27 results on '"Vral, A."'
Search Results
2. Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity.
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Vandersickel, Veerle, Depuydt, Julie, Van Bockstaele, Bram, Perletti, Gianpaolo, Philippe, Jan, Thierens, Hubert, and Vral, Anne
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A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.
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- 2010
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3. Effects of estradiol and progesterone on the variability of the micronucleus assay
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Baeyens, Ans, Vandersickel, Veerle, Thierens, Hubert, Ridder, Leo De, and Vral, Anne
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To investigate chromosomal radiosensitivity of lymphocytes the micronucleus (MN) assay has been used for many years. The results of these studies suggest the use of the MN assay as a biomarker for cancer predisposition. However, the MN assay has still some limitations associated with the reproducibility and sensitivity. Especially a high intra-individual variability has been observed. An explanation for this high intra-individual variability is not yet available. In literature it is suggested that the high variability among females is attributable to hormonal status.
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- 2005
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4. A chromosomal radiosensitivity study of a population of radiation workers using the micronucleus assay
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Thierens, H., Aousalah, B., Vral, A., Ridder, L. De, and Barbe, M.
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The micronucleus (MN) assay already applied to the biomonitoring of radiation workers is a relatively easy and fast technique allowing assessment of the individual chromosomal radiosensitivity status of radiation workers on a large scale. A pilot study was set up to evaluate the chromosomal radiosensitivity in a group of 99 male radiation workers from the nuclear power plant Doel in Belgium. Heparinized blood samples of the workers were divided into three parts: one served for the assessment of a possible effect of occupational exposure on the number of micronuclei, the two others served for the assessment of individual chromosomal radiosensitivity by the increase of the number of micronuclei after 1 and 2 Gy in vitro γ-irradiation. For each worker, date of birth, smoking habit and previous medical exposures were registered. Furthermore the external radiation burden accumulated over the last 10 years was calculated from the personnel dosimetry records. Concerning the MN frequency as a biomarker for effect, the present study confirms the strong increase of MN frequency with age and the tendency of an increase with an accumulated radiation burden, observed in previous studies. Using the MN frequency after in vitro irradiation as a biomarker for chromosomal radiosensitivity, the data of the present study do not support a lowered radiosensitivity in radiation workers due to the adaptive response mechanism induced by occupational exposure. As the overall in vitro radiation induced MN frequency distributions can be represented by a normal distribution without a high frequency tail, none of the workers can be considered as exceptionally by radiosensitive with the test. Further validation of the MN assay as a radiosensitivity assay for a population of workers is necessary.
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- 2003
5. Patient dosimetry after 131I-MIBG therapy for neuroblastoma and carcinoid tumours
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MONSIEURS, M. A., THIERENS, H. M., VRAL, A., BRANS, B., DE RIDDER, L., and DIERCKX, R. A.
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AimThe aim of the study was to determine the equivalent total body dose (ETBD) using the cytokinesis-blocked micronucleus assay in 22 131I-meta-iodobenzylguanidine (131I-MIBG) therapies (18 neuroblastoma, mean 5097 MBq, SD 1591; and four carcinoid tumours, mean 7681 MBq, SD 487). The results are correlated with the total body radiation dose according to the Medical Internal Radiation Dosimetry (MIRD) formalism.MethodsFor each patient, blood samples were taken immediately before and 1 week after 131I-MIBG therapy. The first blood sample was irradiated in vitrowith 60Co γ-rays to determine the dose-response curve. Micronuclei were scored in 1000 binucleated cells. By using the dose-response curve the ETBD was derived from the increase in micronuclei after 131I-MIBG therapy (second blood sample). Based on three consecutive biplanar scans taken at 3, 6 and 9 days post-administration respectively, the total body dose following the MIRD formalism was calculated.ResultsThe micronucleus assay was evaluable in only 14 out of 22 131I-MIBG therapies due to cell division inhibition caused by previous chemotherapy treatments and lymphocyte dilution due to blood transfusions given shortly after 131I-MIBG therapy. For these 14 therapies, the mean micronucleus yield after 131I-MIBG therapy was significantly increased (P<0.01) with a mean of 92 (SD 77) for neuroblastoma patients and with a mean of 35 (SD 8) for carcinoid patients. The increase observed in the present study is greater than previously observed after 131I therapy and 89Sr therapy but much lower than after external beam radiotherapy. For all patients treated with multiple therapies, the initial increase in micronucleus yield had at least partially recovered by the time of the next therapy. This might be explained by an increased turnover of lymphocytes. A mean ETBD of 0.95 Gy (SD 0.55) for neuroblastoma patients and a mean of 0.46 Gy (SD 0.09) for carcinoid patients was calculated. A reasonable correlation (R0.87) between the ETBD and the MIRD dose was obtained. The slope value of 0.75 can be explained by the low dose rate effect.ConclusionsThe observation in the present study of important inter-individual variability in the total body dose, with the possibility of high dose values, suggests the necessity of individual dosimetry when administering 131I-MIBG therapy, especially considering that generally more than one therapy is given to each patient.
