Förderzeitraum 2020
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Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies.
Exon-4 Mutations in KRAS Affect MEK/ERK and PI3K/AKT Signaling in Human Multiple Myeloma Cell Lines
(2020)
Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS. However, there is still no final consent on whether KRAS-mutations are associated with disease outcome. Specifically, no data exist on whether KRAS-mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRAS\(^{p.G12C}\) entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRAS\(^{p.G12A}\) and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRAS\(^{WT}\), KRAS\(^{p.G12A}\), KRAS\(^{p.A146T}\), and KRAS\(^{p.A146V}\) were overexpressed in HEK293 cells and the KRAS\(^{WT}\) MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS-mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.