Background: A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease‐induced substances is highly desirable.Methods: Bone marrow‐derived equine MSCs were transduced with a lentiviral vector expressing interleukin‐1 receptor antagonist (IL‐1Ra) gene under the control of an inducible nuclear factor‐kappa B‐responsive promoter and IL‐1Ra production upon pro‐inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)‐1β] was analysed. To assess the biological activity of the IL‐1Ra protein that was produced and the therapeutic effect of IL‐1Ra‐expressing MSCs (MSC/IL‐1Ra), cytokine‐based two‐ and three‐dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real‐time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin‐1β, interleukin‐6, interleukin‐8, matrix metalloproteinase‐1 and matrix metalloproteinase‐13.Results: A dose‐dependent increase in IL‐1Ra expression was found in MSC/IL‐1Ra cells upon TNFα administration, whereas stimulation using IL‐1β did not lead to IL‐1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL‐1Ra protein present in the conditioned medium from MSC/IL‐1Ra cells blocks OA onset in cytokine‐treated equine chondrocytes and co‐cultivation of MSC/IL‐1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes.Conclusions: Thus, pro‐inflammatory cytokine induced IL‐1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.

Corticosteroid administration prior to the application of chemotherapy in small animal lymphoma patients is a concern, as it is discussed to negatively influence the therapeutic outcome due to corticosteroid-induced drug resistance. Using feline lymphoma cell lines FT-1 and MS4 we have shown, that prednisolone pre-treatment alters the susceptibility of these cells towards doxorubicin or vincristine treatment in vitro. The observed effect was negative as for the killing potential and it was cell line and drug (doxorubicin or vincristine) dependent. Furthermore, increase in mRNA expression of selected proteins with multidrug resistance potential (MDR1, BCRP, LRP, MT) was observed after prednisolone pre-treatment. Administration of chemical inhibitors of these proteins did not lead to reversal in sensitivity of tested cell lines to doxorubicin or vincristine.

Lentiviruses are suitable to transfer potential therapeutic genes into non-replicating cells such as neurons, but systematic in vivo studies on transduction of neural cells within the complete brain are missing. We analysed the distribution of transduced cells with respect to brain structure, virus tropism, numbers of transduced neurons per brain, and influence of the Vpx or Vpr accessory proteins after injection of vectors based on SIVsmmPBj, HIV-2, and HIV-1 lentiviruses into the right striatum of the mouse brain. Transduced cells were found ipsilaterally around the injection canal, in corpus striatum and along corpus callosum, irrespective of the vector type. All vectors except HIV-2SEW transduced also single cells in the olfactory bulb, hippocampus, and cerebellum. Vector HIV-2SEW was the most neuron specific. However, vectors PBjSEW and HIV-1SEW transduced more neurons per brain (means 41,299 and 32,309) than HIV-2SEW (16,102). In the presence of Vpx/Vpr proteins, HIV-2SEW(Vpx) and HIV-1SEW(Vpr) showed higher overall transduction efficiencies (30,696 and 27,947 neurons per brain) than PBjSEW(Vpx) (6636). The distances of transduced cells from the injection canal did not differ among the viruses but correlated positively with the numbers of transduced neurons. The presence of Vpx/Vpr did not increase the numbers of transduced neurons. Parental virus type and the vector equipment seem to influence cellular tropism and transduction efficiency. Thus, precision of injection and choice of virus pseudotype are not sufficient when targeted lentiviral vector transduction of a defined brain cell population is required.

The present study aimed to establish novel canine osteosarcoma cell lines (COS3600, COS3600B, COS4074) and characterize the recently described COS4288 cells. The established D-17 cell line served as a reference. Analyzed cell lines differed notably in their biological characteristics. Calculated doubling times were between 22 h for COS3600B and 426 h for COS4074 cells. COS3600B and COS4288 cells produced visible colonies after anchorage-independent growth in soft agar. COS4288 cells were identified as cells with the highest migratory capacity. All cells displayed the ability to invade through an artificial basement membrane matrix. Immunohistochemical analyses revealed the mesenchymal origin of all COS cell lines as well as positive staining for the osteosarcoma-relevant proteins alkaline phosphatase and karyopherin α2. Expression of p53 was confirmed in all tested cell lines. Gene expression analyses of selected genes linked to cellular immune checkpoints (CD270, CD274, CD276), kinase activity (MET, ERBB2), and metastatic potential (MMP-2, MMP-9) as well as selected long non-coding RNA (MALAT1) and microRNAs (miR-9, miR-34a, miR-93) are provided. All tested cell lines were able to grow as multicellular spheroids. In all spheroids except COS4288, calcium deposition was detected by von Kossa staining. We believe that these new cell lines serve as useful biological models for future studies.

The ezrin/radixin/moesin proteins play a central role in cross-linking plasma membrane proteins with the actin cytoskeleton. Despite intensive ERM protein research in many tissues and pathologies, little is known about these proteins in healthy tissues of reproductive organs. Therefore, we examined ezrin, phosphorylated ezrin/radixin/moesin (pan-pERM), radixin, and moesin distribution at the cellular level by means of immunohistochemistry in ovaries of the following animal species: mouse, dog, cat, sheep, pig, horse, and cynomolgus monkey. Ezrin was expressed in oocytes, ovarian surface, granulosa cells and corpus luteum. A characteristic, predominantly membranous pan-pERM staining pattern was observed in ovarian surface epithelium, oocyte, granulosa cells and corpus luteum. Moesin immunoreactivity was present in all ovarian structures with a prominent signal in endothelial cells of blood vessels. Oocytes, granulosa cells and corpus luteum revealed mainly nuclear radixin staining. Staining pattern and subcellular localization (membranous, cytoplasmic, nuclear) varied between different animal species and between particular ERM proteins as well. This data may help gain new insights into the physiological function of ERM proteins in biological events in the female reproductive system.