Glycogen synthase kinase-3β negatively regulates group IIA phospholipase A2 expression in human aortic smooth muscle and HepG2 hepatoma cells

AbstractThe present study shows that the IFN-γ-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by indirubin-3′-monoxime, 5-iodo or AR-A014418. The effect of GSK-3β inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3β inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-κB-signaling

Veränderung von Diagnostik und Therapie der akuten und chronischen Sigmadivertikulitis durch Implementierung einer neuen Leitlinie und Klassifikation der Divertikelkrankheit/Divertikulitis

Die Divertikulitis des Sigmas stellt eine der häufigsten Erkrankung der westlichen Welt mit steigender Inzidenz dar. Das Erscheinungsbild ist vielgestaltig und der Ausprägungsgrad der Erkrankung therapieentscheidend. Eine einheitliche und konzertierte Klassifikation existierte nicht. Erst mit Etablierung der interdisziplinären S2k Leitlinie Divertikelkrankheit /Divertikulitis wurde 2013 ein Instrument in Form einer Klassifikation (Classification of Diverticular Disease = CDD) geschaffen, das deutschlandweit die verschiedenen Typen der Divertikelkrankheit/ Divertikulitis einteilt und somit Diagnostik- und Therapieentscheidungen konform ermöglicht. Untersucht wurde der Einfluss der neuen Klassifikation CDD auf die Diagnostik und Therapie der Sigmadivertikulitis. Hierzu wurden die Daten von 466 Patienten, die im Zeitraum 2009-2015 in den Helios-Kliniken Schwerin stationär unter der Diagnose Sigmadivertikulitis behandelt wurden, mittels Aktenrecherche erfasst, analysiert und statistisch ausgewertet. Explizit erfolgte die Gegenüberstellung der Klassifikationen Hansen-Stock und CDD, beide angewandt auf das betrachtete Patientengut. Im beobachteten Zeitraum konnte in einigen Bereichen ein signifikanter Wandel sowohl in der Diagnostik als auch in der Therapie belegt werden, insbesondere der Einfluss auf die Therapieentscheidung unter Anwendung der beiden Klassifikationen. Im Ergebnis zeigte sich, dass primäre diagnostische Mittel bei der Akutvorstellung mit abdominellen Beschwerden Anamneseerhebung, klinische Untersuchung, Laboruntersuchung und die Sonographie des Abdomens bleiben. Allerdings nimmt die Computertomographie des Abdomens als sekundäres diagnostisches Mittel zur exakten prätherapeutischen Stadienbestimmung einen immer höheren Stellenwert ein. Wesentliches Fazit der Arbeit ist, dass durch den Wandel in der Betrachtung der Krankheitstypen der Divertikelkrankheit auch komplizierte Formen der Divertikulitis abhängig von Klinik und Allgemeinzustand des Patienten primär konservativ/interventionell behandelt werden. Die Therapie hat sich somit zu Gunsten der konservativen Therapie verschoben. Durch den häufigeren und früheren Einsatz der Computertomographie wird das Ausmaß der Entzündung sichtbarer und erhält einen anderen Stellenwert in der Therapieentscheidung. Überdies hat sich die Anzahl der Elektivoperationen durch die Differenzierung der chronisch rezidivierenden Form in komplizierte und unkomplizierte Formen und Abrücken von der Betrachtung der Anzahl der Schübe verringert. Operativ hat sich die Sigmakontinuitätsresektion als Standardverfahren etabliert. Ein protektives Ileostoma oder eine Diskontinuitätsresektion nach Hartmann stellen nur noch Ausnahmen dar. In Abhängigkeit vom Operationszeitpunkt wird durch den Operateur, ausgerichtet an den vorliegenden Untersuchungsbefunden und dem Allgemeinzustand des Patienten, der operative Zugang gewählt. Im Notfall wird der offene Zugang bedingt durch die Schwere der vorliegenden Krankheitsbilder mit den Hauptindikationen perforierende abszedierende Divertikulitis und dem Vorliegen von eitrigen und kotigen Peritonitiden favorisiert. Elektiv ist es die Laparoskopie mit all ihren Vorteilen einschließlich der Verringerung der stationären Verweildauer.Diverticulitis of the sigmoid colon is one of the most common diseases of the western world with increasing incidence. The appearance is multifaceted and the degree of manifestation of the disease is decisive for therapy. A uniform and concerted classification for this disease did not exist in Germany until the establishment of the interdisciplinary S2k guideline and its own classification (Classification of Diverticular Disease = CDD). This classification divides the various types of diverticular disease/ diverticulitis and thus makes diagnostic and therapeutic decisions conformable. In this thesis the influence of the implementation of the new classification on the diagnosis and treatment of sigmoid diverticulitis was investigated. For this purpose, the data of 466 inpatients with sigmoid diverticulitis treated at the Helios-Kliniken Schwerin between 2009- 2015 were recorded and statistically analyzed. Explicitly the influence of the new classification on the diagnosis and therapy of patients was investigated. In the observed period, a significant change in both diagnostics and therapy could be demonstrated, especially on the treatment decision. CT-scans of the abdomen were increasingly important as a diagnostic tool for accurate pre-therapeutic staging. The main conclusion of the work is that an significant change occurred in the indication to surgery. Complicated forms of diverticulitis are primarily treated conservatively/ interventionally, depending on the patient's clinical condition and general condition. The therapy has thus shifted in favor of conservative therapy. Due to the more frequent and earlier use of computer tomography, the extent of the inflammation becomes more visible and receives a different status in the therapy decision. Moreover, the total number of elective operations has been reduced due to the more restrictive classification of complicated disease. Operatively, sigmoid continuity resection has become established as a standard procedure. A protective ileostomy or a Hartmanns procedure were only random events. In case of emergency, open access was still favored due to the severity of the underlying conditions with the main indications of perforating abscessing diverticulitis and the presence of peritonitis. In the elective situation laparoscopic resection has become the standard procedure leading to a significant reduction in length of stay

