Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

A novel sulfur mustard (HD) vapor inhalation exposure model of pulmonary toxicity for the efficacy evaluation of candidate medical countermeasures.

OBJECTIVE: To develop a novel inhalation exposure system capable of delivering a controlled inhaled HD dose through an endotracheal tube to anesthetized rats to investigate the lung pathophysiology and evaluate potential medical countermeasures. MATERIALS AND METHODS: Target HD vapor exposures were generated by a temperature-controlled vapor generator, while concentration was monitored near real-time by gas chromatography. Animal breathing parameters were monitored real-time by in-line EMKA/SciReq pulmonary analysis system. Individual exposures were halted when the target inhaled doses were achieved. Animals were observed daily for clinical observations and lethality with scheduled termination at 28 days post-exposure. Upon scheduled or unscheduled death, animals underwent a gross necropsy and lung and trachea were collected for histopathology. RESULTS: Controlled HD concentrations ranged from 60 to 320 mg/m3. Delivered inhaled doses range from 0.3 to 3.20 mg/kg with administered doses within 3% of the target. The 28-day inhaled LD50 is 0.80 mg/kg (95% CI = 0.42-1.18 mg/kg). Post exposure respiratory abnormalities were observed across all dose levels though the higher dose levels had earlier onset and higher frequency of occurrence. Histopathologic alterations were not qualitatively altered in accordance with dose but instead showed a relationship to an animals' time of death, with early deaths demonstrating acute damage and later deaths displaying signs of repair. DISCUSSION/CONCLUSION: This novel exposure system administers targeted HD inhaled doses to generate a small animal model that can be used to evaluate physiological toxicities of inhaled HD on the lungs and for evaluation of potential medical countermeasure treatments.

Development of a guinea pig inhalational anthrax model for evaluation of post-exposure prophylaxis efficacy of anthrax vaccines.

A next-generation anthrax vaccine candidate, AV7909, is being developed for post-exposure prophylaxis (PEP) of inhalational anthrax in combination with the recommended course of antimicrobial therapy. Clinical efficacy studies of anthrax countermeasures in humans are not ethical or feasible, therefore, licensure of AV7909 for PEP is being pursued under the US Food and Drug Administration (FDA) Animal Rule, which requires that evidence of effectiveness be demonstrated in an animal model of anthrax, where results of studies in such a model can establish reasonable likelihood of AV7909 to produce clinical benefit in humans. Initial development of a PEP model for inhalational anthrax included evaluation of post-exposure ciprofloxacin pharmacokinetics (PK), tolerability and survival in guinea pigs treated with various ciprofloxacin dosing regimens. Three times per day (TID) intraperitoneal (IP) dosing with 7.5 mg/kg of ciprofloxacin initiated 1 day following inhalational anthrax challenge and continued for 14 days was identified as a well tolerated partially curative ciprofloxacin treatment regimen. The added benefit of AV7909 vaccination was evaluated in guinea pigs given the partially curative ciprofloxacin treatment regimen. Groups of ciprofloxacin-treated guinea pigs were vaccinated. 1 and 8 days post-challenge with serial dilutions of AV7909, a 1:16 dilution of AVA, or normal saline. A group of untreated guinea pigs was included as a positive control to confirm lethal B. anthracis exposure. Post-exposure vaccination with the AV7909 anthrax vaccine candidate administered in combination with the partially curative ciprofloxacin treatment significantly increased survival of guinea pigs compared to ciprofloxacin treatment alone. These results suggest that the developed model can be useful in demonstrating added value of the vaccine for PEP.

Rabbitpox in New Zealand White Rabbits: A Therapeutic Model for Evaluation of Poxvirus Medical Countermeasures Under the FDA Animal Rule.

The elimination of smallpox as an endemic disease and the obvious ethical problems with clinical challenge requires the efficacy evaluation of medical countermeasures against smallpox using the FDA Animal Rule. This approach requires the evaluation of antiviral efficacy in an animal model whose infection recapitulates the human disease sufficiently well enough to provide predictive value of countermeasure effectiveness. The narrow host range of variola virus meant that no other animal species was sufficiently susceptible to variola to manifest a disease with predictive value. To address this dilemma, the FDA, after a public forum with virologists in December 2011, suggested the development of two animal models infected with the cognate orthopoxvirus, intradermal infection of rabbits and intranasal infection of mice, to supplement the non-human primate models in use. In this manuscript, we describe the development of an intradermal challenge model of New Zealand White rabbits with rabbitpox virus (RPXV) for poxvirus countermeasure evaluation. Lethality of RPXV was demonstrated in both 9 and 16-weeks old rabbits with an LD50 < 10 PFU. The natural history of RPXV infection was documented in both ages of rabbits by monitoring the time to onset of abnormal values in clinical data at a lethal challenge of 300 PFU. All infected animals became viremic, developed a fever, exhibited weight loss, developed secondary lesions, and were euthanized after 7 or 8 days. The 16-weeks RPXV-infected animals exhibiting similar clinical signs with euthanasia applied about a day later than for 9-weeks old rabbits. For all animals, the first two unambiguous indicators of infection were detection of viral copies by quantitative polymerase chain reaction and fever at 2 and 3 days following challenge, respectively. These biomarkers provide clinically-relevant trigger(s) for initiating therapy. The major advantage for using 16-weeks NZW rabbits is that older rabbits were more robust and less subject to stress-induced death allowing more reproducible studies.

Development of a hydrogen cyanide inhalation exposure system and determination of the inhaled median lethal dose in the swine model.

OBJECTIVE: Cyanide is a highly toxic chemical, and acute exposure depletes cells and tissue of oxygen, depressing the respiratory, cardiovascular and neurological systems and potentially leading to death. Cyanide has been used as a weapon since ancient Rome and continues to pose a potential threat today. A well-characterized animal model is necessary for the development of novel methods of rapid detection and treatment. This manuscript describes the development of an inhalation exposure system designed to evaluate the lethality of acute cyanide inhalation in the porcine model. MATERIALS AND METHODS: A custom designed hydrogen cyanide (HCN) inhalation exposure system provided stable cyanide concentrations to un-anesthetized swine while monitoring respiratory parameters. Real-time respiratory monitoring, cyanide concentration and body weight were used to calculate inhaled doses. RESULTS: The inhalation exposure system generated controlled HCN ranging from 260 to 986 ppm to achieve inhaled doses between 1.78 and 3.97 mg/kg. Based on survival outcomes, the median lethal dose was determined to be 2.21 mg/kg, and the median lethal exposure level was 5893 mg min/m3. DISCUSSION: The ability of the HCN inhalation exposure system to deliver target inhaled doses and the determination of the inhaled median lethal dose in swine support the use of the exposure system and animal model for the evaluation of medical countermeasures of acute inhaled HCN toxicity.

An inhalational swine model for the characterization of physiological effects and toxicological profile associated with cyanide poisoning.

Cyanides are highly toxic compounds that have been used as weapons of terrorism throughout history. Cyanide (CN) is acutely toxic by all routes of administration; however, inhalation is the main exposure route. To adequately test effective countermeasures against inhalational CN threats, robust and well-characterized animal models are needed. This paper describes the initial development of a hydrogen cyanide (HCN) exposure swine model for documenting the physiological effects and toxicological profile during and after HCN inhalation exposure. Animals were implanted with telemetry transmitters for heart rate (HR), blood pressure, and electrocardiogram monitoring, and vascular access ports for serial blood collections. Nine female swine were exposed to HCN concentrations of 500 ± 6 ppm while breathing parameters were monitored real-time. Inhaled HCN doses ranged from 2.02 to 2.83 mg/kg. Clinical signs included vocalization, agitation, salivation, respiratory distress and apnea. After HCN exposure initiation, systemic arterial pressure fell dramatically with a concomitant increase in HR. Blood samples were collected to determine CN blood levels using LC-MS/MS and blood gas analysis. In summary, the developed HCN inhalation swine model permitted documentation of the physiological effects associated with CN poisoning. This model could be used to evaluate potential CN medical countermeasures in the event of a public health emergency stemming from inhalational CN threats.

Characterization of a nose-only inhaled phosgene acute lung injury mouse model.

CONTEXT: Phosgene's primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6-24 h post-exposure. OBJECTIVE: To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures. METHODS: A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life. RESULTS AND DISCUSSION: The 24-hour LCt50 was 226 ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215 ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies. CONCLUSION: An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant.

Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

A novel sulfur mustard (HD) vapor inhalation exposure system for accurate inhaled dose delivery.

