Development and Validation of a Plaque Assay to Determine the Titer of a Recombinant Live-Attenuated Viral Vaccine for SARS-CoV-2.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than seven million deaths worldwide. To reduce viral spread, the Israel Institute for Biological Research (IIBR) developed and produced a new rVSV-SARS-CoV-2-S vaccine candidate (BriLife®) based on a platform of a genetically engineered vesicular stomatitis virus (VSV) vector that expresses the spike protein of SARS-CoV-2 instead of the VSV-G protein on the virus surface. Quantifying the virus titer to evaluate vaccine potency requires a reliable validated assay that meets all the stringent pharmacopeial requirements of a bioanalytical method. Here, for the first time, we present the development and extensive validation of a quantitative plaque assay using Vero E6 cells for the determination of the concentration of the rVSV-SARS-CoV-2-S viral vector. Three different vaccine preparations with varying titers (DP_low, DP_high, and QC sample) were tested according to a strict validation protocol. The newly developed plaque assay was found to be highly specific, accurate, precise, and robust. The mean deviations from the predetermined titers for the DP_low, DP_high, and QC preparations were 0.01, 0.02, and 0.09 log10, respectively. Moreover, the mean %CV values for intra-assay precision were 18.7%, 12.0%, and 6.0%, respectively. The virus titers did not deviate from the established values between cell passages 5 and 19, and no correlation was found between titer and passage. The validation results presented herein indicate that the newly developed plaque assay can be used to determine the concentration of the BriLife® vaccine, suggesting that the current protocol is a reliable methodology for validating plaque assays for other viral vaccines.

Efficacy of a combined anti-seizure treatment against cholinergic established status epilepticus following a sarin nerve agent insult in rats.

The development of refractory status epilepticus (SE) following sarin intoxication presents a therapeutic challenge. Here, we evaluated the efficacy of delayed combined double or triple treatment in reducing abnormal epileptiform seizure activity (ESA) and the ensuing long-term neuronal insult. SE was induced in rats by exposure to 1.2 LD50 sarin followed by treatment with atropine and TMB4 (TA) 1 min later. Double treatment with ketamine and midazolam or triple treatment with ketamine, midazolam and levetiracetam was administered 30 min post-exposure, and the results were compared to those of single treatment with midazolam alone or triple treatment with ketamine, midazolam, and valproate, which was previously shown to ameliorate this neurological insult. Toxicity and electrocorticogram activity were monitored during the first week, and behavioral evaluations were performed 2 weeks post-exposure, followed by biochemical and immunohistopathological analyses. Both double and triple treatment reduced mortality and enhanced weight recovery compared to TA-only treatment. Triple treatment and, to a lesser extent, double treatment significantly ameliorated the ESA duration. Compared to the TA-only or the TA+ midazolam treatment, both double and triple treatment reduced the sarin-induced increase in the neuroinflammatory marker PGE2 and the brain damage marker TSPO and decreased gliosis, astrocytosis and neuronal damage. Finally, both double and triple treatment prevented a change in behavior, as measured in the open field test. No significant difference was observed between the efficacies of the two triple treatments, and both triple combinations completely prevented brain injury (no differences from the naïve rats). Delayed double and, to a greater extent, triple treatment may serve as an efficacious delayed therapy, preventing brain insult propagation following sarin-induced refractory SE.

Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-In Vivo Determination of Sarin Metabolite in Mice.

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40-80%) were obtained in the range of 10-300 ng/mL, with a linear response (R2 > 0.964, R > 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice's (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

Energy Transport for Thick Holographic Branes.

Universal properties of two-dimensional conformal interfaces are encoded by the flux of energy transmitted and reflected during a scattering process. We develop an innovative method that allows us to use results for the energy transmission in thin-brane holographic models to find the energy transmission for general smooth domain-wall solutions of three-dimensional gravity. Our method is based on treating the continuous geometry as a discrete set of branes. As an application, we compute the transmission coefficient of a Janus interface in terms of its deformation parameter.

Complexity for Conformal Field Theories in General Dimensions.

