Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology

<p>Abstract</p> <p>Background</p> <p>During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology.</p> <p>Results</p> <p>A statistically highly significant correlation (Spearman r: 0.78 – 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology.</p> <p>Conclusion</p> <p>Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.</p

Hypoxia-induced down-regulation of microRNA-449a/b impairs control over targeted SERPINE1 (PAI-1) mRNA - a mechanism involved in SERPINE1 (PAI-1) overexpression

After publication of our article [1], we realized the need for posting a correction note in order to prevent i) overinterpretation of some results by the readers and ii) concerns about potentially unintended misguidance by the authors

Risk Factors and Prognosis in T-Cell Posttransplantation Lymphoproliferative Diseases: Reevaluation of 163 Cases

Background. Posttransplantation lymphoproliferative diseases (PTLD) are mainly Epstein-Barr virus (EBV)-associated disorders of B-cell origin. Due to the rarity of monomorphic T-cell PTLD (T-PTLD), knowledge about pathogenesis, risk factors, therapy, and prognosis relies predominantly on case reports and small series. Therefore, we aimed to provide an overview and a retrospective analysis of this rare PTLD subtype. Methods. We analyzed all available articles on T-PTLD in the PubMed database as well as in our own databases (Institute of Pathology/Department of Paediatric Haematology and Oncology, Hannover Medical School) from 1988 to 2010. Reevaluated parameters were gender, age, transplanted organ, immunosuppressant regimen, time between transplantation and T-PTLD manifestation, T-PTLD subtype, virus positivity, localization, therapy, and follow-up. Results. A total of 163 cases were evaluated. We found that hematopoietic stem cell transplantation was associated with early-onset T-PTLD, whereas late onset occurred after immunosuppression with steroids and azathioprine without administration of calcineurin inhibitors. The major independent favorable prognostic factors were T-PTLD of the large granular lymphocytic leukemia subtype, young age, and a combination of radiotherapy/radiochemotherapy and reduced immunosuppression, whereas the hepatosplenic T-cell lymphoma subtype and cases with involvement of bone marrow, the central nervous system, or graft had an adverse prognosis. Conclusion. T-PTLD is a heterogeneous group of different aberrant T-cell proliferations and represents a significant complication following transplantation, showing a uniformly poor prognosis

"Hypoxia-induced down-regulation of microRNA-449a/b impairs control over targeted SERPINE1 (PAI-1) mRNA - a mechanism involved in SERPINE1 (PAI-1) overexpression"

<p>Abstract</p> <p>Background</p> <p>In damaged organs tissue repair and replacement of cells by connective tissue provokes a response of fibroblasts to cellular stress factors such as hypoxia.</p> <p>MicroRNAs (miRNA) are small non-coding RNA molecules which bind to their mRNA targets which eventually lead to repression of translation. Whether the response of fibroblasts to stress factors also involves the miRNA system is largely unknown.</p> <p>Results</p> <p>By miRNA profiling we identified down-regulation of miRNA-449a/b expression in hypoxic fibroblasts. Specific miRNA inhibitors and mimics showed direct evidence for targeting the serine protease inhibitor (serpin) protein (SERPINE1; plasminogen activator inhibitor-1, PAI-1) by miRNA-449a/b leading to SERPINE1 mRNA and protein up- and down-regulation, respectively. SERPINE1 expression <it>in vivo </it>could be located predominantly in areas of fibrosis and remodeling.</p> <p>Conclusions</p> <p>Our study offers serious lines of evidence for a novel hypoxia-dependent mechanism involving hypoxia-induced decrease of clustered miRNA-449a/b, hypoxia-induced amplification of concomitant increase of targeted SERPINE1 (PAI-1) and its overexpression in tissues showing a hypoxic environment.</p

Tumour angiogenesis in Epstein-Barr virus-associated post-transplant smooth muscle tumours

