Recapitulation of human pathophysiology and identification of forensic biomarkers in a translational model of chlorine inhalation injury.

Chlorine gas (Cl2) has been repeatedly used as a chemical weapon, first in World War I and most recently in Syria. Life-threatening Cl2 exposures frequently occur in domestic and occupational environments, and in transportation accidents. Modeling the human etiology of Cl2-induced acute lung injury (ALI), forensic biomarkers, and targeted countermeasures development have been hampered by inadequate large animal models. The objective of this study was to develop a translational model of Cl2-induced ALI in swine to understand toxico-pathophysiology and evaluate whether it is suitable for screening potential medical countermeasures and to identify biomarkers useful for forensic analysis. Specific pathogen-free Yorkshire swine (30-40 kg) of either sex were exposed to Cl2 (≤240 ppm for 1 h) or filtered air under anesthesia and controlled mechanical ventilation. Exposure to Cl2 resulted in severe hypoxia and hypoxemia, increased airway resistance and peak inspiratory pressure, and decreased dynamic lung compliance. Cl2 exposure resulted in increased total leucocyte and neutrophil counts in bronchoalveolar lavage fluid, vascular leakage, and pulmonary edema compared with the air-exposed group. The model recapitulated all three key histopathological features of human ALI, such as neutrophilic alveolitis, deposition of hyaline membranes, and formation of microthrombi. Free and lipid-bound 2-chlorofatty acids and chlorotyrosine-modified proteins (3-chloro-l-tyrosine and 3,5-dichloro-l-tyrosine) were detected in plasma and lung tissue after Cl2 exposure. In this study, we developed a translational swine model that recapitulates key features of human Cl2 inhalation injury and is suitable for testing medical countermeasures, and validated chlorinated fatty acids and protein adducts as biomarkers of Cl2 inhalation.NEW & NOTEWORTHY We established a swine model of chlorine gas-induced acute lung injury that exhibits several features of human acute lung injury and is suitable for screening potential medical countermeasures. We validated chlorinated fatty acids and protein adducts in plasma and lung samples as forensic biomarkers of chlorine inhalation.

Pharmacologic Inhibition of Transient Receptor Potential Ion Channel Ankyrin 1 Counteracts 2-Chlorobenzalmalononitrile Tear Gas Agent-Induced Cutaneous Injuries.

Deployment of the tear gas agent 2-chlorobenzalmalononitrile (CS) for riot control has significantly increased in recent years. The effects of CS have been believed to be transient and benign. However, CS induces severe pain, blepharospasm, lachrymation, airway obstruction, and skin blisters. Frequent injuries and hospitalizations have been reported after exposure. We have identified the sensory neuronal ion channel, transient receptor potential ankyrin 1 (TRPA1), as a key CS target resulting in acute irritation and pain and also as a mediator of neurogenic inflammation. Here, we examined the effects of pharmacologic TRPA1 inhibition on CS-induced cutaneous injury. We modeled CS-induced cutaneous injury by applying 10 µl CS agent [200 mM in dimethyl sulfoxide (DMSO)] to each side of the right ears of 8- to 9-week-old C57BL/6 male mice, whereas left ears were applied with solvent only (DMSO). The TRPA1 inhibitor HC-030031 or A-967079 was administered after CS exposure. CS exposure induced strong tissue swelling, plasma extravasation, and a dramatic increase in inflammatory cytokine levels in the mouse ear skin. We also showed that the effects of CS were not transient but caused persistent skin injuries. These injury parameters were reduced with TRPA1 inhibitor treatment. Further, we tested the pharmacologic activity of advanced TRPA1 antagonists in vitro. Our findings showed that TRPA1 is a crucial mediator of CS-induced nociception and tissue injury and that TRPA1 inhibitors are effective countermeasures that reduce key injury parameters when administered after exposure. Additional therapeutic efficacy studies with advanced TRPA1 antagonists and decontamination strategies are warranted. SIGNIFICANCE STATEMENT: 2-Chlorobenzalmalononitrile (CS) tear gas agent has been deployed as a crowd dispersion chemical agent in recent times. Exposure to CS tear gas agents has been believed to cause transient acute toxic effects that are minimal at most. Here we found that CS tear gas exposure causes both acute and persistent skin injuries and that treatment with transient receptor potential ion channel ankyrin 1 (TRPA1) antagonists ameliorated skin injuries.

