Cytoprotective mechanisms in cultured cardiomyocytes

Tumor necrosis factor-α (TNF-α), a potent cytokine mainly secreted by macrophages exerts pleiotropic effects on different cell types. However, the intracellular mediators of its action are not yet well characterized. To get an insight into endogenous cytoprotective mechanisms, we developed an in vitro model based on cultured cardiomyocytes treated with TNF-α at which we examined gene expression of heat shock proteins (HSP-27, HSP-70 and ubiquitin). Cardiomyocytes were isolated from the hearts of 18 day old fetal mice by enzymatic dissociation and grown in minimum essential medium containing 10% fetal calf serum. Spontaneously contractile cells were serum deprived for 24 h and treated with TNF-α(25 ng/ml) for 1, 2, 4, 6, 8, 12, and 24 h After each incubation, cells were processed to extract total proteins for Western and total RNA for Northern blot analyses. TNF-α induced arrhythmias and cessation of spontaneous contractions in a concentration and time dependent manner. Steady state (ubiquitin) or undetectable mRNA levels (HSP-27, HSP-70) were drastically induced (> 4 fold for all three genes vs untreated control cells) by TNF-α, reaching maximal values between 6-8 h of stimulation. Thereafter, the expression of these stress genes declined but remained elevated as compared to control. By Western blot analysis, we found increased multiple bands of ubiquitin protein conjugates in TNF-α treated cells whereas no significant change in HSP-27 protein accumulation until 12 h was observed as compared to control. 24 h of TNF-α incubation resulted in partial cellular necrosis. Our results indicate that TNF-α induces in cardiomyocytes transiently gene expression for cytoprotective molecules like HSP-27, HSP-70 and ubiquitin, suggesting these stress proteins to participate in subsequent defense mechanisms, for example in postischemic myocardial recovery

t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

A Modular Cell-Type Focused Inflammatory Process Network Model for Non-Diseased Pulmonary Tissue

Exposure to environmental stressors such as cigarette smoke (CS) elicits a variety of biological responses in humans, including the induction of inflammatory responses. These responses are especially pronounced in the lung, where pulmonary cells sit at the interface between the body's internal and external environments. We combined a literature survey with a computational analysis of multiple transcriptomic data sets to construct a computable causal network model (the Inflammatory Process Network (IPN)) of the main pulmonary inflammatory processes. The IPN model predicted decreased epithelial cell barrier defenses and increased mucus hypersecretion in human bronchial epithelial cells, and an attenuated pro-inflammatory (M1) profile in alveolar macrophages following exposure to CS, consistent with prior results. The IPN provides a comprehensive framework of experimentally supported pathways related to CS-induced pulmonary inflammation. The IPN is freely available to the scientific community as a resource with broad applicability to study the pathogenesis of pulmonary disease

Integrated Testing Strategies (ITS) for safety assessment.

International audienceIntegrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and "Good ITS Practices"