The pathway of the biologically active molecule hydrogen peroxide (H 2 O 2 ) from the plasma generation in the gas phase by an atmospheric pressure argon plasma jet, to its transition into the liquid phase and finally to its inhibiting effect on human skin cells is investigated for different feed gas humidity settings. Gas phase diagnostics like Fourier transformed infrared spectroscopy and laser induced fluorescence spectroscopy on hydroxyl radicals ( • OH) are combined with liquid analytics such as chemical assays and electron paramagnetic resonance spectroscopy. Furthermore, the viability of human skin cells is measured by Alamar Blue ® assay. By comparing the gas phase results with chemical simulations in the far field, H 2 O 2 generation and destruction processes are clearly identified. The net production rate of H 2 O 2 in the gas phase is almost identical to the H 2 O 2 net production rate in the liquid phase. Moreover, by mimicking the H 2 O 2 generation of the plasma jet with the help of an H 2 O 2 bubbler it is concluded that the solubility of gas phase H 2 O 2 plays a major role in generating hydrogen peroxide in the liquid. Furthermore, it is shown that H 2 O 2 concentration correlates remarkably well with the cell viability. Other species in the liquid like • OH or superoxide anion radical (O •− 2 ) do not vary significantly with feed gas humidity.

We report on multi-level atomistic simulations for the interaction of reactive oxygen species (ROS) with the head groups of the phospholipid bilayer, and the subsequent effect of head group and lipid tail oxidation on the structural and dynamic properties of the cell membrane. Our simulations are validated by experiments using a cold atmospheric plasma as external ROS source. We found that plasma treatment leads to a slight initial rise in membrane rigidity, followed by a strong and persistent increase in fluidity, indicating a drop in lipid order. The latter is also revealed by our simulations. This study is important for cancer treatment by therapies producing (extracellular) ROS, such as plasma treatment. These ROS will interact with the cell membrane, first oxidizing the head groups, followed by the lipid tails. A drop in lipid order might allow them to penetrate into the cell interior (e.g., through pores created due to oxidation of the lipid tails) and cause intracellular oxidative damage, eventually leading to cell death. This work in general elucidates the underlying mechanisms of ROS interaction with the cell membrane at the atomic level.In recent years, cold atmospheric plasmas (CAPs) are gaining increasing interest for cancer treatment, i.e., so-called "plasma oncology" [1][2][3][4] . In a recent review, Schlegel et al. 1 summarize the results of several studies on plasma oncology and show the progress in the potential use of CAPs to effectively kill cancer cells (either by apoptosis or necrosis), in vitro as well as in vivo. CAP sources seem to be a powerful tool for cancer treatment, either alone or in combination with other conventional therapies. Indeed, recent experimental results showed that CAPs could enhance the effects of conventional chemotherapy even in resistant tumorous cells; the resistant cell population, if pre-treated with CAP, becomes sensitive to treatment with chemotherapy 5,6 . Moreover, it was demonstrated that plasma treatment, both in vitro and in vivo, is able to attack a wide range of cancer cell lines without damaging their normal counterparts 1, 2 . Thus, preliminary results seem very promising. Nevertheless, the application of CAPs for cancer treatment is still in its initial stage, and there is an enormous need for a better understanding of the underlying mechanisms.CAPs generate reactive oxygen species (ROS, e.g., OH, HO 2 , H 2 O 2 ) and reactive nitrogen species (RNS, e.g., NO, NO 2 , ONOO − ), which are generally believed to play a key role in plasma treatment 3,4,7 . Several studies showed that CAPs elevate intracellular ROS levels, thereby inducing oxidative damage in cancer cells, which can lead to cell death, i.e., apoptosis 3,8 . Normal cells, on the other hand, are able to defend themselves from this harmful effect of ROS by activating multiple anti-oxidative systems that reduce the increased oxidative stress and restore the balance 9, 10 . Besides, also the RNS generated by CAP might play an important role in cancer therapy (see ref. 4 and references therein). ...

The lipopolysaccharide-binding protein (LBP) is critically involved in innate immune responses to Gram-negative infections. We show here that human peripheral blood-derived monocytes, but not lymphocytes, stain positive for endogenous LBP on the cell surface. Studies on human macrophages demonstrate LBP binding at normal serum concentrations of 1-10 μg/ml. Binding was increased in a concentration-dependent manner by lipopolysaccharide (LPS). Fluorescence quenching experiments and confocal microscopy revealed constitutive and LPS-induced internalization of LBP by macrophages. Experiments with macrophages and HEK293 cell lines showed that binding and uptake of LBP do not depend on the LPS receptors CD14 and TLR4/MD-2. Fractionation of Triton X-100 solubilized cytoplasmic membranes revealed that LBP was primarily localized in non-raft domains under resting conditions. Cellular LPS stimulation elevated LBP levels and induced enrichment in fractions marking the transition between non-raft and raft domains. LBP was found to colocalize with LPS at the cytoplasmic membrane and in intracellular compartments of macrophages. In macrophages stimulated with LPS and ATP for inflammasome activation, LBP was observed in close vicinity to activated caspases. Furthermore, LBP conferred IL-1β production by LPS in the absence of ATP. These data establish that LBP serves not only as an extracellular LPS shuttle but in addition facilitates intracellular transport of LPS. This observation adds a new function to this central immune regulator of LPS biology and raises the possibility for a role of LBP in the delivery of LPS to TLR4-independent intracellular receptors.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-β1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

The membrane of both pro-and eukaryotic cells is the cell's interface with the environment. It is the first interaction site of any substance that is externally applied, including reactive species in the liquid cell environment created by plasma medical treatments. Therefore, the liquid surrounding the cell is, due to its influence on the chemical paths, an important mediator for plasma-borne reactive species, and the cellular membrane is their primary target structure. A cellular membrane consists, according to the Singer-Nicolson model, of a lipid bilayer with embedded proteins. Here, we describe experiments of plasma treatments of lipids and liposomal model membranes. The investigations show membrane activity of plasmaborne reactive species against lipids and lipid structures. The methods applied are Raman microscopy and chromophore-based light spectroscopy. Results of dynamic light scattering (DLS) and fluorophore-based assays show that, during the applied plasma treatment, neither macroscopic collapse of the lipid superstructure nor liposome fusion was observed. Raman spectroscopy reveals increased fluidity of lipid layers due to plasma treatment. The results are discussed based on our observations and published results. We propose a detailed molecular mechanism for the formation of lesions that allow a "self-mediated in-and efflux" of plasmaborne reactive species and cell signaling molecules. Resulting consequences for cellular membranes and the cell as a whole are discussed.