INTRODUCTION: A custom designed HD exposure system was used to deliver controlled inhaled doses to an animal model through an endotracheal tube. METHODS: Target HD vapor challenges were generated by a temperature controlled bubbler/aerosol trap, while concentration was monitored near real-time by gas chromatography. Animal breathing parameters were monitored real-time by an in-line pneumotach, pressure transducer, and Buxco pulmonary analysis computer/software. For each exposure, the challenge atmosphere was allowed to stabilize at the desired concentration while the anesthetized animal was provided humidity controlled clean air. Once the target concentration was achieved and stable, a portion of the challenge atmosphere was drawn past the endotracheal tube, where the animal inhaled the exposure ad libitum. During the exposure, HD vapor concentration and animal weight were used to calculate the needed inhaled volume to achieve the target inhaled dose (µg/kg). The exposures were halted when the inhaled volume was achieved. RESULTS: The exposure system successfully controlled HD concentrations from 22.2 to 278mg/m(3) and accurately delivered inhaled doses between 49.3 and 1120µg/kg with actual administered doses being within 4% of the target level. DISCUSSION: This exposure system administers specific HD inhaled doses to evaluate physiological effects and for evaluation of potential medical countermeasure treatments.

A dynamic system for delivering controlled bromine and chlorine vapor exposures to weanling swine skin.

CONTEXT: Assessing the hazards of accidental exposure to toxic industrial chemical (TIC) vapors and evaluating therapeutic compounds or treatment regimens require the development of appropriate animal models. OBJECTIVE: The objective of this project was to develop an exposure system for delivering controlled vapor concentrations of TICs to the skin of anesthetized weanling pigs. Injury levels targeted for study were superficial dermal (SD) and deep dermal (DD) skin lesions as defined histopathologically. MATERIALS AND METHODS: The exposure system was capable of simultaneously delivering chlorine or bromine vapor to four, 3-cm diameter exposure cups placed over skin between the axillary and inguinal areas of the ventral abdomen. Vapor concentrations were generated by mixing saturated bromine or chlorine vapor with either dried dilution air or nitrogen. RESULTS: Bromine exposure concentrations ranged from 6.5 × 10(-4) to 1.03 g/L, and exposure durations ranged from 1 to 45 min. A 7-min skin exposure to bromine vapors at 0.59 g/L was sufficient to produce SD injuries, while a 17-min exposure produced a DD injury. Chlorine exposure concentrations ranged from 1.0 to 2.9 g/L (saturated vapor concentration) for exposures ranging from 3 to 90 min. Saturated chlorine vapor challenges for up to 30 min did not induce significant dermal injuries, whereas saturated chlorine vapor with wetted material on the skin surface for 30-60 min induced SD injuries. DD chlorine injuries could not be induced with this system. CONCLUSION: The vapor exposure system described in this study provides a means for safely regulating, quantifying and delivering TIC vapors to the skin of weanling swine as a model to evaluate therapeutic treatments.

Toxicogenomic analysis of chlorine vapor-induced porcine skin injury.

Chlorine is an industrial chemical that can cause cutaneous burns. Understanding the molecular mechanisms of tissue damage and wound healing is important for the selection and development of an effective post-exposure treatment. This study investigated the effect of cutaneous chlorine vapor exposure using a weanling swine burn model and microarray analysis. Ventral abdominal sites were exposed to a mean calculated chlorine vapor concentration of 2.9 g/L for 30 min. Skin samples were harvested at 1.5 h, 3 h, 6 h, and 24 h post-exposure and stored in RNAlater(®) until processing. Total RNA was isolated, processed, and hybridized to Affymetrix GeneChip(®) Porcine Genome Arrays. Differences in gene expression were observed with respect to sampling time. Ingenuity Pathways Analysis revealed seven common biological functions among the top ten functions of each time point, while canonical pathway analysis revealed 3 genes (IL-6, IL1A, and IL1B) were commonly shared among three significantly altered signaling pathways. The transcripts encoding all three genes were identified as common potential therapeutic targets for Phase II/III clinical trial, or FDA-approved drugs. The present study shows transcriptional profiling of cutaneous wounds induced by chlorine exposure identified potential targets for developing therapeutics against chlorine-induced skin injury.

An assessment of transcriptional changes in porcine skin exposed to bromine vapor.

