Differential evolution of peripheral cytokine levels in symptomatic and asymptomatic responses to experimental influenza virus challenge.

Exposure to influenza virus triggers a complex cascade of events in the human host. In order to understand more clearly the evolution of this intricate response over time, human volunteers were inoculated with influenza A/Wisconsin/67/2005 (H3N2), and then had serial peripheral blood samples drawn and tested for the presence of 25 major human cytokines. Nine of 17 (53%) inoculated subjects developed symptomatic influenza infection. Individuals who will go on to become symptomatic demonstrate increased circulating levels of interleukin (IL)-6, IL-8, IL-15, monocyte chemotactic protein (MCP)-1 and interferon (IFN) gamma-induced protein (IP)-10 as early as 12-29 h post-inoculation (during the presymptomatic phase), whereas challenged patients who remain asymptomatic do not. Overall, the immunological pathways of leucocyte recruitment, Toll-like receptor (TLR)-signalling, innate anti-viral immunity and fever production are all over-represented in symptomatic individuals very early in disease, but are also dynamic and evolve continuously over time. Comparison with simultaneous peripheral blood genomics demonstrates that some inflammatory mediators (MCP-1, IP-10, IL-15) are being expressed actively in circulating cells, while others (IL-6, IL-8, IFN-α and IFN-γ) are probable effectors produced locally at the site of infection. Interestingly, asymptomatic exposed subjects are not quiescent either immunologically or genomically, but instead exhibit early and persistent down-regulation of important inflammatory mediators in the periphery. The host inflammatory response to influenza infection is variable but robust, and evolves over time. These results offer critical insight into pathways driving influenza-related symptomatology and offer the potential to contribute to early detection and differentiation of infected hosts.

Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage.

Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a 'step-arrest' mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells.

A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans.

The development of the nervous system requires the coordinated activity of a variety of regulatory factors that define the individual properties of specific neuronal subtypes. We report a regulatory cascade composed of three homeodomain proteins that act to define the properties of a specific interneuron class in the nematode C. elegans. We describe a set of differentiation markers characteristic for the AIY interneuron class and show that the ceh-10 paired-type and ttx-3 LIM-type homeobox genes function to regulate all known subtype-specific features of the AIY interneurons. In contrast, the acquisition of several pan-neuronal features is unaffected in ceh-10 and ttx-3 mutants, suggesting that the activity of these homeobox genes separates pan-neuronal from subtype-specific differentiation programs. The LIM homeobox gene ttx-3 appears to play a central role in regulation of AIY differentiation. Not only are all AIY subtype characteristics lost in ttx-3 mutants, but ectopic misexpression of ttx-3 is also sufficient to induce AIY-like features in a restricted set of neurons. One of the targets of ceh-10 and ttx-3 is a novel type of homeobox gene, ceh-23. We show that ceh-23 is not required for the initial adoption of AIY differentiation characteristics, but instead is required to maintain the expression of one defined AIY differentiation feature. Finally, we demonstrate that the regulatory relationship between ceh-10, ttx-3 and ceh-23 is only partially conserved in other neurons in the nervous system. Our findings illustrate the complexity of transcriptional regulation in the nervous system and provide an example for the intricate interdependence of transcription factor action.