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- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (22) (remove)
Цель: Оценить влияние локализации точки разрыва в геномной ДНК гена MLL на прогноз острых лейкозов (ОЛ) у детей первого года жизни.
Методы: В исследование было включено 68 детей первого года жизни (29 мальчиков и 39 девочек с медианой возраста 4,8 мес.) с MLL-позитивными острым лимфобластным лейкозом (ОЛЛ) (n = 46), острым миелоидным лейкозом (ОМЛ) (n = 20) и ОЛ смешанной линейности (n = 2).
Результаты: 5-летняя бессобытийная выживаемость (БСВ) детей первого года жизни с ОЛЛ, включенных в исследование MLL-Baby, с точкой разрыва в интроне 11 ДНК гена MLL (n = 29) была статистически значимо ниже, чем у пациентов c локализацией точек разрыва, начиная с интрона 7 по экзон 11 (n = 17; 0,16 ± 0,07 и 0,38 ± 0,14; p = 0,039), а кумулятивная вероятность развития рецидива была значительно выше в группе с точкой разрыва в интроне 11 (0,74 ± 0,09 и 0,52 ± 0,17; p = 0,045). В то же время многофакторный анализ показал, что единственным значимым фактором, связанным с неблагоприятным прогнозом, остается сохранение минимальной остаточной болезни (МОБ) в точке наблюдения 4 протокола MLL-Baby (отношение опасности 5,994; 95%-й доверительный интервал 2,209–16,263; p < 0,001). У 22 пациентов с ОМЛ связи между прогнозом и локализацией точки разрыва в ДНК гена MLL не выявлено.
Заключение: Наличие точки разрыва в интроне 11 гена MLL у детей первого года жизни с ОЛЛ, получавших лечение по протоколу MLL-Baby, вело к статистически значимо более низким показателям БСВ и более высокой кумулятивной вероятности развития рецидива. Однако в многофакторной модели риска это нивелировалось сохранением МОБ в точке наблюдения 4. У детей первого года жизни с ОМЛ взаимосвязи между локализацией точки разрыва в ДНК гена MLL и прогнозом не выявлено.
In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin
(2015)
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.
Background: A delta and C fibers are the major pain-conducting nerve fibers, activate only partly the same brain areas, and are differently involved in pain syndromes. Whether a stimulus excites predominantly A delta or C fibers is a commonly asked question in basic pain research but a quick test was lacking so far. Methodology/Principal Findings: Of 77 verbal descriptors of pain sensations, "pricking", "dull" and "pressing" distinguished best (95% cases correctly) between A delta fiber mediated (punctate pressure produced by means of von Frey hairs) and C fiber mediated (blunt pressure) pain, applied to healthy volunteers in experiment 1. The sensation was assigned to A delta fibers when "pricking" but neither "dull" nor "pressing" were chosen, and to C fibers when the sum of the selections of "dull" or "pressing" was greater than that of the selection of "pricking". In experiment 2, with an independent cohort, the three-descriptor questionnaire achieved sensitivity and specificity above 0.95 for distinguishing fiber preferential non-mechanical induced pain (laser heat, exciting A delta fibers, and 5-Hz electric stimulation, exciting C fibers). Conclusion: A three-item verbal rating test using the words "pricking", "dull", and "pressing" may provide sufficient information to characterize a pain sensation evoked by a physical stimulus as transmitted via A delta or via C fibers. It meets the criteria of a screening test by being easy to administer, taking little time, being comfortable in handling, and inexpensive while providing high specificity for relevant information.
BACKGROUND: Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies.
RESULTS: We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA.
CONCLUSION: Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.
Novel chalcone-based fluorescent human histamine H 3 receptor ligands as pharmacological tools
(2012)
Novel fluorescent chalcone-based ligands at human histamine H(3) receptors (hH(3)R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH(3)R in the same concentration range like the reference antagonist ciproxifan (hH(3)R pK(i) value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH(3)R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH(3)R visualization in different tissues.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
I-kappaB-Kinase epsilon - ein neues Zielprotein für die Pharmakotherapie bei Schmerz und Entzündung?
(2010)
Der Transkriptionsfaktor NF-kappaB spielt eine wichtige Rolle bei der Regulation von Immunantworten, Apoptose und Entzündungen sowie bei der Entstehung und Verarbeitung von Schmerzen. Ein pharmakologischer Eingriff in die NF-kappaB-Aktivierungskaskade könnte daher eine Schmerzhemmung bewirken und so Ansätze für die Entwicklung neuer Therapien für pathophysiologische Schmerzen liefern. Die NF-kappaB-Signalübertragungskaskade bietet verschiedene Angriffspunkte für Pharmaka, wobei zurzeit IkappaB Kinasen (IKK) als hoffnungsvolle Zielmoleküle im Fokus der Untersuchungen stehen. Verschiedene IKKs regulieren die Aktivität von NF-kappaB über die Phosphorylierung des inhibitorischen Proteins IkappaB oder über die direkte Phosphorylierung von NF-kappaB. In der vorliegenden Arbeit wurde die Rolle der neu entdeckten IKK epsilon bei der Schmerzentstehung und -verarbeitung sowie deren Eignung als neues Zielmolekül für die Schmerztherapie näher untersucht. Es konnte gezeigt werden, dass IKK epsilon konstitutiv in Geweben der Maus, welche an der Entstehung und Verarbeitung von Schmerzen beteiligt sind, exprimiert ist. Im Rückenmark konnte die Lokalisation von IKK epsilon in den schmerzrelevanten Laminae I und II des Dorsalhorns nachgewiesen werden und auch in den Hinterwurzelganglien (Dorsal Root Ganglia (DRG’s)) war IKK epsilon in kleinen, nozizeptiven Neuronen exprimiert. Nach peripherer entzündlich-nozizeptiver Stimulation mit Formalin oder Zymosan kam es im Lumbalmark und den DRG’s zu einem signifikanten Anstieg der IKK epsilon-Expression sowohl auf mRNA- als auch auf Proteinebene. Diese Beobachtungen machten eine Beteiligung von IKK epsilon an der Prozessierung von Schmerz sehr wahrscheinlich. Um die Rolle von IKK epsilon während der Schmerzentstehung und -verarbeitung besser beurteilen zu können wurde das Verhalten von IKK epsilon defizienten Mäusen in akuten und inflammatorischen Schmerzmodellen charakterisiert. Es konnte gezeigt werden, dass der Knockout von IKK epsilon zu einem signifikant verringerten nozizeptiven Verhalten im Formalintest und einer Hemmung der mechanischen Hyperalgesie nach Zymosaninjektion im Vergleich zu Wildtyp-Mäusen führte. Gleichzeitig konnte kein Unterschied im akut nozizeptiven Verhalten festgestellt werden. Der Knockout von IKK epsilon hatte demnach keine Auswirkung auf den akuten physiologischen Nozizeptorschmerz, zeigte jedoch eine Verbesserung bei pathophysiologischen Schmerzen. Das verringerte nozizeptive Verhalten der IKK epsilon defizienten Mäuse im Formalintest ging mit einer Hemmung der NF-kappaB-Aktivierung im Rückenmark einher. Auch konnte eine verringerte mRNA-Expression der NF-kappaB-abhängigen Gene Cyclooxygenase-2 (COX-2), Matrixmetalloprotease-9 (MMP-9) und induzierbare Stickstoffmonoxid-Synthase (iNOS), die an der Regulation von Entzündungsschmerzen beteiligt sind, im Rückenmark und den DRG’s nachgewiesen werden. Da IKK epsilon bisher hauptsächlich mit der Aktivierung des TypI Interferon-Signalweges in Zusammenhang gebracht wurde, wurde außerdem geprüft, ob es nach Injektion mit Formalin zu einer Aktivierung des Trankriptionsfaktors Interferon-regulierender Faktor (IRF)-3 in Wildtyp-Mäusen kommt, was nicht beobachtet werden konnte. Der Knockout von IKK epsilon scheint demnach seine antinozizeptive Wirkung direkt über eine fehlende Aktivierung von NF-kappaB zu entfalten, wonach IKK epsilon eine bedeutendere Rolle als bisher angenommen bei der Aktivierung von NF-kappaB spielt. Dies konnte durch in vitro Daten untermauert werden. Der Knockdown von IKK epsilon in Makrophagen-Zellkultur mit spezifischer siRNA verhinderte die Phosphorylierung von NF-kappaBp65 am Serinrest 536 nach Stimulation mit LPS. Anhand der vorliegenden Daten lässt sich also schlussfolgern, dass IKK epsilon an der Schmerzentstehung und verarbeitung bei Entzündungen beteiligt zu sein scheint. Eine Hemmung dieser Kinase könnte demnach ein neues, lohnendes Ziel für die Entwicklung neuer Medikamente für die Schmerztherapie sein.
Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.