Selection of non-steroidal anti-inflammatory drug and treatment regimen for sulfur mustard-induced cutaneous lesions.

The inflammatory process plays an important role in sulfur mustard (HD) injury and HD pathogenesis, suggesting that anti-inflammatory treatments applied as soon as possible following HD injury may reduce tissue damage and accelerate healing. This study used the HD dermal weanling swine model to investigate the efficacy of two non-steroidal anti-inflammatory drugs, capsaicin and diclofenac, when applied in combination with the steroid, clobetasol. The therapeutic regimen was also investigated with respect to initiation of treatment post-exposure, frequency and duration. Yorkshire-cross pigs were randomly assigned to experimental groups, corresponding to all combinations of treatment (capsaicin with clobetasol or diclofenac with clobetasol), onset time (1, 2 or 4 h post-exposure), treatment duration (1, 3 or 5 days) and frequency of applications (2, 3 or 4 per day). For each animal, two sites on the ventral abdomen were exposed to 400 µL of neat HD for 8 min to achieve superficial dermal (SD) lesions and two sites were exposed to 400 µL neat HD for 30 min to achieve deep dermal (DD) lesions. Each treatment regimen was tested against a SD and a DD injury. Untreated SD and DD lesion sites served as within-animal controls. Assessments, up to one week post-challenge, included digital photographs, clinical assessments (lesion size measurements and modified Draize scoring), transepidermal water loss (TEWL), reflectance colorimetry and histopathologic evaluations that included an estimate for depth of injury and wound healing parameters. Diclofenac plus clobetasol treatment resulted in significant reductions in lesion contracture and modified Draize scores, increased barrier function (decreased TEWL), and increased healing as determined by histopathology for both SD and DD injury when compared with untreated sites and sites treated with capsaicin plus clobetasol. An increased duration of treatment from 1 to 5 days was most commonly associated with decreased clinical assessment and histopathological severity scores. Therefore, a combination of diclofenac and clobetasol application, when administered for at least five days, shows promise in ameliorating HD-induced lesions.

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.

Transcriptional changes in porcine skin at 7 days following sulfur mustard and thermal burn injury.

Severe cutaneous injuries continue to result from exposure to sulfur mustard [bis(2-chloroethyl)sulfide; HD] and thermal burns. Microarray analysis was utilized in this study to evaluate transcriptional changes in porcine skin assessing the underlying repair mechanisms of HD and thermal injury involved in wound healing. Four ventral abdominal sites on each of 4 weanling swine were exposed to 400 microL undiluted HD or a heated brass rod (70 degrees C) for 8 minutes and 45-60 seconds, respectively. At 7 days postexposure, skin samples were excised and total RNA was isolated, labeled, and hybridized to Affymetrix GeneChip (Santa Clara, CA, USA) Porcine Genome Arrays (containing 20,201 genes). Based on the gene expression patterns in HD- and thermal-exposed skin at 7 days, the transcriptional profiles do not differ greatly. HD and thermal exposures promoted similar changes in transcription, where 270 and 283 transcripts were increased with HD and thermal exposures, respectively. Both exposures promoted decreases in 317 and 414 transcripts, respectively. Of the significantly increased transcripts, at least 77% were commonly expressed in both HD- and thermal-exposed skin, whereas at least 67% of decreased transcripts were common between both exposure types. Six of the top 10 biological functions were common to HD and thermal injury in which 9 canonical pathways were shared. The present study illustrates the similarities found between HD and thermal injury with respect to transcriptional response and wound healing and identifies specific genes (CXCL2, CXCR4, FGFR2, HMOX1, IGF1, PF4, PLAU, PLAUR, S100A8, SPP1, and TNC) that may be useful as potential therapeutic targets to promote improved wound healing.

Gene expression analysis of bromine-induced burns in porcine skin.

Bromine is an industrial chemical that is irritating to the skin and causes cutaneous burns. An important factor in selecting or developing an effective treatment is to understand the underlying molecular mechanisms of tissue damage and wound healing. This study used a weanling swine burn model and microarray analysis to evaluate the effect of exposure length and sampling times on the transcriptional changes in response to cutaneous bromine injury. Ventral abdominal sites (N=4/treatment group) were exposed to 600microL undiluted bromine for 45 s or 8 min. At 24 h and 7d post-exposure, total RNA from skin samples was isolated, processed, and hybridized to Affymetrix GeneChip Porcine Genome Arrays. Expression analysis revealed that bromine exposure duration appeared to have less effect on the transcript changes than the sampling time. The percent transcripts changed at 24h were similar (30%) whether having a 45 s or 8 min bromine exposure; percent transcripts changed at 7d were also similar (62%) regardless of exposure length. However, only 13-14% of the transcripts were similar when comparing samples analyzed at 24h and 7d. Ingenuity Pathways Analysis (IPA) revealed six common biological functions among the top 10 functions of each experimental group, while canonical pathway analysis revealed 11 genes that were commonly shared among 24 significantly altered signaling pathways. Additionally, there were 11 signaling pathways in which there were no commonly shared transcripts. The present study is an initial assessment of the transcriptional responses to cutaneous bromine exposure identifying molecular networks and genes that could serve as targets for developing therapeutics for bromine-induced skin injury.

