Beate Haertel

Non-Thermal Atmospheric-Pressure Plasma Possible Application in Wound Healing

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology

Modern non‐thermal atmospheric pressure plasma sources enable controllable interaction with biological systems. Their future applications – e.g. wound management – are based on their unique mixture of reactive components sparking both stimulatory as well as inhibitory processes. To gain detailed understanding of plasma–cell interaction and with respect to risk awareness, key mechanisms need to be identified. This study focuses on the impact of an argon non‐thermal atmospheric pressure plasma jet (kINPen 09) on human HaCaT keratinocytes. With increasing duration, cell viability decreased. In accordance, cells accumulated in G2/M phase within the following 24 h. DNA single‐strand breaks were detected immediately after treatment and receded in the aftermath, returning to control levels after 24 h. No directly plasma‐related DNA double‐strand breaks were detected over the same time. Concurrently, DNA synthesis decreased. Coincident with treatment time, an increase in intracellular 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) conversion increased reactive oxygen species (ROS) levels. The radical scavenging activity of culture medium crucially influenced these effects. Thus, ROS changed DNA integrity, and the effectiveness of cellular defence mechanisms characterises the interaction of non‐thermal plasma and eukaryotic cells. Effects were time‐dependent, indicating an active response of the eukaryotic cells. Hence, a stimulation of eukaryotic cells using short‐term non‐thermal plasma treatment seems possible, eg in the context of chronic wound care. Long‐term plasma treatments stopped in cell proliferation and apoptosis, which might be relevant in controlling neoplastic conditions.

Surface molecules on HaCaT keratinocytes after interaction with non-thermal atmospheric pressure plasma.

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α2‐ and β1‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H2DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.

Non-thermal atmospheric-pressure plasma can influence cell adhesion molecules on HaCaT-keratinocytes

Abstract:  Non‐thermal atmospheric‐pressure plasmas provide new hope for improvement in chronic wound management because of their potency in killing microorganisms. However, the effectiveness of the procedure has to be verified and negative effects on healthy tissues have to be excluded. In wound healing adhesion molecules play a crucial role for cell migration and proliferation. We investigated whether an atmospheric‐pressure plasma jet (kINPen09) influences the expression of adhesion molecules responsible for cell‐cell and cell‐matrix interactions after treatment of HaCaT‐keratinocytes for 10 and 30 s. Twenty‐four hours after plasma treatment expression of α2‐ and β1‐integrin, E‐cadherin and the epidermal growth factor receptor (EGFR) was determined by flow cytometry. Plasma‐treated HaCaT‐cells were characterized by normal α2‐integrin and increased β1‐integrin expression. E‐cadherin and EGFR expression was reduced after the 30‐s treatment. We did not observe any effects following the 10‐s plasma treatment. In conclusion, short‐term plasma treatment can be applied without effects for cell‐cell and cell‐matrix adhesion.

Influence of non-thermal atmospheric pressure plasma on cellular structures and processes in human keratinocytes (HaCaT)

BACKGROUND The use of non-thermal atmospheric pressure plasma in dermatology to improve the healing of chronic wounds is a promising application. The antimicrobial properties of physical plasma offer on the one hand the killing of bacteria, which are often a problem in chronic wounds. On the other hand, plasma can activate cells which are involved in the wound closure. OBJECTIVE To guarantee a safe application it is essential to understand basic interactions between physical plasma and human skin cells. METHODS In our study, human keratinocytes (HaCaT cells) were directly plasma treated with a dielectric barrier discharge (DBD) plasma source and effects on viability, DNA, cell cycle, intracellular concentration of reactive oxygen species and induction of apoptosis were observed. RESULTS A treatment time-dependent loss of recovered adherent HaCaT cells after 24h and a linear increase of DNA damage were observed, which was no longer evident 24h after plasma stimulation, except for long treatment times. An accumulation of HaCaT cells in G2/M phase and a decrease in the G1 phase was caused by DBD plasma. The increasing formation of intracellular ROS is also attributed to plasma treatment. In contrast to other studies we did not find clear evidences for apoptosis in adherent HaCaT cells. A culture medium exchange subsequently after plasma treatment weakened the observed effects. CONCLUSION DBD plasma treatment resulted in oxidative stress in human keratinocytes which is related to deficient cell performance.

