Upregulation of gamma-2 laminin-332 in the mouse ear vesicant wound model.

Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.

Transcriptional responses in spleens from mice exposed to Yersinia pestis CO92.

Yersinia pestis is one of the most threatening biological agents due to the associated high mortality and history of plague pandemics. Identifying molecular players in the host response to infection may enable the development of medical countermeasures against Y. pestis. In this study, microarrays were used to identify the host splenic response mechanisms to Y. pestis infection. Groups of Balb/c mice were injected intraperitoneally with 2-257CFU of Y. pestis strain CO92 or vehicle. One group was assessed for mortality rates and another group for transcriptional analysis. The time to death at the 8 and 257CFU challenge doses were 5.0+/-2.3 and 3.8+/-0.4 days, respectively. Gene profiling using Affymetrix Mouse Genome 430 2.0 Arrays revealed no probe sets were significantly altered for all five mice in the low-dose group when compared to the vehicle controls. However, 534 probe sets were significantly altered in the high dose versus vehicle controls; 384 probe sets were down-regulated and 150 probe sets were up-regulated. The predominant biological processes identified were immune function, cytoskeletal, apoptosis, cell cycle, and protein degradation. This study provides new information on the underlying transcriptional mechanisms in mice to Y. pestis infection.

Transcriptional analysis of protective antigen-stimulated PBMC from non-human primates vaccinated with the anthrax vaccine absorbed.

The transcriptional responses in recombinant protective antigen (PA)-stimulated peripheral blood mononuclear cells (PBMCs) from Anthrax Vaccine Absorbed (AVA)-vaccinated rhesus macaques were evaluated using Affymetrix HGU133 Plus 2.0 GeneChips. PBMCs from animals vaccinated at 0, 4, and 26 weeks were harvested at week 30, stimulated with PA, and RNA isolated. The expression of 295 unigenes was significantly increased in PA-stimulated compared to non-stimulated PBMCs; no significant decrease in gene expression was observed. These upregulated transcripts encoded for proteins functioning in both innate and adaptive immunity. Results were corroborated for several genes by real-time RT-PCR. This study provides information on the potential underlying transcriptional mechanisms in the immune response to PA in AVA-vaccinated rhesus macaques.

Localization of substance P gene expression for evaluating protective countermeasures against sulfur mustard.

Sulfur mustard [bis(2-chloroethyl)sulfide; SM] is a chemical warfare agent that produces edema and blister formation with a severe inflammatory reaction. The mouse ear vesicant model for SM injury has been used to evaluate pharmacological agents for countering SM dermal injury. The vanilloid olvanil reduces SM-induced edema and mRNA expression of cytokines and chemokines, suggesting that blocking the inflammatory effects of neuropeptides, such as substance P (SP), may provide protection against SM-induced dermal injury. This study examined SP expression in mice exposed to SM (0.16 mg) on the inner surface of the right ear, with or without olvanil pretreatment at 1, 10, 30, 60, and 360 min following exposure. In naïve skin, SP mRNA localization was associated with blood vessels and sebaceous glands. In SM-exposed skin, SP mRNA was also detected in perivascular dermal cells. Immunohistochemical localization of SP protein was observed in the ear skin of naïve, SM-, olvanil/SM-, and vehicle-treated mice. Quantification of SP+ perivascular dermal cells revealed that SM exposure led to a significant increase (P < or = 0.05) in SP+ cells over the observed time period. Olvanil pretreatment significantly reduced (P < or = 0.05) the mean number of SP+ cells at 60 and 360 min. This study demonstrates that SP expression could provide an additional endpoint for evaluating the effectiveness of vanilloid drugs on SM-induced skin inflammation.

Microarray analysis of gene expression in murine skin exposed to sulfur mustard.