We study circuit complexity for conformal field theory states in an arbitrary number of dimensions. Our circuits start from a primary state and move along a unitary representation of the Lorentzian conformal group. Different choices of distance functions can be understood in terms of the geometry of coadjoint orbits of the conformal group. We explicitly relate our circuits to timelike geodesics in anti-de Sitter space and the complexity metric to distances between these geodesics. We extend our method to circuits in other symmetry groups using a group theoretic generalization of the notion of coherent states.

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase.

Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.

Neuroprotection by delayed triple therapy following sarin nerve agent insult in the rat.

The development of refractory status epilepticus (SE) induced by sarin intoxication presents a therapeutic challenge. In our current research we evaluate the efficacy of a delayed combined triple treatment in ending the abnormal epileptiform seizure activity (ESA) and the ensuing of long-term neuronal insult. SE was induced in male Sprague-Dawley rats by exposure to 1.2LD50 sarin insufficiently treated by atropine and TMB4 (TA) 1 min later. Triple treatment of ketamine, midazolam and valproic acid was administered 30 min or 1 h post exposure and was compared to a delayed single treatment with midazolam alone. Toxicity and electrocorticogram activity were monitored during the first week and behavioral evaluation performed 3 weeks post exposure followed by brain biochemical and immunohistopathological analyses. The addition of both single and triple treatments reduced mortality and enhanced weight recovery compared to the TA-only treated group. The triple treatment also significantly minimized the duration of the ESA, reduced the sarin-induced increase in the neuroinflammatory marker PGE2, the brain damage marker TSPO, decreased the gliosis, astrocytosis and neuronal damage compared to the TA+ midazolam or only TA treated groups. Finally, the triple treatment eliminated the sarin exposed increased open field activity, as well as impairing recognition memory as seen in the other experimental groups. The delayed triple treatment may serve as an efficient therapy, which prevents brain insult propagation following sarin-induced refractory SE, even if treatment is postponed for up to 1 h.

Energy Reflection and Transmission at 2D Holographic Interfaces.

Scattering from conformal interfaces in two dimensions is universal in that the flux of reflected and transmitted energy does not depend on the details of the initial state. In this Letter, we present the first gravitational calculation of energy reflection and transmission coefficients for interfaces with thin-brane holographic duals. Our result for the reflection coefficient depends monotonically on the tension of the dual string anchored at the interface and obeys the lower bound recently derived from the averaged-null-energy condition in conformal field theory. The boundary-conformal-field-theory limit is recovered for infinite ratio of the central charges.

Efficacy of retigabine in ameliorating the brain insult following sarin exposure in the rat.

BACKGROUND: Sarin is an irreversible organophosphate cholinesterase inhibitor. Following toxic signs, an extensive long-term brain damage is often reported. Thus, we evaluated the efficacy of a novel anticonvulsant drug retigabine, a modulator of neuronal voltage gated K+ channels, as a neuroprotective agent following sarin exposure. METHODS: Rats were exposed to 1 LD50 or 1.2 LD50 sarin and treated at onset of convulsions with retigabine (5 mg/kg, i.p.) alone or in combination with 5 mg/kg atropine and 7.5 mg/kg TMB-4 (TA) respectively. Brain biochemical and immunohistopathological analyses were processed 24 h and 1 week following 1 LD50 sarin exposure and at 4 weeks following exposure to 1.2 LD50 sarin. EEG activity in freely moving rats was also monitored by telemetry during the first week following exposure to 1.2 LD50 and behavior in the Open Field was evaluated 3 weeks post exposure. RESULTS: Treatment with retigabine following 1 LD50 sarin exposure or in combination with TA following 1.2 LD50 exposure significantly reduced mortality rate compared to the non-treated groups. In both experiments, the retigabine treatment significantly reduced gliosis, astrocytosis and brain damage as measured by translocator protein (TSPO). Following sarin exposure the combined treatment (retigabine+ TA) significantly minimized epileptiform seizure activity. Finally, in the Open Field behavioral test the non-treated sarin group showed an increased mobility which was reversed by the combined treatment. CONCLUSIONS: The M current modulator retigabine has been shown to be an effective adjunct therapy following OP induced convulsion, minimizing epileptiform seizure activity and attenuating the ensuing brain damage.

Instantaneous monitoring of free sarin in whole blood by dry blood spot-thermal desorption-GC-FPD/MS analysis.