Differential sensitivity of lymphocyte subpopulations to non-thermal atmospheric-pressure plasma

Non-thermal atmospheric-pressure plasmas can possibly be used for several applications in particular in medicine. Plasma treatment can be applied to living tissues and cells, e.g., to induce apoptosis and growth arrest in tumour cells or to improve wound healing. However, detailed investigations of plasma-cell interactions are strongly needed. It is not yet clear whether plasmas will be useful in stimulating immune cells to change their behaviour or function. Therefore, this study focused on the influence of non-thermal atmospheric pressure plasma on cell surface molecules of rat spleen mononuclear cells (MNC) as first important step to gain insight into plasma-immune cells interactions. Rat spleen MNC were treated with plasma by surface dielectric barrier discharge (DBD) at atmospheric pressure in air or argon. Lymphocyte subpopulations and expression of L-selectin, ICAM-1 and LFA-1α expression on T-cells were analysed by flow cytometry 1-48 h after plasma treatment. Plasma changed the ratio of T- and B-cells in favour of B-cells. Of the T-cells the helper T-cells were reduced while cytotoxic T-cells were less affected. L-selectin expressing T-cells were significantly reduced already 1h after plasma treatment and that of ICAM-1(+) and LFA-1α(+)T-cells only after 4h. These effects were time dependent and less dramatic when using DBD/argon plasma. In conclusion, different lymphocyte subpopulations show different sensitivity to plasma. Adhesion molecules as L-selectin, ICAM-1 and LFA-1α are down regulated by plasma. Whether these results can be used to modify lymphocyte homing or to activate MNC for different applications remains to be clarified.

Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.

Computational prediction of immune cell cytotoxicity

Immunotoxicity, defined as adverse effects of xenobiotics on the immune system, is gaining increasing attention in the approval process of industrial chemicals and drugs. In-vivo and ex-vivo experiments have been the gold standard in immunotoxicity assessment so far, so the development of in-vitro and in-silico alternatives is an important issue. In this paper we describe a widely applicable, easy-to use computational approach which can serve as an initial immunotoxicity screen of new chemical entities. Molecular fingerprints describing chemical structure were used as parameters in a machine-learning approach based on the Naïve-Bayes learning algorithm. The model was trained using blood-cell growth inhibition data from the NCI database and validated externally with several in-house and literature-derived data sets tested in cytotoxicity assays on different types on immune cells. Both cross-validations and external validations resulted in areas under the receiver operator curves (ROC/AUC) of 75% or higher. The classification of the validation data sets occurred with excellent specificities and fair to excellent selectivities, depending on the data set. This means that the probability of actual immunotoxicity is very high for compounds classified as immunotoxic, while the fraction of false negative predictions might vary. Thus, in a multistep immunotoxicity screening scheme, the classification as immunotoxic can be accepted without additional confirmation, while compounds classified as not immunotoxic will have to be subjected to further investigation.

Investigations on synthesis and structure elucidation of novel [1,2,4]triazolo[1,2-a]pyridazine-1-thiones and their inhibitory activity against inducible nitric oxide synthase.

The inducible nitric oxide synthase (iNOS) is a target of great research interest due to its importance in a number of diseases, for example, septic shock and inflammatory lung diseases. A variety of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine derivatives was synthesized by ring closure with hexahydropyridazine-1-carbothioamide by using aliphatic and aromatic aldehydes. The activity of the new substances was tested on the insulin-secreting rat insulinoma cell line RINm5F. iNOS was expressed through exposure to interleukin-1β (IL-1β) and interferon-γ (IFN-γ). A number of the investigated compounds were more active than the reference inhibitor aminoguanidine (AG). Structure-activity relationships showed that a phenyl substituent in position 3 is apparently essential for inhibition.

Differential effects of Helenalin, an anti-inflammatory sesquiterpene lactone, on the proteome, metabolome and the oxidative stress response in several immune cell types.

Extracts of Arnica spp. are traditionally used due to their anti-inflammatory effects for the topical treatment of e.g. haematoma or muscle distortions. One of the main active compounds is Helenalin, a sesquiterpene lactone that can be found in various Asteraceae. However, immunotoxic effects of the compound are only poorly analysed. In this study, a 2D gel electrophoresis based proteomic approach together with a membrane based proteomic assay, metabolomics and the detection of intracellular reactive oxygen species (iROS) were used to investigate potential immunotoxic properties of Helenalin on the human immune cell lines Jurkat and THP-1 and on human peripheral blood mononuclear cells (PBMC). The study revealed a dose-dependent cytotoxicity towards both tested cell lines and the PBMC. However, the cell lines were less sensitive to the Helenalin treatment than the PBMC. The proteomic assays showed strong effects on the carbohydrate metabolism and the protein folding in THP-1 cells but only weak impact on Jurkat cells. Metabolomic studies as well as iROS detection in THP-1 cells verified the results of the proteomic analysis. In summary, the approaches used in this study were able to identify target pathways of Helenalin especially in THP-1 monocytes and thus enable a risk assessment of the substance.