The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

A vaccine that would engage the mucosal immune system against a broad range of HIV-1 subtypes and prevent epithelial transmission is highly desirable. Here we report fusing the mucosal targeting B subunit of cholera toxin to the conserved galactosylceramide-binding domain (including the ELDKWA-neutralizing epitope) of the HIV-1 gp41 envelope protein, which mediates the transcytosis of HIV-1 across the mucosal epithelia. Chimeric protein expressed in bacteria or plants assembled into oligomers that were capable of binding galactosyl-ceramide and G M1 gangliosides. Mucosal (intranasal) administration in mice of the purified chimeric protein followed by an i.p. boost resulted in transcytosis-neutralizing serum IgG and mucosal IgA responses and induced immunological memory. Plant production of mucosally targeted immunogens could be particularly useful for immunization programs in developing countries, where desirable product traits include low cost of manufacture, heat stability, and needle-free delivery.

SummaryOrganophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.

Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the 'Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid beta (Abeta) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Abeta oligomers and fibrils and abolished Abeta toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Abeta-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic events.

The Tax transforming protein encoded by human Tcell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-B and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of IB kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the IB inhibitory subunit of NF-B. We now demonstrate that the IKK␤ catalytic subunit and IKK␥ regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKK␤ protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of IB kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKK␤ that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKK␥ substrate in vitro. We conclude that Tax-induced phosphorylation of IKK␤ is required for IKK␤ activation, phosphoryl group transfer to IKK␥, and acquisition of the deregulated IKK phenotype.

Objective-To identify critical Emergency Medicine (EM)-focused firearm injury research questions and to develop an evidence-based research agenda. Results-Fifty-nine final EM-relevant research questions were identified, including questions that cut across all firearm injury topics and questions specific to self-directed violence (suicide and attempted suicide); intimate partner violence; peer (non-partner) violence; mass violence; and unintentional ("accidental") injury. Some questions could be addressed through research conducted in emergency departments (EDs); others would require work in other settings. HHS Public AccessConclusions-The technical advisory group identified key EM-relevant firearm injury research questions. EM-specific data is limited for most of these questions. Funders and researchers should consider increasing their attention to firearm injury prevention and control, particularly to the questions identified here and in other recently developed research agendas.

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Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChES variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R ER produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R ER purified to homogeneity showed indistinguishable biochemical properties, with IC 50 = 10 −7 M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R ER of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R ER as a bi-modal agent, conferring both short-and long-term protection from organophosphate intoxication. Keywords paraoxon intoxication; neuromuscular junction; transgenic plants; long-term protection; bioscavenger Accidental (environmental or occupational) and self-inflicted (suicide) exposure to organophosphate (OP) pesticides is encountered frequently in the emergency room, especially in the developing world (1,2). These perennial public health issues are compounded by a growing concern over the potential use of OP nerve agents such as sarin as a means of terror and nonconventional warfare (3,4). OPs disrupt neuro-transmission by inhibiting synaptic acetylcholinesterase (AChE-S), leading to accumulation of acetylcholine in the synapse and 2Correspondence: T.S.M., neural overstimulation (5). The severity of the ensuing nicotinic and muscarinic symptoms is dose dependent and can result in death due to cardiovascular and respiratory collapse (3,4). Those surviving the initial insult often suffer long-term sequelae, including OP-induced delayed neuropathy, muscle weakness, permanent brain dismorphology, and social/behavioral deficits (3,4,6). The mechanisms underlying OP-delayed toxicity and other stressful insults (e.g., psychological stress or head trauma) involve rapid elevation of c-fos, followed by up-regulation of the ACHE gene (7) and rapid but long-lasting shifted alternative splicing from AChES to the otherwise rare "read-through" variant (AChE-R, 8-10). Up-regulation and isoform switching are associated with short-term neuroprotec...