Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.
Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.
Key Points• Administration of donorspecific regulatory T cells prevents chronic rejection of BM and skin allografts in the mouse.• Injected regulatory T cells induce the emergence of host regulatory T cells with similar specificity thus ensuring persistence of tolerance.Despite the use of immunosuppressive drugs, chronic allograft rejection remains a major hurdle in transplantation medicine. Induction of specific immunologic tolerance to antigens expressed by the graft would avoid its eventual functional loss and the severe side effects of paralyzing the immune system. We previously showed that donorspecific regulatory T-lymphocytes prevent rejection of fully allogeneic bone marrow (BM) grafts in mice. Thus generated hematopoietic chimeras then accepted skin and heart allografts of the same donor. We noticed that injected regulatory T-cells (Tregs) disappeared with time and investigated the mechanisms involved in the nevertheless longterm persistence of allograft tolerance. Using Tregs that can be depleted in vivo with diphtheria toxin, we show that injected cells are required for induction but not for maintenance of tolerance to BM allografts. We observed progressive deletion of donorspecific T-lymphocytes, accounting at least in part for maintenance of tolerance. Toxin-induced depletion of administered as well as host Tregs did not affect hematopoietic chimerism but it led to rapid loss of skin allografts. Therefore, our data show that newly generated host Tregs can prevent chronic allograft rejection. Long-lasting tolerance to allografts is thus achieved. (Blood. 2013;121(21):4303-4310)
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