Brian C. Geyer

Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

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.

Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase.

Organophosphorous 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.

Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath

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 acetylcho‐linesterase variant, AChE‐R. AChE‐R shares its active site with the synaptic AChE‐S 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‐RER produced from plant‐optimized cDNA in Nicotiana benthamiana plants. AChE‐RER purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 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‐RER 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 organophos‐phate‐induced neuromuscular junction dismorphology. Our findings present plant‐produced AChE‐RER as a bimodal agent, conferring both short‐ and long‐term protection from organophosphate intoxication.—Evron T., Geyer, B. C., Cherni, I., Muralidharan, M., Kilbourne, J., Fletcher, S. P., Soreq, H., Mor T. S. Plant‐derived human acetylcholinesterase‐R provides protection from lethal organophosphate poisoning and its chronic aftermath. FASEB J. 21, 2961–2969 (2007)

Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from α-tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.

Increased organophosphate scavenging in a butyrylcholinesterase mutant

Nicotiana benthamiana plant lines expressing a reengineered human butyrylcholinesterase (BChE) with enhanced cocaine hydrolase activity were created. Subsequent purification and biochemical analysis revealed that compared to wild-type butyrylcholinesterase, the cocaine hydrolase displayed increased affinity to the organophosphate (OP) pesticides paraoxon (6.8 4x 10(-10)M vs. 1.11 x 10(-8)M) and malaoxon (9.81 x 10(-8)M vs. 5.99 x 10(-7)M). Furthermore, the cocaine hydrolase retained identical anticholinesterase binding profiles for all other compounds tested. Thus we have demonstrated a potential large-scale production platform for a multivalent antidote for cocaine and anticholinesterase poisoning.

Patient Factors Associated with Failure to Diagnose Tuberculosis in the Emergency Department

BACKGROUND Emergency department (ED) presentation of pulmonary tuberculosis (TB) can be highly atypical and an ED visit might be the only health care interaction for high-risk patients. OBJECTIVE Our objective was to identify patient factors associated with discharge without a diagnosis of TB during an infectious ED visit. METHODS The study population consisted of 150 patients from 2000 to 2009 with 190 infectious ED visits. Patients were initially identified from the state registry of confirmed TB cases and epidemiological characteristics were identified prospectively during case investigation. A retrospective review was performed for clinical characteristics of visits dichotomized according to whether the diagnosis of TB was made during the ED visit. RESULTS Analysis revealed that 77% of all infectious-patient visits ended with a diagnosis of TB. A TB diagnosis was more likely when patients presented with pulmonary or infectious chief complaints, endorsed cough, subjective fever, chills, dyspnea, previous TB infection, or had an abnormal lung examination or chest x-ray study. Patients were significantly less likely to be diagnosed with TB when they were unresponsive during clinical evaluation or when they reported a history of both homelessness and any substance abuse during the last year. In addition, these characteristics were independent predictors of nondiagnosis when traditional TB risk factors or abnormal vital signs were considered. CONCLUSIONS Patients with atypical presentations, as well as those who were unresponsive or reported a history of homelessness and substance abuse, were at greater risk for nondiagnosis of TB during an infectious ED visit.

Nicotinic stimulation induces Tristetraprolin over-production and attenuates inflammation in muscle

Cholinergic signaling suppresses inflammation in blood and brain and attenuates apoptosis in other tissues, but whether it blocks inflammation in skeletal muscle under toxicant exposure, injuries and diseases remained unexplored. Here, we report nicotinic attenuation of inflammation and alteration of apoptotic protein expression pattern in murine muscle tissue and cultured myotubes, involving the RNA-binding protein, Tristetraprolin, and the anti-apoptotic protein, Mcl-1. In muscles and C2C12 myotubes, cholinergic excitation by exposure to nicotine or the organophosphorous pesticide, Paraoxon, induced Tristetraprolin overproduction while reducing pro-inflammatory transcripts such as IL-6, CXCL1 (KC) and CCL2 (MCP-1). Furthermore, nicotinic excitation under exposure to the bacterial endotoxin LPS attenuated over-expression of the CCL2 and suppressed the transcriptional activity of NF-ĸB and AP-1. Tristetraprolin was essential for this anti-inflammatory effect of nicotine in basal conditions. However, its knockdown also impaired the pro-inflammatory response to LPS. Finally, in vivo administration of Paraoxon or recombinant Acetylcholinesterase, leading respectively to either gain or loss of cholinergic signaling, modified muscle expression of key mRNA processing factors and several of their apoptosis-related targets. Specifically, cholinergic imbalances enhanced the kinase activators of the Serine-Arginine splicing kinases, Clk1 and Clk3. Moreover, Paraoxon raised the levels of the anti-apoptotic protein, Mcl-1, through a previously unrecognized polyadenylation site selection mechanism, producing longer, less stable Mcl-1 mRNA transcripts. Together, our findings demonstrate that in addition to activating muscle function, acetylcholine regulates muscle inflammation and cell survival, and point to Tristetraprolin and the choice of Mcl-1 mRNA polyadenylation sites as potential key players in muscle reactions to insults.

Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine. Methods Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed. Results Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD100 of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications. Conclusions Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.

Organophosphate intoxication: Molecular consequences, mechanisms and solutions

Publisher Summary The essential role of acetylcholinesterase (AChE) in the neuromuscular junction (NMJ) predicts severe consequences following loss of function by its irreversible inhibition. The vital role of the acetylcholine-hydrolyzing enzyme AChE in terminating cholinergic neurotransmission has been recognized for almost as many years. Anticholinesterases can be commonly encountered as industrial pesticides, weapons of war, Alzheimers medications as well as the natural toxins of many organisms including fungi, plants, and animals. Exposure to clinically relevant doses of anticholinesterases results in immediate and multisystem physiological disturbances that underscore the broad anatomical distribution of the mammalian cholinergic system. The body adapts to the insult and attempts to compensate for the cholinergic dysregulation by inhibitor–enzyme interactions, NMJ remodeling, and changes in circulating cytokine profiles. In addition the loss of synaptic cholinergic regulation by AChE inhibition has immediate negative consequences for mammalian physiology. The severe multisystem clinical presentation of anti-ChE intoxication demonstrates the essential and ubiquitous nature of the mammalian cholinergic system. Current medical intervention in the case of acute exposure to anticholinesterase agents includes use of the muscarinic receptor antagonist atropine to block overstimulation, and oximes to reactivate the OP-modified AChE. The reversible carbamate inhibitor, pyridostigmine bromide, is also used for prophylaxis.