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Die Plasmamembrancalcium-ATPase (PMCA) konnte als Interaktionspartner und Inhibitor der neuronalen NO-Synthase (nNOS) in Kardiomyozyten identifiziert werden. PMCA und nNO-Synthase kommen in Caveolae von Kardiomyocyten vor. Humane PMCA4b überexprimierende Rattenherzen zeigen ex vivo eine signifikant höhere cGMP-Konzentration als wildtypische Rattenherzen. Das Ergebnis kann in mit PMCA4b und nNOS transfizierten wildtypischen und PMCA4b-überexprimierenden Kardiomyozyten bestätigt werden. Je höher die PMCA-Konzentration während der Transfektion ist, desto niedriger wird die Induktion von cGMP bei gleichbleibender nNOS-Transfektionskonzentration. Auffällig sind die absolut höheren Werte bei einer deutlich niedrigeren x-fachen Induktion von cGMP in PMCA4b-überexprimierenden Zellen. Dies deutet auf eine Anpassungsreaktion der transgenen Kardiomyozyten auf den inhibitorischen Effekt der PMCA auf die neuronale NO-Synthase.
Cutaneous leishmaniasis is an infectious disease that is endemic especially in tropical and desert regions with an incidence of 1.5 million cases per year and a prevalence of 12 million people infected worldwide. The infection can be caused by the intracellular parasite Leishmania major. The disease has been studied extensively in the murine model. It has become apparent that the induction of a class of interferon (IFN)--producing CD4+ T helper cells (TH1 cells) that activate macrophages to kill the parasites they harbor is desicive for the establishment of immunity. The redirection of the host’s immune response towards a protective TH1 phenotype will also be the key to an effective vaccine. Dendritic cells (DC) loaded with leishmanial antigens ex vivo were lately described as vaccines against L. major infections. One single recombinant Leishmania antigen, LeIF (Leishmania homologue of eukaryotic ribosomal initiation factor 4a), which was identified as a protein that stimulates DC to secrete interleukin (IL)-12 and discussed as a pattern-associated molecular pattern (PAMP), was found to mediate a protective TH1-dependent effect when used for pulsing of DC. The application of recombinant proteins is tied to many disadvantages, which is why other methods of antigen administration have been developed. RNA electroporation of DC has recently emerged from tumor research as a safe and versatile method of antigen delivery, by which a large number of RNA molecules encoding a specific antigen gains access to the cytosol of DC by an electrical impulse. The present study describes, for the first time, transfection of DC with RNA encoding a molecularly defined parasite antigen. Initially, a standardized protocol for RNA transfection was established, using the enhanced green fluorescent protein (EGFP) as reporter antigen. EGFP-RNA was well translatable in an in vitro translation system, and both a DC cell line (fetal skin-derived DC; FSDC) and murine primary bone marrow-derived DC (BMDC) could be transfected efficiently, with a yield of up to 90% and 75%, respectively. In both cell types, maximal transfection efficiency was attained with 20 µg RNA and could not be further increased with larger amounts of RNA. The level of antigen expression, measured as the mean fluorescence intensity (MFI) by flow cytometry, was directly proportional to the amount of RNA used for transfection. In FSDC, transfection efficiency and MFI were generally higher than in BMDC when the same amounts of RNA were used. Furthermore, the kinetics was shown to be sensitive to treatment with lipopolysaccharide (LPS): the expression peak was higher and was reached sooner, followed by a more rapid decline. In transfection experiments with LeIF, two variants of LeIF-RNA were used: LeIF(fl)-RNA, encoding the complete LeIF sequence, and LeIF(226)-RNA, encoding only the aminoterminal half of the LeIF sequence (226 amino acids), the immunogenic part of LeIF. Only LeIF(fl) was detectable by Western Blot in whole cell lysates of BMDC after LeIF(fl)-RNA transfection, whereas LeIF(226) could never be detected in LeIF(226)-transfected BMDC. However, as both constructs were well translatable in a cell-free system, the failure to detect LeIF(226) in BMDC lysates did not represent a failure in RNA translation, but rather a rapid antigen degradation. It was therefore expected that LeIF(226)-transfected BMDC should nevertheless be able to present LeIF(226)-derived antigenic peptides to T cells from BALB/c mice primed with recombinant LeIF (rLeIF). This hypothesis was confirmed by measuring IFN- production in BMDC-T cell co-incubation assays, showing that rLeIF-pulsed, LeIF(226)- and LeIF(fl)-transfected day 7 BMDC did indeed activate T cells from LeIF-immunized mice in an antigen-specific manner. In contrast, IL-4 was not produced, which was consistent with the fact that T cells found in lymph nodes from LeIF-primed mice are primarily of the TH1 type. In the supernatants of LeIF-transfected BMDC cultures, in contrast to rLeIF-pulsed BMDC, the proinflammatory cytokines IL-1β, IL-6, IL-10 and IL-12 were not detected. This effect was not due to the electroporation procedure, as cytokine production by BMDC electroporated with rLeIF was only partially impaired. Also, the expression levels of CD86 were lower upon LeIF transfection than after pulsing with rLeIF. Thus, LeIF transfection did not induce maturation of DC. In conclusion, LeIF-transfected BMDC may have acted as semi-mature antigen-specific tolerance inducers, with regulatory T cells as responders. The effect of LeIF transfection on the immunostimulatory capacity of BMDC was not significantly increased when day 8 or 9 BMDC were used. However, day 8, and even more day 9 BMDC pulsed with rLeIF mounted a vigorous T cell response. Day 9 BMDC were able to activate naïve T cells. In conclusion, before a strong T cell response against LeIF can be induced, DC need to – besides presenting antigen and expressing co-stimulatory molecules – exhibit a susceptibility to the innate signaling molecule LeIF which is linked to their maturation age. This third signal is provided by extracellular rLeIF, but it is not conveyed – or is suppressed – by intracellular LeIF after LeIF-RNA transfection. Furthermore, electroporation of rLeIF abrogated IL-12 production by BMDC completely, the production of IL-1 was reduced with higher antigen doses, and the production of IL-10 was partially increased. The IL-6 production was unaffected. This altered cytokine profile suggests that LeIF as a PAMP might have a bipartite nature: besides exhibiting the capacity to stimulate IL-12 production upon extracellular presence, thereby enhancing host resistance against L. major, LeIF could also contribute to parasitic host evasion mechanisms from intracellular compartments of DC, possibly by interfering with mitogen-activated protein (MAP) kinase signaling pathways. Thus, the adjuvant properties of LeIF depend both on its mode of delivery (transfection with RNA vs. pulsing with the recombinant protein) and the targeted compartment (extra- vs. intracellular). From this work, it can be summarized that BMDC are well transfectable with a parasite antigen. The antigen is processed and presented, but it is not recognized as a PAMP by DC. Hence, transfection with antigen-encoding mRNA by itself does not convey all necessary signals for the elicitation of a potent immune response.
