The selective in vitro anti-tumor mechanisms of cold atmospheric plasma (CAP) and plasma-activated media (PAM) follow a sequential multi-step process. The first step involves the formation of primary singlet oxygen (¹O₂) through the complex interaction between NO₂⁻ and H₂O_2.¹O₂ then inactivates some membrane-associated catalase molecules on at least a few tumor cells. With some molecules of their protective catalase inactivated, these tumor cells allow locally surviving cell-derived, extracellular H₂O₂ and ONOO^─ to form secondary ¹O₂. These species continue to inactivate catalase on the originally triggered cells and on adjacent cells. At the site of inactivated catalase, cell-generated H₂O₂ enters the cell via aquaporins, depletes glutathione and thus abrogates the cell’s protection towards lipid peroxidation. Optimal inactivation of catalase then allows efficient apoptosis induction through the HOCl signaling pathway that is finalized by lipid peroxidation. An identical CAP exposure did not result in apoptosis for nonmalignant cells. A key conclusion from these experiments is that tumor cell-generated RONS play the major role in inactivating protective catalase, depleting glutathione and establishing apoptosis-inducing RONS signaling. CAP or PAM exposure only trigger this response by initially inactivating a small percentage of protective membrane associated catalase molecules on tumor cells.

Nitrite and H₂O₂ are long-lived species in cold atmospheric plasma and plasma-activated medium. It is known that their synergistic interaction is required for selective apoptosis induction in tumor cells that are treated with plasma-activated medium. This study shows that the interaction between nitrite and H₂O₂ leads to the formation of peroxynitrite, followed by singlet oxygen generation through the interaction between peroxynitrite and residual H₂O₂. This primary singlet oxygen causes local inactivation of few catalase molecules on the surface of tumor cells. As a consequence, H₂O₂ and peroxynitrite that are constantly produced by tumor cells and are usually decomposed by their protective membrane-associated catalase, are surviving at the site of locally inactivated catalase. This leads to the generation of secondary singlet oxygen through the interaction between tumor cell-derived H₂O₂ and peroxynitrite. This selfsustained process leads to autoamplification of secondary singlet oxygen generation and catalase inactivation. Inactivation of catalase allows the influx of H₂O₂ through aquaporins, leading to intracellular glutathione depletion and sensitization of the cells for apoptosis induction through lipid peroxidation. It also allows to establish intercellular apoptosis-inducing HOCl signaling, driven by active NOX1 and finalized by lipid peroxidation through hydroxyl radicals that activates the mitochondrial pathway of apoptosis. This experimentally established model is based on a triggering function of CAP and PAM-derived H₂O₂/nitrite that causes selective cell death in tumor cells based on their own ROS and RNS. This model explains the selectivity of CAP and PAM action towards tumor cells and is in contradiction to previous models that implicated that ROS/RNS from CAP or PAM were sufficient to directly cause cell death of tumor cells.

Purpose: This review is focused on the potential impact of low dose radiation effects on intercellular induction of apoptosis and the underlying reactive-oxygen species (ROS)-mediated signaling pathways. Results: Transformed cells are subject to ROS-mediated apoptosis induction by non-transformed cells (‘intercellular induction of apoptosis’) and by ROS-mediated autocrine self-destruction. Sensitivity to intercellular induction of apoptosis and autocrine self-destruction are strictly correlated to the expression of the transformed state. Extracellular superoxide anions generated by transformed target cells drive the selectivity and sensitivity of this signaling system which is based on four different signaling pathways. Low dose irradiation of non-transformed cells enhances intercellular induction of apoptosis in transformed cells. This process is controlled by TGF-beta and seems to depend on the induction of peroxidase release. In addition, low dose radiation enhances superoxide anion generation of transformed target cells. Conclusions: Low dose radiation-triggered enhancement of intercellular induction of apoptosis and autocrine selfdestruction might represent a potential control system during carcinogenesis. It might be the underlying mechanism for the well-known inhibitory effect of low dose radiation on detectable transformation events. However, modifications of the complex intercellular ROS-based signaling system may also lead to configurations in which low dose radiation attenuates ROS-mediated apoptosis induction.

Avidity is defined as the strength of binding between IgG and its specific target epitope. IgG of high avidity is established during affinity maturation. A failure to achieve high avidity IgG may result in the lack of protective immunity towards infection and disease. It is known that the interaction between SARS-CoV-2 spike protein and its cellular receptor is driven by high affinity. Therefore it is predictable that protective antibodies towards SARS-CoV-2 should show high affinity/avidity. Recent findings by several groups demonstrate that the serological response towards infection with SARS-CoV-2 as well as with seasonal coronaviruses is characterized by incomplete avidity maturation, followed by a decline of the serological response. This might facilitate reinfection, prevent herd immunity and potentially allow repeated cycles of infection. Therefore, the sole focus on antibody titers reached after vaccination towards SARS-CoV-2 might not be sufficient to evaluate the degree of achieved protection. Rather, it is suggested to include avidity determination into the optimization of vaccination protocols and to try to achieve high avidity IgG directed towards SARS-CoV-2 through vaccination. Avidity determination also might be useful to control for truly protective immunity towards SARS-CoV-2 in individual cases.

Tumor cells are protected against intercellular reactive oxygen species (ROS)-mediated apoptosis signaling mediated by the HOCl and/or the nitric oxide (NO)/peroxynitrite signaling pathway. We have recently shown that tumor cell resistance against HOCl signaling can be abrogated through inhibition of catalase. The protection of tumor cells against the NO/peroxynitrite signaling pathway has remained enigmatic so far. Here, we show that suboptimal inhibition of catalase by 3-aminotriazole or a monoclonal antibody against catalase, as well as partial knockdown of catalase by specific siRNA allows selective reactivation of the NO/peroxynitrite pathway in MKN 45 gastric carcinoma cells, followed by the HOCl pathway at higher inhibitor or siRNA concentrations. In SKN-MC Ewing sarcoma cells, catalase inhibition causes apoptosis induction solely based on the NO/peroxynitrite pathway. Protection against NO/peroxynitrite signaling is shown to be due to the potential of catalase to decompose peroxynitrite. The direct interaction of catalase with peroxynitrite is verified through the detection of compound I (CAT Fe^IV=O⁺·) after the interaction of peroxynitrite with catalase. In a complementary experiment, addition of catalase protects sensitive transformed cells against ROS-mediated apoptosis induction. Thus, the expression of membrane-associated catalase is sufficient to protect tumor cells against multiple intercellular ROS-mediated signaling pathways.