A rationally designed mutant of plasma platelet-activating factor acetylhydrolase hydrolyzes the organophosphorus nerve agent soman.

Organophosphorus compounds (OPs) such as sarin and soman are some of the most toxic chemicals synthesized by man. They exert toxic effects by inactivating acetylcholinesterase (AChE) and bind secondary target protein. Organophosphorus compounds are hemi-substrates for enzymes of the serine hydrolase superfamily. Enzymes can be engineered by amino acid substitution into OP-hydrolyzing variants (bioscavengers) and used as therapeutics. Some enzymes associated with lipoproteins, such as human plasma platelet-activating factor acetylhydrolase (pPAF-AH), are also inhibited by OPs; these proteins have largely been ignored for engineering purposes because of complex interfacial kinetics and a lack of structural data. We have expressed active human pPAF-AH in bacteria and previously solved the crystal structure of this enzyme with OP adducts. Using these structures as a guide, we created histidine mutations near the active site of pPAF-AH (F322H, W298H, L153H) in an attempt to generate novel OP-hydrolase activity. Wild-type pPAF-AH, L153H, and F322H have essentially no hydrolytic activity against the nerve agents tested. In contrast, the W298H mutant displayed novel somanase activity with a kcat of 5min(-1) and a KM of 590µM at pH7.5. There was no selective preference for hydrolysis of any of the four soman stereoisomers.

Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter.

Design, synthesis, and characterization of novel, nonquaternary reactivators of GF-inhibited human acetylcholinesterase.

The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.

EMS provider compliance with infection control recommendations is suboptimal.

INTRODUCTION: Standard precautions are disease transmission prevention strategies recommended by both the World Health Organization (WHO) and by the Centers for Disease Control and Prevention (CDC). Emergency medical services (EMS) personnel are expected to utilize standard precautions. METHODS: This was a prospective observational study of the use of standard precautions by EMS providers arriving at a large urban emergency department (ED). Research assistants (RAs) observed EMS crews throughout their arrival and delivery of patients and recorded data related to the use of gloves, hand hygiene, and equipment disinfection. RESULTS: A total of 423 EMS deliveries were observed, allowing for observation of 899 EMS providers. Only 512 (56.9%) EMS providers arrived wearing gloves. Hand washing was observed in 250 (27.8%) of providers. Reusable equipment disinfection was noted in only 31.6% of opportunities. The most commonly disinfected item was the stretcher (55%). CONCLUSION: EMS provider compliance with standard precautions and equipment disinfection recommendations is suboptimal. Strategies must be developed to improve EMS provider compliance with internationally recognized infection control guidelines. Key words: Emergency medical services, hand washing, hygiene, disinfection, disease prevention.

Trauma Center Hospitals Charged Higher Prices For Some Nontrauma Care Than Non-Trauma Center Hospitals, 2012-18.

Rising prices are a major cause of increased health care spending and health insurance premiums in the US. Hospital prices, specifically-for both inpatient and outpatient care-are the largest driver of rising health care spending in the commercial insurance market. As a result, policy makers and employers are increasingly interested in understanding the determinants of hospital prices. Hospitals serving as trauma centers are often endowed by regulators with monopoly power over trauma services in their geographic areas, and this monopoly power may spill over to nontrauma services. This study focused on the growing number of designated trauma centers and how trauma center status affects hospital prices for other, nontrauma services. We found that hospitals designated as trauma centers charged higher prices for nontrauma inpatient admissions and nontrauma emergency department visits when compared with hospitals that were not designated as trauma centers, even after controlling for potential confounders.

Characterization of vancomycin pharmacokinetics in the adult acute myeloid leukemia population.

Current vancomycin dosing guidelines in our acute myeloid leukemia population too often achieve suboptimal initial drug concentrations. Our aim was to assess vancomycin pharmacokinetic parameters in acute myeloid leukemia patients and develop an improved dosing equation to attain more accurate initial therapeutic trough levels. Acute myeloid leukemia patients receiving vancomycin for a presumed or documented gram positive infection were eligible. Patients hospitalized in the intensive care unit, those with creatinine clearance <30 mL/min or with limb amputation were excluded. Three samples were collected at the following post-infusion time ranges: 1 h, 3-8 h, and 8-24 h post-infusion, contingent on the dosing interval. Pharmacokinetic data were then fit using a Bayesian-based population pharmacokinetic model. A total of 25 acute myeloid leukemia patients were studied with a mean volume in the central compartment (Vc; L/65 kg), volume of distribution at steady state (Vss; L/65 kg) and distributional clearance (CLd; L/h/65 kg) of 15, 38.9, and 9.32, respectively. CLslope was 0.59 (mg of vancomycin clearance per unit of creatinine clearance in mL/min); this value is 21.4% lower than the established literature value (0.75). The derived equation, based upon these values, was reasonably precise at achieving the desired trough concentration using a priori dosing. The mean (CV%) of the achieved trough was 94% (29%) with a range of 66-188%; 3/25 at <75% and >125%]. We have established that the derived dosing equation can place ≈ 75% of adult acute myeloid leukemia patients at vancomycin trough levels within 75-125% of the target trough level.

Role of acetylcholinesterase on the structure and function of cholinergic synapses: insights gained from studies on knockout mice.

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.

A structure-activity analysis of the variation in oxime efficacy against nerve agents.

A structure-activity analysis was used to evaluate the variation in oxime efficacy of 2-PAM, obidoxime, HI-6 and ICD585 against nerve agents. In vivo oxime protection and in vitro oxime reactivation were used as indicators of oxime efficacy against VX, sarin, VR and cyclosarin. Analysis of in vivo oxime protection was conducted with oxime protective ratios (PR) from guinea pigs receiving oxime and atropine therapy after sc administration of nerve agent. Analysis of in vitro reactivation was conducted with second-order rate contants (k(r2)) for oxime reactivation of agent-inhibited acetylcholinesterase (AChE) from guinea pig erythrocytes. In vivo oxime PR and in vitro k(r2) decreased as the volume of the alkylmethylphosphonate moiety of nerve agents increased from VX to cyclosarin. This effect was greater with 2-PAM and obidoxime (>14-fold decrease in PR) than with HI-6 and ICD585 (<3.7-fold decrease in PR). The decrease in oxime PR and k(r2) as the volume of the agent moiety conjugated to AChE increased was consistent with a steric hindrance mechanism. Linear regression of log (PR-1) against log (k(r2)[oxime dose]) produced two offset parallel regression lines that delineated a significant difference between the coupling of oxime reactivation and oxime protection for HI-6 and ICD585 compared to 2-PAM and obidoxime. HI-6 and ICD585 appeared to be 6.8-fold more effective than 2-PAM and obidoxime at coupling oxime reactivation to oxime protection, which suggested that the isonicotinamide group that is common to both of these oximes, but absent from 2-PAM and obidoxime, is important for oxime efficacy.

Nilotinib: a new tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder arising from a single genetic mutation that leads to an increase in immature myeloid cells in the bone marrow and the accumulation of these cells in the blood. Typically, CML represents 15-20% of all adult leukemias, with 4830 new cases expected in 2008. The cytogenetic hallmark of CML is the Philadelphia chromosome, which is the result of the reciprocal translocation and conjugation of the breakpoint cluster region (BCR) gene, BCR, on chromosome 22 and the Abelson (ABL) kinase gene, ABL, on chromosome 9. Current treatment is aimed at inhibiting BCR and ABL kinase with novel agents, the first being imatinib in 2003, and more recently dasatinib in 2006. Nilotinib is a new small-molecule inhibitor of tyrosine kinase rationally developed from the crystalline structure of the imatinib-ABL complex. It represents an aminopyrimidine derivative of imatinib with approximately 30 times more potency in vitro against imatinib-sensitive BCR-ABL-expressing cell lines and activity against 32 of 33 point mutations conferring resistance to imatinib. Data from phase I and II studies show that nilotinib has activity against all phases of CML in patients who are intolerant or have failed therapy with imatinib or dasatinib. Nilotinib represents a new therapeutic option for patients with CML who are intolerant or have failed therapy with imatinib. Ongoing clinical trials are assessing nilotinib's role in the treatment of patients with newly diagnosed CML and its long-term efficacy and safety.

A gas chromatographic-mass spectrometric approach to examining stereoselective interaction of human plasma proteins with soman.

The organophosphorus (OP) nerve agent soman (GD) contains two chiral centers (a carbon and a phosphorus atom), resulting in four stereoisomers (C+P+, C-P+, C+P-, and C-P-); the P- isomers exhibit a mammalian toxicity that is approximately 1000-fold greater than that of the P+ isomers. The capacity to assess the binding or hydrolysis of each of the four stereoisomers is an important tool in the development of enzymes with the potential to protect against GD intoxication. Using a gas chromatography-mass spectrometry-based approach, we have examined the capacity of plasma-derived human serum albumin, plasma-purified human butyrylcholinesterase, goat milk-derived recombinant human butyrylcholinesterase, and recombinant human paraoxonase 1 to interact with each of the four stereoisomers of GD in vitro at pH 7.4 and 25 degrees C. Under these experimental conditions, the butyrylcholinesterase samples were found to bind GD with a relative preference for the more toxic stereoisomers (C-P- > C+P- > C-P+ > C+P+), while human serum albumin and paraoxonase 1 interacted with GD with a relative preference for the less toxic isomers (C-P+/C+P+ > C+P-/C-P-). The results indicate that these human proteins exhibit distinct stereoselective interactions with GD. The approach described presents a means to rapidly assess substrate stereospecificity, supporting future efforts to develop more effective OP bioscavenger proteins.

Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase.

Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.

Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review.

Currently fielded treatments for nerve agent intoxication promote survival, but do not afford complete protection against either nerve agent-induced motor and cognitive deficits or neuronal pathology. The use of human plasma-derived butyrylcholinesterase (HuBuChE) to neutralize the toxic effects of nerve agents in vivo has been shown to both aid survival and protect against decreased cognitive function after nerve agent exposure. Recently, a commercially produced recombinant form of human butyrylcholinesterase (r-HuBuChE; PharmAthene Inc.) expressed in the milk of transgenic goats has become available. This material is biochemically similar to plasma-derived HuBuChE in in vitro assays. The pharmacokinetic characteristics of a polyethylene glycol coated (pegylated) form of r-HuBuChE were determined in guinea pigs; the enzyme was rapidly bioavailable with a half-life (t(1/2)) and pharmacokinetic profile that resembled that of plasma-derived huBuChE. Guinea pigs were injected with 140mg/kg (i.m.) of pegylated r-HuBuChE 18h prior to exposure (sc) to 5.5xLD(50) VX or soman. VX and soman were administered in a series of three injections of 1.5xLD(50), 2.0xLD(50), and 2.0xLD(50), respectively, with injections separated by 2h. Pretreatment with pegylated r-HuBuChE provided 100% survival against multiple lethal doses of VX and soman. Guinea pigs displayed no signs of nerve agent toxicity following exposure. Assessments of motor activity, coordination, and acquisition of spatial memory were performed for 2 weeks following nerve agent exposure. There were no measurable decreases in motor or cognitive function during this period. In contrast, animals receiving 1.5xLD(50) challenges of soman or VX and treated with standard atropine, 2-PAM, and diazepam therapy showed 50 and 100% survival, respectively, but exhibited marked decrements in motor function and, in the case of GD, impaired spatial memory acquisition. The advances in this field have resulted in the decision to select both the plasma-derived and the recombinant form of BuChE for advanced development and transition to clinical trials. Efforts have now been expanded to identify a catalytic protein capable of not only binding, but also rapidly hydrolyzing the standard threat nerve agents. Recent work has focused on paraoxonase-1 (PON1), a naturally occurring human serum enzyme with the capacity to catalyze the hydrolysis of nerve agents, albeit too slowly to afford dramatic protection. Using rational design, several amino acids involved in substrate binding have been identified and site-directed mutations have revealed that residue H115 plays an important role in binding. In addition, the stereospecificity of PON1 for the catalytic hydrolysis of soman has been examined. The enzyme exhibits a slight stereospecificity for the C+P+ isomer of soman, which is due more to preferential binding than to selective hydrolysis of this isomer. The results suggest that it may be possible to engineer a mutant form of PON1 with enhanced activity and stereospecificity for the most toxic nerve agent isoforms.

