Génsebészeti módszerekkel sugárérzékenyített daganatsejtek sugárterápiája és angiogenezist gátló terápia kombinációja kísérletes agytumorok kezelésében

A pályázat keretében a daganatok sugárézékenységét kívántuk fokozni génterápiás módszerekkel. A daganatsejtbe olyan enzimek génjét juttattuk, amelyek sugárérzékenyítő kemoterápiás szerek intracelluláris aktivációjáért felelősek. Célunk az volt, hogy lokálisan egyaránt javítsuk a kemoterápiás szerek toxikus és sugárérzékenyítő hatását.Elsőként a gemcitabin intracelluláris aktivációjáért felelős deoxicitidin kináz (dCK) fokozott expressziójával próbáltuk meg a gemcitabin terápiás hatását javítani. Kísérleteinket az egér Gl261, a humán U373, a patkány C6 és 9L glioma modellekben végeztük el. Eredményeink szerint az alap dCK expresszió jelentősen különbözött a négy sejtvonalban, mindazonáltal a génbevitel nagy mértékben fokozta a dCK expressziót valamennyi sejtvonalban. A fokozott dCK expresszió javította a gemcitabin toxikus és sugárérzékenyítő hatását is in vitro és in vivo. Mindazonáltal a hatás jelentős mértékben sejttípus függő volt.Továbbiakban bebizonyítottuk, hogy az 5-fluorouracil (5-FU) aktiválására képes foszforibozil transzferáz expressziójának fokozása a Gl261 sejtekben jelentős mértékben javította az egerek túlélését akkor, ha az 5-FU terápiát sugárterápiával és ganciklovir kezeléssel is kombináltuk. Jelentős bystander hatást is kimutattunk.Kimutattuk továbbá, hogy a nitrogén oxid szintáz (NOS) fokozott expressziója jelentős sugárérzékenyítő hatással rendelkezik, és képes fokozni a ciszplatin daganatpusztító hatását a Gl261 modellen. | Our task was to increase tumor radiosensitivity by the means of gene therapy. We introduced genes encoding for enzymes responsible for the intracellular activation of radiosensitizing drugs in the tumor cells. Our aim was to to improve both the toxic and radiosensitizing effect of the corresponding drugs in the tumor.First, we tried to improve the therapeutic efficacy of gemcitabine by overexpressing the deoxycytidine kinase (dCK) gene, which is responsible for the intracellular activation of gemcitabine, locally in the tumor cells. We used the following four glioma models: murine Gl261, human U373, rat C6 and 9L. We found that, despite a substantial difference in the basal dCK level among the cell lines, gene transfer could increase dCK overexpression in all four cell lines. Increased dCK expression could improve both the toxic and radiosensitizing effect of gemcitabine in vitro and in vivo. However, the effect was cell type specific. Next, we showed in a Gl261 murine model that overexpression of the phosphoribosyltransferase enzyme, which can activate the 5-fluorouracil (5-FU) within the cells, can dramatically increase survival of mice if 5-FU treatment was combined with local radiotherapy and gancyclovir treament. A substantial bystander effect could also be shown.Finally, we showed that overexpression of the NOS gene has a well defined radiosensitizing effect on its own and is able to improve the cytotoxic effect of cisplatin in a murine Gl261 model

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

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

RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay)

Purpose: In the framework of the ‘Realizing the European Network of Biodosimetry’ (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Materials and methods: Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. Results: The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (== 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. Conclusion: The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories

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

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans : joint RENEB and EURADOS inter-laboratory comparisons

Purpose: RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. Materials and methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation induced thermoluminescent signals in glass screens taken from mobile phones. Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios