Structure and transcription of integrated HPV DNA in vulvar carcinomas.

HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1, C5orf67, and LRP1B. Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.

PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

Expression of Laminin γ2 Proteolytic Fragments in Murine Skin Following Exposure to Sulfur Mustard.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure.

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

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.

Differential gene expression profiling of mouse skin after sulfur mustard exposure: Extended time response and inhibitor effect.

Sulfur mustard (HD, SM), is a chemical warfare agent that within hours causes extensive blistering at the dermal-epidermal junction of skin. To better understand the progression of SM-induced blistering, gene expression profiling for mouse skin was performed after a single high dose of SM exposure. Punch biopsies of mouse ears were collected at both early and late time periods following SM exposure (previous studies only considered early time periods). The biopsies were examined for pathological disturbances and the samples further assayed for gene expression profiling using the Affymetrix microarray analysis system. Principal component analysis and hierarchical cluster analysis of the differently expressed genes, performed with ArrayTrack showed clear separation of the various groups. Pathway analysis employing the KEGG library and Ingenuity Pathway Analysis (IPA) indicated that cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), and hematopoietic cell lineage are common pathways affected at different time points. Gene ontology analysis identified the most significantly altered biological processes as the immune response, inflammatory response, and chemotaxis; these findings are consistent with other reported results for shorter time periods. Selected genes were chosen for RT-PCR verification and showed correlations in the general trends for the microarrays. Interleukin 1 beta was checked for biological analysis to confirm the presence of protein correlated to the corresponding microarray data. The impact of a matrix metalloproteinase inhibitor, MMP-2/MMP-9 inhibitor I, against SM exposure was assessed. These results can help in understanding the molecular mechanism of SM-induced blistering, as well as to test the efficacy of different inhibitors.