Transcriptional responses associated with sulfur mustard and thermal burns in porcine skin.

In military and civilian environments, serious cutaneous damage can result from thermal burns or exposure to the blistering agent sulfur mustard [bis (2-chloroethyl) sulfide; HD]. Similar therapies have historically been used to treat cutaneous thermal and HD injuries; however, the underlying molecular mechanisms of tissue damage and wound healing may differ between the types of burns. Using microarray analysis, this study assessed the transcriptional responses to cutaneous HD and thermal injury at 48 hours post-exposure to identify molecular networks and genes associated with each type of skin injury. Ventral abdominal sites on each of 4 weanling swine were exposed to 400 mul of undiluted HD or a heated brass rod (70 degrees C) for 8 minutes and 45-60 seconds, respectively. At 48 hours post-exposure, total RNA was isolated from excised skin samples and hybridized to Affymetrix GeneChip Porcine Genome Arrays (containing 20,201 genes). Both HD and thermal exposure promoted significant transcriptional changes where 290 and 267 transcripts were increased and 197 and 707 transcripts were decreased with HD and thermal exposure, respectively. HD- and thermal-injured skin expressed 149 increased and 148 decreased common transcripts. Comparison of the 10 most significantly changed biological functions for HD and thermal exposures identified 7 overlapping functional groups. Canonical pathways analysis revealed 15 separate signaling pathways containing transcripts associated with both HD and thermal exposure. Within these pathways, 5 transcripts (CXCR4, FGFR2, HMOX1, IL1R1, and TLR4) were identified as known targets for existing phase II/III clinical trial or Food and Drug Administration (FDA)-approved drugs. This study is the first to directly assess transcriptional changes in porcine skin subjected to HD or thermal injury over the same time period.

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.

Biomechanical monitoring of cutaneous sulfur mustard-induced lesions in the weanling pig model for depth of injury.

BACKGROUND/PURPOSE: A sulfur mustard (SM)-induced cutaneous injury model was developed in weanling swine to evaluate the efficacy of candidate treatment regimens. Lesions were assessed clinically and histopathologically. Histopathologic evaluation of lesions was a subjective and invasive assessment. Biomechanical engineering methods offer an objective and less invasive method to evaluate lesions. The purpose of this study was to use biomechanical engineering instruments to assess SM-induced lesions for depth of injury and to correlate those assessments with histopathology. METHODS: Two groups of six animals each were exposed to 400 microL undiluted SM applied at each of six abdominal sites for either 2 or 30 min. An additional seven animals received a sham treatment (control; 400 microL deionized water applied to each of six sites for 30 min). Each site was evaluated before exposure and 2 days after exposure. Biomechanical engineering techniques used to assess each lesion were reflectance colorimetry, evaporimetry [transepidermal water loss (TEWL)], laser Doppler perfusion imaging, and high-frequency (20 MHz) two-dimensional ultrasound. Injury depth and lesion severity were assessed and correlated to biomechanical methods using special histopathologic staining techniques. RESULTS: Two- and 30-min cutaneous lesions were significantly different from controls at the 0.05 probability level for redness (chroma meter) and TEWL (evaporimeter), but were not significantly different from each other. The 2-min lesions had a significant increase (2.11 AU, SE=0.06) and the 30-min lesions had a decrease (0.96 AU, SE=0.04) from controls (1.31 AU, SE=0.03) in microcirculatory blood flux (laser Doppler). The 2-min lesions and controls were significantly different at the 0.05 level from 30-min lesions in skin thickness (ultrasound). The 2- and 30-min groups were significantly different from controls and from each other at the 0.05 level in histopathologic assessment of injury depth, basal cell necrosis, depth of necrosis, and vascular necrosis, with the 30-min injuries being most severe. CONCLUSION: There was mixed evidence that the bioengineering techniques tested could differentiate between controls, 2-min (partial-thickness) cutaneous injuries and 30-min (full-thickness) cutaneous injuries at day 2. Both biomechanical and histopathologic assessments are useful methods of characterizing SM lesions in the weanling pig model. Biomechanical methods are non-invasive and quantitative, and multiple readings over shorter and longer periods of time may improve differentiation in depth of injury. Histopathologic assessments are important for confirmation of lesion depth and severity, and for assisting interpretation when a single assessment using bioengineering methods is used.