Dysregulation of the mTOR pathway by mechlorethamine.

Mechlorethamine (HN2) is a derivative of the chemical warfare agent sulfur mustard (SM) and cutaneous exposure to HN2 is associated with dermal-epidermal junction (DEJ) disruption (vesication). The primary purpose of the present study was to investigate the effect of HN2 on the mammalian target of rapamycin (mTOR) signaling pathway using an in vivo mouse ear vesicant model (MEVM). To this end, the ears of male C57BL/ 6 J mice were exposed to a single topical dose of HN2 (100 mM) or vehicle control (DMSO). Mice were then euthanized 30 min, 1 h or 24 h following exposure. Mouse ear skin exposed to HN2 and biopsied 24 h thereafter exhibited increased tissue expression of Raptor, an important member of the mTORC1 complex, relative to vehicle treated samples. HN2 reduced the downstream effectors phospho S6 (Ser 240/244) ribosomal protein and phospho 4E-BP1 (Thr 37/46) of the mTOR pathway in the epidermis at 30 min, 1 h and 24 h following HN2 exposure but not in the dermis. These results support the hypothesis that HN2-mediated cutaneous toxicity involves dysregulation of the mTOR signaling pathway in the epidermis.

Inhibition of NETosis by a Nuclear-Penetrating Anti-DNA Autoantibody.

Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase-dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.

Combination of ebselen and hydrocortisone substantially reduces nitrogen mustard-induced cutaneous injury.

The purpose of the present study was to investigate the vesicant countermeasure effects of hydrocortisone (HC) and ebselen (EB-1), administered as monotherapy or as a combination treatment. The mouse ear vesicant model (MEVM) was utilized and test doses of HC (0.016, 0.023, 0.031, 0.047, 0.063, 0.125 or 0.250 mg/ear), EB-1 (0.125, 0.187, 0.250, 0.375 or 0.500 mg/ear) or the combination of HC + EB-1 were topically applied at 15 min, 4 h and 8 h after nitrogen mustard exposure. Ear punch biopsies were obtained 24 h after mechlorethamine (HN2) exposure. Compared to control ears, ear tissues exposed topically to HN2 (0.500 µmol/ear) presented with an increase in ear thickness, vesication, TUNEL fluorescence and expression of matrix metalloproteinase 9 (MMP-9) and inducible nitric oxide synthase (iNOS). In contrast, HN2 exposed ears treated topically with EB-1 showed a significant decrease in morphometric thickness and vesication vs. HN2 alone. Ear tissues exposed to HN2 and then treated with HC also demonstrated reductions in morphometric thickness and vesication. Combination treatment of HC + EB-1 was found to be the most effective at reducing HN2-induced ear edema and vesication. The combination also dramatically decreased HN2-mediated cutaneous expression of iNOS and MMP-9 and decreased HN2-induced TUNEL staining. Taken together, our study demonstrates that the combination of HC + EB-1 is an efficacious countermeasure to HN2.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Ebselen oxide attenuates mechlorethamine dermatotoxicity in the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard mimetic. Topical exposure to HN2 is associated with tissue blistering. Previous work in our laboratory has shown that ebselen (EB-1) possesses anti-vesicant, anti-inflammatory, anti-bacterial, anti-fungal, and cytoprotective properties, both in vivo and in vitro. We recently reported that ebselen oxide (EB-2), an analog of EB-1 with a tetravalent selenium atom, also possesses anti-bacterial and anti-fungal activity and confers cytoprotection against HN2 in vitro. The purpose of the present study was to determine the vesicant countermeasure potential of EB-2 using the mouse ear vesicant model (MEVM). Compared to control ears, mouse ears exposed to a single dose of HN2 (0.500 µmol/ear) showed an increase in wet weights, ear thickness, hyperplasia, vesication, and inflammatory cell infiltration after 24 h. Fluorescence microscopy of terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-stained sections showed that the occurrence of apoptosis extended from the epidermis of the HN2-treated side, all the way to the contralateral epidermis. In contrast, HN2-exposed ears treated topically with EB-2 at a test dose of 0.250 mg/ear showed a significant decrease in wet weight (12% less vs. HN2 alone), morphometric thickness (13% less vs. HN2 alone), and vesication. In addition, TUNEL staining revealed that HN2 ears treated with EB-2 (0.250 mg/ear) showed a decrease in apoptosis as compared to the HN2 group. EB-2 also reduced the abundance of matrix metalloproteinase-9 (MMP-9) in ear tissues exposed to HN2. Taken together, our study demonstrates that EB-2 is an efficacious countermeasure to HN2.

Assessment of the time-dependent dermatotoxicity of mechlorethamine using the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 µmol/ear) or DMSO vehicle (5 µl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures.