Cost savings associated with transfer of trauma patients within an accountable care organization.

BACKGROUND: The Patient Protection and Affordable Care Act supports the establishment of accountable care organizations (ACOs) as care delivery models designed to save costs. The potential for these cost savings has been demonstrated in the primary care and inpatient populations, but not for patients with emergency conditions or traumatic injuries. METHODS: Our study evaluated adult trauma patients transferred to the tertiary care hospitals of a pioneer ACO, comparing those who were transferred from within the ACO to those from outside the ACO in terms of overall cost of hospitalization. Hospital length of stay and number of imaging studies were predetermined secondary outcomes. RESULTS: The study population included 7696 hospitalizations for traumatic injuries over a 5-year period, 85.1% of which were for patients transferred from outside the ACO. Patients transferred from within the ACO had a 7.2% lower overall cost of hospitalization (P = .032). Mean injury severity scores were not significantly different between groups. Differences in mortality, intensive care unit length of stay, and overall hospital length of stay were not significant. However, analysis of radiology studies performed during the hospitalization revealed that patients transferred from within the ACO had, on average, 0.47 fewer advanced imaging studies per hospitalization than did those transferred from outside the ACO (3.55 vs 4.02 studies per hospitalization, P = .003). CONCLUSIONS: Adult trauma patients transferred from within an ACO have significantly lower total costs of hospitalization than do those transferred from outside the system, without significant differences in disease burden, hospital length of stay, or mortality.

Patient preferences for testing for pulmonary embolism in the ED using a shared decision-making model.

INTRODUCTION: Shared decision making (SDM) is a process whereby patients and clinicians work together to make informed medical decisions that incorporate patient values. Recent data suggest that, for patients with low pretest probability of pulmonary embolism (PE), doubling the standard d-dimer cutoff may reduce the need for imaging with minimal increase in missed PE diagnoses. We used an SDM approach to determine patient preferences regarding this diagnostic approach. METHODS: We prospectively enrolled a consecutive sample of emergency department (ED) patients presenting with chest pain or dyspnea. We provided patients with a standardized description of the diagnostic workup for PE. We also provided image arrays describing the risks of computed tomography in low pretest probability patients and the risks of deferring imaging assuming a d-dimer was less than twice the value normally considered positive. We surveyed patients for their preference to undergo or defer imaging in this scenario. RESULTS: We enrolled 203 ED patients. Mean age was 55 ± 17 years, and 61% were male. Seventy-four patients (37%) elected to defer computed tomography of the pulmonary arteries testing. Patients with a previous PE diagnosis were less likely to defer computed tomography of the pulmonary arteries testing (P = .007). There was no association between the decision to defer testing and age, sex, family history of PE, or self-assessed risk-taking tendency. CONCLUSIONS: When presented with a hypothetical scenario, more than one-third of patients deferred imaging for PE based on low clinical probability and a d-dimer less than twice the normal threshold. An SDM approach is acceptable to patients and may decrease imaging for PE.

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.

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.

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.

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.

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology.

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.

Hairy-root organ cultures for the production of human acetylcholinesterase.

BACKGROUND: Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified. RESULTS: As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4 degrees C for up to 5 months. CONCLUSION: Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.

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 acetylcholinesterase 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-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) 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-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 bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.

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.

Translational control of recombinant human acetylcholinesterase accumulation in plants.

BACKGROUND: Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content. RESULTS: We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA. CONCLUSION: Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.

Purification of transgenic plant-derived recombinant human acetylcholinesterase-R.

Nicotiana benthamiana plants were engineered to express a codon-optimized gene encoding the human acetylcholinesterase-R (AChE) isoform. The transgenic plants expressed the protein at >0.4% of total soluble protein, and the plant-produced enzyme was purified to homogeneity. Following lysis, procainamide affinity chromatography and anion-exchange chromatography, more than 400-fold purification was achieved and electrophoretic purity was obtained. This pure protein is kinetically indistinguishable from the only commercially available source of human acetylcholinesterase, which is produced in mammalian cell culture. Thus, we have demonstrated a model system for the production of acetylcholinesterase, which is not susceptible to the quantitative limitations or mammalian pathogens associated with purification from mammalian cell culture or human serum.

