Characterization of the Stem Cell Fraction in Pancreatobiliary Carcinomas: The Notch Signaling Pathway as a Potential Therapeutic Target

Das duktale Adenokarzinom des Pankreas (PDAC) und das extrahepatische Cholangiokarzinom (eCC) sind hochmaligne Tumoren und mit einer sehr schlechten Prognose vergesellschaftet. Im klinischen Alltag zeigen beide Tumorentitäten eine ausgeprägte Resistenz gegenüber den aktuell zur Verfügung stehenden medikamentösen Therapieansätzen. Diverse Publikationen diskutieren den Notch-Signalweg als mitursächlich für die Entstehung des PDACs und eCCs. Diese Signalkaskade beeinflusst im Rahmen der Embryonalentwicklung die Mobilisierung und Differenzierung gewebsspezifischer Vorläuferzellen. Im adulten Organismus steuert sie grundlegende zelluläre Prozesse, deren Dysregulation die maligne Entartung verschiedener Gewebe verursachen kann. Weiterhin wird angenommen, dass so genannte Tumorstammzellen, beziehungsweise Cancer Stem Cells (CSC) die Tumorformation induzieren können. CSCs werden als eine Subpopulation des zellulären Netzwerks eines Karzinoms beschrieben, denen eine führende Rolle in dessen Chemoresistenz, Metastasierungs- und Rezidivfähigkeit zugesprochen wird. In dieser Arbeit wurde die Durchflusszytometrie beziehungsweise die Fluorescence-activated Cell Sorting (FACS)-Analyse zur Charakterisierung der CSCs und Notch-assoziierten Proteinen in fünf etablierten PDAC- und eCC-Zelllinien durchgeführt. Des Weiteren wurde der therapeutische Effekt eines spezifischen Notch-Inhibitors (GSI-IX beziehungsweise DAPT) auf die CSCs untersucht und mit dem des Chemotherapeutikums Gemcitabin verglichen. Zusätzlich wurde der apoptotische und zytotoxische Effekt der Therapeutika auf die eCC- und PDAC-Zellpopulationen in Form des TUNEL- und CellToxGreen Cytotoxity Assays untersucht. Die FACS-Analyse ergab, dass die eCC-Zellpopulationen einen signifikant höheren CSC-Anteil aufweist als die PDAC-Zelllinien (p<0.0001). Zusätzlich wurde die CSC-Population der eCC-Zelllinien unter der Therapie mit DAPT (p<0.008) und Gemcitabin (p<0.04) signifikant reduziert. Dahingegen zeigte die CSC-Fraktion der PDAC-Zelllinien ein schwaches Ansprechen auf die Therapie. Das CellToxGreen Cytotoxity Assay zeigte eine signifikante Verringerung der Zellviabilität in der gesamten eCC-Zellpopulation unter der DAPT- und Gemcitabin-Therapie (p<0.05), wohingegen nur moderate Effekte in den PDAC-Zelllinien beobachtet wurden. Die Auswertung des TUNEL-Apoptoseassays bestätigte diese Erkenntnisse. Zusammenfassend zeigten die CSC-Populationen beider Tumorentitäten ein unterschiedliches Ansprechen auf die Blockade des Notch-Signalweges. Diese Erkenntnis eröffnet neue Perspektiven auf die Therapie des eCCs. Bezogen auf die Therapieoptionen des PDACs erscheint dieser Ansatz jedoch weniger erfolgversprechend. Weitere Studien sind notwendig, um die Rolle der Notch-Signalkaskade und die klinische Relevanz der CSCs in Hinblick auf das Therapieansprechen beider Tumorentitäten zu determinieren.Pancreatic cancer and cholangiocellular carcinoma are aggressive tumors with high mortality rates and poor treatment options. Recent reports describe the Notch pathway as highly relevant for a variety of fundamental cellular processes in the embryonic and adult organism. Therefore its deregulation can induce tumorigenesis. Moreover, cancer stem cells (CSC) are discussed as a source for the formation, chemotherapeutical resistance and recurrence of tumors. Through Fluorescence-activated Cell Sorting (FACS)-Analysis, the existence of cancer stem cells in eCCA- and PDAC cell lines as well as the presence of five Notch-associated proteins in this population was examined. Additionally, the effect of a specific Notch-inhibitor (DAPT-IX/GSI) and the chemotherapeutic gemcitabine on CSCs in eCCA- and PDAC cell lines was explored. To investigate whether these pharmalogical agents also affect the entire cell population, TUNEL-staining indicated the rate of apoptosis in eCCA- und PDAC cell lines. Additionally, CellToxGreen Cytotxity Assay was used to demonstrate the cytotoxity potential of gemcitabine and GSI in PDAC- and eCCA cell lines. In the here presented study, FACS-analysis showed that the CSC population in eCCA cell lines is significantly larger than in PDAC cell lines (p<0.0001). Contrary to the effect on PDAC cell lines, GSI-IX/DAPT (p<0.008) and Gemcitabin (p<0.04) treatment caused a significant decrease of the eCCA-CSC populations. The CellToxGreen Cytotoxity Assay indicated that GSI-IX/DAPT and Gemcitabin treatment decreases cell viability of both ECC cell lines significantly (p<0.05), but reveals only moderate effects on PDAC cell lines. The rate of apoptosis underlines these results. In conclusion, the eCCA und PDAC cell lines are different due to the percentage of CSCs and the expression of Notch-associated proteins. Particularly in regard to its effect on the CSC population, Notch-Signaling may be of therapeutic value in eCC, but seems to show no effect on more aggressive PDAC. We could hereby further support our hypothesis, that it is required to modulate cancer therapy in regard to the CSC fraction to achieve a more effective therapy against those tumour entities. As could be essential for the improvement in outcomes of the eCC patients, other trials are needed to determine the role of further Notch components

Zellvitalität von Glioblastomzellen und Fibroblasten in Anwesenheit von imidazolhaltigen Stoffen

Das Glioblastom ist der häufigste maligne Hirntumor. Unter der aktuellen Standardtherapie bestehend aus möglichst kompletter Resektion, kombinierter Radiochemotherapie und einer Erhaltungstherapie mit Temozolomid bleibt die Prognose mit einer 5-Jahresüberlebensrate von 7,2% schlecht. Das natürlich vorkommende Dipeptid L-Carnosin (β-Alanyl-L-Histidin) schwächt das Wachstum von Tumorzellen ab. In der vorliegenden Arbeit sind wir der Frage nachgegangen, ob andere kleine imidazolhaltige Substanzen auch die Vitalität von Glioblastomzellen beeinflussen ohne dabei nicht-maligne Zellen zu beeinflussen und ob diesem Effekt die Bildung von Histamin zugrunde liegt. Primäre Fibroblastenkulturen und Glioblastomzellen wurden mit L-Carnosin, L-Alanyl-L-Histidin, β-Alanyl-L-Alanin, L-Histidin, Histamin, Imidazol, β-Alanin und L-Alanin für 48 Stunden behandelt. Über zellbasierte Vitalitätsassays und mikroskopische Analysen wurde die Zellvitalität unter dem Einfluss der verschiedenen Substanzen sowohl von den primären Fibroblastenkulturen als auch den Glioblastomzellen bestimmt. Zudem wurde die intrazelluläre Freisetzung von L-Histidin und die Bildung von Histamin nach Behandlung der Zellen entweder mit L-Carnosin oder mir L-Alanyl-L-Histidin mittels Hochleistungsflüssigkeitschromatographie (HPLC) bestimmt. L-Carnosin und L-Alanyl-L-Histidin hemmten das Tumorwachstums bei gleichzeitig nur geringem Effekt auf die Vitalität der primären Fibroblastenkulturen. L-Histidin, Histamin und Imidazol hingegen reduzierten die Zellvitalität beider Zelltypen. Substanzen die keinen Imidazolanteil besaßen hatten keinen Einfluss auf die Zellvitalität. In Anwesenheit von L-Alanyl-L-Histidin aber nicht von L-Carnosin kam es zu einem signifikanten Anstieg der intrazellulären L-Histidinmenge. Die Bildung von Histamin konnte weder nach Behandlung der Zellen mit L-Carnosin und L-Alanyl-L-Histidin noch mit L-Histidin nachgewiesen werden. Zusammenfassend trägt der Imidazolanteil zum antineoplastischen Effekt von L-Carnosin bei, was auch auf den Effekt von L-Alanyl-L-Histidin und L-Histidin zutrifft. Obwohl Histamin einen starken Einfluss auf die Zellvitalität ausübt, ist die Bildung von Histamin nicht für den antineoplastischen Effekt von L-Carnosin, L-Alanyl-L-Histidin und L-Histidin verantwortlich

Conus Medullaris Enterogenous Cyst

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147142/1/pmr2698.pd

Viability of Glioblastoma Cells and Fibroblasts in the Presence of Imidazole-Containing Compounds

The naturally occurring dipeptide carnosine (-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), -alanyl-L-alanine, L-histidine, histamine, imidazole, -alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine

Facile Synthesis of a Stable Side-on Phosphinyne Complex by Redox Driven Intramolecular Cyclisation

