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Brain Struct Funct
January, 2015

Acute neuroinflammation in a clinically relevant focal cortical ischemicstroke model in rat: longitudinal positron emission tomography andimmunofluorescent tracking.

Miklos Toth, Philip Little, Fabian Arnberg, Jenny Haeggkvist, Jan Mulder, Christer Halldin, Balazs Gulyas, Staffan Holmin

Adequate estimation of neuroinflammatory processes following ischemic stroke is essential for better understanding of disease mechanisms, and for the development of treatment strategies. With the TSPO (18 kDa translocator protein) positron emission tomography (PET) radioligand [(11)C]PBR28, we monitored longitudinally the inflammatory response post-transient cerebral ischemia in rats, using a recently developed rat stroke model that produces isolated focal cortical infarcts with clinical relevance in size and pathophysiology. Six Sprague-Dawley rats were subjected to 90 min transient endovascular occlusion of the M2 segment of the middle cerebral artery (M2CAO). Animals were imaged with a nanoScan(®) PET/MRI system at 1, 4, 7 and 14 days after M2CAO with a bolus injection of [(11)C]PBR28. In the infarct region, we found a significantly increased uptake of [(11)C]PBR28 on day 4, 7 and 14 compared to day 1 as well as compared to the contralateral cortex. No significant increase was detected in the contralateral cortex during the 14 days of imaging. The activation in the infarct region gradually decreased between day 4 and day 14. In an additional group of animals (n = 26), immunofluorescence studies were performed with antibodies for activated microglia/monocytes (Cd11b), phagocytes (Cd68), astrocytes (glial fibrillary acidic protein) and TSPO. The TSPO immunofluorescence signal indicated reactive microgliosis post injury, corresponding to PET findings. The present clinically relevant animal model and TSPO PET ligand appear to be well suited for studies on neuroinflammation after ischemic stroke.

Cytogenet Genome Res, 147(2-3), 144–153

Comparative Cytogenetics of the Congo African Grey Parrot (Psittacus erithacus).

Seibold-Torres, Cassandra, Owens, Elaine, Chowdhary, Renuka, Ferguson-Smith, Malcolm A., Tizard, Ian, Raudsepp, Terje

The Congo African grey parrot (Psittacus erithacus, PER) is an endemic species of Central Africa, valued for its intelligence and listed as vulnerable due to poaching and habitat destruction. Improved knowledge about the P. erithacus genome is needed to address key biological questions and conservation of this species. The P. erithacus genome was studied using conventional and molecular cytogenetic approaches including Zoo-FISH. P. erithacus has a 'typical' parrot karyotype with 2n = 62-64 and 8 pairs of macrochromosomes. A distinct feature was a sharp macro-microchromosome boundary. Telomeric sequences were present at all chromosome ends and interstitially in PER2q, the latter coinciding with a C-band. NORs mapped to 4 pairs of microchromosomes which is in contrast to a single NOR in ancestral type avian karyotypes. Zoo-FISH with chicken macrochromosomes GGA1-9 and Z revealed patterns of conserved synteny similar to many other avian groups, though neighboring synteny combinations of GGA6/7, 8/9, and 1/4 were distinctive only to parrots. Overall, P. erithacus shared more Zoo-FISH patterns with neotropical macaws than Australian species such as cockatiel and budgerigar. The observations suggest that Psittaciformes karyotypes have undergone more extensive evolutionary rearrangements compared to the majority of other avian genomes.

Digital object identifier (DOI): 10.1159/000444136

Cancers, 281-295

A Novel Three-Colour Fluorescence in Situ Hybridization Approach for the Detection of t(7;12)(q36;p13) in Acute Myeloid Leukaemia Reveals New Cryptic Three Way Translocation t(7;12;16)

Abdulbasit Naiel, Michael Vetter, Olga Plekhanova, Elena Fleischman, Olga Sokova, Grigory Tsaur, Jochen Harbott, Sabrina Tosi

The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20-30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting.