Bromine is an industrial chemical that can cause severe cutaneous burns. This study was a preliminary investigation into the effect of cutaneous exposure to bromine vapor using a weanling swine burn model and microarray analysis. Ventral abdominal sites were exposed to a mean calculated bromine vapor concentration of 0.69 g L(-1) for 10 or 20 min. At 48 h postexposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine vapor exposure for 10 or 20 min promoted similar transcriptional changes in the number of significantly modulated probe sets. A minimum of 83% of the probe sets was similar for both exposure times. Ingenuity pathways analysis revealed eight common biological functions among the top 10 functions of each experimental group, in which 30 genes were commonly shared among 19 significantly altered signaling pathways. Transcripts encoding heme oxygenase 1, interleukin-1ß, interleukin 2 receptor gamma chain, and plasminogen activator inhibitor-1 were identified as common potential therapeutic targets for Phase II/III clinical trial or FDA-approved drugs. The present study is an initial assessment of the transcriptional responses to cutaneous bromine vapor exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Temporal effects in porcine skin following bromine vapor exposure.

Bromine is an industrial chemical that causes severe cutaneous burns. When selecting or developing effective treatments for bromine burns, it is important to understand the molecular mechanisms of tissue damage and wound healing. This study investigated the effect of cutaneous bromine vapor exposure on gene expression using a weanling swine burn model by microarray analysis. Ventral abdominal sites were exposed to a mean calculated bromine vapor concentration of 0.51 g/L for 7 or 17 min. At 6 h, 48 h, and 7 days post-exposure, total RNA from skin samples was isolated, processed, and analyzed with Affymetrix GeneChip® Porcine Genome Arrays (N = 3 per experimental group). Differences in gene expression were observed with respect to exposure duration and sampling time. Ingenuity Pathways Analysis (IPA) revealed four common biological functions (cancer, cellular movement, cell-to-cell signaling and interaction, and tissue development) among the top ten functions of each experimental group, while canonical pathway analysis revealed 9 genes (ARG2, CCR1, HMOX1, ATF2, IL-8, TIMP1, ESR1, HSPAIL, and SELE) that were commonly shared among four significantly altered signaling pathways. Among these, the transcripts encoding HMOX1 and ESR1 were identified using IPA as common potential therapeutic targets for Phase II/III clinical trial or FDA-approved drugs. The present study describes the transcriptional responses to cutaneous bromine vapor exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Evaluation of Acute Immunotoxicity of Aerosolized Aflatoxin B(1) in Female C57BL/6N Mice.

There is evidence for immunotoxicity of aflatoxin B1 (AFB(1)) in chronic animal feeding studies; however, little information is available as to the effects of inhalation exposure. This study evaluated the acute affects of aerosolized AFB(1) on systemic immune function of female C57BL/6N mice following a single aerosol exposure. Mice were exposed in nose-only inhalation tubes to 0, 2.86, 6.59 and 10 mug AFB(1) aerosol/L air for 90 minutes. A negative control group of untreated mice and a positive control group of cyclophosphamide-treated mice were included to account for day to day variation. Three days following exposure, mice were sacrificed and body, liver, lung, thymus and spleen weights, and complete blood counts and white blood cell differentials were measured. Splenocytes were isolated for flow cytometric analysis of CD4(+) and CD8(+) lymphocytes, CD19(+) B-cells and natural killer cells (NK 1.1(+)). The effect of AFB(1) on humoral immunity was assessed by measuring serum anti-keyhole limpet hemocyanin (KLH) IgM levels. Of the tissues examined, only the thymus weight of AFB(1) exposed mice decreased significantly compared to naive mice; however, the decrease was not dose related and was also observed in the 0 AFB(1) aerosol control group. A decrease in the mean white blood cell count of treated vs. naive mice was observed at all dose levels but was clearly not dose related and was statistically significant only in the 0 and 2.86 mug/L groups. Red blood cell and platelet counts and white blood cell differentials were not significantly affected by AFB(1). The number of CD4(+) (helper T-cells), CD8(+) (cytotoxic T-cells) and CD19(+) (B-cells) decreased in spleens of AFB(1) aerosol exposed mice compared to naive mice; however, the decrease was not dose-related and was also observed in the 0 AFB(1) exposure group. Dose-related changes in the CD4(+)/CD8(+) T-lymphocyte ratios were not observed. The IgM response to KLH was not significantly different in AFB(1) compared to naive mice, suggesting that AFB(1) did not effect antigen-specific antibody production. Based on the results of this study, a single AFB(1) inhalation exposure up to 10 mug/L for 90 minutes (CxT = 900 mug .min/L) did not significantly alter the immune parameters measured in this study. The aerosol vehicle (ethanol) and/or stress could have masked subtle AFB(1)-dependent changes in thymus and spleen weights, and in splenic lymphocyte subpopulations. However, for other immunological parameters, such as the IgM response to KLH, there was clearly no significant effect of AFB(1) aerosol exposure.