Dry blood spot (DBS), a micro whole-blood sampling technique, enables rapid and self-blood collection; it is stable and economical. Currently, DBS filters require various sample preparation procedures specifically tailored for the target compounds, which are followed by GC-MS or LC-MS analysis. However, the small amounts of blood make the approach analytically challenging, mostly in terms of sensitivity and quantification. Herein, we introduce a new DBS concept for GC-compatible volatile to semi-volatile compounds in which DBS is directly coupled with thermal desorption analysis, thus eliminating time consuming treatments. Furthermore, to stabilize the target compound over the sampling DBS substrate, a commercial filter based on an extremely efficient trapping adsorption phase, styrene-divinylbenzene (SDVB), is first used. The performance of the new SDVB-DBS concept was demonstrated herein for monitoring the most volatile chemical warfare agent, sarin, which might be present in blood and the detection of which is usually challenging due to its rapid metabolism. This study encompasses adequate sampling and analysis method parametrization and validation, leading to a detection sensitivity of 100 pg sarin per 30 µL whole blood in 5-day-old samples, with a linear dynamic range of two orders of magnitude, adequate precision, and acceptable accuracy. Applying the method to an in-vivo mouse intranasal exposure experiment (3LD50 GB) enabled the successful detection of 25-90 ng mL-1 free sarin in blood samples drawn 2 min after exposure. The method's performance clearly emphasizes the potential of the new concept in "freezing the clock" for reactive whole blood media in pharmacokinetics and pharmacodynamics studies, as well as in applications in which informative and reliable monitoring of unstable target compounds and biomarkers is desired.

Lung damage following whole body, but not intramuscular, exposure to median lethality dose of sarin: findings in rats and guinea pigs.

Objective: Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic, volatile warfare agent. Rats and guinea pigs exposed to sarin display cholinergic excitotoxicity which includes hyper-salivation, respiratory distress, tremors, seizures, and death. Here we focused on the characterization of the airways injury induced by direct exposure of the lungs to sarin vapor and compared it to that induced by the intramuscularly route. Materials and methods: Rats were exposed to sarin either in vapor (∼1LCT50, 34.2 ± 0.8 µg/l/min, 10 min) or by i.m. (∼1LD50, 80 µg/kg), and lung injury was evaluated by broncho-alveolar lavage (BAL). Results and discussion: BAL analysis revealed route-dependent effects in rats: vapor exposed animals showed elevation of inflammatory cytokines, protein, and neutrophil cells. These elevations were seen at 24 h and were still significantly higher compared to control values at 1 week following vapor exposure. These elevations were not detected in rats exposed to sarin i.m. Histological evaluation of the brains revealed typical changes following sarin poisoning independent of the route of administration. The airways damage following vapor exposure in rats was also compared to that induced in guinea pigs. The latter showed increased eosinophilia and histamine levels that constitutes an anaphylactic response not seen in rats. Conclusions: These data clearly point out the importance of using the appropriate route of administration in studying the deleterious effects of volatile nerve agents, as well as the selection of the appropriate animal species. Since airways form major target organs for the development of injury following inhalation toxicity, they should be included in any comprehensive evaluation of countermeasures efficacy.

Time dependent dual effect of anti-inflammatory treatments on sarin-induced brain inflammation: Suggested role of prostaglandins.

A common consequence of exposure to organophosphate nerve agents is the centrally mediated seizure activity that appears even after conventional treatment with atropine and oximes. We have previously demonstrated a major inflammatory response with subsequent brain damage which was correlated with the duration of the sarin-induced seizures (Chapman et al., 2006). In the present work seizures were induced by the nerve agent sarin (1.2 LD50) insufficiently treated 1 min later by atropine and trimedoxime bromide (TA), with additional midazolam treatment either 5 or 30 min after continuous seizure activity. The efficacy of both steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs), as well as other drugs that were reported as beneficial in neuroprotection, were evaluated for their contribution as adjunct treatment against sarin induced seizures and the ensuing inflammatory brain damage. Results show that both steroids and NSAIDs were harmful when administered during convulsions, and steroids were at best ineffective if administered at their termination. However, if administered at termination of convulsions, the NSAID ibuprofen, the selective COX 2 inhibitor nimesulide and the PLA2 inhibitor quinacrine were partially effective in reducing brain inflammatory markers. Administration of exogenous analogs of prostaglandins (PGE2) immediately following sarin-induced convulsions was found to have a beneficial effect in reducing brain inflammatory markers measured at 24 h and one week post sarin exposure. These findings support the hypothesis that elevated levels of PGE2 have a beneficial role immediately following sarin induced seizures, and that early inhibition of PGE2 production by both steroids and NSAID is contraindicative.

Dry Blood Spot sample collection for post-exposure monitoring of chemical warfare agents - In vivo determination of phosphonic acids using LC-MS/MS.

Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots (DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1-100 ng/ml for GB and GD metabolites, and 3-100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment, after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and compatible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this technique is highly suitable for mass-casualty incidents.

Toward a Definition of Complexity for Quantum Field Theory States.

We investigate notions of complexity of states in continuous many-body quantum systems. We focus on Gaussian states which include ground states of free quantum field theories and their approximations encountered in the context of the continuous version of the multiscale entanglement renormalization ansatz. Our proposal for quantifying state complexity is based on the Fubini-Study metric. It leads to counting the number of applications of each gate (infinitesimal generator) in the transformation, subject to a state-dependent metric. We minimize the defined complexity with respect to momentum-preserving quadratic generators which form su(1,1) algebras. On the manifold of Gaussian states generated by these operations, the Fubini-Study metric factorizes into hyperbolic planes with minimal complexity circuits reducing to known geodesics. Despite working with quantum field theories far outside the regime where Einstein gravity duals exist, we find striking similarities between our results and those of holographic complexity proposals.

Early changes in M2 muscarinic acetylcholine receptors (mAChRs) induced by sarin intoxication may be linked to long lasting neurological effects.

The effect of sarin on the binding parameters (KD & Bmax) of M2 muscarinic acetylcholine receptor (mAChR) was studied 24h and 1 week post exposure. Male & female Sprague-Daweley rats were poisoned with 1XLD50 sarin (80µg/kg, im) followed by treatment of trimedoxime bromide and atropine (7.5:5mg/kg, im) 1min later. Brains were removed and analyzed for M2 mAChR binding, using [3H]AFDX384, an M2 selective antagonist. A significant increase in KD of M2 mAChR was found in the cortex 24h post poisoning, displaying elevation from 4.65±1.16 to 8.45±1.06nM and 5.24±0.93 to 9.29±1.56nM in male and female rats, respectively. A rise in KD was also noted 1 week following exposure from 5.04±1.20 to 11.75±2.78 and from 5.37±1.02 to 11.66±1.73nM, presenting an added increase of 51 and 40% (compared to 24h) in males and females, respectively. Analysis of M2 receptor density (Bmax) revealed a significant reduction of 68% in males and insignificant reduction of 22% in females, 24h after sarin exposure which was followed by 37% recovery in males and 100% recovery in females, 1 week later. These results indicate that sarin induces a long-term decreased affinity in M2 mAChR (elevated KDs) and a transient effect on the number of this receptor subtype (Bmax). We hypothesize that the reduced affinity of the M2 receptors (negative auto-regulatory receptors) may cause long-term brain deficits by impairing the normal regulation release of ACh into the synaptic cleft.

Propagation of damage in the rat brain following sarin exposure: Differential progression of early processes.

Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic warfare agent. Following the overt, dose-dependent signs (e.g. tremor, hyper secretion, seizures, respiratory depression and eventually death), brain damage is often reported. The goal of the present study was to characterize the early histopathological and biochemical events leading to this damage. Rats were exposed to 1LD50 of sarin (80µg/kg, i.m.). Brains were removed at 1, 2, 6, 24 and 48h and processed for analysis. Results showed that TSPO (translocator protein) mRNA increased at 6h post exposure while TSPO receptor density increased only at 24h. In all brain regions tested, bax mRNA decreased 1h post exposure followed by an increase 24h later, with only minor increase in bcl2 mRNA. At this time point a decrease was seen in both anti-apoptotic protein Bcl2 and pro-apoptotic Bax, followed by a time and region specific increase in Bax. An immediate elevation in ERK1/2 activity with no change in JNK may indicate an endogenous "first response" mechanism used to attenuate the forthcoming apoptosis. The time dependent increase in the severity of brain damage included an early bi-phasic activation of astrocytes, a sharp decrease in intact neuronal cells, a time dependent reduction in MAP2 and up to 15% of apoptosis. Thus, neuronal death is mostly due to necrosis and severe astrocytosis. The data suggests that timing of possible treatments should be determined by early events following exposure. For example, the biphasic changes in astrocytes activity indicate a possible beneficial effects of delayed anti-inflammatory intervention.

Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity.

The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via (CH2)n, (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3-10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (kr = 3.6 × 102 vs. 0.2 × 102 M-1min-1, respectively, 37 °C). These hybrids inhibited AChE reversibly, IC50 = 16-48 µM, thereby decreasing the inhibition rates by OPs. The LD50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 µmol/kg in rats and 144, 203 and >506 µmol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD50 sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs.

Sarin-induced brain damage in rats is attenuated by delayed administration of midazolam.

Sarin poisoned rats display a hyper-cholinergic activity including hypersalivation, tremors, seizures and death. Here we studied the time and dose effects of midazolam treatment following nerve agent exposure. Rats were exposed to sarin (1.2 LD50, 108 µg/kg, im), and treated 1 min later with TMB4 and atropine (TA 7.5 and 5 mg/kg, im, respectively). Midazolam was injected either at 1 min (1 mg/kg, im), or 1 h later (1 or 5 mg/kg i.m.). Cortical seizures were monitored by electrocorticogram (ECoG). At 5 weeks, rats were assessed in a water maze task, and then their brains were extracted for biochemical analysis and histological evaluation. Results revealed a time and dose dependent effects of midazolam treatment. Rats treated with TA only displayed acute signs of sarin intoxication, 29% died within 24h and the ECoG showed seizures for several hours. Animals that received midazolam within 1 min survived with only minor clinical signs but with no biochemical, behavioral, or histological sequel. Animals that lived to receive midazolam at 1h (87%) survived and the effects of the delayed administration were dose dependent. Midazolam 5 mg/kg significantly counteracted the acute signs of intoxication and the impaired behavioral performance, attenuated some of the inflammatory response with no effect on morphological damage. Midazolam 1mg/kg showed only a slight tendency to modulate the cognitive function. In addition, the delayed administration of both midazolam doses significantly attenuated ECoG compared to TA treatment only. These results suggest that following prolonged seizure, high dose midazolam is beneficial in counteracting adverse effects of sarin poisoning.

Lipopolysaccharide induced protection against sulfur mustard cytotoxicity in RAW264.7 cells through generation of TNF-alpha.

Sulfur mustard (HD), a very potent alkylating agent and lipopolysacchride (LPS), are both well characterized inflammatory factors. We have found that concomitant exposure of murine macrophage cells (RAW264.7) to LPS and HD induced protection against HD induced cytotoxicity. Both HD and LPS induce release of inflammatory markers in RAW264.7 cells. However, there are marked differences in the repertoire of inflammatory factors released by the two toxins: While exposure to HD, induced a dose-dependant death of these cells, no significant change in survival rate was observed following LPS (1-100 ng/ml) exposure. Additionally, LPS elicited a robust nitric oxide (NO) and TNF-alpha secretion whereas HD was practically ineffective. Both toxins increased PGE(2) secretion in a concentration dependent manner. Treatment of HD-exposed RAW264.7 cells with anti-inflammatory drugs such as dexamethazone (5 muM), voltaren (diclofenac) (8 muM) or doxycycline (5 muM), decreased the release of cytokines but had no effect on cell viability. Simultaneous application of LPS (100 ng/ml) and HD (20-100 muM) resulted in an amelioration of HD cytotoxicity. Adding the NO generator S-nitrosoglutathione (GSNO) or inhibiting NO production using L-N(G)-monomethyl Arginine, had no effect on cell viability. Moreover, addition of PGE(2) (20 ng/ml) failed to induce any changes in cell viability under basal or HD-induced toxicity. In contrast, TNF-alpha (20 ng/ml) provided remarkable protection against HD-induced cell death. These findings strongly suggest that LPS exerts its protective action against HD toxicity through the generation of TNF-alpha and may provide better understanding of the mechanism of cytoprotection.