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Sulfur mustard (SM) is a lipid soluble alkylating agent that causes genotoxic injury. The eye is highly sensitive to SM toxicity and exposures exceeding 400 mg min/m3 can elicit irreversible corneal pathophysiologies. Development of medical countermeasures for ocular SM exposure has been hindered by a limited understanding of dose-dependent effects of SM on corneal injury. Here, clinical, histological and ultrastructural analyses were used to characterize the effects of SM dose on corneal injury progression. Corneas were evaluated for up to 20 wk following exposure to saturated SM vapor for 30−150 s, which corresponds to 300−1,500 mg min/m3. In acute studies, a ceiling effect on corneal edema developed at doses associated with full-thickness corneal lesions, implicating endothelial toxicity in corneal swelling. Recurrent edematous lesions (RELs) transiently emerged after 2 wk in a dose-dependent fashion, followed by the development of secondary corneal pathophysiologies such as neovascularization, stromal scarring and endothelial abnormalities. RELs appeared in 96 % of corneas exposed for ≥ 90 s, 52 % of corneas exposed for 60 s and 0 % of corneas exposed for 30 s. While REL latency was variable in corneas exposed for 60 s, REL emergence was synchronized at exposures ≥ 90 s. Corneas did not exhibit more than one REL, suggesting RELs are part of a programmed pathophysiological response to severe alkylating lesions. In post-mortem studies at 12 wk, corneal edema was positively correlated to severity of endothelial pathologies, consistent with previous findings that endothelial toxicity influences long-term outcomes. These results provide novel insight into long-term corneal pathophysiological responses to acute toxicity and identify exposure conditions suitable for therapeutic testing.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25–16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Purpose Ocular exposure to sulfur mustard (SM) vapor causes acute loss of corneal endothelial cells (CECs). Persistent corneal endothelial pathologies are observed in eyes that do not recover from SM exposure, suggesting that endothelial toxicity contributes to mustard gas keratopathy (MGK). Here, we evaluated the contributions of endothelial loss to acute and chronic corneal injuries in SM-exposed eyes. Methods Rabbit eyes were exposed in vivo to equivalent doses of SM using 9-, 11-, or 14-mm vapor caps. The effects of exposure area on corneal injury progression were longitudinally evaluated over 12 weeks using clinical evaluations. The effects of exposure area on CEC morphology, endothelial and epithelial ultrastructure, and endothelial barrier function were determined from 1 day to 12 weeks. Results SM exposure caused loss of CECs and failure of endothelial barrier integrity at 1 day, independent of exposure cap size. By 3 weeks, eyes exposed with the 14-mm vapor cap exhibited increased corneal permeability, repopulation of the endothelium by cells with fibroblastic morphology, and abnormal deposition of extracellular matrix. Eyes exposed with 9- or 11-mm vapor caps exhibited transient symptoms of injury that fully resolved, with the rate of recovery correlated with cap size. Conclusions The nonlinear correlation between endothelial lesion size and probability of developing MGK suggests that the CEC loss is a determinative factor for emergence of MGK. These studies illustrate the importance of endothelial repair in preventing MGK. Furthermore, they exclude chemical modification of basement membrane as a mechanistic cause of recurrent epithelial erosions in MGK eyes.

Single domain antibodies (sdAbs) have substantial biophysical advantages over conventional antibodies and are increasingly being employed as components of immunotherapeutic agents. One particularly favorable property is the ability to link different sdAbs into heteromultimers. This feature allows production of single molecules capable of simultaneously targeting more than one antigen. In particular, cooperative binding of multiple linked sdAbs to non-overlapping epitopes on the same target can produce synergistic improvements in target affinity, variant specificity, and in vivo potencies. Here we seek to test the option of increased component sdAbs in these heteromultimers by testing different sdAb heterohexamers in which each of the six camelid sdAb components (VHHs) can neutralize one of three different Botulinum neurotoxin (BoNT) serotypes, A, B or E. Each heterohexamer bound all three targeted BoNT serotypes and protected mice from at least 100 MIPLD50 of each serotype. To test the potential of mRNA therapeutics encoding long sdAb heteromultimers, one heterohexamer was encoded as replicating RNA (repRNA), formulated with a cationic nanocarrier, and delivered to mice via intramuscular injection. Heterohexamer antitoxin serum expression levels were easily detected by 8 hours post-treatment, peaked at 5-10 nM around two days, and persisted for more than three days. Mice treated with the formulated repRNA one day post-treatment survived challenge with 100 MIPLD50 of each toxin serotype, demonstrating the function of all six component VHHs. Use of long sdAb multimers, administered as proteins or repRNA, offer the potential for substantially improved versatility in the development of antibody-based therapeutics.