Interatrial Shunt Devices.

Elevated left atrial pressure during exercise is a hallmark of heart failure (HF) and is associated with adverse left atrial remodeling and poor outcomes. To decompress the pressure-overloaded left atrium in patients with HF, several device-based approaches have been developed to create a permanent, pressure-dependent, left-to-right interatrial shunt. Such approaches are currently in various stages of investigations in both HF with reduced ejection fraction (EF) and HF with preserved EF. This review discusses the evolution of the concept of left atrial decompression and summarizes the current landscape of device-based approaches used for left atrial decompression.

Toxic effects of chlorine gas and potential treatments: a literature review.

Chlorine gas is one of the highly produced chemicals in the USA and around the world. Chlorine gas has several uses in water purification, sanitation, and industrial applications; however, it is a toxic inhalation hazard agent. Inhalation of chlorine gas, based on the concentration and duration of the exposure, causes a spectrum of symptoms, including but not limited to lacrimation, rhinorrhea, bronchospasm, cough, dyspnea, acute lung injury, death, and survivors develop signs of pulmonary fibrosis and reactive airway disease. Despite the use of chlorine gas as a chemical warfare agent since World War I and its known potential as an industrial hazard, there is no specific antidote. The resurgence of the use of chlorine gas as a chemical warfare agent in recent years has brought speculation of its use as weapons of mass destruction. Therefore, developing antidotes for chlorine gas-induced lung injuries remains the need of the hour. While some of the pre-clinical studies have made substantial progress in the understanding of chlorine gas-induced pulmonary pathophysiology and identifying potential medical countermeasure(s), yet none of the drug candidates are approved by the U.S. Food and Drug Administration (FDA). In this review, we summarized pathophysiology of chlorine gas-induced pulmonary injuries, pre-clinical animal models, development of a pipeline of potential medical countermeasures under FDA animal rule, and future directions for the development of antidotes for chlorine gas-induced lung injuries.

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy.

Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2 Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury.

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

Development and evaluation of a mechanical ventilator-sharing system.

Background: During the COVID-19 pandemic surge in the hospitalization of critically ill patients and the global demand for mechanical ventilators, alternative strategies for device sharing were explored. We developed and assessed the performance of a system for shared ventilation that uses clinically available components to individualize tidal volumes under a variety of clinically relevant conditions. The feasibility of remote monitoring of ventilators was also assessed. Methods: By using existing resources and off-the-shelf components, a ventilator-sharing system (VSS) that ventilates 2 patients simultaneously with a single device, and a ventilator monitoring system (VMS) that remotely monitors pulmonary mechanics were developed. The feasibility and effectiveness of VSS and VMS were evaluated in benchtop testing using 2 test lungs on a single ventilator, and then performance was assessed in translational swine models of normal and impaired lung function. Results: In benchtop testing, VSS and VMS delivered the set individualized parameters with minimal % errors in test lungs under pressure- and volume-regulated ventilation modes, suggesting the highest precision and accuracy. In animal studies, the VSS and VMS successfully delivered the individualized mechanical ventilation parameters within clinically acceptable limits. Further, we found no statistically significant difference between the target and measured values. Conclusion: The VSS adequately ventilated 2 test lungs or animals with variable lung conditions. The VMS accurately displayed mechanical ventilation settings, parameters, and alarms. Both of these systems could be rapidly assembled for scaling up to ventilate several critically ill patients in a pandemic or mass casualty disaster situations by leveraging off-the-shelf and custom 3D printed components.

Protocol for non-invasive assessment of spontaneous movements of group-housed animals using remote video monitoring.

Frequent monitoring of laboratory animals is critical for ensuring animal welfare and experimental data collection. To minimize the adverse and confounding effects caused by current monitoring protocols and human presence, we developed a low-cost, non-invasive, remotely accessible, extensible infrared video monitoring system. This protocol describes the construction and operation of the system, followed by applying deep-learning neural networks to track group-housed, unmarked mice for objective behavioral quantification. This system can be adapted to a variety of home-cage environments and species.

Development of a Pleural Pressure Catheter via Continuous Fiberoptic Esophageal Pressure Measurements.

There is growing research showing the importance of measuring esophageal pressure as a surrogate for pleural pressure for patients on mechanical ventilators. The most common measurement method uses a balloon catheter, whose accuracy can vary based on patient anatomy, balloon position, balloon inflation, and the presence of other tubes in the esophagus. The authors present the development and initial testing results of a new combination catheter, utilizing fiberoptic pressure sensing to provide more accurate esophageal pressure measurements and allowing for the incorporation of a feeding tube and temperature sensor.

Transient receptor potential channels in pulmonary chemical injuries and as countermeasure targets.

The lung is highly sensitive to chemical injuries caused by exposure to threat agents in industrial or transportation accidents, occupational exposures, or deliberate use as weapons of mass destruction (WMD). There are no antidotes for the majority of the chemical threat agents and toxic inhalation hazards despite their use as WMDs for more than a century. Among several putative targets, evidence for transient receptor potential (TRP) ion channels as mediators of injury by various inhalational chemical threat agents is emerging. TRP channels are expressed in the respiratory system and are essential for homeostasis. Among TRP channels, the body of literature supporting essential roles for TRPA1, TRPV1, and TRPV4 in pulmonary chemical injuries is abundant. TRP channels mediate their function through sensory neuronal and nonneuronal pathways. TRP channels play a crucial role in complex pulmonary pathophysiologic events including, but not limited to, increased intracellular calcium levels, signal transduction, recruitment of proinflammatory cells, neurogenic inflammatory pathways, cough reflex, hampered mucus clearance, disruption of the integrity of the epithelia, pulmonary edema, and fibrosis. In this review, we summarize the role of TRP channels in chemical threat agents-induced pulmonary injuries and how these channels may serve as medical countermeasure targets for broader indications.

TRPA1 and CGRP antagonists counteract vesicant-induced skin injury and inflammation.

The skin is highly sensitive to the chemical warfare agent in mustard gas, sulfur mustard (SM) that initiates a delayed injury response characterized by erythema, inflammation and severe vesication (blistering). Although SM poses a continuing threat, used as recently as in the Syrian conflict, no mechanism-based antidotes against SM are available. Recent studies demonstrated that Transient Receptor Potential Ankyrin 1 (TRPA1), a chemosensory cation channel in sensory nerves innervating the skin, is activated by SM and 2-chloroethyl ethyl sulfide (CEES), an SM analog, in vitro, suggesting it may promote vesicant injury. Here, we investigated the effects of TRPA1 inhibitors, and an inhibitor of Calcitonin Gene Related Peptide (CGRP), a neurogenic inflammatory peptide released upon TRPA1 activation, in a CEES-induced mouse ear vesicant model (CEES-MEVM). TRPA1 inhibitors (HC-030031 and A-967079) and a CGRP inhibitor (MK-8825) reduced skin edema, pro-inflammatory cytokines (IL-1ß, CXCL1/KC), MMP-9, a protease implicated in skin damage, and improved histopathological outcomes. These findings suggest that TRPA1 and neurogenic inflammation contribute to the deleterious effects of vesicants in vivo, activated either directly by alkylation, or indirectly, by reactive intermediates or pro-inflammatory mediators. TRPA1 and CGRP inhibitors represent new leads that could be considered for validation and further development in other vesicant injury models.

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol.

