Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

Triterpenoid CDDO-Me induces ROS generation and up-regulates cellular levels of antioxidative enzymes without induction of DSBs in human peripheral blood mononuclear cells.

Ionizing radiation produces reactive oxygen species (ROS) leading to cellular DNA damage. Therefore, patients undergoing radiation therapy or first responders in radiological accident scenarios could both benefit from the identification of specifically acting pharmacological radiomitigators. The synthetic triterpenoid bardoxolone-methyl (CDDO-Me) has previously been shown to exert antioxidant, anti-inflammatory and anticancer activities in several cell lines, in part by enhancing the DNA damage response. In our study, we examined the effect of nanomolar concentrations of CDDO-Me in human peripheral blood mononuclear cells (PBMC). We observed increased cellular levels of the antioxidative enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone1) and mitochondrial superoxide dismutase 2 by immunoblotting. Surprisingly, we found increased intracellular ROS-levels using imaging flow-cytometry. However, the radiation-induced DNA double-strand break (DSB) formation using the γ-H2AX + 53BP1 DSB focus assay and the cytokinesis-block micronucleus assay both revealed, that nanomolar CDDO-Me pre-treatment of PBMC for 2 h or 6 h ahead of X irradiation with 2 Gy did neither significantly affect γ-H2AX + 53BP1 DSB foci formation nor the frequency of micronuclei. CDDO-Me treatment also failed to alter the nuclear division index and the frequency of IR-induced PBMC apoptosis as investigated by Annexin V-labeled live-cell imaging. Our results indicate that pharmacologically increased cellular concentrations of antioxidative enzymes might not necessarily exert radiomitigating short-term effects in IR-exposed PBMC. However, the increase of antioxidative enzymes could also be a result of a defensive cellular mechanism towards elevated ROS levels.

Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro.

The chemical warfare agent sulfur mustard (SM) alkylates a multitude of biomacromolecules including DNA and proteins. Cysteine residues and nucleophilic nitrogen atoms in purine DNA bases are typical targets of SM but potentially every nucleophilic structure may be alkylated by SM. In the present study, we analyzed potential SM-induced alkylation of glucocorticoid (GC) hormones and functional consequences thereof. Hydrocortisone (HC), the synthetic betamethasone (BM) and dexamethasone (DEX) were chosen as representative GCs. Structural modifications were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hypothesized alkylation was verified and structurally allocated to the OH-group of the C21 atom. The biological function of SM-alkylated GCs was investigated using GC-regulated dual-luciferase reporter gene assays and an ex vivo GC responsiveness assay coupled with real-time quantitative polymerase chain reaction (RT-qPCR). For the reporter gene assays, HEK293-cells were transiently transfected with a dual-luciferase reporter gene that is transcriptional regulated by a GC-response element. These cells were then incubated either with untreated or SM-derivatized HC, BM or DEX. Firefly-luciferase (Fluc) activity was determined 24 h after stimulation. Fluc-activity significantly decreased after stimulation with SM-pre-exposed GC dependent on the SM concentration. The ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to DEX revealed a transcriptional dysregulation of GC-regulated genes (FKBP5, IL1R2, and GILZ) after stimulation with SM-alkylated DEX. Our results present GCs as new biological targets of SM associated with a disturbance of hormone function.

Immediate responses of the cockroach Blaptica dubia after the exposure to sulfur mustard.

The chemical agent sulfur mustard (SM) causes erythema, skin blisters, ulcerations, and delayed wound healing. It is accepted that the underlying molecular toxicology is based on DNA alkylation. With an expected delay, DNA damage causes impairment of protein biosynthesis and disturbance of cell division. However, using the cockroach model Blaptica dubia, the presented results show that alkylating compounds provoke immediate behavior responses along with fast changes in the electrical field potential (EFP) of neurons, suggesting that lesions of DNA are probably not the only effect of alkylating compounds. Blaptica dubia was challenged with SM or 2-chloroethyl-ethyl sulfide (CEES). Acute toxicity was objectified by a disability score. Physiological behavior responses (antennae pullback reflex, escape attempts, and grooming) were monitored after exposure. To estimate the impact of alkylating agents on neuronal activity, EFP recordings of the antennae and the thoracic ganglion were performed. After contact to neat SM, a pullback reflex of the antennae was the first observation. Subsequently, a striking escape behavior occured which was characterized by persistent movement of the legs. In addition, an instantaneous processing of the electrical firing pattern from the antennae to the descending ganglia was detectable. Remarkably, comparing the toxicity of the applied alkylating agents, effects induced by CEES were much more pronounced compared to SM. In summary, our findings document immediate effects of B. dubia after exposure to alkylating substances. These fast responses cannot be interpreted as a consequence of DNA alkylation. Therefore, the dogma that DNA alkylation is the exclusive cause for SM toxicity has to be questioned.

