Regulatory T Cell Extracellular Vesicles Modify T-Effector Cell Cytokine Production and Protect Against Human Skin Allograft Damage

Regulatory T cells (Tregs) are a subpopulation of CD4⁺ T cells with a fundamental role in maintaining immune homeostasis and inhibiting unwanted immune responses using several different mechanisms. Recently, the intercellular transfer of molecules between Tregs and their target cells has been shown via trogocytosis and the release of small extracellular vesicles (sEVs). In this study, CD4⁺CD25⁺CD127ˡᵒ human Tregs were found to produce sEVs capable of inhibiting the proliferation of effector T cells (Teffs) in a dose dependent manner. These vesicles also modified the cytokine profile of Teffs leading to an increase in the production of IL-4 and IL-10 whilst simultaneously decreasing the levels of IL-6, IL-2, and IFNγ. MicroRNAs found enriched in the Treg EVs were indirectly linked to the changes in the cytokine profile observed. In a humanized mouse skin transplant model, human Treg derived EVs inhibited alloimmune-mediated skin tissue damage by limiting immune cell infiltration. Taken together, Treg sEVs may represent an exciting cell-free therapy to promote transplant survival

Pattern Recognition Analysis of Proton Nuclear Magnetic Resonance Spectra of Brain Tissue Extracts from Rats Anesthetized with Propofol or Isoflurane

BACKGROUND: General anesthesia is routinely used as a surgical procedure and its safety has been endorsed by clinical outcomes; however, its effects at the molecular level have not been elucidated. General anesthetics influence glucose metabolism in the brain. However, the effects of anesthetics on brain metabolites other than those related to glucose have not been well characterized. We used a pattern recognition analysis of proton nuclear magnetic resonance spectra to visualize the changes in holistic brain metabolic phenotypes in response to the widely used intravenous anesthetic propofol and the volatile anesthetic isoflurane. METHODOLOGY/PRINCIPAL FINDINGS: Rats were randomized into five groups (n = 7 each group). Propofol and isoflurane were administered to two groups each, for 2 or 6 h. The control group received no anesthesia. Brains were removed directly after anesthesia. Hydrophilic compounds were extracted from excised whole brains and measured by proton nuclear magnetic resonance spectroscopy. All spectral data were processed and analyzed by principal component analysis for comparison of the metabolite profiles. Data were visualized by plotting principal component (PC) scores. In the plots, each point represents an individual sample. The propofol and isoflurane groups were clustered separately on the plots, and this separation was especially pronounced when comparing the 6-h groups. The PC scores of the propofol group were clearly distinct from those of the control group, particularly in the 6-h group, whereas the difference in PC scores was more subtle in the isoflurane group and control groups. CONCLUSIONS/SIGNIFICANCE: The results of the present study showed that propofol and isoflurane exerted differential effects on holistic brain metabolism under anesthesia

Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure

Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF) providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use.Human hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.The optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10(6) cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.An optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation

Use of indocyanine green for functional assessment of human hepatocytes for transplantation

Hepatocyte transplantation is a promising alternative to liver transplantation in children with liver metabolic disorders and acute liver failure. Currently, it is difficult to assess rapidly hepatocyte function before transplantation. The aim of this study was to investigate whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used. Human hepatocytes (106 cells) isolated from unused donor livers were incubated at 37°C for 30 minutes with ICG (0–2 mg/mL) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 hours with supernatants collected at 1, 2 and 3 hours for measurement of ICG release. Cell viability was determined by trypan blue exclusion, (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (mitochondrial dehydrogenase activity) and sulforhodamine B (SRB) assay (cell attachment). HepG2 cells were also used. ICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG > 1.0 mg/mL had toxic effects on hepatocytes. Hepatocytes incubated with 1.0 mg/mL ICG had higher mitochondrial dehydrogenase activity compared to 0.5 mg/mL ICG or control cells (0.025 ± 0.0004 OD unit vs. 0.019 ± 0.0008 OD unit or 0.020 ± 0.002 OD unit, p < 0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9 ± 0.02 ng/mL, 66.6 ± 0.05 ng/mL and 39.1 ± 0.4 ng/mL for control cells, and 0.5 mg/mL and 1.0 mg/mL ICG, respectively), and decreased [3H]-thymidine incorporation in a dose-dependent manner. Addition of taurine (20 mM) to plated hepatocytes gave greater release of ICG and hepatocyte attachment compared to controls, at all ICG concentrations (SRB 1.360 ± 0.083 optical density units vs. 0.908 ± 0.159 optical density units, p = 0.011 at 1.0 mg/mL). With further refinement, ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes

Fibroblast state switching orchestrates dermal maturation and wound healing

SummaryMurine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual-tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue-scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.Standfirst textWe show that fibroblast behaviour switching between two distinct states – proliferating and depositing ECM - is necessary and sufficient to define dermal architecture. Understanding this interdependence is critical for identifying new therapeutic strategies to enhance skin regeneration.HighlightsTissue-scale coordination in murine dermis is driven by the interdependence of cell proliferation and ECM depositionThe tissue architecture is set by a negative feedback loop between ECM deposition/remodelling and proliferationFibroblast lineages lose segregation with ageFibroblast migration is the critical discriminator between dermal development and wound healing</jats:sec

Use of indocyanine green for functional assessment of human hepatocytes for transplantation

SummaryBackground/ObjectiveHepatocyte transplantation is a promising alternative to liver transplantation in children with liver metabolic disorders and acute liver failure. Currently, it is difficult to assess rapidly hepatocyte function before transplantation. The aim of this study was to investigate whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used.MethodsHuman hepatocytes (106 cells) isolated from unused donor livers were incubated at 37°C for 30 minutes with ICG (0–2mg/mL) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 hours with supernatants collected at 1, 2 and 3 hours for measurement of ICG release. Cell viability was determined by trypan blue exclusion, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (mitochondrial dehydrogenase activity) and sulforhodamine B (SRB) assay (cell attachment). HepG2 cells were also used.ResultsICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG > 1.0mg/mL had toxic effects on hepatocytes. Hepatocytes incubated with 1.0mg/mL ICG had higher mitochondrial dehydrogenase activity compared to 0.5mg/mL ICG or control cells (0.025±0.0004 OD unit vs. 0.019±0.0008 OD unit or 0.020±0.002 OD unit, p<0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9±0.02ng/mL, 66.6±0.05ng/mL and 39.1±0.4ng/mL for control cells, and 0.5mg/mL and 1.0mg/mL ICG, respectively), and decreased [3H]-thymidine incorporation in a dose-dependent manner. Addition of taurine (20mM) to plated hepatocytes gave greater release of ICG and hepatocyte attachment compared to controls, at all ICG concentrations (SRB 1.360±0.083 optical density units vs. 0.908±0.159 optical density units, p=0.011 at 1.0mg/mL).ConclusionWith further refinement, ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes