Mesna Improves Outcomes of Sulfur Mustard Inhalation Toxicity in an Acute Rat Model.

Inhalation of high levels of sulfur mustard (SM), a potent vesicating and alkylating agent used in chemical warfare, results in acutely lethal pulmonary damage. Sodium 2-mercaptoethane sulfonate (mesna) is an organosulfur compound that is currently Food and Drug Administration (FDA)-approved for decreasing the toxicity of mustard-derived chemotherapeutic alkylating agents like ifosfamide and cyclophosphamide. The nucleophilic thiol of mesna is a suitable reactant for the neutralization of the electrophilic group of toxic mustard intermediates. In a rat model of SM inhalation, treatment with mesna (three doses: 300 mg/kg intraperitoneally 20 minutes, 4 hours, and 8 hours postexposure) afforded 74% survival at 48 hours, compared with 0% survival at less than 17 hours in the untreated and vehicle-treated control groups. Protection from cardiopulmonary failure by mesna was demonstrated by improved peripheral oxygen saturation and increased heart rate through 48 hours. Additionally, mesna normalized arterial pH and pACO2 Airway fibrin cast formation was decreased by more than 66% in the mesna-treated group at 9 hour after exposure compared with the vehicle group. Finally, analysis of mixtures of a mustard agent and mesna by a 5,5'-dithiobis(2-nitrobenzoic acid) assay and high performance liquid chromatography tandem mass spectrometry demonstrate a direct reaction between the compounds. This study provides evidence that mesna is an efficacious, inexpensive, FDA-approved candidate antidote for SM exposure. SIGNIFICANCE STATEMENT: Despite the use of sulfur mustard (SM) as a chemical weapon for over 100 years, an ideal drug candidate for treatment after real-world exposure situations has not yet been identified. Utilizing a uniformly lethal animal model, the results of the present study demonstrate that sodium 2-mercaptoethane sulfonate is a promising candidate for repurposing as an antidote, decreasing airway obstruction and improving pulmonary gas exchange, tissue oxygen delivery, and survival following high level SM inhalation exposure, and warrants further consideration.

Identification and determination of phenyl methyl carbamate released from adducted hemoglobin for methyl isocyanate exposure verification.

Methyl isocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, is a toxic industrial chemical that causes irritation and damage to the eyes, respiratory tract, and skin. Due to the high reactivity of MIC, it binds to proteins to form protein adducts. While these adducts can be used as biomarkers to verify exposure to MIC, methods to detect MIC adducts are cumbersome, typically involving enzymatic (pronase) or strong acid (Edman degradation) hydrolysis of hemoglobin. Hence, in this study, a simple method was developed which utilizes base hydrolysis of MIC-tyrosine adducts from isolated hemoglobin to form phenyl methyl carbamate (PMC), followed by rapid liquid-liquid extraction, and liquid chromatography tandem mass spectrometry analysis. The hydrolysis chemistry is the first report of base hydrolysis of a tyrosine-ß-C-hydroxo phenol bond in aqueous solution. The method produced excellent sensitivity (detection limit of 0.02 mg/kg), linearity (R2 = 0.998, percent residual accuracies > 96), and dynamic range (0.06‒15 mg/kg). The accuracy and precision (100 ± 9% and < 10% relative standard deviation, respectively) of the method were outstanding compared to existing techniques. The validated method was able to detect significantly elevated levels of PMC from hemoglobin isolated from MIC-exposed rats.

Acute vaping in a golden Syrian hamster causes inflammatory response transcriptomic changes.

E-cigarette vaping is a major aspect of nicotine consumption, especially for children and young adults. Although it is branded as a safer alternative to cigarette smoking, murine and rat models of subacute and chronic e-cigarette vaping exposure have shown many proinflammatory changes in the respiratory tract. An acute vaping exposure paradigm has not been demonstrated in the golden Syrian hamster, and the hamster is a readily available small animal model that has the unique benefit of becoming infected with and transmitting respiratory viruses, including SARS-CoV-2, without genetic alteration of the animal or virus. Using a 2-day, whole body vaping exposure protocol in male golden Syrian hamsters, we evaluated serum cotinine, bronchoalveolar lavage cells, lung, and nasal histopathology, and gene expression in the nasopharynx and lung through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Depending on the presence of nonnormality or outliers, statistical analysis was performed by ANOVA or Kruskal-Wallis tests. For tests that were statistically significant (P < 0.05), post hoc Tukey-Kramer and Dunn's tests, respectively, were performed to make pairwise comparisons between groups. In nasal tissue, RT-qPCR analysis revealed nicotine-dependent increases in gene expression associated with type 1 inflammation (CCL-5 and CXCL-10), fibrosis [transforming growth factor-ß (TGF-ß)], nicotine-independent increase oxidative stress response (SOD-2), and a nicotine-independent decrease in vasculogenesis/angiogenesis (VEGF-A). In the lung, nicotine-dependent increases in the expression of genes involved in the renin-angiotensin pathway [angiotensin-converting enzyme (ACE), ACE2], coagulation (tissue factor, Serpine-1), extracellular matrix remodeling (MMP-2, MMP-9), type 1 inflammation (IL-1ß, TNF-α, and CXCL-10), fibrosis (TGF-ß and Serpine-1), oxidative stress response (SOD-2), neutrophil extracellular traps release (ELANE), and vasculogenesis and angiogenesis (VEGF-A) were identified. To our knowledge, this is the first demonstration that the Syrian hamster is a viable model of e-cigarette vaping. In addition, this is the first report that e-cigarette vaping with nicotine can increase tissue factor gene expression in the lung. Our results show that even an acute exposure to e-cigarette vaping causes significant upregulation of mRNAs in the respiratory tract from pathways involving the renin-angiotensin system, coagulation, extracellular matrix remodeling, type 1 inflammation, fibrosis, oxidative stress response, neutrophil extracellular trap release (NETosis), vasculogenesis, and angiogenesis.

Analysis of sodium 2-mercaptoethane sulfonate in rat plasma using high performance liquid chromatography tandem-mass spectrometry.

Sodium 2-mercaptoethane sulfonate (MESNA) is a thiol-containing compound that has proven to be effective in inactivating acrolein, the toxic metabolite of some anti-cancer drugs (e.g., cyclophosphamide and ifosphamide). Also, it scavenges free radicals which cause numerous disorders by attacking biological molecules. Current methods available to analyze MESNA in biological matrices include colorimetry and high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection. These methods have several limitations including low sensitivity, poor selectivity, a high degree of difficulty, and long analysis times. Hence, a rapid, simple, and sensitive HPLC tandem mass spectrometry (MS/MS) method was developed and validated to quantify MESNA in rat plasma following IP administration. The analysis of MESNA was accomplished via plasma protein precipitation, centrifugation, supernatant evaporation, reconstitution, and HPLC-MS/MS analysis. The method showcases an outstanding limit of detection (20 nM), excellent linearity (R2 = 0.999, and percent residual accuracy >90%) and a wide linear range (0.05-200 µM). The method also produced good accuracy and precision (100 ± 10% and <10% relative standard deviation, respectively). The validated method was successfully used to analyze MESNA from treated animals and will allow easier development of MESNA for therapeutic purposes.

Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure.

Quantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.

Alleviation of methyl isocyanate-induced airway obstruction and mortality by tissue plasminogen activator.

Methyl isocyanate (MIC, "Bhopal agent") is a highly reactive, toxic industrial chemical. Inhalation of high levels (500-1000 ppm) of MIC vapor is almost uniformly fatal. No therapeutic interventions other than supportive care have been described that can delay the onset of illness or death due to MIC. Recently, we found that inhalation of MIC caused the appearance of activated tissue factor in circulation with subsequent activation of the coagulation cascade. Herein, we report that MIC exposure (500 ppm for 30 min, nose-only) caused deposition of fibrin-rich casts in the conducting airways resulting in respiratory failure and death within 24 h in a rat model (LC90-100 ). We thus investigated the effect of airway delivery of the fibrinolytic agent tissue plasminogen activator (tPA) on mortality and morbidity in this model. Intratracheal administration of tPA was initiated 11 h post MIC exposure and repeated every 4 h for the duration of the study. Treatment with tPA afforded nearly 60% survival at 24 h post MIC exposure and was associated with decreased airway fibrin casts, stabilization of hypoxemia and respiratory distress, and improved acidosis. This work supports the potential of airway-delivered tPA therapy as a useful countermeasure in stabilizing victims of high-level MIC exposure.