The chemical warfare agent sulfur mustard [bis-(2-chloroethyl)-sulfide; SM] produces a delayed inflammatory response followed by blister formation in skin of exposed individuals. Studies are underway evaluating the efficacy of pharmacological compounds to protect against SM skin injury. Microarray analysis provides the opportunity to identify multiple transcriptional biomarkers associated with SM exposure. This study examined SM-induced changes in gene expression in skin from mice cutaneously exposed to SM using cDNA microarrays. Ear skin from five mice, paired as SM-exposed right ear and dichloromethane vehicle-exposed left ear at six dose levels (0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 mg; 6 mM to 195 mM range), was harvested at 24 h post-exposure. SM-induced gene expression was analyzed using cDNA microarrays that included 1,176 genes. Genes were selected on the basis of all mice (N=5) in the same dose group demonstrating a > or =2-fold increase or decrease in gene expression for the SM-exposed tissue compared to the dichloromethane vehicle control ear tissue at all six SM doses. When skin exposed to all six concentrations of SM was compared to controls, a total of 19 genes within apoptosis, transcription factors, cell cycle, inflammation, and oncogenes and tumor suppressors categories were found to be upregulated; no genes were observed to be downregulated. Differences in the number and category of genes that were up- or down-regulated in skin exposed to low (0.005-0.01 mg) and high (0.08-0.16 mg) doses of SM were also observed. The results of this study provide a further understanding of the molecular responses to cutaneous SM exposure, and enable the identification of potential diagnostic markers and therapeutic targets for treating SM injury.

Time- and dose-dependent analysis of gene expression using microarrays in sulfur mustard-exposed mice.

The chemical warfare agent sulfur mustard (SM) produces blister formation with a severe inflammatory reaction in skin of exposed individuals. The development of efficacious countermeasures against SM vesication requires an understanding of the cellular and molecular mechanism of SM-induced tissue injury. This study examined SM-induced alterations in gene expression using Atlas Mouse 5K DNA microarrays (5002 genes) to identify transcriptional events associated with SM skin injury. Mice (N=3) were exposed topically to SM (0.04, 0.08, and 0.16 mg; 48.8, 97.5, and 195 mM) on the inner surface of the right ear and skin tissues were harvested at 1.5, 3, 6, and 12 h. Genes were selected based on the three mice in the same dose group demonstrating a > or =2-fold increase or decrease in gene expression for the SM-exposed tissue when compared to the dichloromethane vehicle control ear at all three doses and four time points. At the 0.04 mg SM dose, the genes observed were primarily involved in inflammation, apoptosis, and cell cycle regulation. Exposure to 0.08 mg SM increased the expression of genes related to inflammation and cell cycle regulation. Exposure to 0.16 mg SM led to a total of six genes that were changed at all observed time periods; however, these genes do not appear to be directly influential in biological mechanisms such as inflammation, apoptosis, and cell cycle regulation as was observed at the lower SM doses of 0.04 and 0.08 mg. These functional categories have been observed in previous studies utilizing both in vivo and in vitro model systems of SM-induced dermal injury, suggesting that molecular mechanisms associated with inflammation, apoptosis, and cell cycle regulation may be appropriate targets for developing prophylactic/therapeutic treatments for SM skin injury.

Cytokine, chemokine, and matrix metalloproteinase response after sulfur mustard injury to weanling pig skin.

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide; SM] produces a delayed inflammatory skin response and severe tissue injury. Pig skin has organ similarities to human skin that is characterized by the content and types of epidermal lipids, the density of hair follicles and presence of sweat glands, which together afford penetration of topically applied compounds, complex inflammatory responses, and subsequent wound healing. The goal of this study was to identify in vivo proinflammatory biomarkers of the SM porcine skin injury within 72 h after SM challenge, using the weanling pig model. Changes in gene expression of inflammatory mediators were examined at 3, 6, 24, 48, and 72 h, using subtraction library analyses and by quantitation of selected transcripts by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis of subtraction libraries identified up-regulation of IL-8 at 24, 48, and 72 h. No other specific proinflammatory gene transcripts were isolated from the libraries. Specific transcript RT-PCR analysis showed increased production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase-9 (MMP-9, gelatinase B) mRNA levels in response to SM exposure. Tumor necrosis factor-alpha (TNF-alpha) expression was only slightly increased and no change in the levels of expression was observed for monocyte chemoattractant protein-1 and MMP-2. This study identifies the main proinflammatory mediators involved in SM-induced skin injury in a weanling pig model. The results suggest transcriptional activity in the inflammatory response proteins IL-8, IL-6, IL-1beta, and MMP-9 and modest changes in TNF-alpha that together produce inflammation and contribute to the pathogenesis of SM dermatotoxicity. Therefore, drugs preventing SM-induced inflammation should be prime candidates for medical intervention to lessen collateral inflammation associated with tissue destruction.