Blood Cancer J, 5, e374

Four genetic lymphoma-specific events (MYC, BCL2, BCL6 and CCND1) identified in a high grade B lymphoma case.

Ittel, A., Hélias, C., Wissler, M. P., Toussaint, E., Miguet, L., Chenard, M. P., Monier, L., Gervais, C., Mauvieux, L.

In the WHO classification, double or triple-hit lymphoma depicts rare and aggressive lymphomas displaying BCL2 and/or MYC and/or BCL6 gene rearrangements that are categorized as B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma. Bacher et al.2 described an interesting series of 10 cases of such neoplasms. In addition, they reported the two first cases displaying four different lymphoma-specific events (quadruple hit) involving the genes MYC, BCL2, BCL6 and CCND1. We describe here a third case occurring in a 79-year-old male patient suffering from paraesthesias for 4 months who was referred for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathies, neuro-meningeal invasion and skin infiltration. The biopsy of a left arm skin nodule revealed large proliferating cells (Ki-67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, CD10 and CD23 remaining negative, consistent with the diagnosis of diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Blood cell counts showed 8.1 × 109/l leukocytes, 13.2 g/dl hemoglobin, 166 × 109/l platelets. LDH and β-2 microglobulin were elevated (989 U/I and 9.14 mg/l, respectively). Blood cell film examination showed the presence of 28% abnormal lymphocytes (medium sized, with intense basophilia, irregular nuclear contours, slightly clumped chromatin and frequent prominent nucleoli) suggestive of a high grade lymphoma. Flow cytometry revealed a lambda immunoglobulin light chain restriction. These cells expressed pan-B markers such as CD19, CD20, FMC7, CD22, with weak CD5 and CD43 positivity. CD10 and 23 were negative. Both the morphology and immunophenotype of the blood cells favored a pleomorphic mantle cell lymphoma (MCL) aggressive variant diagnosis. Cytogenetic study performed in the WBCs found a complex hyperdiploid karyotype (47 chromosomes, Figure 1) with a t(3;22) translocation involving the BCL6 and IGL genes, a structural abnormality of chromosome 8 resulting in juxtaposition of 5′ MYC and BCL2 in fluorescence in situ hybridization (with break of the MYC probe), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5′IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (Figure 2). Other numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected (Figure 1). Overexpression of cyclin D1 was detected in the WBCs by real-time quantitative PCR, as well as in the skin lesion using immunochemistry. Anti-SOX11 antibody staining was found to be negative. Chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate was initiated, but the patient died 4 months after the diagnosis. This third case of quadruple-hit lymphoma underlines the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbor in very rare cases additional rearrangements of MYC or BCL6, but histological transformation to typical large cell lymphoma is not retained in the WHO classification. In addition, cyclin D1 overexpression is considered to be a rare feature in DLBCL. Recently, Ok et al.3 proposed to reclassify DLBCL with expression of cyclin D1, CCND1 chromosomal rearrangement and CD5 positivity as an aggressive pleomorphic MCL variant. However, no observation of multiple lymphoma-specific gene rearrangements was described in that study. Juskevicius et al.4 suggest the existence of a ‘gray zone’ in which morphologic, clinical and genetic features are insufficient to segregate lymphomas with overexpression of cyclin D1/translocations involving CCND1 between blastoid MCL and cyclin D1-positive DLBCL. Regarding the immunophenotyping and molecular data, our case is possibly a genetically unstable aggressive pleomorphic MCL variant, which acquired three additional genetic hits.

Digital object identifier (DOI): 10.1038/bcj.2015.99

Molecular cytogenetics, 8, 79

Karyotype alteration generates the neoplastic phenotypes of SV40-infected human and rodent cells.

Bloomfield, Mathew, Duesberg, Peter

Despite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don't explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes. As an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations. Since all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and phenotypes, we conclude that these karyotypes have generated and since maintained these neoplastic clones. Thus SV40 causes cancer indirectly, like carcinogens, by inducing aneuploidy from which new cancer-specific karyotypes evolve automatically at low rates. This theory explains the (1) low probability of carcinogenesis per virus-infected cell, (2) the individuality and clonal flexibility of cancer karyotypes, (3) recurrence of neoplasias without viral T-antigens, and (4) the individual clonal karyotypes, transcriptomes and immortality of virus-induced neoplasias - all unexplained by current viral theories.

Digital object identifier (DOI): 10.1186/s13039-015-0183-y

Trends in Cancer Research

Fluorescence in situ hybridisation assays designed for del(7q) detection uncover more complex rearrangements in myeloid leukaemia cell lines

Yasser Mostafa Kamel, Abdulbasit Naiel, Areej Alshehri, Michael Vetter, Salvatore Saccone, Rhona Anderson, Sabrina Tosi

Fluorescence in situ hybridisation assays designed for del(7q) detection uncover more complex rearrangements in myeloid leukaemia cell lines ABSTRACT Chromosome 7 abnormalities are associated with poor prognosis in myeloid leukaemia. The pathogenetic mechanisms chromosome 7 rearrangements and lead to malignancy are still poorly understood. The use of leukaemia- derived cell lines might be a useful tool to shed some light on these mechanisms. The cytogenetic characterisation of these cell lines is therefore important for the understanding of the genetic alterations leading to the disease. We carried out fluorescence in situ hybridisation (FISH) on three different myeloid leukaemia-derived cell lines (GDM-1, GF-D8 and K562). These were selected on the basis of harbouring rearrangements of chromosome 7. The probes used in these experiments were whole and partial chromosome paints, Multiplex-fluorescence in situ hybridisation (M-FISH) probes as well as locus specific probes for the 7q22, 7q31 and 7q36 regions. Our study confirmed the chromosome 7 abnormalities previously reported in the cell lines GDM-1 and GF-D8. We refined one of the rearrangements of chromosome 7 in the K562 cell line and reported some discrepancies with the data published in earlier reports. With this study, we confirm the importance of using a series of FISH that arise from probes to characterise chromosomal abnormalities in detail, as some rearrangements might go under-detected or mis-interpreted. Moreover, we highlight the importance of monitoring cell lines broadly used in research, as these can lose or acquire characteristics as they evolve in time in different laboratories.

Dental materials : official publication of the Academy of Dental Materials, 31, 1335--1344

Dental composite components induce DNA-damage and altered nuclear morphology in gingiva fibroblasts.

Styllou, Marianthi, Reichl, Franz-Xaver, Styllou, Panorea, Urcan, Ebru, Rothmund, Lena, Hickel, Reinhard, Högg, Christof, Scherthan, Harry

Released dental composite components can damage human gingival fibroblasts (HGFs) and their DNA. The cytotoxicity, chromatin condensation and the induction of DNA double strand breaks (DSBs) by different compounds of dental composites was investigated using an improved γ-H2AX focus assay. HGFs were incubated with the monomers: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), bisphenol-A-glycerolate-dimethacrylate (BisGMA), ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA), and acrylamide (ACR). DSBs were determined by enumerating γ-H2AX and 53BP1 foci colocalized at DSBs. A concentration-dependent induction of DSBs was found in the order: GMA>BisGMA>ACR>Bis-DMA>HDDMA>TMPTA>ETEGMA. HGFs exposure to GMA (0.3mM) and to BisGMA (0.09mM) induced the highest rate of DSB foci, i.e. 12-fold and 8-fold, respectively, relative to control (0.33 DSB foci/cell). At the highest concentrations (EC50) prominent changes in the chromatin morphology of HGF cell nuclei, i.e. compaction of nuclear chromatin and reduction of the area covered by the ovoid fibroblast nuclei, were observed. Nuclear condensation was significantly induced by GMA (1.7-fold at 0.3mM) and BisGMA (1.6-fold at 0.09mM), which correlated with the highest numbers of induced DSB foci (GMA, BisGMA, 3.9 and 2.6 foci/cell, respectively). The improved γ-H2AX/53BP1 focus assay revealed a concentration-dependent increase in DSBs for all tested substances. Furthermore, concentration-dependent changes in HGF cell nucleus morphology was noted, demonstrating genotoxic effects of the substances tested.

Digital object identifier (DOI): 10.1016/j.dental.2015.08.156

Nat Genet, 46(11), 1239--1244
November, 2014

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomicinstability and progeroid features.

Davor Lessel, Bruno Vaz, Swagata Halder, Paul J. Lockhart, Ivana Marinovic-Terzic, Jaime Lopez-Mosqueda, Melanie Philipp, Joe C H. Sim, Katherine R. Smith, Judith Oehler, Elisa Cabrera, Raimundo Freire, Kate Pope, Amsha Nahid, Fiona Norris, Richard J. Leventer, Martin B. Delatycki, Gotthold Barbi, Simon {von Ameln}, Josef H{\"o}gel, Marina Degoricija, Regina Fertig, Martin D. Burkhalter, Kay Hofmann, Holger Thiele, Janine Altm{\"u}ller, Gudrun N{\"u}rnberg, Peter N{\"u}rnberg, Melanie Bahlo, George M. Martin, Cora M. Aalfs, Junko Oshima, Janos Terzic, David J. Amor, Ivan Dikic, Kristijan Ramadan, Christian Kubisch

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Prenat Diagn, 34(11), 1066--1072
November, 2014

A new marker set that identifies fetal cells in maternal circulationwith high specificity.

Lotte Hatt, Marie Brinch, Ripudaman Singh, Kristine M{\o}ller, Rune Hoff Lauridsen, Jacob M{\o}rup Schl{\"u}tter, Niels Uldbjerg, Britta Christensen, Steen K{\o}lvraa

Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking.Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes.Fetal cell candidates could be detected in 91\% of the samples, and in 85\% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100\% if three or more fetal cells with FISH signals could be found in a sample.The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic.

Toxicol Sci, 140(1), 103--117
July, 2014

The gamma-H2AX Assay for Genotoxic and Nongenotoxic Agents: Comparisonof H2AX Phosphorylation with Cell Death Response.

Teodora Nikolova, Mirek Dvorak, Fabian Jung, Isabell Adam, Elisabeth Kr{\"a}mer, Aslihan Gerhold-Ay, Bernd Kaina

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, gamma-H2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if gamma-H2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified gamma-H2AX foci manually and automatically. In addition, total gamma-H2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced ?H2AX dose-dependently whereas nongenotoxic agents do not; (2) gamma-H2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and gamma-H2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to gamma-H2AX; (3) manual scoring of gamma-H2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that gamma-H2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) gamma-H2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of gamma-H2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of gamma-H2AX by genotoxicants is related to loss of viability and support gamma-H2AX as a reliable bio-indicator for pretoxic DNA damage.

Mutagenesis, 29(3), 165--175
May, 2014

Automation and validation of micronucleus detection in the 3D EpiDerm?human reconstructed skin assay and correlation with 2D dose responses.

K. E. Chapman, A. D. Thomas, J. W. Wills, S. Pfuhler, S. H. Doak, G J S. Jenkins

Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay's fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 ?g/ml and methyl methanesulfonate (MMS) at 1750 ?g/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 ?g/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm?. Our preliminary validation here demonstrates that the RSMN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives.

J Radiat Res
April, 2014

Biodosimetry estimation using the ratio of the longest:shortest lengthin the premature chromosome condensation (PCC) method applying autocaptureand automatic image analysis.

