Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay)

Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy c-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Results: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Conclusions: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay

7. Biological Effectiveness of 12 C and 20 Ne Ions with Very High LET

Knowledge of radiobiological effects of heavy ions at the cellular and molecular level is of fundamental importance in the field of radiation therapy (for example C ions) and space radiation biology (for example Ne ions). One of the issues that require deeper investigations is a determination of RBE values for a wide range of LET, for all relevant doses, for many cell types and various kinds of radiations During recent years, the biological effectiveness of heavy ions has been widely investigated with the aim to identify physical characteristics relevant to biological actions. These investigations are pertinent to the use of heavy ions in radiosurgery and radiotherapy. What has not been investigated so thoroughly is the biological effectiveness of heavy ions at low energies and very high LET values. The LET, which is equal to the stopping power of heavy particles, increases sharply at the end of the particle's path, forming a so-called Bragg peak. The shape of the Bragg peak depends on the particle type. Because overlying beams with different energies and components of primary and secondary particles are used in radiotherapy, the knowledge of RBE values of very high LET radiation need to be well characterized. An experimental set-up designed for such investigations was constructed at the isochronic cyclotron in Heavy Ion Laboratory. A more detailed description of the set-up can be found in Ref. CHO-K1 cells have been used as a suitable biological system for our studies. The cell line is characterized by genetic stability, the ability to form colonies, a relatively rapid growth rate with a cell cycle of 12-14 hours. For exposure to ions the cells were seeded in specially designed Petri dishes, which were filled with medium, sealed by a parafilm cover and placed in a vertical sample holder mounted in an x-y-z table that was connected to a special stepping motor. The irradiated sample moved under the beam according to a planned route. Movement was initiated when the number of counts detected by the 20 o particle detector reached the preset value. When all fields have been exposed the sample holder returned to the start position. Stored information enabled to evaluate the beam stability and intensity. The whole set-up was surveyed by a digital camera. The total time of exposure per dish was between 1-5 min. depending on the dose and beam intensity. The dose rates were changed from 0.05 Gy/min. to 1 Gy/min depending on the dose. Cell survival was estimated according to standard procedure

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

Longitudinal Auxological recovery in a cohort of children with Hyperinsulinaemic Hypoglycaemia

Background: Hypoglycaemia due to hyperinsulinism (HI) is the commonest cause of severe, recurrent hypoglycaemia in childhood. Cohort outcomes of HI remain to be described and whilst previous follow up studies have focused on neurodevelopmental outcomes, there is no information available on feeding and auxology. Aim: We aimed to describe HI outcomes for auxology, medications, feeding and neurodevelopmental in a cohort up to age 5 years. Method: We reviewed medical records for all patients with confirmed HI over a three-year period in a single centre to derive a longitudinal dataset. Results: Seventy patients were recruited to the study. Mean weight at birth was - 1.0 standard deviation scores (SDS) for age and sex, while mean height at 3 months was - 1.5 SDS. Both weight and height trended to the population median over the follow up period. Feeding difficulties were noted in 17% of patients at 3 months and this reduced to 3% by 5 years. At age 5 years, 11 patients (15%) had neurodevelopmental delay and of these only one was severe. Resolution of disease was predicted by lower maximum early diazoxide dose (p = 0.007) and being born SGA (p = 0.009). Conclusion: In a three-year cohort of HI patients followed up for 5 years, in spite of feeding difficulties and carbohydrate loading in early life, auxology parameters are normal in follow up. A lower than expected rate of neurodevelopmental delay could be attributed to prompt early treatment.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.The study was supported by the Northern Congenital Hyperinsulinism (NORCHI) charitable fund, by the Manchester Academic Health Sciences Centre and by The University of Manchester MRC Confidence in Concept (CiC) Award (MC_PC_18056). KEC was funded by a Research Councils UK Academic Fellowship (https://www.ukri.org/). SEF has a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (105636/Z/14/Z).published version, accepted versio

Relationships between EGFR-initiated signalling, DNA double-strand break rejoining and survival in X-irradiated human glioma M059 cells

The aim of this study was to investigate the effect of signalling inhibition on survival and double-strand break (DSB) rejoining in cells differing in sensitivity to inhibitors, X-rays and bleomycin. Human glioma M059 cells, K (relatively radioresistant) and J (radiosensitive, defective in DSB rejoining for lack of DNA-dependent protein kinase catalytic subunit, DNA-PKcs) were pretreated with signalling inhibitors: tyrphostin AG 1478, specific for epidermalgrowth- factor-receptor (EGFR) kinase or PD 98059, specific for kinase MEK 1/2 (mitogen-activated, extracellular signal-activated kinases 1 and 2). Subsequently, the cells were X-irradiated or treated with bleomycin. Cell survival was determined by clonogenicity test. DSB rejoining was monitored with the use of pulsed-field gel electrophoresis (PFGE). We found that in X-irradiated M059 K cells EGFR kinase activity was necessary for efficient DSB rejoining and the kinase inhibitor, tyrphostin AG 1478, acted as radiosensitizer in the dose range that reduced cell survival to 0.7-0.8. Inhibition of EGFR kinase, however, did not decrease survival or affect DSB rejoining in DNA-PKcs-deficient M059 J cells. These results indicated that the decrease in cell survival was due to a disturbed DSB rejoining by the DNA-PK dependent system. In contrast, inhibition of MEK 1/2 kinase on EGFR downstream signalling pathway by PD 98059 did not affect DSB rejoining in either cell line and exerted a radioprotective effect

Modified neutral comet assay for human lymphocytes

Comet assay under neutral conditions allows the detection of DNA double-strand breaks, considered to be the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA double strand breaks in human lymphocytes irradiated with doses of 60Co gamma-rays (from 10 to 100 Gy). The analysis carried out according to this protocol takes less time than those published so far. Also, the use of lysis at 50°C is avoided; this is important in view of the presence of heat-labile sites in the DNA of irradiated cells, recently reported by Rydberg [12]. The comets have well defined, sharp limits, are suitable for computer image analysis and chromatin of the control cells remains condensed