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- 2001
6. HUman MicroNucleus project: international database comparison for results with the cytokinesis‐block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei
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Bonassi, Stefano, Fenech, Michael, Lando, Cecilia, Lin, Yi‐ping, Ceppi, Marcello, Chang, Wushou Peter, Holland, Nina, Kirsch‐Volders, Micheline, Zeiger, Errol, Ban, Sadayuki, Barale, Roberto, Bigatti, Maria Paola, Bolognesi, Claudia, Jia, Cao, Di Giorgio, Marina, Ferguson, Lynnette R., Fucic, Aleksandra, Lima, Omar Garcia, Hrelia, Patrizia, Krishnaja, Ayyathan P., Lee, Tung‐Kwang, Migliore, Lucia, Mikhalevich, Ludmilla, Mirkova, Ekaterina, Mosesso, Pasquale, Müller, Wolfgang‐Ulrich, Odagiri, Youichi, Scarffi, Maria Rosaria, Szabova, Elena, Vorobtsova, Irena, Vral, Anne, and Zijno, Andrea
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Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis‐block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin‐B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5‰ and the interquartile range was between 3 and 12‰. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14–24%). Statistical analyses were performed using random‐effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods. Environ. Mol. Mutagen. 37:31–45, 2001 © 2001 Wiley‐Liss, Inc.
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- 2001
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7. Estimation of risk based on biological dosimetry for patients treated with radioiodine
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MONSIEURS, M. A., THIERENS, H. M., VAN DE WIELE, C., VRAL, A. M., MEIRLAEN, I. A., WINTER, H. A. DE, SADELEER, C. J. DE, RIDDER, L. I. DE, KAUFMAN, J.M., and DIERCKX, R. A.
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A multicentre study was undertaken to assess the cytogenetic damage to peripheral blood lymphocytes in 31 patients treated with 131I for thyrotoxicosis using the cytokinesisblocked micronucleus assay. The results were compared to those for eight thyroid carcinoma patients using the same method. For each patient, blood samples were taken immediately before and 1 week after iodine administration. The first blood sample was divided into three fractions and each fraction was subsequently irradiated in vitrowith 0, 0.5 and 1 Gy 60Co gamma rays, respectively. After blood culture for 70 h, cells were harvested, stained with RomanowskyGiemsa and the micronuclei scored in 1000 binucleated cells. For both patient groups, a linearquadratic doseresponse curve was fitted through the data set of the first blood sample by a least squares analysis. The mean increase in micronuclei after 131I therapy second blood sample was fitted to this curve and the mean equivalent total body dose ETBD calculated. Surprisingly, in view of the large difference in administered activity between thyroid carcinoma patients and thyrotoxicosis patients, the increase in micronuclei after therapy mean ± S.D. 32 ± 30 and 32 ± 23, respectively and the equivalent total body dose 0.34 and 0.32 Gy, respectively were not significantly different P> 0.1. The small number of micronuclei induced by 131I therapy 32 ± 29, compared with external beam radiotherapy for Hodgkin's disease 640 ± 381 or cervix carcinoma 298 ± 76 1, gave a cancer mortality estimate of less than 1. This also explains why late detrimental effects in patients after 131I treatment have not been reported in the literature.
