Biodistribution Investigations of Technetium-Labelllled Murine Bone Marrow-Derived Extracellllular Vesicles by Nanospect/Ct

The in vivo tracing of the biodistribution of extracellular vesicles (EVs) is a pre-requisite in identifying their target cells and understanding their function. Although luorescent labelling of EVs is already used, radiolabelling can provide more details in understanding biodistribution of EVs. In the present paper we report radiolabelling of bone marrow-derived EVs and in vivo tracing of their biodistribution. EVs isolated from the bone marrow supernatant of űő7ŰL/6 mice were labelled with the technetium-99m (99mTc) isotope. Labelling was eficient and labelled EVs were stable during the 2Ő hours follow-up. Detection of labelled EVs after intravenous injection in mice was performed using ex vivo measurements and in vivo imaging. Ex vivo examinations revealed that at Ő hours post-injection, the highest accumulation rate was in the liver, kidney, spleen and femur epiphysis. In vivo imaging using nanoSPEűT/űT conirmed the ex vivo examinations and demonstrated slow elimination of the radioactivity, 2Ő hours post- injection the bone marrow-containing epiphysis and lymph nodes showed the highest retention values; liver, spleen and kidney were also clearly detectable. In summary, labelling of bone marrow-derived EVs with 99mTc coupled with SPEűT/űT detection was a reliable method for quantitative distribution studies of EVs in vivo

Extracellular vesicles mediate radiation-induced systemic bystander signals in the bone marrow and spleen

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EV) in mediating radiation-induced bystander signals but in vivo investigations are still lacking. Here we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), 24 hours later EVs were isolated from the bone marrow and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similarly to direct radiation EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of 8 differentially expressed miRNAs were identified in the EVs originating from both low and high dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. ​ In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms

Realising the European network of biodosimetry: RENEB-status quo

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed

Effects of Low Dose Radiation on the Main Immune Parameters in Healthy Mice

Objectives: We investigated the effect of low dose ionizing radiation on the quantitative and qualitative changes of major immune parameters in healthy mice. Methods: To study radiation effects on various lymphocyte subsets, mice were irradiated with different acute doses (0.01, 0.05, 0.1, 0.5, 1, 2 and 4 Gy) of Co60-? rays. Animals were killed at different time points after irradiation, and splenocytes were isolated. Quantitative and functional changes were determined in the Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), Treg (CD4+CD25+), NK (NK1.1+) and DC (MHCII+CD11c+) cells. The ratio of various lymphocyte subsets was determined by flow cytometry. The apoptotic rate of the cells was determined by TUNEL assay 4 hours after the in vivo irradiation of the animals. The proliferative response of lymphocytes to non-specific stimuli (Concanavalin A) was determined. The T-cell activation potential of irradiated DCs was studied in mixed lymphocyte culture. Cytokine expression of irradiated lymphocytes was investigated by real-time RT-PCR. Results: Flow cytometry data show that low dose irradiation affects the main compartments of T-cell immunity, but ample differences exist in the radiosensitivity of various cellular compartments, with the CD3+CD8+ compartment being the most radiosensitive and the CD4+CD25+ compartment being the most radioresistant. Certain lymphocyte subsets presented hypersensitivity to radiation at low doses (10, 50 and 100 mGy). The proliferation rate of ConA stimulated lymphocytes was either not affected or slightly depressed after irradiation with low doses. Higher doses led to a marked decrease in lymphocyte proliferation. Spontaneous apoptosis rate was around 3% and doses up to 0.1 Gy did not change apoptosis frequency. A steep increase in the apoptotic rate was detected after irradiation with doses of 0.5 Gy or above. The capacity of splenic DCs to activate allogeneic T cells was investigated three days after the irradiation of the animals. Preliminary data showed, that an increased T cell activation could be detected, if DCs were irradiated with low doses. The ratio of IFN-? expression in the ConA stimulated versus unstimulated lymphocytes showed a dose-dependent decrease. Conclusion: The experiments suggest that even low doses of ionizing radiation might have substantial impact on various compartments of the immune system. Different lymphocyte subpopulations react in very different ways to irradiation, which clearly point to a heterogeneous radiation response of the immune cells mostly involved in the anti-tumor immune response

Out-of-field effects: Lessons learned from partial body exposure.

Partial body exposure and inhomogeneous dose delivery are features of the majority of medical and occupational exposure situations. However, mounting evidence indicates that the effects of partial body exposure are not limited to the irradiated area but also have systemic effects that are propagated outside the irradiated field. It was the aim of the "Partial body exposure" session within the MELODI workshop 2020 to discuss recent developments and insights into this field by covering clinical, epidemiological, dosimetric as well as mechanistic aspects. Especially the impact of out-of-field effects on dysfunctions of immune cells, cardiovascular diseases and effects on the brain were debated. The presentations at the workshop acknowledged the relevance of out-of-field effects as components of the cellular and organismal radiation response. Furthermore, their importance for the understanding of radiation-induced pathologies, for the discovery of early disease biomarkers and for the identification of high-risk organs after inhomogeneous exposure was emphasized. With the rapid advancement of clinical treatment modalities, including new dose rates and distributions a better understanding of individual health risk is urgently needed. To achieve this, a deeper mechanistic understanding of out-of-field effects in close connection to improved modelling was suggested as priorities for future research. This will support the amelioration of risk models and the personalization of risk assessments for cancer and non-cancer effects after partial body irradiation