The acute toxicity of organophosphorus-based compounds is primarily a result of acetylcholinesterase inhibition in the central and peripheral nervous systems. The resulting cholinergic crisis manifests as seizure, paralysis, respiratory failure and neurotoxicity. Though overstimulation of muscarinic receptors is the mechanistic basis of central organophosphorus (OP) toxicities, short-term changes in synapse physiology that precede OP-induced seizures have not been investigated in detail. To study acute effects of OP exposure on synaptic function, field excitatory postsynaptic potentials (fEPSPs) were recorded from Schaffer collateral synapses in the mouse hippocampus CA1 stratum radiatum during perfusion with various OP compounds. Administration of the OPs paraoxon, soman or VX rapidly and stably depressed fEPSPs via a presynaptic mechanism, while the non-OP proconvulsant tetramethylenedisulfotetramine had no effect on fEPSP amplitudes. OP-induced presynaptic long-term depression manifested prior to interictal spiking, occurred independent of recurrent firing, and did not require NMDA receptor currents, suggesting that it was not mediated by activity-dependent calcium uptake. Pharmacological dissection revealed that the presynaptic endocannabinoid type 1 receptor (CB1R) as well as postsynaptic M1 and M3 muscarinic acetylcholine receptors were necessary for OP-LTD. Administration of CB1R antagonists significantly reduced survival in mice after a soman challenge, revealing an acute protective role for endogenous CB1R signaling during OP exposure. Collectively these data demonstrate that the endocannabinoid system alters glutamatergic synaptic function during the acute response to OP acetylcholinesterase inhibitors.

Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.

Author(s): Cao, Zhijian; Wang, Jing-Lin; McNutt, Patrick Michael; Utkin, Yuri N; Shahbazzadeh, Delavar; Wulff, Heike; Kovacic, Herve; Sabatier, Jean-Marc

Botulinum neurotoxins (BoNTs) are highly potent, neuroparalytic protein toxins that block the release of acetylcholine from motor neurons and autonomic synapses. The unparalleled toxicity of BoNTs results from the highly specific and localized cleavage of presynaptic proteins required for nerve transmission. Currently, the only pharmacotherapy for botulism is prophylaxis with antitoxin, which becomes progressively less effective as symptoms develop. Treatment for symptomatic botulism is limited to supportive care and artificial ventilation until respiratory function spontaneously recovers, which can take weeks or longer. Mechanistic insights into intracellular toxin behavior have progressed significantly since it was shown that toxins exploit synaptic endocytosis for entry into the nerve terminal, but fundamental questions about host-toxin interactions remain unanswered. Chief among these are mechanisms by which BoNT is internalized into neurons and trafficked to sites of molecular toxicity. Elucidating how receptor-bound toxin is internalized and conditions under which the toxin light chain engages with target SNARE proteins is critical for understanding the dynamics of intoxication and identifying novel therapeutics. Here, we discuss the implications of newly discovered modes of synaptic vesicle recycling on BoNT uptake and intraneuronal trafficking.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Purpose To develop and characterize a mouse model of spontaneous recurrent seizures following nerve agent-induced status epilepticus (SE) and test the efficacy of existing antiepileptic drugs. Methods SE was induced in telemeterized male C57Bl6/J mice by soman exposure, and electroencephalographic activity was recorded for 4–6 weeks. Mice were treated with antiepileptic drugs (levetiracetam, valproic acid, phenobarbital) or corresponding vehicles for 14 d after exposure, followed by 14 d of drug washout. Survival, body weight, seizure characteristics, and histopathology were used to characterize the acute and chronic effects of nerve agent exposure and to evaluate the efficacy of treatments in mitigating or preventing neurological effects. Results Spontaneous recurrent seizures manifested in all survivors, but the number and frequency of seizures varied considerably among mice. In untreated mice, seizures became longer over time. Moderate to severe histopathology was observed in the amygdala, piriform cortex, and CA1. Levetiracetam provided modest improvements in neurological parameters such as reduced spike rate and improved histopathology scores, whereas valproic acid and phenobarbital were largely ineffective. Conclusions This model of post-SE spontaneous recurrent seizures differs from other experimental models in the brief latency to seizure development, the occurrence of seizures in 100 % of exposed animals, and the lack of damage to CA4/dentate gyrus. It may serve as a useful tool for rapidly and efficiently screening novel therapies that would be effective against severe epilepsy cases.

