Project description:Treatment of tumor cells with H2O2 and nitrite, two long-lived species derived from cold atmospheric plasma, induces a complex autoamplificatory, singlet oxygen-mediated process, which leads to catalase inactivation and reactivation of intercellular apoptosis-inducing signaling. Experimental dissection and quantification of this process is described in this study. When tumor cells were pretreated with H2O2 and nitrite, and then were added to untreated tumor cells, they propaged singlet oxygen mediated catalase inactivation and generation of singlet oxygen to the untreated cell population. This bystander effect allowed to analyze the biochemical requirements of a) induction of the bystander effect-inducing potential, b) transmission of the bystander effect to untreated neighbouring cells, and c) the biochemical consequences of these signaling events. The induction of bystander effect-inducing potential requires the generation of "primary singlet oxygen" through the reactions following the interaction between nitrite and H2O2, followed by local inactivation of a few catalase molecules. This primary effect seems to be very rare, but is efficiently enhanced by the generation of "secondary singlet oxygen" through the interaction between H2O2 and peroxynitrite at the site of inactivated catalase. Transmission of bystander signaling between pretreated and untreated tumor cells depends on the generation of secondary singlet oxygen by the pretreated cells and singlet oxygen-mediated catalase inactivation of the untreated recipient cells. This induces autoamplificatory propagation of secondary singlet oxygen generation in the population. This experimental approach allowed to quantify the efficiencies of primary and secondary singlet oxgen generation after CAP and PAM action, to dissect the system and to study the underlying chemical biology in detail. Our data confirm that CAP and PAM-derived components are merely the trigger for the activation of autoamplificatory mechanisms of tumor cells, whereas the tumor cells efficiently propagate their cell death through their own ROS/RNS signaling potential.

Project description:Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking. The objective of this study is to develop experimental models (monolayer, multilayer, and multicellular spheroid co-cultures) comprising 'activator' cells with high expression of prodrug-activating reductases and reductase-deficient 'target' cells, and to couple these with agent-based models (ABMs) that describe diffusion and reaction of prodrugs and their active metabolites, and killing probability for each cell. HCT116 cells were engineered as activators by overexpressing P450 oxidoreductase (POR) and as targets by knockout of POR, with fluorescent protein and antibiotic resistance markers to enable their quantitation in co-cultures. We investigated two HAPs with very different pharmacology: SN30000 is metabolized to DNA-breaking free radicals under hypoxia, while the dinitrobenzamide PR104A generates DNA-crosslinking nitrogen mustard metabolites. In anoxic spheroid co-cultures, increasing the proportion of activator cells decreased killing of both activators and targets by SN30000. An ABM parameterized by measuring SN30000 cytotoxicity in monolayers and diffusion-reaction in multilayers accurately predicted SN30000 activity in spheroids, demonstrating the lack of bystander effects and that rapid metabolic consumption of SN30000 inhibited prodrug penetration. In contrast, killing of targets by PR104A in anoxic spheroids was markedly increased by activators, demonstrating that a bystander effect more than compensates any penetration limitation. However, the ABM based on the well-studied hydroxylamine and amine metabolites of PR104A did not fit the cell survival data, indicating a need to reassess its cellular pharmacology. Characterization of extracellular metabolites of PR104A in anoxic cultures identified more stable, lipophilic, activated dichloro mustards with greater tissue diffusion distances. Including these metabolites explicitly in the ABM provided a good description of activator and target cell killing by PR104A in spheroids. This study represents the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the power of combining mathematical modeling of pharmacokinetics/pharmacodynamics with multicellular culture models to dissect bystander effects of targeted drug carriers.

