Mammals are co-infected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine gammaherpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine gammaherpesvirus infection in vivo, suggesting a ‘two-signal’ model for viral reactivation. Thus chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K+ channels is enhanced by cytosolic calcium (Ca2+). Activation is sensitive to a range of [Ca2+] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 α subunits at [Ca2+] and [Mg2+] up to 100 mM. The half-activation voltage (V0.5) is steeply dependent on [Ca2+] in the micromolar range, but shows a tendency towards saturation over the range of 60–300 μM Ca2+. As [Ca2+] is increased to millimolar levels, the V0.5 is strongly shifted again to more negative potentials. When channels are activated by 300 μM Ca2+, further addition of either mM Ca2+ or mM Mg2+ produces similar negative shifts in steady-state activation. Millimolar Mg2+ also produces shifts of similar magnitude in the complete absence of Ca2+. The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca2+] increase activation rates, addition of 10 mM Mg2+ to 0 Ca2+ produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg2+ does not participate in Ca2+-dependent steps that influence current activation rate. We conclude that millimolar Mg2+ and Ca2+ concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca2+-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca2+ binding steps, four voltage sensors, and four independent lower affinity Ca2+/Mg2+ binding steps describes well the behavior of G-V curves over a range of Ca2+ and Mg2+. The ability of a broad range of [Ca2+] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

Leukocyte recruitment is a key host defense mechanism to infection and a salient feature of autoimmune diseases such as arthritis. The cell dynamics of these processes are difficult to study due to the challenge of tracking cells flowing in the circulation and migrating through light scattering tissues. Here, we describe a noninvasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. In the absence of inflammation, cells moved > several hundred m/s in vessels and only rarely adhered to endothelium or entered the tissue parenchyma. In response to bacterial infection, neutrophils moved in small capillaries at reduced speeds of (14-45 m/min) and rolled in larger vessels at 5-60 m/min. Within minutes of adoptive transfer, neutrophils entered the connective tissue and crawled with a median velocity of 7.3 m/min. 2P imaging has excellent spatiotemporal resolution and is a promising in vivo approach to study the cellular basis of inflammation.

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