BackgroundThe JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK-STAT signaling pathways.MethodsPorcine lens epithelial cells, pigmented iris epithelial cells and pigmented ciliary body cells were used in this study. These cells were isolated from freshly enucleated porcine eyes by enzymatic digestion. Cultured cells between passages 3–8 were used in all experiments. Electromobility shift assay (EMSA), proliferation assay, immunofluorescence staining and flow cytometry were used to evaluate the JAK-STAT signaling pathway in these cells.ResultsJAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, in pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human interferons and cytokines. All cells showed very strong STAT1 activation upon stimulation with porcine interferon-gamma. Porcine ocular cells also respond to human cytokines; IFN-alpha induced strong activation of STAT1 in EMSA, flow cytometry and immunofluorescence experiments whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL-6 activated STAT3 and human IL-4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of porcine ocular cells with cytokines and interferons. Human IFN-α had an inhibitory effect on porcine ocular cells in proliferation assays.ConclusionOur study demonstrated that some types of human cytokines and interferon activate intracellular JAK-STAT signaling pathways in porcine ocular cells. We hypothesize that direct stimulation of the JAK-STAT pathway in porcine cells in response to human cytokines will lead to complications or failure, if pig-to-human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signaling pathways.
Hydrogels and copolymers of 2-hydroxyethylmethacrylate and 2-hydroxyethylacrylate structured with 3-mercaptopropyltrimethoxysilane nanoparticles of different composition and nanoparticles concentration have been synthesized and characterized using IR-spectroscopy, SEM, small angle X-ray scattering, compression mechanical test, thermogravimetric analysis, iodometry. Mucoadhesive properties of obtained hydrogels were investigated. Study revealed that increase in concentration of hydrophilic component, nanoparticles content and crosslinking agent concentration reflects an increase of adhesion time of obtained hydrogel materials. Drug loading and drug release studies of HEA-HEMA-MPTS samples with metronidazole were conducted. It was observed that thiolated samples absorb more drug amount than non-thiolated ones. As nanoparticles concentration increases metronidazole content rises respectively. Drug release studies revealed that the higher concentration of 3-MPTS nanoparticles in hydrogels results in prolongation of drug release but concentration of discharged metronidazole decreases.
Introduction The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. The use of porcine ocular cells (POC) as putative xenotransplants appears theoretically possible. We investigated cytokine‐induced changes of the JAK/STAT signaling pathways in POC in response to human cytokines. Methods POC were used to study the nuclear translocation of STAT proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy, flow and imaging cytometry. Results JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human cytokines. All POC showed very strong STAT1 activation upon stimulation with porcine interferon‐gamma. POC also respond to human cytokines; IFN‐alpha caused strong activation of STAT1 in all assays whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL‐6 activated STAT3 and human IL‐4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of POC with cytokines and interferons. Human IFN‐α had an inhibitory effect on POC in proliferation assays. Conclusion Our data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in POC. Human cytokines and interferon activate JAK‐STAT signalling pathway in POC. We hypothesize that direct stimulation of the JAK‐STAT pathway in POC in response to human cytokines will lead to complications or failure, if pig‐to‐human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signalling pathways. Grant Funding Source: University of Basel
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