SummaryA colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR ¹ and rpoS ¹ derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37ЊC. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37ЊC and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Type I interferons (IFNs) (alpha/beta interferon [IFN-␣/␤]) are expressed as a first line of defense against viruses and are known to play a critical role in the antiviral response (38). Type I IFNs combat viruses both directly by inhibiting virus replication in the cells and indirectly by stimulating the innate and adaptive immune responses (38). The direct antiviral activity of type I IFNs is exerted by a number of different mechanisms, e.g., blockage of viral entry into the cell, control of viral transcription, cleavage of RNA, and preventing translation (16,31,37). In addition to the direct effects, type I IFNs play immunoregulatory roles and thereby shape the innate and adaptive immune responses. For instance, IFN-␣/␤ induce natural killer cell cytotoxicity and up-regulate expression of major histocompatibility complex class I on most cells and costimulatory molecules on antigen-presenting cells (9, 37). Furthermore, type I IFNs enhance cross-presentation of exogenous antigen in major histocompatibility complex class I and promote T-cell expansion (14,19,36 (11,15,20,23,32), although they exert their action through a receptor complex distinct from the type I IFNs (20,32). Most of the reports demonstrating antiviral activity of IFN-have addressed the issue in an in vitro experimental setup, but one report has shown that a recombinant IFN--expressing vaccinia virus is attenuated in vivo (4), whereas recombinant IFN-had no antiviral effect in vivo in the transgenic hepatitis B virus mouse model (30). Thus, we still do not have a clear picture of the antiviral potential of IFN-in vivo or of the mechanisms of action.The IFN-s have been demonstrated to be induced after stimulation with several single-stranded RNA (ssRNA) viruses, whereas the information on viruses with other genomes (DNA and double-stranded RNA [dsRNA]) is sparse (11). Virtually all cell types are capable of producing type I IFNs in response to viral infections, with the amount of IFN being virus and cell type dependent (7) and with plasmacytoid dendritic cells (pDCs) being the most potent producers of type I IFNs (3). IFN-s can be produced by a number of cell types, although the pattern of expression has not been elucidated. One report has demonstrated that IFN-s are produced by pDCs to a greater extent than by monocyte-derived DCs after influenza A virus (IAV) infection, suggesting that pDCs are the primary IFN--producing cells (13). However, this needs to be confirmed for other virus infections.Here, we have investigated the expression of type I and III IFNs after infection with DNA and RNA viruses in lymphoid, myeloid, and epithelial cell lines, and we have also examined the ability of type I and III IFNs to cross-induce one another. Subsequently, we investigated the antiviral activity of IFN-in

Inflammation is a critical factor for development of hypoxic-ischemic (HI) brain injury. Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in microglia and processed by caspase-1. Our aim was to characterize the expression of IL-18 and its receptor in relation to caspase-1 and IL-1beta after HI and to evaluate to what extent IL-18 contributes to HI brain injury. Seven-day-old rats were subjected to HI, and brain tissue was sampled at different time points (3 hr to 14 d) after insult. The mRNA for IL-18 and caspase-1 were analyzed with reverse transcriptase PCR, protein was analyzed by Western blot (IL-18, caspase-1) or ELISA (IL-1beta), and the regional distribution was assessed by immunohistochemistry. HI was also induced in C57BL/6 mice, and brain injury in IL-18-deficient animals was compared with that in wild-type animals. The expression of mRNA/protein for caspase-1 and IL-18 in brain homogenates increased progressively at 12 hr to 14 d after HI, whereas IL-1beta peaked at 8 hr. A widespread expression of caspase-1 and IL-18 protein in microglia was found in the HI hemisphere. The IL-18 receptor was expressed on neurons of the cerebral cortex and thalamus. IL-1beta was primarily found in microglia in the habenular nucleus of the thalamus. The infarct volume was reduced by 21% (p = 0.01), and the neuropathology score was significantly decreased in the cerebral cortex (-35%), hippocampus (-22%), striatum (-18%), and thalamus (-17%) in mice with IL-18 deficiency compared with wild-type mice. In conclusion, we found that IL-18 expression in microglia was markedly increased after HI and that IL-18 appears to be important for the development of HI brain injury.

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Neurological deficits in children, including cerebral palsy, are associated with prior infection during the perinatal period. Experimentally, we have shown that pre-exposure to the Gram-negative component LPS potentiates hypoxic-ischemic (HI) brain injury in newborn animals. LPS effects are mediated by binding to TLR4, which requires recruitment of the MyD88 adaptor protein or Toll/IL-1R domain-containing adapter inducing IFN-β for signal transduction. In this study, we investigated the role of MyD88 in neonatal brain injury. MyD88 knockout (MyD88 KO) and wild-type mice were subjected to left carotid artery ligation and 10% O2 for 50 min on postnatal day 9. LPS or saline were administered i.p. at 14 h before HI. At 5 days after HI in wild-type mice, LPS in combination with HI caused a significant increase in gray and white matter tissue loss compared with the saline-HI group. By contrast, in the MyD88 KO mice there was no potentiation of brain injury with LPS-HI. MyD88 KO mice exhibited reduced NFκB activation and proinflammatory cytokine-chemokine expression in response to LPS. The number of microglia and caspase-3 activation was increased in the brain of MyD88 KO mice after LPS exposure. Collectively, these findings indicate that MyD88 plays an essential role in LPS-sensitized HI neonatal brain injury, which involves both inflammatory and caspase-dependent pathways.