Jorge E. Gonz{\'a}lez, Ivonne Romero, Eric Gregoire, C{\'e}cile Martin, Ana I. Lamadrid, Philippe Voisin, Joan-Francesc Barquinero, Omar Garc{\'i}a

The combination of automatic image acquisition and automatic image analysis of premature chromosome condensation (PCC) spreads was tested as a rapid biodosimeter protocol. Human peripheral lymphocytes were irradiated with (60)Co gamma rays in a single dose of between 1 and 20 Gy, stimulated with phytohaemaglutinin and incubated for 48 h, division blocked with Colcemid, and PCC-induced by Calyculin A. Images of chromosome spreads were captured and analysed automatically by combining the Metafer 4 and CellProfiler platforms. Automatic measurement of chromosome lengths allows the calculation of the length ratio (LR) of the longest and the shortest piece that can be used for dose estimation since this ratio is correlated with ionizing radiation dose. The LR of the longest and the shortest chromosome pieces showed the best goodness-of-fit to a linear model in the dose interval tested. The application of the automatic analysis increases the potential use of the PCC method for triage in the event of massive radiation causalities.

Int J Radiat Biol, 90(4), 318--324
April, 2014

Effect of hypothermia on radiation-induced micronuclei and delayof cell cycle progression in TK6 cells.

Halina Lisowska, Karl Brehwens, Friedo Z{\"o}lzer, Aneta Wegierek-Ciuk, Joanna Czub, Anna Lankoff, Siamak Haghdoost, Andrzej Wojcik

Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis.Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8°C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8°C or 37.0°C.The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected.The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.

J Trop Pediatr, 60(2), 134–140
April, 2014

Effect of therapeutic hypothermia on DNA damage and neurodevelopmental outcome among term neonates with perinatal asphyxia: a randomized controlled trial.

Gane, Bahubali D., Bhat, Vishnu, Rao, Ramachandra, Nandhakumar, S., Harichandrakumar, K. T., Adhisivam, B.

To study the effect of therapeutic hypothermia (TH) on deoxyribonucleic acid (DNA) damage and the neurodevelopmental outcome in term babies with perinatal asphyxia.Babies in the hypothermia group were cooled for the first 72 h, using gel packs. Rectal temperature of 33-34°C was maintained. Blood sample was collected before, at 36 h and after completion of TH for assessment of comet assay and 8-hydroxy2-deoxyguanosine (8-OHdG). Infants were followed up till 12 months.Baseline parameters were similar. After 72 h, the hypothermia group showed lower olive tail moment (12.88 ± 2.14) than the control group (22.16 ± 5.26) (p < 0.001). 8-HDG levels increased significantly in the control group (1252.87 ± 357.07) as compared to the hypothermia group (757.03 ± 198.49) (p < 0.001). Neurodevelopmental assessment at 12 months showed significantly low motor and mental developmental quotient in the control than hypothermia group.TH reduces oxidative stress-induced DNA damage and improves neurodevelopmental outcome. <Trial registration No: CTRI/2011/10/002094>

Digital object identifier (DOI): 10.1093/tropej/fmt098

Environ Mol Mutagen, 55(2), 114--121
March, 2014

Influence of experimental conditions on data variability in the liver comet assay.

M. Gu{\'e}rard, C. Marchand, U. Plappert-Helbig

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters. Environ. Mol. Mutagen. 55:114-121, 2014. © 2013 Wiley Periodicals, Inc.

Int J Radiat Biol, 90(2), 149--158
February, 2014

Induction and disappearance of gammaH2AX foci and formation of micronuclei after exposure of human lymphocytes to (60)Co gamma-rays and p(66)+ Be(40) neutrons.

Veerle Vandersickel, Philip Beukes, Bram {Van Bockstaele}, Julie Depuydt, Anne Vral, Jacobus Slabbert

To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (gammaH2AX foci) after in vitro exposure of human lymphocytes to either (60)Co gamma-rays or p(66)+ Be(40) neutrons.MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0-4 Gy gamma-rays or 0-2 Gy neutrons. Also, gammaH2AX foci DR curves were obtained following exposure to doses ranging from 0-0.5 Gy of either gamma-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy gamma-rays or neutrons were studied up to 24 hours post-irradiation.Micronuclei yields following neutron exposure were consistently higher compared to that from (60)Co gamma-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced gammaH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from gamma-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from gamma-rays. Foci formations were more likely to be over-dispersed for neutron irradiations.Although neutrons are more effective to induce MN, the absolute number of induced gammaH2AX foci are less at first compared to gamma-rays. With time neutron-induced foci are more persistent. These findings are helpful for using £^H2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

Int J Radiat Biol, 90(2), 193--202
February, 2014

Inter- and intra-laboratory comparison of a multibiodosimetric approachto triage in a simulated, large scale radiation emergency.

Elizabeth A. Ainsbury, Jenna Al-Hafidh, Ainars Bajinskis, Stephen Barnard, Joan Francesc Barquinero, Christina Beinke, Virginie {de Gelder}, Eric Gregoire, Alicja Jaworska, Carita Lindholm, David Lloyd, Jayne Moquet, Reetta Nylund, Ursula Oestreicher, Sandrine Roch-Lef{\'e}vre, Kai Rothkamm, Horst Romm, Harry Scherthan, Sylwester Sommer, Hubert Thierens, Charlot Vandevoorde, Anne Vral, Andrzej Wojcik

The European Union's Seventh Framework Programme-funded project 'Multi-disciplinary biodosimetric tools to manage high scale radiological casualties' (MULTIBIODOSE) has developed a multiparametric approach to radiation biodosimetry, with a particular emphasis on triage of large numbers of potentially exposed individuals following accidental exposures. In November 2012, an emergency exercise took place which tested the capabilities of the MULTIBIODOSE project partners. The exercise described here had a dual purpose: Intercomparison of (i) three biodosimetric assays, and (ii) the capabilities of the seven laboratories, with regards to provision of triage status for suspected radiation exposed individuals.Three biological dosimetry tools - the dicentric, micronucleus and gamma-H2AX (the phosphorylated form of member X of histone H2A, in response to DNA double-strand breaks) foci assays - were tested, in addition to provision of the triage status results (low exposure: 2 Gy) by the MULTIBIODOSE software. The exercise was run in two modes: An initial triage categorisation of samples (based on the first dose estimates for each assay received from each laboratory) followed by collation of the full set of estimated doses (all the results from all modes of each assay carried out by the participating laboratories) calculated using as many modes of operation as possible of the different assays developed during the project. Simulated acute whole body and partial body exposures were included.The results of the initial triage categorisation and the full comparison of assays and methods within and between laboratories are presented here.The data demonstrate that the MULTIBIODOSE approach of applying multiparametric tools to radiation emergencies is valid and effective.

Prenat Diagn
February, 2014

Validation of automatic scanning of microscope slides in recoveringrare cellular events: application for detection of fetal cells inmaternal blood.

Ahmed Emad, Eric F. Bouchard, Jos?e Lamoureux, Annie Ouellet, Aparajita Dutta, Uli Klingbeil, R?gen Drouin

Detection of rare fetal cells (FCs) in the maternal circulation could be used for non-invasive prenatal diagnosis. Considering that FCs in maternal blood are present in extremely low frequency, manual scanning is cumbersome, time-consuming and unsuitable for clinical applications. As an alternative, we optimized a custom-made classifier for automatic detection of FCs.Using MetaSystems' automated platform, we developed a robust detection algorithm and validated its efficiency on retrieval of rare XY cells in a pure population of XX cells. Slides were scanned for presence of predefined XY cells after fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS). Retrieval of FCs was also performed on samples from maternal blood.The efficiency of detection of rare XY cells was 88\% using FISH (117/133) in comparison to 78\% (53/68) with PRINS. FC frequencies per 1?ml of maternal blood ranged from 3-6 FCs in normal pregnancies versus 13-21 FCs in Down syndrome pregnancies.Automatic scanning was more efficient and consistent than manual scanning for detection of rare FCs and required considerably less operator time. Automatic scanning using FISH is more sensitive than that using PRINS. The study validates automatic scanning retrieval of FCs from maternal blood. This article is protected by copyright. All rights reserved.