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- 1999
8. APOPTOSIS INDUCED BY γ IRRADIATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS IS NOT MEDIATED BY CYTOCHROME-C RELEASE AND ONLY PARTIALLY INVOLVES CASPASE-3-LIKE PROTEASES
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Louagie, H., Schotte, P., Vral, A., Cornelissen, M., Thierens, H., Beyaert, R., De Ridder, L., and Philippe, J.
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Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after γ-irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after γ irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (ΔΨm) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5-Gy γ irradiation, is characterized by a decrease in ΔΨm, but surprisingly no release of cytochrome-c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in ΔΨm with cytochrome-c release and a clear caspase 3 activation. We were unable to block the decrease in ΔΨm with the caspase-inhibitors zVAD-fmk or zDEVD-fmk after γ irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in γ irradiated mature PBL, caspase-dependent and -independent pathways, but not cytochrome c, seem to be involved in the apoptotic process.
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- 1999
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9. The effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes: an electron microscopic approach
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Cornelissen, M., Vral, A., Thierens, H., and de Ridder, L.
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Resting lymphocytes are sensitive to radiation damage and die by apoptosis. We investigated the effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes. Lymphocytes were irradiated in vitro with 5 Gy 60Co-γ-rays and cultured for 24 hours in the presence or absence of the caspase-inhibitors zVAD-fmk and zDEVD-fmk. Cell death was evaluated by electron microscopy. Irradiation in the absence of the inhibitors resulted in about 30% dead cells, almost all showing typical apoptotic morphologies. Addition of either one of the inhibitors could not rescue cells from death. Part of the dead lymphocytes (about 65%) still showed typical nuclear characteristics of apoptotic cells: sharply marginated, condensed chromatin, clumped into one sphere or into a crescent shaped mass. The remaining part of the dead cells had ultrastructural characteristics, aberrant from apoptic cells: clumping of the chromatin was less pronounced and less sharply marginated. Irregular clumps were formed. Data indicate that part of the lymphocytes go in apoptosis in a caspase-independent way. The other part shows caspase-dependent apoptosis with respect to the nuclear events.
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- 1999
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10. A cytogenetic study of radiological workers: effect of age, smoking and radiation burden on the micronucleus frequency
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Thierens, H., Vral, A., and De Ridder, L.
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A large scale cytogenetic study of the radiation damage in nuclear power plant workers and medical workers handling X-ray machines (269 individuals) was undertaken using the micronucleus assay for peripheral blood lymphocytes. The micronucleus frequency was found to increase systematically with donor age. After correction for the age-dependence, no correlation of the micronucleus frequency with smoking habits, expressed as cigarette-years and cigarette consumption per day, could be observed. Compared to the group of administrative workers receiving doses below 1 mSv/year, limit recommended by the ICRP for public exposure, the micronucleus frequency was slightly increased in the group of radiation workers, exposed occupationally. However, applying the Mann-Whitney test, the observed differences are not statistically significant. After correction of the dose accumulation pattern for the turn-over of the lymphocyte pool, a weak correlation between the micronucleus frequency and the equivalent dose accumulated over the 10 years preceding the study was obtained. For clear-cut conclusions on the radiation damage of low-dose worker cohorts, an increase in the sensitivity of the assay, e.g., by analysis of the micronuclei for the presence of centromeres is necessary.
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- 1996
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11. FLOW CYTOMETRIC SCORING OF APOPTOSIS COMPARED TO ELECTRON MICROSCOPY IN γ IRRADIATED LYMPHOCYTES
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LOUAGIE, H, CORNELISSEN, M, PHILIPPE, J, VRAL, A, THIERENS, H, and DE RIDDER, L
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One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in γ irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5Gy and 20Gy γ irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5Gy γ irradiation of PBMCs mainly causes apoptosis, whereas a 20Gy γ irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.
