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

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

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

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals: a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

Blood-Derived Biomarkers of Diagnosis, Prognosis and Therapy Response in Prostate Cancer Patients

Prostate cancer is among the most frequent cancers in men worldwide. Despite the fact that multiple therapeutic alternatives are available for its treatment, it is often discovered in an advanced stage as a metastatic disease. Prostate cancer screening is based on physical examination of prostate size and prostate-specific antigen (PSA) level in the blood as well as biopsy in suspect cases. However, these markers often fail to correctly identify the presence of cancer, or their positivity might lead to overdiagnosis and consequent overtreatment of an otherwise silent non-progressing disease. Moreover, these markers have very limited if any predictive value regarding therapy response or individual risk for therapy-related toxicities. Therefore, novel, optimally liquid biopsy-based (blood-derived) markers or marker panels are needed, which have better prognostic and predictive value than the ones currently used in the everyday routine. In this review the role of circulating tumour cells, extracellular vesicles and their microRNA content, as well as cellular and soluble immunological and inflammation- related blood markers for prostate cancer diagnosis, prognosis and prediction of therapy response is discussed. A special emphasis is placed on markers predicting response to radiotherapy and radiotherapy-related late side effects