TLR9. The importance of TLR9 was highlighted by the fact that mice lacking TLR9 showed drastically increased susceptibility to infection with ECTV. In contrast, we found that the strongly attenuated poxvirus modified vaccinia virus Ankara (MVA) activated DCs by both TLR9-dependent and -independent pathways. We therefore tested whether we could use the broader induction of immune responses by MVA to protect mice from a lethal infection with ECTV. Indeed, MVA given at the same time as a lethal dose of ECTV protected mice from death. Importantly, MVA also rescued TLR9-deficient mice if administered 2 full days after an otherwise lethal infection with ECTV. Therefore, these data suggest an essential role for TLR9 in the defense against poxviruses. In addition, postexposure application of MVA may protect against lethal poxvirus infection.

Transformation of mouse fibroblasts alters the induction pattern of type I IFNs after virus infection.

Type I interferons (IFNs) have been shown to be involved in many immune defence and inflammatory responses. We here show that IFN-beta plays an absolute essential role in the efficient induction of all type I IFNs after infection of primary embryonic as well as primary adult fibroblasts with Sendai virus. In contrast, after immortalization of such fibroblasts with SV40 large T antigen, IFN-alpha4 can be induced independently of IFN-beta. However, efficient secretion of type I IFNs even in immortalized fibroblasts is only found when the complete signalling loop is induced by IFN-beta.

Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation.

BACKGROUND: Ex vivo gene therapy in the central nervous system (CNS) holds great promise for diseases such as the neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. This study reports the establishment of an in vitro model of transgene down-regulation in cells grafted to the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B. METHODS: Neural cell lines were transduced at 33 degrees C with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. Cell differentiation in vitro was obtained by culturing the cells at 37 degrees C in serum-free defined media, which halts cell division, and GFP-expression was analysed by FACS. As early as day 3 of culture at 37 degrees C, the transgene expression decreased markedly in most cell lines. To validate the assay, the same clones were grafted to the adult rat striatum and the down-regulation of GFP-expression was evaluated. RESULTS: The temporal pattern of down-regulation was found to be similar in vitro and in vivo. Using this assay, it was shown that addition of inhibitors of histone deacetylation, but not an inhibitor of DNA methylation, reversed the silencing of GFP in quiescent neural progenitors by up to 308% of control values. CONCLUSION: These results suggest that the same mechanisms controlling gene transcription of the host cell's genome are active in controlling transgene expression and that this should be taken into account when constructing vectors for gene therapy. The assay reported in this study could be used as a screening method to evaluate new vectors.