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- 1998
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12. Micronuclei Induced by Fast Neutrons Versus 60Co γ-rays in Human Peripheral Blood Lymphocytes
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Vral, A., Verhaegen, F., Thierens, H., and de Ridder, L.
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Here we compared the effectiveness of neutrons (〈E〉 = 5·5 MeV) versus 60Co γ-rays in producing micronuclei (MN) in human lymphocytes. To obtain dose-response data, blood samples of six donors were irradiated with doses ranging from 0·1 to 5 Gy for γ-rays and 0·1-3 Gy for neutrons. A linear dependence of MN yield with dose was found for fast neutrons while for γ-rays a nonlinear dependence existed. For both radiation qualities no significant interindividual differences were found. Derived relative biological effectiveness values decreased with increasing dose. The MN frequency distributions were overdispersed with respect to the Poisson distribution, with neutrons showing higher dispersion values than with γ-rays. To compare the repair kinetics of both radiation qualities split-dose experiments were performed. A dose of 4 Gy γ-rays (3 Gy neutrons) was delivered either as a single exposure or in two equal fractions separated by time intervals ranging from 30 min to 10h (30 min to 7h for neutrons). The data showed for γ-rays a significant decline (30% ± 10%) in MN yield with interfraction time due to repair of DNA damage. This repair is a continuous process starting almost immediately after the first of the two doses and lasting 3-5h. For fast neutrons no decline was observed indicating irreparable damage.
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- 1994
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13. Micronucleus Induction in Peripheral Blood Lymphocytes of Patients under Radiotherapy Treatment for Cervical Cancer or Hodgkin's Disease
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Thierens, H., Vral, A., Van Eijkeren, M., Speleman, F., and De Ridder, L.
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The genetic damage present in peripheral blood lymphocytes of patients treated with fractionated partial-body radiation therapy for cervical cancer or Hodgkin's disease was followed during treatment by means of the cytokinesis-block micronucleus assay. For each patient a dose - response relationship with respect to the number of micronuclei after in vitro irradiation of blood samples pretreatment was also determined. Comparing the individual in vivo-in vitro data, the micronucleus yields after the equivalent whole-body dose during radiotherapy were found to differ substantially from the in vitro dose - response. Contrary to the linear - quadratic dose dependence after in vitro irradiation the initial increase in the micronucleus yield during radiotherapy levelled off at elevated doses. The observed differences cannot be attributed only to the effects of interphase death and the partial irradiation of the lymphocyte pool. The correlation between the micronucleus yield and the equivalent whole-body dose for values up to 2 Gy, observed for the pooled data of the first part of the radiotherapy treatment, demonstrates the suitability of the cytokinesis-block micronucleus assay as a biological dosemeter after accidents involving partial-body irradiation.
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- 1995
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14. Technical Report Flow cytometry as a quantitative and sensitive method to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes
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HERTVELDT, K., PHILIPPE, J., THIERENS, H., CORNELISSEN, M., VRAL, A., and DE RIDDER, L.
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Human peripheral blood lymphocytes, irradiated in vitro , die by an apoptotic process. The number of apoptotic cells after in vitro gamma -irradiation (0, 0.1, 0.2, 1, 2 and 5 Gy) was measured by flow cytometry using Annexin V and DiOC6 (a cationic dye) after 24 and 48 h incubation. The mean dose-response curves for apoptosis of six healthy volunteers obtained with both methods were steep below 1 Gy and flatter at higher doses. A slightly higher number of apoptotic cells was observed with DiOC6 , compared to Annexin V. This can be assigned to a minor DiOC6-int/PI population. Forty-eight hour cultures contained higher numbers of apoptotic cells compared with 24 h cultures. For both culture times, DiOC6 and Annexin V detected a statistically significant difference between a control sample and a 0.1 Gy irradiated one, illustrating the high sensitivity of the methods.
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- 1997
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15. Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens
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Philippé, Jan, Louagie, Henk, Thierens, Hubert, Vral, Anne, Cornelissen, Maria, and Ridder, Leo De
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Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose γ-irradition prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change. Cytometry 29:242249, 1997. © 1997 Wiley-Liss, Inc.
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- 1997
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16. Study of Dose-rate and Split-dose Effects on the in Vitro Micronucleus Yield in Human Lymphocytes Exposed to X-rays
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Vral, A., Thierens, H., and de Ridder, L.
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This paper reports the effects of changes in dose-rate and dose-fractionation on the micronucleus (MN) yield in human lymphocytes exposed to 250 kV X-rays. For the investigation of dose-rate effects whole blood samples of four healthy donors were irradiated with doses ranging from 1 to 4 Gy given at various dose-rates between 0·2 and 40 Gy/h. For the higher doses (3 and 4 Gy) a decline in the MN yield became apparent when the dose-rate was reduced below 1·6 Gy/h. This effect was enhanced systematically by a further lowering of the dose-rate. For lower doses (1 and 2 Gy) the reduction in the MN yield was less pronounced: only a small effect was observed for two donors when a dose of 2 Gy was administered at a dose-rate of 0·2 Gy/h. In the split-dose experiment a dose of 4 Gy was delivered either as a single exposure or in two fractions of 2 Gy, separated by time intervals ranging from 30 min to 10 h. A continuous decrease of the MN yield with increasing interfraction time is observed: after an initial fast decline a further slight reduction in the MN yield occurs. The observed dose-rate and split-dose effects on the MN yield can be attributed to repair of sublethal damage.
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- 1992
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17. In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes
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VRAL, A., THIERENS, H., and DE RIDDER, L.
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One of the major drawbacks of the in vitro micronucleus MN assay for human lymphocytes is its reduced sensitivity for the detection of damage induced by low radiation doses, due to the high variability among the spontaneous MN frequencies. In this paper we investigated the enhancement of the sensitivity of the MN assay by analysing spontaneous and radiation-induced MN for the presence of centromeres. For this, in situ hybridization FISH with the human pancentromeric DNA probe, p82H, was performed. Our results revealed that a high percentage 73 of the spontaneous MN contain a centromere. These centromerepositive MN indicate the presence of a whole chromosome chromatid. After in vitro irradiation with low doses 0 1-2 Gy 60 Co-rays mainly centromere-negative MN were induced while only a very small number of additional centromerepositive MN were formed. This demonstrates that radiationinduced MN mainly contain acentric fragments, pointing to the clastogenic action of ionizing radiation. Furthermore, our data show that the sensitivity of the MN assay for low dose detection is increased by scoring only centromere-negative MN. .
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- 1997
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18. Micronucleus induction by 60Co gamma-rays and fast neutrons in ataxia telangiectasia lymphocytes
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Vral, A.
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Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by a progressive neuronal degeneration, immunodeficiency, cancer proneness and an extreme sensitivity to ionizing radiation. In this work, micronucleus dose-response curves for lymphocytes of normal and AT individuals, exposed in G 0 to low LET gamma-rays and high LET fast neutrons, are compared. After gamma-irradiation, the micronucleus yields for AT lymphocytes are strongly increased compared with controls. The micronucleus dose-response curve for AT cells shows a linear dependence instead of a linear-quadratic one which is found for normal cells. After neutron irradiation, the increase in micronucleus yield above controls is less pronounced than with gamma-rays and the micronucleus dose-response curves are linear, as expected. The high increase in micronucleus yield compared with controls after gamma-irradiation further suggests the application of the micronucleus assay as a diagnostic tool for ataxia telangiectasia.
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- 1996
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19. Automated micronucleus (MN) scoring for population triage in case of large radiation accidents.
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August, L., Willems, P., Thierens, H., Slabbert, J., and Vral, A.
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In case of a large radiation accident, when hundreds of people may be exposed, it is very important to distinguish the severely exposed individuals, who require early medical treatment, from those less exposed. In such a situation, specific biological indicators should promptly indicate whether the whole-body dose received by the victims exceeded 1Gy, the level above which it is necessary to provide medical care. Once the exposed population has undergone such an early triage, a second step may be indicated for a more accurate assessment of the exposure. Up to now, scoring of dicentrics is the reference to estimate the dose received in case of individual overexposures. However, to obtain a reliable dose estimate, 500 metaphases should be screened for the presence of dicentrics, a very time consuming procedure, that can only be applied to small populations of exposed individuals. The aim of our study was to develop a quick population triage method based on automated micronucleus (MN) scoring. Automated MN scoring was performed using a MN software module specifically developed by Metasystems for the Metafer4 platform. Standard dose response curves were established for manual, automated and semi-automated MN scoring. To this aim, blood samples of 10 individuals were irradiated with in vitro doses of 0-0,2-0,5-1-2-3Gy Co-60 γ-rays. Whole blood cultures were set up and cells were harvested after 70h, applying rigorous culture/harvest conditions. Cells were stained with DAPI for manual and automated scoring. For both scoring methods 500 binucleated (BN) cells were counted. The reproducibility, accuracy and sensitivity of the manual versus automated scoring procedure were analyzed. A comparison between manual and automated scoring of the same slides, revealed a drop in the MN yield for the automated scoring procedure. However, the automated scores were as reproducible as the manual scores and both scores were highly correlated. Scoring of 500 BN cells allows us to detect a dose of 0,5 Gy, with 95% confidence, for both manual and automated MN scoring procedures (Mann-Whitney U test, p<0.05). The semi-automated scoring procedure, taking into account false positives and false negatives in the MN class, didnt result in a better accuracy or reproducibility. With the automated MN scoring procedure, about 80 slides (500 BN cells) can be scored in 1 day with the Metafer4. In conclusion, our preliminary results show that automated MN scoring is a quick and reliable method, useful for a first population triage in case of large radiation accidents.
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- 2008
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20. Variations in the Radiosensitivity of T-lymphocytes of different Individuals to a Therapeutic Neutron Beam
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August, L., Slabbert, J., Vral, A., and Symons, J.
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Purpose: Variations in the radiosensitivty of different tumours pertain to the potential for therapeutic gain for treatment with high energy neutrons. In this work the radiosensitivities of T-lymphocytes of different individuals to the clinical neutron beam at iThemba LABS were measured. The objective of this investigation was to establish if a relationship between neutron RBE and resistance to conventional gamma radiation could be determined.
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- 2008
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21. Lentivirus-mediated RNA Interference of Ku70 to Enhance Radiosensitivity of Human Mammary Epithelial Cells.
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Vandersickel, V., Mancini, M., Marras, E., Perletti, G., Thierens, H., and Vral, A.
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Breast cancer patients are characterised by an enhanced chromosomal radiosensitivity pointing to a defect in the repair of DNA double strand breaks (dsb). In mammalian cells, radiation induced dsb are mainly repaired by the non homologous end-joining (NHEJ) repair pathway, a pathway in which the Ku70/Ku80 heterodimer plays a key role as it binds to the broken DNA ends. In this study we wanted to investigate the radiosensitizing effect of Ku70/80 knockdown, by lentivirus-mediated RNAinterference, in a spontaneous immortalised human mammary epithelial cell line (MCF10A). Several endpoints for measuring radiosensitivity were taken into acount: micronucleus formation (chromosomal radiosensitivity), cell survival, apoptosis and senescence. For all endpoints, MCF10A cells were infected with lentiviral vectors for RNAi of Ku70 (pLVTHM/shKu70/GFP). Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. When a satisfactory knockdown was obtained (70-90% vs. mock-infected (pLVTHM/GFP) cells), the cells were used to perform radiation experiments. For the in vitro MN assay, cells were irradiated with doses of 2 and 4 Gy 60Co gamma-rays. A significantly higher radiation-induced MN yield was obtained in the Ku70/80 knock down cell line compared to the mock-infected cell line, pointing to an increased chromosomal radiosensitivity. This increased chromosomal radiosensitivity demonstrates that the repair genes, Ku70 and Ku80, are involved in the repair of DNA double strand breaks, which are the main DNA lesions resulting in chromosomal aberrations such as micronuclei. Besides chromosomal radiosensitivity we also investigated radiosensitivity at the cellular level by cell survival experiments. Cells were irradiated with doses ranging between 0 and 8 Gy and cultured for 5 days before being analysed. The results of the cell survival assay show that Ku70/80 knockdown cells have a lower survival yield after irradiation compared to mock-infected cells, pointing to an enhanced cellular radiosensitivity. Analysis of the cell death pattern showed that MCF10 cells (Ku70/80 knockdown and mock-infected) do not undergo apoptosis but go into cellular senescence. In conclusion, we can state that knockdown of Ku70 and Ku80 by RNAi of Ku70 resulted in an increased chromosomal and cellular radiosensitivity in an immortalised MCF10 human mammary cell line after irradiation with low LET 60Co gamma-rays. These results may further support the role of DNA dsb repair genes in breast cancer.
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- 2008
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22. Towards Clinical Application of Patients Radiosensitivity Data
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Thierens, H., De Ruyck, K., Werbrouck, J., Willems, P., Vral, A., Duprez, F., Van Eijkeren, M., and De Neve, W.
- Abstract
INTRODUCTION
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- 2008
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23. Acute reactions in IMRT treated H&N cancer patients: association with DVH and SNPs in DNA DSB repair genes
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Werbrouck, J., De Ruyck, K., Duprez, F., Claes, K., Van Eijkeren, M., Boterberg, T., Willems, P., Vral, A., De Neve, W., and Thierens, H.
- Abstract
The aim of present study was to investigate the relation between physical dose related parameters and single nucleotide polymorphisms (SNPs) in DNA DSB repair genes XRCC3 (c.-1843 A>G, c.562-14 A>G, c.722 C>T), Rad51 (c.-3429 G>C, c.-3392 G>T), Lig4 (c.26 C>T, c.1704 T>C), Ku70 (c.-1310 C>G) and Ku80 (c.2110-2804 G>A) and development of acute normal tissue RT reactions. The study population consisted of 89 head and neck cancer patients treated with IMRT. All RT reactions were scored using the CTCAE scale for mucositis, dermatitis and dysphagia. The population was subdivided in a group with low and moderate radiosensitivity (CTC0-2) and a group with high radiosensitivity (CTC3) for each acute normal tissue effect. The polymorphic regions were analysed by PCR- RFLP or single base extension analysis (PCR- Snapshot). Analyses showed that if we compare the CTC0-2 and the CTC3 group, the mean dose to the oral cavity is significantly associated with the development of mucositis (p= 0.042). For dysphagia a statistically significant relation was found with the dose of the upper, mid and lower constrictor pharyngeus (CP) muscles. These physical factors are considered as confounding factors in the radiogenomics analyses of this study. The SNPs in the coding region of the XRCC3 gene (c.722 C>T) and in the 5 UTR region of the Ku70 gene (c.-1310 C>G) were significantly associated with the risk of developing severe dysphagia (CTC3) and can therefore be considered as risk alleles. Heterozygous carriers of the variant allele had a respectively 4.47 (p= 0.033) and 4.17 (p=0.014) increased risk to develop acute swallowing problems. We developed a risk analysis model using logistic regression analysis to predict patients probability to suffer from severe dysphagia as a result of the RT treatment. In this model the CP dose and the information regarding XRCC3 c.722 C>T and Ku70 c.-1310 C>G genotypes were included. Using a cut off value of two times the median, the model provided us a sensitivity of 78.6% and a specificity of 77.6% for prediction of severe dysphagia. No association between the investigated polymorphisms and the development of mucositis or dermatitis was found. Considering the dose to the CP muscles as confounding factor in a logistic regression model, presence of the variant allele of XRCC3 c.722 and Ku70 c.-1310 is significantly associated with development of dysphagia. Using the paramount physical and biological parameters, we developed a risk model for prediction of patients risk for severe dysphagia.