Bacterial Stimulation of Toll-Like Receptor 4 Drives Macrophages To Hemophagocytose.

During acute infection with bacteria, viruses or parasites, a fraction of macrophages engulf large numbers of red and white blood cells, a process called hemophagocytosis. Hemophagocytes persist into the chronic stage of infection and have an anti-inflammatory phenotype. Salmonella enterica serovar Typhimurium infection of immunocompetent mice results in acute followed by chronic infection, with the accumulation of hemophagocytes. The mechanism(s) that triggers a macrophage to become hemophagocytic is unknown, but it has been reported that the proinflammatory cytokine gamma interferon (IFN-γ) is responsible. We show that primary macrophages become hemophagocytic in the absence or presence of IFN-γ upon infection with Gram-negative bacterial pathogens or prolonged exposure to heat-killed Salmonella enterica, the Gram-positive bacterium Bacillus subtilis, or Mycobacterium marinum. Moreover, conserved microbe-associated molecular patterns are sufficient to stimulate macrophages to hemophagocytose. Purified bacterial lipopolysaccharide (LPS) induced hemophagocytosis in resting and IFN-γ-pretreated macrophages, whereas lipoteichoic acid and synthetic unmethylated deoxycytidine-deoxyguanosine dinucleotides, which mimic bacterial DNA, induced hemophagocytosis only in IFN-γ-pretreated macrophages. Chemical inhibition or genetic deletion of Toll-like receptor 4, a pattern recognition receptor responsive to LPS, prevented both Salmonella- and LPS-stimulated hemophagocytosis. Inhibition of NF-κB also prevented hemophagocytosis. These results indicate that recognition of microbial products by Toll-like receptors stimulates hemophagocytosis, a novel outcome of prolonged Toll-like receptor signaling, suggesting hemophagocytosis is a highly conserved innate immune response.

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women.

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.

Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by Salmonella enterica Serovar Typhimurium infection in mice.

The Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium causes persistent systemic inflammatory disease in immunocompetent mice. Following oral inoculation with S. Typhimurium, mice develop a hematopathological syndrome akin to typhoid fever with splenomegaly, microcytic anemia, extramedullary erythropoiesis, and increased hemophagocytic macrophages in the bone marrow, liver, and spleen. Additionally, there is marked loss of iron from the spleen, an unanticipated result, given the iron sequestration reported in anemia of inflammatory disease. To establish why tissue iron does not accumulate, we evaluated multiple measures of pathology for 4 weeks following oral infection in mice. We demonstrate a quantitative decrease in splenic iron following infection despite increased numbers of splenic phagocytes. Infected mice have increased duodenal expression of the iron exporter ferroportin-1, consistent with increased uptake of dietary iron. Liver and splenic macrophages also express high levels of ferroportin-1. These observations indicate that splenic and hepatic macrophages export iron during S. Typhimurium infection, in contrast to macrophage iron sequestration observed in anemia of inflammatory disease. Tissue macrophage export of iron occurs concurrent with high serum concentrations of interferon gamma (IFN-γ) and interleukin 12 (IL-12). In individual mice, high concentrations of both proinflammatory (tumor necrosis factor alpha [TNF-α]) and anti-inflammatory (IL-10) cytokines in serum correlate with increased tissue bacterial loads throughout 4 weeks of infection. These in vivo observations are consistent with previous cell culture studies and suggest that the relocation of iron from tissue macrophages during infection may contribute to anemia and also to host survival of acute S. Typhimurium infection.

MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro.

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype.

Distinct classes of c-Kit-activating mutations differ in their ability to promote RUNX1-ETO-associated acute myeloid leukemia.

The t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO(+) patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-Kit(D814V) or c-Kit(T417IΔ418-419) in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-Kit(D814V) coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-Kit(T417IΔ418-419) coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-Kit(D814V) promotes a more varied and aggressive leukemic phenotype than c-Kit(T417IΔ418-419), which may be the result of differing potencies of the activating c-Kit alleles.