In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation

In the present study a protocol of in situ reverse transcriptasenested polymerase chain reaction (in situ RT-nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by "DNA repair mechanisms" and "endogenous priming", a two-step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin-labeled nucleotides and intrinsic primers. ing PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT-nested PCR, which in comparison to the method of in situ RT-PCRin situ-hybridisation is simpler and less time-consuming, can be used as an alternative approach to identify intracellular nucleic acids

Expression and shedding of endothelial protein C receptor in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.</p> <p>Results</p> <p>EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H₂O₂, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.</p> <p>Conclusions</p> <p>The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.</p

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.Deutsche Forschungsgemeinschaf

A pivotal role for interleuking-4 in Atorvastatin-associated neuroprotection in rat brain.

noInflammatory changes, characterized by an increase in pro-inflammatory cytokine production and up-regulation of the corresponding signaling pathways, have been described in the brains of aged rats and rats treated with the potent immune modulatory molecule lipopolysaccharide (LPS). These changes have been coupled with a deficit in long-term potentiation (LTP) in hippocampus. The evidence suggests that anti-inflammatory agents, which attenuate the LPS-induced and age-associated increase in hippocampal interleukin-1ß (IL-1ß) concentration, lead to restoration of LTP. Here we report that atorvastatin, a member of the family of agents that act as inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, exerts powerful anti-inflammatory effects in brain and that these effects are mediated by IL-4 and independent of its cholesterol-lowering actions. Treatment of rats with atorvastatin increased IL-4 concentration in hippocampal tissue prepared from LPS-treated and aged rats and abrogated the age-related and LPS-induced increases in pro-inflammatory cytokines, interferon-¿ (IFN¿) and IL-1ß, and the accompanying deficit in LTP. The effect of atorvastatin on the LPS-induced increases in IFN¿ and IL-1ß was absent in tissue prepared from IL-4¿/¿ mice. The increase in IL-1ß in LPS-treated and aged rats is associated with increased microglial activation, assessed by analysis of major histocompatibility complex II expression, and the evidence suggests that IFN¿ may trigger this activation. We propose that the primary effect of atorvastatin is to increase IL-4, which antagonizes the effects of IFN¿, the associated increase in microglial activation, and the subsequent cascade of events