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT), are rare complications following organ/stem cell transplantation. Despite the mainly benign behaviour of PTSMT, alternative therapies are needed for those patients with progressive tumours. In tumours not approachable by surgery or reduction of immunosuppression, the angiogenic microenvironment might be a potential target of therapy, an approach that is well utilised in other soft tissue neoplasms. In a previous study, we evaluated the expression of EBV-related genes and the microRNA profile in PTSMT, but so far the characteristics of angiogenesis in PTSMT are not known. Therefore, the aim of this study was to evaluate the expression pattern of angiogenesis-related genes in PTSMT, in order to identify potential target molecules for anti-angiogenic therapy. PTSMT (n = 5 tumours) were compared with uterine leiomyomas (n = 7). Analyses included real-time PCR of 45 angiogenesis-associated genes, immunohistochemistry (CD31, prostaglandin endoperoxide synthase 1/PTGS1) and assessment of tumour vascularisation by conventional histopathology. PTSMT showed similar or fewer vessels than leiomyomas. Of the genes under investigation, 23 were down-deregulated (pro-angiogenic and some anti-angiogenic factors) and five were up-regulated (e.g. PTGS1 which is expressed at very low levels in leiomyomas but moderately higher levels in PTSMT). In summary, no particular target molecule could be identified, because tumour angiogenesis in PTSMT is characterised by low levels of major pro-angiogenic factors and there is no prominent increase in tumour vascularisation. EBV can induce angiogenesis via its viral late membrane protein 1 (LMP1) but PTSMT frequently do not express LMP1, which could be an explanation why, despite EBV infection, PTSMT show no exaggerated tumour angiogenesis

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution

<p>Abstract</p> <p>Background</p> <p>New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.</p> <p>Results</p> <p>To this end the methylation patterns of 12 loci (<it>GSTπ1, p16</it>^{<it>INK4a</it>}<it>, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31</it>, and <it>LINE-1</it>) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r²= 0.927).</p> <p>Conclusions</p> <p>Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify <it>MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 </it>as new targets of aberrant DNA methylation in human hepatocelluar carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.</p

Evaluation of the biological tolerability of the starch-based medical device 4DryField® PH in vitro and in vivo a rat model

Purpose To evaluate in vitro cytotoxicity/biocompatibility as well as in vivo tolerability of the novel polysaccharide 4DryField® PH, certified for haemostasis and adhesion prevention. Methods In vitro cytotoxicity/viability testing according to ISO EN 10,993 using murine and human tumour cell lines incubated with 4DryField® PH (PlantTec Medical GmbH). Using a rat model the impact of 4DryField® PH on animals viability and in vivo effects were macro- and micropathologically assessed. Results In vitro testing revealed no cytotoxic effect of 4DryField® PH nor enhancement of viability to tumour cell lines. In vivo viability of rats was unimpaired by 4DryField® PH. Bodyweight loss in animals with abdominal injury plus treatment with 4DryField® PH was in the range of controls and less than in injured rats without treatment. At day 7 after surgery no formation of adhesions, neither macroscopic nor histological remnants nor signs of foreign body reaction were present in animals without injury. In animals with peritoneal injury and 4DryField® PH application, histopathological observation revealed minor residuals of polysaccharide in the depth of wound cavity embedded in a thickened subperitoneal layer; however, with a suggested intact neoperitoneum. The presence of mononuclear cells surrounding polysaccharide particles in varying states of degradation was observable as well. Conclusion 4DryField® PH is not cytotoxic and does not enhance viability of tumour cell lines. High dose of 4DryField® PH of 1.09 g/kg bodyweight is well tolerated and reduces weight loss in animals with peritoneal injury. The biocompatibility of 4DryField® PH can be rated as being excellent. © SAGE Publications

Практический опыт развития комплексной системы экологического просвещения в образовательной организации

В статье представлены разработанные и апробированные в образовательной организации новые подходы, механизмы и инструменты по одному из приоритетных направлений развития страны в части формирования экологического культуры вузовской молодежи с целью повышения познавательной активности, уровня экологических знаний в области гармоничного развития человека и природы, устойчивого интереса к экологическим проблемам современности, воспитания и привития бережного отношения к окружающей природе.The article presents new approaches, mechanisms and tools developed and tested in the educational organization in one of the priority directions of the country's development in terms of forming the ecological culture of university youth in order to increase cognitive activity, the level of ecological knowledge in the field of harmonious development of man and nature, and a steady interest in environmental problems of modernity, upbringing and inculcation of respect for nature

Exploring the spatial dimension of estrogen and progesterone signaling: detection of nuclear labeling in lobular epithelial cells in normal mammary glands adjacent to breast cancer

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