Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.A s our understanding of physiology grows, we are increasingly aware of its Newtonian aspects in that for every action there is another reaction with opposite consequences (1). Irrespective of whether one thinks in terms of homeostasis, feedback loops, or checkpoints, we increasingly uncover molecular systems that, in terms of function, induce cells to move in opposite ways. For example, although immune T-cell activation is critical in controlling disease, one needs a checkpoint to guard against overactivity that could result in autoimmunity (2, 3).In addition to adding to our general knowledge of cellular physiology, detailed understanding of the molecular mechanisms of activation and checkpoint processes has important therapeutic implications. Such an understanding allows two separate entry points into the regulation of cellular events. Thus, if one wants to promote a cellular function, the same outcome can be achieved by either enhancing the effector or inhibiting the checkpoint pathways, usually by perturbing the molecules that initiate them.One of the most important physiological systems is the cytokine cascade (4). However, here, because the cascade contains large numbers of separate molecules, each operating through different receptors, simple models of regulation break down. While one can imagine that each cytokine is paired with a separate checkpoint system operating in an opposite direction, it seemed to us to be unlikely, if for no other reason than the economical use of genetic information. Given that one had large sets of related molecules with overlapping signal transduction mechanisms and pathways, it seemed more reasonable to assume that, depending on the circumstance, some members of the cytokine repertoire regulate other members of the repertoire. This differs from situations such as PD-1, where the checkpoint mechanistic cascade differs completely from the activation mechanism. By contrast, we propose that cytokine members of a family regulate each other by perturbing common molecular mechanisms. Thus, if gene action economy is proposed for a family, its hallmark should be that some of the same molecules used for...

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Tspan12/β-catenin signaling is critical for the progression of vasoproliferative disease. The newly developed anti-Tspan12 antibody has therapeutic effects in vasoproliferative retinopathy and can enhance the potency of existing anti- vascular endothelial growth factor agents.