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- 2008
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24. Polymorphisms in DNA double-strand break repair genes and breast cancer risk.
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Willems, P., Claes, K., Van Den Broecke, R., Makar, A., Marras, E., Perletti, G., Thierens, H., and Vral, A.
- Abstract
Enhanced chromosomal radiosensitivity (CR) has been observed in a significant number of breast cancer patients. Since ionising radiation induces double-strand breaks (DSB), polymorphisms in DSB repair genes could be involved in genetic predisposition to breast cancer. A family history of breast cancer is a well known risk factor for the disease. Other risk factors are also associated with breast cancer, such as early age of first menarche, nulliparity or late first childbirth, and late menopause. The principle culprit common for these risk factors is said to be the prolonged exposure to elevated levels of estrogens. As non homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells, we investigated the association of 5 SNPs in 3 different NHEJ genes with breast cancer in a population-based case-control setting. The total patient population was composed of a selected group of patients with a family history of the disease and an unselected group. CR was previously studied in both patient groups. SNP analysis showed that the c.2099-2408G>A SNP (Ku80) has a significant odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30-6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the group of familial patients. The He+HV genotypes of the c.2099-2408G>A SNP (Ku80) also showed high and significant ORs in the group of radiosensitive, familial breast cancer patients (OR=4.62 ,95%CI: 1.28-16.74). For the c.-1310 C>G SNP (Ku70) a significant OR of 1.85 (95%CI: 1.01-3.41) was found for the He genotype in the unselected patient group. For the radiosensitive, unselected patients, increased, but non-significant ORs were observed. The c.-1310 C>G SNP (Ku70) SNP exhibits significant results in the unselected patient group only, indicating an influence of other, environmental factors besides genetic factors. As breast epithelium is exposed to endogenous oxidative stress through increased estrogen exposure, the possible effect of hormone exposure was examined in an enlarged, unselected patient population (OR enlarged population =1.68, 95%CI:1.09-2.60). The ORs of the c.-1310 C>G SNP (Ku70) SNP for patients with a longer estrogen exposure were high and significant (early menarche: OR=1.85, 95%CI:1.20-2.92; late menopause: OR=1.94, 95%CI:1.08-3.51) while the ORs for patients with a short hormone exposure were lower and non-significant. These results provide preliminary evidence that the variant allele of c.-1310C>G (Ku70) and c.2099-2408G>A (Ku80) are risk alleles for breast cancer as well as CR. Furthermore, the association of c.-1310C>G (Ku70) with breast cancer risk was stronger in women with a long estrogen exposure.
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- 2008
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25. LIGHT AND ELECTRON MICROSCOPY OF APOPTOSIS IN IRRADIATED HUMAN LYMPHOCYTES
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Cornelissen, M., Vral, A., Thierens, H., and De Ridder, L.
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- 1996
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26. FLOW CYTOMETRIC ANALYSIS OF APOPTOTIC LYMPHOCYTES WITH ANNEXIN V AND DiOC6 AFTER IN VITRO IRRADIATION
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Hertveldt, K., Philippé, J., Thierens, H., Vral, A., and De Ridder, L.
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- 1996
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27. P XIV C.13 Interlaboratory comparison of different cytogenetic endpoints for scoring of radiation damage in peripheral blood lymphocytes after in vitro low dose γ-exposure
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Thierens, Hubert, Vral, Anne, De Ridder, Leo, Kirsch-Volders, Micheline, Touil, Nadia, Laurent, Christian, and Lambert, Vincent
- Published
- 1997
- Full Text
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