Die Insulinbiosynthese in ß-Zellen des endokrinen Pankreas wird auf transkriptioneller Ebene durch die Aktivität des Insulingenpromotors reguliert. Die detaillierte Analyse der Aktivität des humanen Insulingenpromotors erfolgte bisher nur in speziesdifferenten ß-Zelllinien, da glukosesensitive ß-Zelllinien aus dem Pankreas des Menschen nicht verfügbar sind. Es ist jedoch bekannt, dass signifikante Unterschiede in der transkriptionellen Regulation der Genexpression in unterschiedlichen Spezies existieren. Deshalb wurde im Rahmen dieser Arbeit eine Methode entwickelt, mit deren Hilfe die spezifische Untersuchung der Regulation des humanen Insulingenpromotors hochsensitiv in primären humanen ß-Zellen des endokrinen Pankreas des Menschen möglich ist. Dazu wurde ein Vektor kloniert, der das SEAP (secreted alkaline phosphatase)-Reportergen unter der Kontrolle des -336 bp langen humanen Insulingenpromotors enthält. Im Laufe verschiedener Transfektionsexperimente mit dem Vektor p-336hInsP-SEAP, pSEAP2-Control (Positivkontrolle) und pSEAP2-Basic (Negativkontrolle) sowohl in INS-1-ß-Zellen, in beta-TC3-Zellen als auch in primären humanen ß-Zellen, zeigten sich in den luminometrisch bestimmten SEAP-Aktivitäten, die als Maß für die Aktivität des humanen Insulingenpromotors dienen, deutliche Unterschiede zwischen den transkriptionellen Aktivitäten der einzelnen Vektoren. Dieses System eignet sich also ausgezeichnet für die hochsensitive Analyse der Insulingenpromotoraktiviät. Zur detaillierteren Analyse wurden 5’-Deletionskonstrukte des Vektors p-336hInsP-SEAP konstruiert und damit INS-1- und beta-TC3-Zellen transient transfiziert. In beiden Zelllinien wurden Experimente bei unterschiedlichen Glukosekonzentrationen durchgeführt, um daraus Rückschlüsse auf die Glukoseresponsivität des humanen Insulingenpromotors ziehen zu können. Dabei zeigte der humane Insulingenpromotor die aus Versuchen mit dem RattenInsulingenpromotor 1 erwartete Glukoseresponsivität. Allerdings ließ sich keine Abnahme der transkriptionellen Aktivität des Promotors bei Abnahme der Länge der Konstrukte beobachten. Unter Verwendung von Effectene® als Transfektionsreagenz eignet sich das SEAP-System zur Analyse der Aktivität des humanen Insulingenpromotors in primären insulinproduzierenden Zellen aus dem menschlichen Pankreas.
Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation - An Appraisal
(2014)
Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in "hard-to-transfect" MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.
PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling
(2016)
Aim
Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.
Materials and Methods
In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes.
Results
In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.
Conclusions
PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.
Als Teil des angeborenen Immunsystems spielen Natürliche Killer(NK)- Zellen eine entscheidende Rolle in der Interaktion mit Pathogenen und Tumorzellen. Mithilfe der RNA-Interferenz bestimmter Gene wie beispielsweise CD56 könnten Hinweise auf die genaue Funktionsweise der NK-Zellen gewonnen werden. Ziel dieser Arbeit war es eine geeignete Transfektionsmethode zum Einführen von siRNA in NK-Zellen zu finden, welche eine hohe Transfektionseffizienz bei gleichzeitig hoher Zellviabilität aufweist. Hierfür wurden drei verschiedene Lipofektionsreagenzien und sechs verschiedene Elektroporationsprogramme verglichen. Zunächst wurde die Transfektionseffizienz mit an AF488 gekoppelter negativer siRNA per Fluoreszenzmikroskopie und Durchflusszytometrie evaluiert und jeweils das effizienteste Lipofektionsreagenz bzw. Elektroporationsprogramm ausgewählt. Mit diesen wurde anschließend eine Herunterregulation des CD56-Gens mit CD56-siRNA per RNA-Interferenz versucht. Die CD56-Gen-Expression wurde auf Proteinebene per Durchflusszytometrie und auf mRNA-Ebene per PCR (Polymerase-Ketten-Reaktion) evaluiert.