N-Acetyl-L-cysteine inhibits sulfur mustard-induced and TRPA1-dependent calcium influx.

Transient receptor potential family channels (TRPs) have been identified as relevant targets in many pharmacological as well as toxicological studies. TRP channels are ubiquitously expressed in different tissues and act among others as sensors for different external stimuli, such as mechanical stress or noxious impacts. Recent studies suggest that one member of this family, the transient receptor potential ankyrin 1 cation channel (TRPA1), is involved in pain, itch, and various diseases, suggesting TRPA1 as a potential therapeutic target. As a nociceptor, TRPA1 is mainly activated by noxious or electrophilic compounds, including alkylating substances. Previous studies already revealed an impact of 2-chloroethyl-ethyl sulfide on the ion channel TRPA1. In this study, we demonstrate that sulfur mustard (bis-(2-chloroethyl) sulfide, SM) activates the human TRPA1 (hTRPA1) in a dose-dependent manner measured by the increase in intracellular Ca2+ concentration ([Ca2+]i). Besides that, SM-induced toxicity was attenuated by antioxidants. However, very little is known about the underlying mechanisms. Here, we demonstrate that N-acetyl-L-cysteine (NAC) prevents SM-induced hTRPA1-activation. HEK293-A1-E cells, overexpressing hTRPA1, show a distinct increase in [Ca2+]i immediately after SM exposure, whereas this increase is reduced in cells pretreated with NAC in a dose-dependent manner. Interestingly, glutathione, although being highly related to NAC, did not show an effect on hTRPA1 channel activity. Taken together, our results provide evidence that SM-dependent activation of hTRPA1 can be diminished by NAC treatment, suggesting a direct interaction of NAC and the hTRPA1 cation channel. Our previous studies already showed a correlation of hTRPA1-activation with cell damage after exposure to alkylating agents. Therefore, NAC might be a feasible approach mitigating hTRPA1-related dysregulations after exposure to SM.

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/ß-catenin signaling.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/ß-catenin signaling pathway as indicated by the increased stability and nuclear localization of ß-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced ß-catenin transcriptional activity, determined by Wnt/ß-catenin target gene expression analysis and a luciferase-based ß-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on ß-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/ß-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of ß-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of ß-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/ß-catenin activity.

The circadian clock of the bacterium B. subtilis evokes properties of complex, multicellular circadian systems.

Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff's rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

TRPM7 kinase is required for insulin production and compensatory islet responses during obesity.

Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.

Paper-based electrochemical sensor for on-site detection of the sulphur mustard.

Herein, we report a novel paper-based electrochemical sensor for on-site detection of sulphur mustards. This sensor was conceived combining office paper-based electrochemical sensor with choline oxidase enzyme to deliver a sustainable sensing tool. The mustard agent detection relies on the evaluation of inhibition degree of choline oxidase, which is reversibly inhibited by sulphur mustards, by measuring the enzymatic by-product H2O2 in chronoamperometric mode. A nanocomposite constituted of Prussian Blue nanoparticles and Carbon Black was used as working electrode modifier to improve the electroanalytical performances. This bioassay was successfully applied for the measurement of a sulphur mustard, Yprite, obtaining a detection limit in the millimolar range (LOD = 0.9 mM). The developed sensor, combined with a portable and easy-to-use instrumentation, can be applied for a fast and cost-effective detection of sulphur mustards.

Acute radiation syndrome-related gene expression in irradiated peripheral blood cell populations.

PURPOSE: In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes. MATERIALS AND METHODS: EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood. RESULTS: The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude. CONCLUSIONS: T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3. This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.

Effect of sulfur mustard on melanogenesis in vitro.

The chemical warfare agent sulfur mustard (SM) affects all cells in the epidermis including melanocytes which are responsible for melanin synthesis. After exposure to SM, pigment abnormalities like hypo- and hyperpigmentation can occur. The underlying molecular pathomechanisms of SM exposure on human melanogenesis have not been elucidated so far. In our study, we investigated the effect of SM on human melanocytes and melanogenesis. Normal human epidermal melanocytes (NHEM) were used as in vitro model and they were exposed to different concentrations of SM (4.5 µM-100 µM). Melanin production was analyzed by absorption measurements at 405 nm. In addition, quantitative real-time PCR (qPCR) and Western blot experiments were performed to determine the expression of essential melanogenesis-related proteins including tyrosinase (TYR), tyrosinase-related protein (TRP) 1 and 2 and microphthalmia transcription factor (MITF). Our findings demonstrated that exposure to low SM concentrations increased melanin synthesis accompanied with an increase in protein expression. In contrast, high SM concentrations led to decreased melanin content and a downregulation in expression of all investigated melanogenesis-associated proteins. We concluded that low SM concentrations may cause hyperpigmentation while high SM concentrations decreased melanin content which may explain hypopigmented skin areas in SM exposed patients.

Assessment of α-amanitin toxicity and effects of silibinin and penicillin in different in vitro models.

Silibinin (Sil) is used as hepatoprotective drug and is approved for therapeutic use in amanitin poisoning. In our study we compared Sil-bis-succinate (SilBS), a water-soluble drug approved for i.v.-administration, with Sil solved in ethanol (SilEtOH), which is normally used in research. We challenged monocultures or 3D-microtissues consisting of HepG2 cells or primary hepatocytes with α-amanitin and treated with SILBS, SILEtOH, penicillin and combinations thereof. Cell viability and the integrity of the microtissues was monitored. Finally, the expression of the transporters OATP1B1 and B3 was analyzed by qRT-PCR. We demonstrated that primary hepatocytes were more sensitive to α-amanitin compared to HepG2. Primary hepatocytes cultures were protected by SilBS and SilEtOH independent of penicillin from the cytotoxic effects of α-amanitin. Subsequent studies of the expression profile of the transporters OATP1B1/B3 revealed that primary hepatocytes do express both whereas in HepG2 cells they were hardly detectable. Our study showed that SilBS has significant advantage over SilEtOH with no additional benefit of penicillin. Moreover, HepG2 cells may not represent an appropriate model to investigate Amanita phalloides poisoning in vitro with focus on OATP transporters since these cells are lacking sensitivity towards α-amanitin probably due to missing cytotoxicity-associated transporters suggesting that primary hepatocytes should be preferred in this context.

Heterogeneous nuclear ribonucleoprotein K is overexpressed and contributes to radioresistance irrespective of HPV status in head and neck squamous cell carcinoma.

Radiotherapy is a major treatment option for head and neck squamous cell carcinoma (HNSCC). However, the success of radiotherapy is limited by tumor cell resistance to ionizing radiation (IR). Clinical studies have demonstrated an overall improved prognosis and higher susceptibility to radiotherapy of high­risk human papillomavirus (HPV)­associated HNSCC compared with classic HNSCC, as well as worse overall survival for male HNSCC patients. Overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) K has been associated with resistance to radiotherapy in melanoma and colorectal carcinoma. The aim of the present study was to analyze the impact of hnRNP K expression on the aggressiveness and radioresistance of HNSCC with respect to patient sex and HPV status. Immunohistochemical staining of HNSCC tissue specimens revealed elevated hnRNP K levels compared with those in the non­neoplastic epithelium. Cytoplasmic hnRNP K accumulation was associated with advanced tumor stage and male sex. Exposure of HNSCC cells to IR was followed by rapid upregulation of hnRNP K at the protein level, along with re­localization from the tumor cell nucleus to the cytoplasm. siRNA­based knockdown of hnRNP K induced apoptosis and abolished tumor formation after xenotransplantation of HNSCC cells onto the chick egg chorioallantoic membrane (CAM). The observed effects were independent of the respective HPV status of the cell lines. These results indicated a tumorigenic and anti­apoptotic role of hnRNP K in HNSCC, which appeared to be enhanced in male patients and contributed to the radioresistance of these tumors. However, the radioprotective effects of hnRNP K were found to be independent of the tumor's HPV status.

Bardoxolone-Methyl (CDDO-Me) Impairs Tumor Growth and Induces Radiosensitization of Oral Squamous Cell Carcinoma Cells.

Radiotherapy represents a common treatment strategy for patients suffering from oral squamous cell carcinoma (OSCC). However, application of radiotherapy is immanently limited by radio-sensitivity of normal tissue surrounding the tumor sites. In this study, we used normal human epithelial keratinocytes (NHEK) and OSCC cells (Cal-27) as models to investigate radio-modulating and anti-tumor effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid methyl ester (CDDO-Me). Nanomolar CDDO-Me significantly reduced OSCC tumor xenograft-growth in-ovo applying the chick chorioallantoic membrane (CAM) assay. In the presence of CDDO-Me reactive oxygen species (ROS) were found to be reduced in NHEK when applying radiation doses of 8 Gy, whereas ROS levels in OSCC cells rose significantly even without radiation. In parallel, CDDO-Me was shown to enhance metabolic activity in malignant cells only as indicated by significant accumulation of reducing equivalents NADPH/NADH. Furthermore, antioxidative heme oxygenase-1 (HO-1) levels were only enhanced in NHEK and not in the OSCC cell line, as shown by immunoblotting. Clonogenic survival was left unchanged by CDDO-Me treatment in NHEK but revealed to be abolished almost completely in OSCC cells. Our results indicate anti-cancer and radio-sensitizing effects of CDDO-Me treatment in OSCC cells, whereas nanomolar CDDO-Me failed to provoke clear detrimental consequences in non-malignant keratinocytes. We conclude, that the observed differential aftermath of CDDO-Me treatment in malignant OSCC and non-malignant skin cells may be utilized to broaden the therapeutic range of clinical radiotherapy.

Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression.

Based on the capacity of mesenchymal stem cells (MSC) to differentiate into multiple cell types in vitro and in vivo, MCS may be a suitable source for cell therapy and regeneration strategies. A prerequisite for effective clinical applications of human MSC (hMSC) is a profound knowledge of signal transduction cascades that mediate processes like proliferation, targeted migration and differentiation. Recently, we identified the canonical Wnt signal transduction pathway as a key player in hMSC proliferation and invasion. To evaluate whether those findings are transferable to the equivalent counterparts in mice, we studied important steps in the wingless/int-1 (Wnt) signal transduction pathway in mouse MSC (mMSC) and mMSC carrying a T cell specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF)-reporter transgene. We found that the induction of the canonical Wnt pathway resulted in the up-regulation of the known Wnt target gene cyclin D1, closely associated with an enhanced proliferation capacity of mMSC. Interestingly, the expression of the Wnt target gene membrane type 1-matrix metalloproteinase (MT1-MMP) was diminished in mMSC upon Wnt3a stimulation, which came along with an impaired invasion. In line with these findings, MMP-2 and MMP-9 expression levels in mMSC were also decreased after Wnt3a treatment. In contrast, inhibition of Wnt signalling by the knockdown of the transcriptional activator beta-catenin resulted in an up-regulation of MT1-MMP and mMSC invasion. By comparing these findings with the settings in hMSC, major differences in Wnt-regulated MMP expression were observed in mMSC. Thus, our data advice caution when mouse model systems represent the pre-clinical validation of MSC-mediated therapeutical approaches.

Evaluation of selective and non-selective cyclooxygenase inhibitors on sulfur mustard-induced pro-inflammatory cytokine formation in normal human epidermal keratinocytes.

Sulfur mustard (SM) is a highly toxic chemical warfare agent, which produces blisters after skin contact. Treatment of SM-induced adverse health effects, such as cutaneous blistering, ulceration, and inflammation remains a challenging task. Antidotes or specific therapeutic measures are lacking. Some drugs (e.g. cyclooxygenase (COX) inhibitors) exhibited beneficial effects after SM poisoning in vivo. However, in vitro studies that evaluate and compare the potency of COX inhibitors are missing. In the presented study, non-specific (acetylsalicylic acid, ibuprofen, diclofenac, indomethacin, and piroxicam), COX-2-specific (celecoxib and parecoxib) inhibitors and COX-independent drugs (paracetamol and tofacitinib) were compared regarding anti-inflammatory and cytoprotective effects after SM exposure in post-exposure treatment settings. Normal human epidermal keratinocytes (NHEK) were used as a surrogate model. Prostaglandin E2 (PGE2) formation, a direct indicator for COX activity, was determined by ELISA. Changes in pro-inflammatory cytokine levels after SM exposures were assessed by quantitative determination of 27 inflammatory cytokines using a multiplex method. Cytotoxicity was determined using an XTT viability assay. The results demonstrated that SM highly increased PGE2 production and release of pro-inflammatory cytokines, predominantly IL-6, IL-8 and TNF-α. In general, all COX inhibitors and paracetamol were able to reduce the PGE2 formation, while tofacitinib, an inhibitor of Janus kinase, had no influence on PGE2 levels. In addition, IL-6, IL-8, and TNF-α formation were also inhibited, but sometimes independently of PGE2. The COX-2 specific celecoxib was identified as the most potent drug to reduce IL-6, IL-8 and TNF-α formation after SM exposures in vitro. However, cell viability was not improved significantly by any of the investigated drugs in our experiments.

Necrosulfonamide - Unexpected effect in the course of a sulfur mustard intoxication.

Although its first military use in Ypres was 100 years ago, no causal therapy for sulfur mustard (SM) intoxications exists so far. To improve the therapeutic options for the treatment of SM intoxications, we developed a co-culture of keratinocytes (HaCaT cells) and immunocompetent cells (THP-1 cells) to identify potential substances for further research. Here, we report on the influence of necrosulfonamide (NSA) on the course of a SM intoxication in vitro. The cells were challenged with 100, 200 and 300 µM SM and after 1 h treated with NSA (1, 5, 10 µM). NSA was chosen for its known ability to inhibit necroptosis, a specialized pathway of programmed necrosis. However, in our settings NSA showed only mild effects on necrotic cell death after SM intoxication, whereas it had an immense ability to prevent apoptosis. Furthermore, NSA was able to reduce the production of interleukin-6 and interleukin-8 at certain concentrations. Our data highlight NSA as a candidate compound to address cell death and inflammation in SM exposure.

A wearable origami-like paper-based electrochemical biosensor for sulfur mustard detection.

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m3 for liquid and aerosol phase, respectively.