Alkyne complexes with vicinal substitution by a Lewis acid and a Lewis base at the coordinated alkyne are prospective frustrated Lewis pairs exhibiting a particular mutual distance and, hence, a specific activation potential. In this contribution, investigations on the generation of a WII alkyne complex bearing a phosphine as Lewis base and a carbenium group as Lewis acid are presented. Independently on potential substrates added, an intramolecular cyclisation product was always isolated. A subsequent deprotonation step led to an unprecedented side-on λ5-phosphinyne complex, which is interpreted as highly zwitterionic according to visible absorption spectroscopy supported by TD-DFT. Low-temperature 31P NMR and EPR spectroscopic measurements combined with time-dependent IR-spectroscopic monitoring provided insights in the mechanism of the cyclisation reaction. Decomposition of the multicomponent IR spectra by multivariate curve resolution and a kinetic hard-modelling approach allowed the derivation of kinetic parameters. Assignment of the individual IR spectra to potential intermediates was provided by DFT calculations. © 2020 The Authors. Published by Wiley-VCH Gmb

Decrease of a specific biomarker of collagen degradation in osteoarthritis, Coll2-1, by treatment with highly bioavailable curcumin during an exploratory clinical trial

BACKGROUND: The management of osteoarthritis (OA) remains a challenge. There is a need not only for safe and efficient treatments but also for accurate and reliable biomarkers that would help diagnosis and monitoring both disease activity and treatment efficacy. Curcumin is basically a spice that is known for its anti-inflammatory properties. In vitro studies suggest that curcumin could be beneficial for cartilage in OA. The aim of this exploratory, non-controlled clinical trial was to evaluate the effects of bio-optimized curcumin in knee OA patients on the serum levels of specific biomarkers of OA and on the evaluation of pain. METHODS: Twenty two patients with knee OA were asked to take 2x3 caps/day of bio-optimized curcumin (Flexofytol®) for 3 months. They were monitored after 7, 14, 28 and 84 days of treatment. Pain over the last 24 hours and global assessment of disease activity by the patient were evaluated using a visual analog scale (100 mm). The serum levels of Coll-2-1, Coll-2-1NO(2), Fib3-1, Fib3-2, CRP, CTX-II and MPO were determined before and after 14 and 84 days of treatment. RESULTS: The treatment with curcumin was globally well tolerated. It significantly reduced the serum level of Coll2-1 (p < 0.002) and tended to decrease CRP. No other significant difference was observed with the other biomarkers. In addition, curcumin significantly reduced the global assessment of disease activity by the patient. CONCLUSION: This study highlighted the potential effect of curcumin in knee OA patient. This effect was reflected by the variation of a cartilage specific biomarker, Coll2-1 that was rapidly affected by the treatment. These results are encouraging for the qualification of Coll2-1 as a biomarker for the evaluation of curcumin in OA treatment. TRIAL REGISTRATION: NCT01909037 at clinicaltrials.go

Die Zeit der großen Gräben: Modelle zur Chronologie des Michelsberger Fundplatzes von Heilbronn-Klingenberg „Schlossberg“, Stadtkreis Heilbronn, Baden-Württemberg

This paper presents an attempt to establish more precise dating of the Michelsberg enclosure of Klingenberg-Schlossberg in the Neckar valley. The approach used is advocated as the basis on which to explore the timing and duration of other Michelsberg enclosures of the later fifth&ndash;earlier fourth millennium cal BC. Excavated extensively in 1986&ndash;1987, the Klingenberg enclosure has two ditches across a loess promontory, traces of a palisade inside the inner ditch, remains of burnt superstructure in both ditches, numerous pits both inside and outside, and numerous dog remains. There are some signs of pre-enclosure occupation in the MK II and III/IV phases, but the bulk of activity belongs to the MK V/Munzingen phase. A formal chronological approach combines the detailed archaeological information from the excavation with radiocarbon dates on carefully selected samples, here mainly charred cereals or articulated or articulating animal bones, in a Bayesian statistical framework.&nbsp; No samples were found to date MK II activity. Samples for two MK III/IV pits suggest a date in the 40th&ndash;39thcenturies cal BC. MK V/Mz activity began at the very end of the 39thand the start of the 38thcentury cal BC. Unlike in the previously published interpretation of the sequence, this activity probably began with the construction of the enclosure, both ditches being dug either together or in very quick succession. This was followed by pits inside the enclosure, from the earlier 38thcentury cal BC. Probably after a few decades, pits began to be dug outside the enclosure, in the middle part of the 38thcentury cal BC. Both ditches probably went out of use in the mid-37thcentury cal BC, probably simultaneously, after the burning of the rampart between them, and the ending of the external pits could be of the same date; the internal pits might have continued a little longer. A duration of 120&ndash;150 years for MK V/Mz activity is estimated. Alternative models are considered.&nbsp; The local and wider implications of these formal date estimates are discussed, topics covered including the circumstances in which the enclosure may have been constructed and those in which it ended, the chronology of MK pottery, and the wider development of MK and other enclosures