Quantitative Comparison of HSF1 Activators.

The heat shock response (HSR) pathway is a highly conserved rescue mechanism, which protects the cells from harmful insults disturbing the cellular protein homeostasis via expression of chaperones. Furthermore, it was demonstrated to play crucial roles in various diseases like neurodegeneration and cancer. For neurodegenerative diseases, an overexpression of chaperones is a potential therapeutic approach to clear the cells from non-functional protein aggregates. Therefore, activators of the HSR pathway and its master regulator HSF1 are under close observation. There are numerous HSR activators published in the literature using different model systems, experimental designs, and readout assays. The aim of this work was to provide a quantitative comparison of a broad range of published activators using a newly developed HSF responsive dual-luciferase cell line. Contrary to natural target genes, which are regulated by multiple input pathways, the artificial reporter exclusively reacts to HSF activity. In addition, the results were compared to endogenous heat shock protein expression. As a result, great differences in the intensity of pathway activation were observed. In addition, a parallel viability assessment revealed high variability in the specificity of the drugs. Furthermore, the differences seen compared to published data indicate that some activators exhibit tissue-specific differences leading to interesting assumptions about the regulation of HSF1.

A dual luciferase assay for evaluation of skin sensitizing potential of medical devices.

According to standing regulations animal tests are still state of the art for the evaluation of the sensitization potential of medical devices. The aim of our study was to develop an in vitro method that can be used for testing of extracts of medical devices. The novel MDA-ARE assay is a cell based reporter gene assay focused on the ARE-Nrf2 pathway, which is involved in the dermal sensitization process. Optimization of the reporter construct and the cell line resulted in an improvement of the detection limit and a reduction of the incubation time to 6 h, which lowers cytotoxic side effects of the extracts on the cells. Using the assay, 21 out of 22 pure chemicals were identified correctly as skin sensitizers or non-sensitizers. All sensitizers could be detected at far lower concentrations compared to the local lymph node assay, the state-of-the-art animal test. To evaluate the assay's suitability for the testing of medical devices, medical grade silicone containing 0.1% of known skin sensitizers was prepared as positive controls and extracts of these positive controls were tested in comparison to extracts from pure silicone samples. All silicone samples were correctly and reproducibly identified as sensitizing or non-sensitizing demonstrating that the MDA-ARE assay is a sensitive and reliable tool for the detection of skin sensitizers in extracts of medical devices. The developed and validated test protocol was used for medical device extracts and showed its applicability for real samples and thus can contribute to reduce or even to replace the need for animal tests.

HSF1 mediated stress response of heavy metals.

The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1.

Functionalization of 3D scaffolds with protein-releasing biomaterials for intracellular delivery.

Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300µm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80% of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups.