Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans, the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-, strain-, and species-specific, which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line, Jurkat, is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells, whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover, anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well, cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT, but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically, anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells, thereby blocking functions that are pivotal in the regulation of immune responses.

Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5). Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7). We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1␤ and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1. Studies of the processing of IL-1␤ reveal that the levels of activated/cleaved IL-1␤ in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT. Enhanced processing of IL-1␤ directly correlates with increased levels in the activation of its upstream regulator, IL-1␤-converting enzyme/Caspase-1 (ICE). The extracellular release of IL-1␤ and IL-18 in response to anthrax LT is ICE-dependent, as an ICEspecific inhibitor blocks this process. These data indicate that ICE, IL-1␤, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.Patients with anthrax infection recognized at late stages have high mortality even with appropriate antibiotic therapy (8), which is likely due to the effects of bacterial toxins that persist following death of the pathogen. One of these toxins, anthrax LT, 1 comprises anthrax protective antigen (PA) and anthrax lethal factor (LF). Anthrax PA binds target cells and allows entry of the enzymatically active anthrax LF (9). LF, in turn, inactivates mitogen-activated protein kinase kinases (MAPKKs) through cleavage at specific recognition sites (1-5).MAPKKs are critical intermediates in signal transduction cascades that ultimately lead to activation of the NF-B family of transcription factors that promote macrophage survival (4). Although some of the elements underlying the mechanism of action of anthrax LT-induced apoptosis have now been elucidated, the etiology of species-and cell-specific differences in sensitivity to anthrax LT remains unclear. In addition, the role of other downstream effectors, such as cytokines, is disputed. In this regard, seemingly contradictory reports have been published that either support or reject roles for proinflammatory cytokines in responses to anthrax LT in vitro (6 -7). We now report that anthrax LT treatment induces rapid activation of ICE, a caspase family enzyme responsible for the processing of IL-1␤ and IL-18 into active forms. Anthrax LT treatment results in the extracellular release of IL-1␤ and IL-18 by murine macrophage cell lines in a manner dependent upon the activation of ICE. Studies of the action of anthrax lethal toxin on the regulation of IL-1␤ reveal that anthrax lethal toxin likely enhances both the cleavage of cytokine proforms and the release of intracellular stores of preprocessed cytokine. These data provide the basis for developing new anthrax LT bioassays an...

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca 2؉ -dependent K ؉ current. This current produces a post-spike hyperpolarization (AHP slow ) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHP slow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHP slow in nodose neurons displays a slow rise time to peak (0.3-0.5 s) and a long duration (3-15 s Activation and sensitization of primary afferent nerve fibers during allergic inflammation are orchestrated by inflammatory mediators released from various cells, including tissue mast cells. Inf lammatory mediators provoke excitability changes in sensory nerves through diverse mechanisms, including (i) modification of the density and coupling efficacy of ligand-gated ionic channels; (ii) alteration in voltage-gated sodium, potassium, and calcium channels; and (iii) manipulation of cellular mechanisms that control spike-frequency adaptation.After immunologic activation of mast cells in airway in vivo or in sensory ganglia in vitro, a wide range of electrophysiological changes can be detected in peripheral sensory nerve terminals of the vagus (1) and in vagal primary afferent somata (located in the nodose and jugular ganglia) (2). These changes range from transient (minutes) membrane depolarizations that sometimes reach action potential (AP) threshold (3) to a sustained (days) unmasking of functional NK-2 tachykinin receptors (4, 5). One electrical membrane property that is particularly sensitive to inflammatory mediators is a slow post-spike afterhyperpolarization (AHP slow ; see Fig. 1 ) (3).This slow afterpotential influences neuronal excitability and determines the frequency and pattern of neuronal discharge. We have found that the amplitude and duration of the AHP slow are exquisitely sensitive to known inflammatory mediators such as prostanoids, amines, and kinins applied exogenously (Table 1) or released endogenously (i.e., after immunologic activation of mast cells) (3, 6). Inhibition of the AHP slow is accompanied by a loss of spike-frequency adaptation. Thus, modulation of the AHP slow amplitude and duration provides a mechanism for neuronal sensitization.We are interested in identifying the ionic channels and second-messenger transduction pathways that participate in the initiation and maintenance of the AHP slow in vagal primary afferent neurons. In this report, we describe the general properties of this slow afterpotential and our progress in its characterization. Our working hypothesis is that a close functional proximity between three separate channels [N t...

Purpose: To describe the Food and Drug Administration review and marketing approval considerations for panitumumab (Vectibix) for the third-line treatment of patients with epidermal growth factor receptor^expressing metastatic colorectal carcinoma. Experimental Design: Food and Drug Administration reviewed a single, open-label, multicenter trial in which 463 patients with epidermal growth factor receptor^expressing metastatic colorectal cancer who had progressed on or following treatment with a regimen containing a fluoropyrimidine, oxaliplatin, and irinotecan were randomized (1:1) to receive best supportive care (BSC) with or without panitumumab (6 mg/kg every other week) administered until disease progression or intolerable toxicity. Progression and response were confirmed by an independent review committee masked to treatment assignment. At progression, patients in the BSC-alone arm were eligible to receive panitumumab. Results: Although median progression-free survival (PFS) was similar in both treatment arms (f8 weeks), the mean PFS was f50% longer among patients receiving panitumumab than among those receiving BSC alone (96 versus 60 days, respectively) and the objective response rate in patients receiving panitumumab was 8%. However, no difference in overall survival was shown between the two study arms. Conclusions: Panitumumab received accelerated approval based on improvement in PFS and an independently confirmed response rate of 8%, similar to that observed with other active agents at this advanced stage of disease. Confirmation of clinical benefit will be required for full approval. 5 This accelerated approval was based on the surrogate end point of tumor response. The clinical benefit (i.e., improved PFS or OS) of cetuximab in a study of 572 patients who had failed both irinotecan-and oxaliplatin-based chemotherapy regimens randomized (1:1) to receive best supportive care (BSC) with or without cetuximab administered as an i.v. infusion of 400 mg/m 2 on the first dose and then 250 mg/m 2 . Patients randomized to receive cetuximab showed a statistically 5