SUMMARY Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties., Graphical Abstract, In Brief Botulinum neurotoxins (BoNTs) are extremely toxic biothreats. Lam et al. report the crystal structures and neutralizing mechanisms of six unique antitoxin VHHs against BoNT/A1 and BoNT/B1, the two major human pathogenic BoNTs. They then develop a platform for structure-based rational design of bifunctional VHH heterodimers with superior antitoxin potencies.

Sulfur mustard is one of the most toxic chemical warfare agents worldwide. We report the use of 4,4-difluoro-4-bora-3a,4a-diaza- s-indacene (BODIPY) photosensitizers as a fast and effective sulfur mustard decontaminant and their incorporation into various polymer coatings and fabrics, including army combat uniform. These BODIPY-embedded materials are capable of generating singlet oxygen under visible light irradiation and effectively detoxifying sulfur mustard by converting it into nontoxic sulfoxides as the major products. The rate of decontamination is found to be affected by the photosensitizer structure and concentration as well as the excitation wavelength. The most effective BODIPY-embedded self-decontamination material observed in this study shows a half-life of only 0.8 min. In comparison to the current methods, which use activated carbon as the adsorbent layer, these self-detoxifying coatings and fabrics provide constant destruction of and real-time protection against sulfur mustard.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that prevent neurotransmitter release from peripheral nerve terminals by cleaving presynaptic proteins required for synaptic vesicle fusion. The ensuing neuromuscular paralysis causes death by asphyxiation. Although no antidotal treatments exist to block toxin activity within the nerve terminal, aminopyridine antagonists of voltage-gated potassium channels have been proposed as symptomatic treatments for botulism toxemia. However, clinical evaluation of aminopyridines as symptomatic treatments for botulism has been inconclusive, in part because mechanisms responsible for reversal of paralysis in BoNT-poisoned nerve terminals are not understood. Here we measured the effects of 3,4-diaminopyridine (DAP) on phrenic nerve-elicited diaphragm contraction and end-plate potentials at various times after intoxication with BoNT serotypes A, B, or E. We found that DAP-mediated increases in quantal content promote neurotransmission from intoxicated nerve terminals through two functionally distinguishable mechanisms. First, DAP increases the probability of neurotransmission at non-intoxicated release sites. This mechanism is serotype-independent, becomes less effective as nerve terminals become progressively impaired, and remains susceptible to ongoing intoxication. Second, DAP elicits persistent production of toxin-resistant endplate potentials from nerve terminals fully intoxicated by BoNT/A, but not serotypes B or E. Since this effect appears specific to BoNT/A intoxication, we propose that DAP treatment enables BoNT/A-cleaved SNAP-25 to productively engage in fusogenic release by increasing the opportunity for low-efficiency fusion events. These findings have important implications for DAP as a botulism therapeutic by defining conditions under which DAP may be clinically effective in reversing botulism symptoms.

Botulinum neurotoxins (BoNTs) are potent neuroparalytic toxins that cause mortality through respiratory paralysis. The approved medical countermeasure for BoNT poisoning is infusion of antitoxin immunoglobulins. However, antitoxins have poor therapeutic efficacy in symptomatic patients; thus, there is an urgent need for treatments that reduce the need for artificial ventilation. We report that the US Food and Drug Administration–approved potassium channel blocker 3,4-diaminopyridine (3,4-DAP) reverses respiratory depression and neuromuscular weakness in murine models of acute and chronic botulism. In ex vivo studies, 3,4-DAP restored end-plate potentials and twitch contractions of diaphragms isolated from mice at terminal stages of BoNT serotype A (BoNT/A) botulism. In vivo, human-equivalent doses of 3,4-DAP reversed signs of severe respiratory depression and restored mobility in BoNT/A-intoxicated mice at terminal stages of respiratory collapse. Multiple-dosing administration of 3,4-DAP improved respiration and extended survival at up to 5 LD(50) BoNT/A. Finally, 3,4-DAP reduced gastrocnemius muscle paralysis and reversed respiratory depression in sublethal models of serotype A–, B–, and E–induced botulism. These findings make a compelling argument for repurposing 3,4-DAP to symptomatically treat symptoms of muscle paralysis caused by botulism, independent of serotype. Furthermore, they suggest that 3,4-DAP is effective for a range of botulism symptoms at clinically relevant time points.

Cornea, a highly specialized transparent tissue, is the major refractive element of the eye. The cornea is highly susceptible to chemotoxic injury through topical exposure to vapors, microparticles, and aqueous drops, as well as through systemically absorbed chemicals that access the cornea via tear film, aqueous humor, and limbal vasculature. Corneal injury activates a carefully orchestrated series of repair processes capable of resolving minor lesions over time, but it often fails to overcome the menace of moderate, severe, and chronic injuries and secondary pathophysiologies that permanently impair vision. The most serious complications of chemical injuries-persistent corneal edema, neovascularization, scarring/haze, limbal stem cell deficiency, and corneal melting-often manifest over months to years, suggesting that a better understanding of endogenous regenerative mechanisms of corneal repair can lead to the development of improved treatments that may attenuate or prevent corneal defects and protect vision.