A National Evaluation of the Scholarly Activity Requirement in Residency Programs: A Survey of Emergency Medicine Program Directors.

OBJECTIVES: The Review Committee for Emergency Medicine (RC-EM) requirement for scholarly activity, which programs may define as an original research project or some other form of scholarly activity, applies to all EM residents. The objectives of this study were to: 1) describe the percentage of residency programs that require an original research project to meet the RC-EM requirement for scholarly activity, 2) describe specific challenges and resources for residents completing the RC-EM scholarly activity requirement, and 3) identify associations between the interpretation of the requirement and early career outcomes. METHODS: This was a cross-sectional online survey of program or research directors from all U.S. allopathic EM residency programs. Respondents were queried about key demographics and domains relating to research curriculum, resources, expectations, outcomes, challenges, and future opportunities. Data were analyzed using descriptive statistics. RESULTS: The overall response rate was 113 of 156 (72%) EM residency programs. Respondents were more likely to represent university-based programs, but otherwise did not differ from nonrespondents across key demographic criteria. An original research project was required by 39% of responding programs, with a minimum deliverable in 93% of these programs. Program directors listed data collection and study design as the principle challenges residents face while completing their scholarly activities. Faculty mentorship, biostatistical support, and travel support were common resources reportedly available to residents. Comparison of programs with an original research requirement to those without revealed many differences in outcomes. Programs with a research requirement were more likely to have residents with oral or poster presentations (46% vs. 25%, mean difference = 21%, 95% confidence interval [CI] = 16% to 28%), published manuscripts (25% vs. 18%, mean difference = 7%, 95% CI = 2% to 10%), entering fellowship training after residency (27% vs. 20%, mean difference = 7%, 95% CI = 4% to 10%), and using a biostatistician (64% vs. 28%, median difference = 26%, 95% CI = 24% to 28%). There were no statistically significant differences in other evaluations of resources or outcome measures, including resident choice of academic career after leaving residency. CONCLUSIONS: There is no consistent interpretation and implementation of the RC-EM requirement for scholarly activity among EM residency programs. Residency programs requiring an original research project were more likely to have residents with accepted oral or poster presentations, published manuscripts, and entering fellowships after residency training.

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.

Humoral immune responses by prime-boost heterologous route immunizations with CTB-MPR(649-684), a mucosal subunit HIV/AIDS vaccine candidate.

CTB-MPR(649-684) is a translational fusion protein consisting of the cholera toxin B subunit and a 36-residue peptide, MPR(649-684), corresponding to the conserved membrane proximal ectodomain of gp41. CTB-MPR(649-684) was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice. In this report, we describe an effective immunization regimen for this novel anti HIV-1 vaccine-candidate. Bacterially-produced CTB-MPR(649-684) was intranasally and/or intraperitoneally administered to investigate several prime-boost heterologous route immunization regimens. Mucosal priming with the adjuvant cholera toxin elicited significant levels of vaginal IgA and serum IgG specific to MPR(649-684). Systemic boosting after mucosal priming enhanced the levels of serum and mucosal antibodies. Systemic priming induced a strong serum anti-MPR(649-684) IgG response, which was efficiently recalled and augmented by either systemic or mucosal boosting. However, this regimen was less effective in inducing secretory anti-MPR(649-684) IgA. The serum anti-MPR(649-684) IgG subtype profile revealed that both IgG1 and IgG2a were induced in all the immunization regimens, and that mucosal co-administration of cholera toxin shifted the bias to the latter subtype. We concluded that, of the various immunization regimens examined here, mucosal priming with adjuvant followed by systemic boosting exhibited the best response in respect to either systemic or mucosal anti-MPR(649-684) antibodies. Most importantly, mucosal antibodies elicited by this regimen significantly inhibited HIV-1 transcytosis in a human tight epithelium model.

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 alpha-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.

A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs.

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.