BACKGROUND AND PURPOSE: Eucalyptol (1,8-cineol), the major ingredient in the essential oil of eucalyptus leaves and other medicinal plants, has long been known for its anti-inflammatory properties. Eucalyptol interacts with the TRP cation channels among other targets, but it is unclear which of these mediates its anti-inflammatory effects. EXPERIMENTAL APPROACH: Effects of eucalyptol were compared in wild-type and TRPM8 channel-deficient mice in two different models: footpad inflammation elicited by complete Freund's adjuvant (CFA) and pulmonary inflammation following administration of LPS. Oedema formation, behavioural inflammatory pain responses, leukocyte infiltration, enzyme activities and cytokine and chemokine levels were measured. KEY RESULTS: In the CFA model, eucalyptol strongly attenuated oedema and mechanical allodynia and reduced levels of inflammatory cytokines (IL-1ß, TNF-α and IL-6), effects comparable with those of ibuprofen. In the LPS model of pulmonary inflammation, eucalyptol treatment diminished leukocyte infiltration, myeloperoxidase activity and production of TNF-α, IL-1ß, IFN-γ and IL-6. Genetic deletion of TRPM8 channels abolished the anti-inflammatory effects of eucalyptol in both models. Eucalyptol was at least sixfold more potent on human, than on mouse TRPM8 channels. A metabolite of eucalyptol, 2-hydroxy-1,8-cineol, also activated human TRPM8 channels. CONCLUSION AND IMPLICATIONS: Among the pharmacological targets of eucalyptol, TRPM8 channels were essential for its anti-inflammatory effects in mice. Human TRPM8 channels are more sensitive to eucalyptol than rodent TRPM8 channels explaining the higher potency of eucalyptol in humans. Metabolites of eucalyptol could contribute to its anti-inflammatory effects. The development of more potent and selective TRPM8 agonists may yield novel anti-inflammatory agents.

Comparison of Glucosamine Absorption After Administration of Oral Liquid, Chewable, and Tablet Formulations to Dogs.

Glucosamine (GS) is commonly administered as a nutritional supplement to support joint function. Although many supplements are available, the effect of formulation on oral absorption in dogs is unknown. The purpose of this study was to determine the relative bioavailability of GS for liquid, chewable, and tablet formulations containing GS sulfate or hydrochloride and chondroitin sulfate. In a randomized cross-over design, supplements were administered daily for 8 days with a 1 wk washout period between treatments. Liquid or Tablet A was administered to four dogs, whereas Liquid or Tablet B was administered to four additional dogs. When nutraceutical exposure was normalized to the administered dose of GS free base, similar relative bioavailabilities were determined for all three formulations. However, the dose-normalized maximum plasma GS concentration was higher for the liquid supplement (5.5 ± 0.5 µg/mL) than for the two tablets (3.1 ± 0.6 and 2.1 ± 0.6 µg/mL, P < 0.001). Similarly, the time at which maximal plasma GS concentrations occurred was shorter for the liquid formulation (0.7 ± 0.5 hr) than for the two tablets (4.2 ± 0.6 and 5.0 ± 0.6 hr, P < 0.001). These data show that the formulation of joint supplements affects the oral absorption of GS in dogs.

Tear gas: an epidemiological and mechanistic reassessment.

Deployments of tear gas and pepper spray have rapidly increased worldwide. Large amounts of tear gas have been used in densely populated cities, including Cairo, Istanbul, Rio de Janeiro, Manama (Bahrain), and Hong Kong. In the United States, tear gas was used extensively during recent riots in Ferguson, Missouri. Whereas tear gas deployment systems have rapidly improved-with aerial drone systems tested and requested by law enforcement-epidemiological and mechanistic research have lagged behind and have received little attention. Case studies and recent epidemiological studies revealed that tear gas agents can cause lung, cutaneous, and ocular injuries, with individuals affected by chronic morbidities at high risk for complications. Mechanistic studies identified the ion channels TRPV1 and TRPA1 as targets of capsaicin in pepper spray, and of the tear gas agents chloroacetophenone, CS, and CR. TRPV1 and TRPA1 localize to pain-sensing peripheral sensory neurons and have been linked to acute and chronic pain, cough, asthma, lung injury, dermatitis, itch, and neurodegeneration. In animal models, transient receptor potential inhibitors show promising effects as potential countermeasures against tear gas injuries. On the basis of the available data, a reassessment of the health risks of tear gas exposures in the civilian population is advised, and development of new countermeasures is proposed.

Effect of Multi Drug Resistance Protein 4 (MRP4) Inhibition on the Pharmacokinetics and Pharmacodynamics of Ciprofloxacin in Normal and Rats with LPS-Induced Inflammation.

BACKGROUND AND OBJECTIVES: Infection and inflammation are known to cause wide variability in disposition of drugs through modulation of drug transporters. However, the effects of inhibition of multidrug resistance protein 4 (MRP4) on pharmacokinetics and pharmacodynamics are poorly understood in normal and inflamed conditions. We hypothesized that inflammation alters the pharmacokinetic parameters of ciprofloxacin; and Pharmacokinetic/Pharmacodynamic indices, such as ratio of peak plasma concentration to minimum inhibitory concentration (C max/MIC) and ratio of area under the plasma drug concentration-time curve to minimum inhibitory concentration (AUC/MIC) of ciprofloxacin will be improved with the co-administration of a MRP4 inhibitor, dipyridamole, in inflammatory conditions. METHODS: In this study, the role of MRP4 on the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin was investigated by the co-administration of dipyridamole in rats with or without lipopolysaccharide (LPS)-induced inflammation. The pharmacokinetic parameters for ciprofloxacin were calculated by non-compartmental approach. MIC of ciprofloxacin was determined using broth microdilution technique. RESULTS: Induction of inflammation in rats resulted in marked reduction in C max and AUC; and an increase in the volume of distribution (V d/F) and clearance (Cl/F) of ciprofloxacin, compared to normal rats. Co-administration of dipyridamole with ciprofloxacin in inflamed rats resulted in a threefold increase in AUC, a twofold decrease in V d/F and a threefold decrease in Cl/F of ciprofloxacin with significantly prolonged half-life compared to inflamed rats who received ciprofloxacin alone. Co-administration of dipyridamole enhanced AUC/MIC values of ciprofloxacin in both normal and inflamed rats. CONCLUSIONS: The results suggest that MRP4 inhibition increases the systemic exposure of ciprofloxacin in both normal and inflammatory conditions.

Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice.

Oxidation products of the naturally occurring phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), which are known as OxPAPC, accumulate in atherosclerotic lesions and at other sites of inflammation in conditions such as septic inflammation and acute lung injury to exert pro- or anti-inflammatory effects. It is currently unknown whether OxPAPC also contributes to inflammatory pain and peripheral neuronal excitability in these conditions. Here, we observed that OxPAPC dose-dependently and selectively activated human TRPA1 nociceptive ion channels expressed in HEK293 cells in vitro, without any effect on other TRP channels, including TRPV1, TRPV4 and TRPM8. OxPAPC agonist activity was dependent on essential cysteine and lysine residues within the N-terminus of the TRPA1 channel protein. OxPAPC activated calcium influx into a subset of mouse sensory neurons which were also sensitive to the TRPA1 agonist mustard oil. Neuronal OxPAPC responses were largely abolished in neurons isolated from TRPA1-deficient mice. Intraplantar injection of OxPAPC into the mouse hind paw induced acute pain and persistent mechanical hyperalgesia and this effect was attenuated by the TRPA1 inhibitor, HC-030031. More importantly, we found levels of OxPAPC to be significantly increased in inflamed tissue in a mouse model of chronic inflammatory pain, identified by the binding of an OxPAPC-specific antibody. These findings suggest that TRPA1 is a molecular target for OxPAPC and OxPAPC may contribute to chronic inflammatory pain through TRPA1 activation. Targeting against OxPAPC and TRPA1 signaling pathway may be promising in inflammatory pain treatment.

Simultaneous quantification of vinblastine and desacetylvinblastine concentrations in canine plasma and urine samples using LC-APCI-MS/MS.

A highly sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS/MS) method has been developed and validated for simultaneous quantification of vinblastine and its metabolite, desacetylvinblastine, in canine plasma and urine samples. Plasma and urine samples were processed by a solid phase extraction procedure. The optimal chromatographic behavior of these analytes was achieved on pentafluorophenyl (PFP) propyl analytical column (5µm, 50×2.1mm) under isocratic elution of 0.75mL/min with a mobile phase of 5mM ammonium acetate and methanol. The samples were analyzed in positive ion, multiple reaction monitoring mode. The calibration curves were linear over 0.125-2ng/mL (lower calibration curve); 2-100ng/mL (higher calibration curve) and 0.125-5ng/mL for vinblastine and desacetylvinblastine in plasma, and over 1-2000ng/mL and 0.5-100ng/mL for vinblastine and desacetylvinblastine in urine samples, respectively. The limits of quantitation of vinblastine and desacetylvinblastine were 0.125ng/mL in both matrices. The intra and interday accuracy was above 89% and precision below 8.6% for both analytes in both matrices. The developed method was successfully applied to ongoing in vivo vinblastine pharmacokinetic studies in dogs.