Mod Pathol, 27(1), 107--112
January, 2014

EGFR alterations and EML4-ALK rearrangement in primary adenocarcinomaof the urinary bladder.

Riley E. Alexander, Rodolfo Montironi, Antonio Lopez-Beltran, Sean R. Williamson, Mingsheng Wang, Kristin M. Post, Joyashree D. Sen, Ashley K. Arnold, Shaobo Zhang, Xiaoyan Wang, Michael O. Koch, Noah M. Hahn, Timothy A. Masterson, Gregory T. Maclennan, Darrell D. Davidson, Eva Comp{\'e}rat, Liang Cheng

The identification of mutations in epidermal growth factor receptor (EGFR) and translocations involving anaplastic lymphoma kinase (ALK) in lung adenocarcinoma has drastically changed understanding of the disease and led to the development of targeted therapies. Adenocarcinoma of the urinary bladder is rare and poorly understood at the molecular level. We undertook this study to determine whether EGFR mutations, increases in EGFR copy number, or ALK translocations are present in these tumors. Twenty-eight cases of primary bladder adenocarcinoma were analyzed. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits. All cases were analyzed via fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probes for detection of ALK chromosomal translocation and Vysis Dual Color Probes to assess for increased gene copy number of EGFR. None of the 28 cases examined showed mutational events in EGFR or ALK rearrangements. EGFR polysomy was seen in 10 out of 28 (36\%) cases. No correlation with EGFR polysomy was seen in the tumors with respect to age, histologic subtypes, pathologic stage, or lymph node metastasis. In summary, EGFR mutations and ALK rearrangements do not appear to be involved in the development of primary adenocarcinoma of the urinary bladder. A subgroup of cases (36\%), however, demonstrated increased gene copy number of EGFR by FISH.

EJNMMI Res, 4(1), 17

Visualization of angiogenesis during cancer development in the polyoma middle T breast cancer model: molecular imaging with (R)-[11C]PAQ.

Samén, Erik, Lu, Li, Mulder, Jan, Thorell, Jan-Olov, Damberg, Peter, Tegnebratt, Tetyana, Holmgren, Lars, Rundqvist, Helene, Stone-Elander, Sharon

Vascular endothelial growth factor receptor 2 (VEGFR2) is a crucial mediator of tumour angiogenesis. High expression levels of the receptor have been correlated to poor prognosis in cancer patients. Reliable imaging biomarkers for stratifying patients for anti-angiogenic therapy could therefore be valuable for increasing treatment success rates. The aim of this study was to investigate the pharmacokinetics and angiogenesis imaging abilities of the VEGFR2-targeting positron emission tomography (PET) tracer (R)-[11C]PAQ.(R)-[11C]PAQ was evaluated in the mouse mammary tumour virus-polyoma middle T (MMTV-PyMT) model of metastatic breast cancer. Mice at different stages of disease progression were imaged with (R)-[11C]PAQ PET, and results were compared to those obtained with [18 F]FDG PET and magnetic resonance imaging. (R)-[11C]PAQ uptake levels were also compared to ex vivo immunofluorescence analysis of tumour- and angiogenesis-specific biomarkers. Additional pharmacokinetic studies were performed in rat and mouse.A heterogeneous uptake of (R)-[11C]PAQ was observed in the tumorous mammary glands. Ex vivo analysis confirmed the co-localization of areas with high radioactivity uptake and areas with elevated levels of VEGFR2. In some animals, a high focal uptake was observed in the lungs. The lung uptake correlated to metastatic and angiogenic activity, but not to uptake of [18 F]FDG PET. The pharmacokinetic studies revealed a limited metabolism and excretion during the 1-h scan and a distribution of radioactivity mainly to the liver, kidneys and lungs. In rat, a high uptake was additionally observed in adrenal and parathyroid glands.The results indicate that (R)-[11C]PAQ is a promising imaging biomarker for visualization of angiogenesis, based on VEGFR2 expression, in primary tumours and during metastasis development.

Digital object identifier (DOI): 10.1186/2191-219X-4-17