Botulism outbreak due to consumption of food contaminated with botulinum neurotoxins (BoNTs) is a public health emergency. The threat of bioterrorism through deliberate distribution in food sources and/or aerosolization of BoNTs raises global public health and security concerns due to the potential for high mortality and morbidity. Rapid and reliable detection methods are necessary to support clinical diagnosis and surveillance for identifying the source of contamination, performing epidemiological analysis of the outbreak, preventing and responding to botulism outbreaks. This review considers the applicability of various BoNT detection methods and examines their fitness-for-purpose in safeguarding the public health and security goals.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca 2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca 2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca 2+ -modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

Corneal injuries resulting from ocular exposure to sulfur mustard (SM) vapor are the most prevalent chemical warfare injury. Ocular exposures exhibit three distinct, dose-dependent clinical trajectories: complete injury resolution, immediate transition to a chronic injury, or apparent recovery followed by the subsequent development of persistent ocular manifestations. These latter two trajectories include a constellation of corneal symptoms that are collectively known as mustard gas keratopathy (MGK). The etiology of MGK is not understood. Here, we synthesize recent findings from in vivo rabbit SM vapor studies, suggesting that tissue-specific damage during the acute injury can decrement the regenerative capacities of corneal endothelium and limbal stem cells, thereby predisposing the cornea to the chronic or delayed forms of MGK. This hypothesis not only provides a mechanism to explain the acute and MGK injuries but also identifies novel therapeutic modalities to mitigate or eliminate the acute and long-term consequences of ocular exposure to SM vapor.

PURPOSE: Sulfur mustard (SM) exposure results in dose-dependent morbidities caused by cytotoxicity and vesication. Although lesions resulting from ocular exposure often resolve clinically, an idiopathic delayed mustard gas keratopathy (MGK) can develop after a moderate or severe exposure. Sequelae include persistent keratitis, recurring epithelial lesions, corneal neovascularization, and corneal degeneration, which can lead to impaired vision or loss of sight. The purpose of this effort is to correlate structural changes with injury progression during the development of MGK. METHODS: New Zealand White rabbit corneas were exposed to SM using a vapor cup delivery system. The transition from acute to delayed injury was characterized by clinical, histological, and ultrastructural metrics over 8 weeks. RESULTS: Exposure dose was correlated to the likelihood of developing MGK but not to its severity. In a 56-animal cohort, a 2.5-minute exposure generated a corneal lesion, with 89% of corneas developing MGK within 5 weeks. A significant decrease in corneal edema at 2 weeks was predictive of the 11% of corneas that underwent asymptomatic recovery. Ultrastructural comparison of asymptomatic and MGK corneas at 8 weeks indicates that MGK is characterized by persistent edema and profound disorganization of the basement membrane zone. CONCLUSIONS: Ultrastructural changes associated with the delayed pathophysiology of corneal SM vapor exposure involve severe degeneration of the basement membrane zone and persistent edema. The mechanisms underlying MGK pathogenesis seem to alter injury progression as soon as 2 weeks after exposure. These data suggest that the vapor cup model system is suitable for therapeutic evaluation. Language: en

There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use.

Exposure of tissues to sulfur mustard (SM) results in the formation of protein and nucleotide adducts that disrupt cellular metabolism and cause cell death. Subsequent pathologies involve a significant proinflammatory response, disrupted healing, and long-term defects in tissue architecture. Following ocular exposure, acute corneal sequelae include epithelial erosions, necrosis, and corneal inflammation. Longer term, a progressive injury becomes distributed throughout the anterior chamber, which ultimately causes a profound remodeling of corneal tissues. In many cases, debilitating and vision-threatening injuries reoccur months to years after the initial exposure. Preliminary data in humans suffering from chronic epithelial lesions suggest that thymosin β4 (Tβ4) may be a viable candidate to mitigate acute or long-term ocular SM injury. To evaluate therapeutic candidates, we have developed a rabbit ocular exposure model system. In this paper, we report molecular, histological, ultrastructural, and clinical consequences of rabbit ocular SM injury, which can be used to assess Tβ4 efficacy, including timepoints at which Tβ4 will be assessed for therapeutic utility.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1(+) pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT.

The use of fold-change (FC) to prioritize differentially expressed genes (DEGs) for post-hoc characterization is a common technique in the analysis of RNA sequencing datasets. However, the use of FC can overlook certain population of DEGs, such as high copy number transcripts which undergo metabolically expensive changes in expression yet fail to exceed the ratiometric FC cut-off, thereby missing potential important biological information. Here we evaluate an alternative approach to prioritizing RNAseq data based on absolute changes in normalized transcript counts (ΔT) between control and treatment conditions. In five pairwise comparisons with a wide range of effect sizes, rank-ordering of DEGs based on the magnitude of ΔT produced a power curve-like distribution, in which 4.7-5.0% of transcripts were responsible for 36-50% of the cumulative change. Thus, differential gene expression is characterized by the high production-cost expression of a small number of genes (large ΔT genes), while the differential expression of the majority of genes involves a much smaller metabolic investment by the cell. To determine whether the large ΔT datasets are representative of coordinated changes in the transcriptional program, we evaluated large ΔT genes for enrichment of gene ontologies (GOs) and predicted protein interactions. In comparison to randomly selected DEGs, the large ΔT transcripts were significantly enriched for both GOs and predicted protein interactions. Furthermore, enrichments were were consistent with the biological context of each comparison yet distinct from those produced using equal-sized populations of large FC genes, indicating that the large ΔT genes represent an orthagonal transcriptional response. Finally, the composition of the large ΔT gene sets were unique to each pairwise comparison, indicating that they represent coherent and context-specific responses to biological conditions rather than the non-specific upregulation of a family of genes. These findings suggest that the large ΔT genes are not a product of random or stochastic phenomenon, but rather represent biologically meaningful changes in the transcriptional program. They furthermore imply that high abundance transcripts are associated with particularly cellular states, and as cells change in response to internal or external conditions, the relative distribution of the abundant transcripts changes accordingly. Thus, prioritization of DEGs based on the concept of metabolic cost is a simple yet powerful method to identify biologically important transcriptional changes and provide novel insights into cellular behaviors.

Objective The purpose of this study was to characterize effect of progesterone (P4) on interleukin-6 (IL-6) production by fetoplacental artery explants, fetal granulocytes, and fetal and maternal mononuclear cells. Study design Arteries and cord blood were obtained from 5 term pregnancies undergoing repeat cesarean section. Maternal blood was obtained from another 6 women at 16 to 20 weeks' gestation. Tissues were fractionated by dissection or Histopaque gradient. Specimens were incubated in physiologic media then exposed to lipopolysaccharide (LPS) or P4 alone, or pretreated with P4 and then exposed to LPS. Samples were evaluated for IL-6 by enzyme-linked immunosorbent assay (ELISA). Results Arteries and fetal and maternal mononuclear cells exposed to LPS increased IL-6 secretion by 9-, 27-, and 29-fold, respectively. P4 pretreatment blocked LPS induction of IL-6. Fetal granulocytes did not increase IL-6 production in response to LPS exposure. Conclusion LPS induces IL-6 in arteries and fetal and maternal mononuclear cells. P4 pretreatment significantly blocks this effect in these cell populations, suggesting possible targets for anti-inflammatory actions of P4 in prevention of preterm birth.

The eye and the accessory visual structures (e.g., the conjunctiva, lacrimal apparatus, and eyelids) are arguably the most important sensory organ of the body. It is estimated that 75% of perception and cognition incorporates visual information. The ocular structure is highly complex, containing a variety of tissues with distinct functions that are critically dependent on the structural and cellular environment for proper function. Consequently, any injury that interferes with vision can be extremely disruptive, incapacitatiing or even life-threatening. Due in large part to its exposed location and intricate internal structure of the eye, it is highly sensitive to injury by a wide array of chemical warfare agents (CWAs). Ocular manifestations caused by exposure to CWAs can range from mild conjunctivitis to corneal ulceration and loss of vision. The severity of response is highly dependent on a combination of dose, agent, and environmental conditions and is mediated through the specific interactions of each agent with the physiological and biochemical environment of the eye. In some cases, the recovery from intoxication may be significantly influenced by the regenerative capacity of individual ocular tissues. This chapter discusses what is known about the toxicological and toxicokinetic effects of ocular exposure to a wide array of traditional CWAs, including several biological toxins.

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.

Cadherins — a family of cell–cell adhesion molecules — are linked to the actin cytoskeleton via intervening proteins. Recent results address molecular explanations for observed cadherin behavior, point to signals that regulate adhesion by modulating elements of the cadherin-associated complex, challenge the belief that different cadherins generally cannot cross-adhere, and highlight instructive roles for cadherins in cell signaling and differentiation.