Project description:BACKGROUND:Violence against women and girls is a public health epidemic. Campus-based research has found bystander programmes show promise as effective primary prevention of sexual violence. However, evidence regarding domestic violence and abuse bystander prevention specifically, and in community settings generally, is still in development. Further, research has predominantly emanated from the US. Examining proof of concept in differing cultural contexts is required. This study evaluates the feasibility and potential for effectiveness of a domestic violence and abuse bystander intervention within UK general communities-Active Bystander Communities. METHODS:Participants recruited opportunistically attended a three-session programme facilitated by experts in the field. Programme feasibility was measured using participant attendance and feedback across nine learning objectives. Myth acceptance, bystander efficacy, behavioural intent and bystander behaviours were assessed using validated scales at baseline, post-intervention, and four-month follow-up. Results were examined for potential backlash. Analyses used a paired sample t-test and effect size was quantified with Cohen's d. RESULTS:58/70 participants attended all programme sessions. Participant feedback consistently rated the programme highly and significant change (p???0·001) was observed in the desired direction across behavioural intent, bystander efficacy, and myth acceptance scores at post and follow-up. Effect size was generally large and, with the exception of Perception of Peer Myth Acceptance, improved at follow-up. Backlash was minimal. CONCLUSIONS:To our knowledge this is the first UK-based study to examine the potential of bystander intervention as a community-level intervention for domestic violence and abuse. Findings are promising and indicate the translatability of the bystander approach to domestic violence and abuse prevention as well as community contexts. This is likely to be of great interest to policymakers and may help shape future community-based interventions. Further research is now needed using experimental designs engaging diverse community audiences.

Project description:Radiotherapy has extensively been employed as a curative or palliative intervention against cancer throughout the last century, with a varying degree of success. For a long time, the antineoplastic activity of X- and ?-rays was entirely ascribed to their capacity of damaging macromolecules, in particular DNA, and hence triggering the (apoptotic) demise of malignant cells. However, accumulating evidence indicates that (at least part of) the clinical potential of radiotherapy stems from cancer cell-extrinsic mechanisms, including the normalization of tumor vasculature as well as short- and long-range bystander effects. Local bystander effects involve either the direct transmission of lethal signals between cells connected by gap junctions or the production of diffusible cytotoxic mediators, including reactive oxygen species, nitric oxide and cytokines. Conversely, long-range bystander effects, also known as out-of-field or abscopal effects, presumably reflect the elicitation of tumor-specific adaptive immune responses. Ionizing rays have indeed been shown to promote the immunogenic demise of malignant cells, a process that relies on the spatiotemporally defined emanation of specific damage-associated molecular patterns (DAMPs). Thus, irradiation reportedly improves the clinical efficacy of other treatment modalities such as surgery (both in neo-adjuvant and adjuvant settings) or chemotherapy. Moreover, at least under some circumstances, radiotherapy may potentiate anticancer immune responses as elicited by various immunotherapeutic agents, including (but presumably not limited to) immunomodulatory monoclonal antibodies, cancer-specific vaccines, dendritic cell-based interventions and Toll-like receptor agonists. Here, we review the rationale of using radiotherapy, alone or combined with immunomodulatory agents, as a means to elicit or boost anticancer immune responses, and present recent clinical trials investigating the therapeutic potential of this approach in cancer patients.

Project description:Background: Workplace bullying is an important and prevalent risk factors for health impairment, reduced workability and lowered efficiency among both targets and observers. Development and tests of effective organizational intervention strategies are therefore highly important. The present study describes the background, design, and protocol of a cluster randomized controlled trial evaluating the effectiveness of an organization-wide intervention on preventing workplace bullying with a focus on promoting active and constructive bystander behavior. The main overarching goal is to develop an easy to use and standardized organizational intervention based on theory and research in the role of bystanders in bullying situations with the potential of reducing the prevalence of workplace bullying. The theoretical framework of the study is theory of planned behavior (TPB; Ajzen, 1991).Methods/Design: Using a full randomized control trial (RCT) design, this project will empirically test the outcomes of an intervention program targeting bullying and harassment as the main distal outcomes and perceived behavioral control and helping behavior among bystanders as the main proximal outcome. A 1-year cluster randomized controlled design will be utilized, in which controls will also receive the intervention. About 1,500 workers from two different locations of a Norwegian industrial company will be randomized into one intervention group and two control groups with at least 400 workers in each group. A survey will be conducted electronically. With a total of three assessments over 10–12 months, the time interval between the measurement times will be 4 months. Thus, the data collection will take place at baseline, completion of the intervention and at 4 months follow-up.Discussion: This study primarily aims to develop, implement, and evaluate an intervention based on the abovementioned features with the ultimate aim of reducing the prevalence of workplace bullying, by awareness raising and training of bystanders. Manager involvement and involvement of the union representative and the elected health and safety representatives is an important feature of the program. Results of the intervention study will provide important information regarding the effectiveness of preventive interventions against workplace bullying when focusing on bystanders, particularly so regarding the role of bystander awareness, bystander self-efficacy, and bystander behavioral control on the one hand and the prevalence of bullying and harassment on the other.

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