Use of V agents and V-analogue compounds to probe the active site of atypical butyrylcholinesterase from Oryzias latipes.

The atypical butyrylcholinesterase (aBuChE) from Oryzias latipes shares approximately 65% sequence similarity to both acetylcholinesterase and butyrylcholinesterase and was studied for its capacity to spontaneously reactivate following inhibition by organophosphorus nerve agents. Like other cholinesterases, aBuChE was inhibited by all G- and V-type nerve agents. Interestingly, aBuChE was able to undergo spontaneous reactivation after inhibition with VR (t1/2 = 5.5 ± 0.2 h). Mass spectrometry of aBuChE after VR inhibition confirmed the presence of a covalently bound adduct of the size expected for non-aged VR on the peptide containing the active site serine. To understand the effect of substrate volume on rates of reactivation, the capacity of aBuChE to bind and spontaneously reactivate after inhibition with five V-agent analogues was examined. No appreciable reactivation was detected for enzyme inhibited by V2 (VX with O-isopropyl on retained group), V4 (VX with N-diethyl leaving group termination), or V5 (VX with N-dimethyl leaving group termination). Minimal reactivation was detected with V1 (VX with O-propyl on retained group). Conversely, spontaneous reactivation was observed when aBuChE was inhibited by V3 (VX with O-isobutyl on retained group; t1/2 = 6.3 ± 0.4 h). The data suggest that the ability of aBuChE to spontaneously reactivate after inhibition by V-agent analogues is related to the structure of the retained group. These results provide structural information that may shed light on the design of improved small molecule reactivators of nerve agent-inhibited acetylcholinesterase or butyrylcholinesterase, and further suggest that re-engineering the active site of a cholinesterase could result in enzymes with clinically relevant rates of nerve agent hydrolysis.

The constitution of mineral trioxide aggregate.

OBJECTIVES: The aim of this study was to determine the constitution of a commercially available root-end filling material, mineral trioxide aggregate, (MTA) (ProRoot MTA, Tulsa Dental, Tulsa, OK, USA). The surface morphology of the material with various treatment conditions was also evaluated. METHODS: The constitution of two commercial versions of MTA was determined before and after mixing with water. The unset material was analysed using Energy Dispersive Analysis by X-ray (EDAX) in a scanning electron microscope (SEM) and X-ray diffraction (XRD). The first technique identified the constituent elements while XRD analysis identified the compounds or phases present. The set material was evaluated using EDAX. The surface morphology of the material stored under various conditions (100% humidity, immersion in water, or immersion in phosphate solution) was evaluated using SEM. RESULTS: The EDAX showed the white MTA to be composed primarily of calcium, silicon, bismuth and oxygen, with the gray MTA also having small peaks for iron and aluminum. The XRD analysis showed gray MTA to be composed primarily of tricalcium silicate and dicalcium silicate. The surface morphology of the materials differed under the various conditions, particularly following immersion in phosphate solution with crystal formation. SIGNIFICANCE: The commercial versions of MTA were shown to have broadly similar constitution to ordinary Portland cement except for the addition of bismuth compounds. The white MTA did not contain iron.

Behavioral data management using Visual Basics for Applications to automate data capture and analysis.

Many researchers are familiar with the spreadsheet capabilities of Microsoft Excel, but have never explored using customized VISUAL BASIC FOR APPLICATIONS (VBA) macros embedded in the program. At the United States Army Medical Research Institute of Chemical Defense (USAMRICD), the implementation of VBA program code to carry out repetitive operations has resulted in a tremendous savings in both the time and manpower required to reliably capture, analyze, and plot data from research protocols. A set of "template" workbooks was developed and is used to organize data from different types of studies. At the heart of the concept is a "setup information" worksheet onto which the user enters information about the study (i.e. the number of subjects, groups, graphs). Clicking a control button on this worksheet launches the VBA code that creates worksheets for each subject, group and chart specified. A "pairmatch" function allows the user to create groups either randomly or by pair matching based on user-specified variables. Controls are provided that run VBA program code to parse subject data files into the proper worksheets and perform group averages. Charts are updated automatically whenever group averages change so that the researcher always has an up-to-date plot available. The ability to reformat the captured data onto a "statistical output" worksheet allows data to be imported easily to statistical software packages. The concept is being used for several different types of studies at USAMRICD and has significantly reduced not only amount of time spent on data management, but also the number of data entry errors. Readers interested in acquiring an electronic copy of the startle workbook example, which contains the complete VBA code, should send the request to the authors at reseco@erols.com or maurice.sipos@us.army.mil.

A theoretical expression for the protection associated with stoichiometric and catalytic scavengers in a single compartment model of organophosphorus poisoning.

The ability of certain organophosphorus (OP) compounds to inhibit acetylcholinesterase (AChE) has made them useful for industrial (insecticides) and military (nerve agents) purposes. We have previously published a single compartment mathematical model of the interactions between OP nerve agents and the enzymes affected by these agents. That model, which could be used to predict the LD50 of seven nerve agents in rats, has been extended to include the protective actions of stoichiometric and catalytic OP-scavenger enzymes (delivered as pretreatments) so that protective ratios attributable to the scavengers may be predicted. Prediction of expected human protection from in vitro rate constant and initial enzyme level measurements is the ultimate goal for this work. The enhanced model predicts the LD50 from rate constants of the OP agent's binding reactions with AChE, carboxylesterase (CaE) and a stoichiometric scavenger (S); a first-order OP elimination rate (including a contribution due to a catalytic scavenger); and whole body estimates of AChE, CaE and S. The ratio of the scavenger-treated LD50 estimate to the scavenger-free LD50 estimate provided a theoretical expression describing the scavenger's contributions to the protective ratio. Published in vivo protective ratios for two stoichiometric scavengers (fetal bovine serum AChE and human utyrylcholinesterase) against challenge by several OP agents in mice were compared with ratios predicted by the model. A linear regression analysis of in vivo protective ratios in mice versus the ratios predicted by the model from the in vitro measurements resulted in an R(2) value of 0.902. The catalytic scavenger portion of the theory could not be validated due to a lack of published data. We conclude that the one-compartment model can be used to make reasonable estimates of the protective ratio attributable to stoichiometric scavengers, but can make no conclusions regarding the ability of the model to predict catalytic scavenger protection ratios.

Neutralization effects of interleukin-6 (IL-6) antibodies on sulfur mustard (HD)-induced IL-6 secretion on human epidermal keratinocytes.

The proinflammatory cytokine human interleukin-6 (hIL-6) plays an important role in the early and late courses of inflammation, trauma, and wound healing caused by sulfur mustard (HD). Previously, we demonstrated that hIL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein, which indirectly initiates the cascade of events, such as skin irritation and blister formation observed in the pathophysiology of HD injury. In this present work, we focus on the neutralization effect of IL-6 antibodies with regard to the modulation of hIL-6 secretion. Levels of secreted cytokine hIL-6 in normal human epidermal keratinocytes (NHEK) stimulated with HD (10(-4)M) and incubated for 24h at 37°C were determined by enzyme immunoassay, protein immunocytologic assay and reverse-transcriptase-polymerase chain reaction (RT-PCR). The ratio of HD-treated NHEK to constitutive non-stimulated NHEK controls (S/C) on the induction of hIL-6 is reported. S/C was four-fold higher than non-stimulated NHEK controls as determined by ELISA. By using a more sensitive immunocytologic assay, Luminex(100)™, the increment was verified. hIL-6 levels in NHEK stimulated with HD were 21±11ng/mL as measured by Luminex(100)™. The messenger RNA expression of the cytokine (hIL-6) gene was analyzed semiquantitatively. RT-PCR demonstrated that HD induced an increase in the transcription of hIL-6 gene. Selective immunosuppression, using IL-6 neutralizing antibodies, led to a reduction of such expression of HD-induced transcription of hIL-6 in human keratinocytes. The neutralization by pre-incubating NHEK with monoclonal anti-IL6 antibodies decreased hIL-6 secretion by 76%±1.8 ((*)P<0.05).

A common mechanism for resistance to oxime reactivation of acetylcholinesterase inhibited by organophosphorus compounds.

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate.