Generierung von hochproduktiven CHO Zelllinien für die Produktion von rekombinanten Antikörpern durch die Verwendung optimierter Signalpeptide und eines neuartigen ER Stress basierten Selektionssystems

The suitability of several signal peptides was tested in transiently transfected cell lines in the first part of this thesis. Two promising signal peptides were identified and these candidates were optimized by introducing specific mutations. After that, the performance of the best signal peptides was analyzed in stably transfected cell lines. Clones with a very high productivity were selected and their potency was evaluated in a fed-batch experiment. Three different very potent Signal peptides (B, E and E3) were identified as by this way which can be used to generate cell lines with clearly improved production rates (up to 4.0 g/L antibody in 13 days) suitable for commercial purposes. The aim of the second part of the present thesis was to establish a novel selection system based on ER stress, which allows the rapid identification and isolation of high-producers. Therefore, regulatory elements involved in ER stress responses were identified in various production clones. By using a Recombinase mediated cassette exchange approach, regulatory elements involved in ER stress responses (e.g.: ERSE I, ERSE II, UPRE and AARE) as well as ER stress regulated promoters (Calreticulin (CALR), Glucose-regulated Protein 78 kDa and 94 kDa (GRP78, GRP94)) and the intron-containing X-box binding protein 1 (XBP1) fragment were analyzed and a very strong correlation was observed between the amount of secreted antibody and the activity of the GRP78 Reporter construct. Furthermore, a selection system based on the GRP78 Promoter was tested. The obtained results indicate that the novel ER stress based selection system, developed during this thesis, should be suitable to identify and isolate clones expressing high amounts of antibody.Im ersten Teil der Arbeit wurde die Eignung verschiedener Signalpeptide zu Expressionssteigung in transient transfizierten Zelllinien getestet. Zwei vielversprechende Signalpeptide wurden auf diese Weise gefunden und danach durch spezifische Mutationen optimiert. Danach wurden stabile Zelllinien generiert und ihr Potenzial in Fed-batch Experimenten untersucht. Auf diese Weise wurden drei sehr starke Signalpeptide (B, E und E3) identifiziert, welche zur Erzeugung von kommerziellen Zelllinien mit erhöhter Produktivität (bis zu 4.0 g/L Antikörper in 13 Tagen) eingesetzt werden können. Im zweiten Teil der Arbeit wurde ein neuartiges ER Stress basiertes Selektionssystem etabliert, welches die schnelle Identifizierung und Isolierung von hochproduktiven Klonen erlaubt. Dafür wurden vielversprechende regulatorische Elemente, welche in der ER Stress Regulation beteiligt sind, identifiziert. Mittels eines Rekombinase-vermittelten Kassettenaustausches wurden regulatorischen Elemente welche in der ER Stress-Antwort involviert sind (z.B.: ERSE I, ERSE II, UPRE und AARE) ebenso wie ER stress regulierte Promotoren (Calreticulin (CALR), Glukose-reguliertes Protein 78 kDa und 94 kDa (GRP78, GRP94)) und das intron-enthaltende X-Box bindende Protein 1 (XBP1) analysiert und eine sehr starke Korrelation zwischen der sekretierten Antikörpermenge und der Aktivität des GRP78 Reportes festgestellt. Darüberhinaus wurde ein Selektionssystem basierend auf dem GRP78 Promoter getestet. Die erhaltenen Ergebnisse zeigen, dass das in diese Arbeit neuentwickelte Selektionssystem geeignet ist hochproduktive Klone zu isolieren

Exercise-based cardiac rehabilitation for adults after heart valve surgery:review

Exercise-based cardiac rehabilitation may benefit heart valve surgery patients. We conducted a systematic review to assess the evidence for the use of exercise-based intervention programmes following heart valve surgery.To assess the benefits and harms of exercise-based cardiac rehabilitation compared with no exercise training intervention, or treatment as usual, in adults following heart valve surgery. We considered programmes including exercise training with or without another intervention (such as a psycho-educational component).We searched: the Cochrane Central Register of Controlled Trials (CENTRAL); the Database of Abstracts of Reviews of Effects (DARE); MEDLINE (Ovid); EMBASE (Ovid); CINAHL (EBSCO); PsycINFO (Ovid); LILACS (Bireme); and Conference Proceedings Citation Index-S (CPCI-S) on Web of Science (Thomson Reuters) on 23 March 2015. We handsearched Web of Science, bibliographies of systematic reviews and trial registers (ClinicalTrials.gov, Controlled-trials.com, and The World Health Organization International Clinical Trials Registry Platform).We included randomised clinical trials that investigated exercise-based interventions compared with no exercise intervention control. The trial participants comprised adults aged 18 years or older who had undergone heart valve surgery for heart valve disease (from any cause) and received either heart valve replacement, or heart valve repair.Two authors independently extracted data. We assessed the risk of systematic errors ('bias') by evaluation of bias risk domains. Clinical and statistical heterogeneity were assessed. Meta-analyses were undertaken using both fixed-effect and random-effects models. We used the GRADE approach to assess the quality of evidence. We sought to assess the risk of random errors with trial sequential analysis.We included two trials from 1987 and 2004 with a total 148 participants who have had heart valve surgery. Both trials had a high risk of bias.There was insufficient evidence at 3 to 6 months follow-up to judge the effect of exercise-based cardiac rehabilitation compared to no exercise on mortality (RR 4.46 (95% confidence interval (CI) 0.22 to 90.78); participants = 104; studies = 1; quality of evidence: very low) and on serious adverse events (RR 1.15 (95% CI 0.37 to 3.62); participants = 148; studies = 2; quality of evidence: very low). Included trials did not report on health-related quality of life (HRQoL), and the secondary outcomes of New York Heart Association class, left ventricular ejection fraction and cost. We did find that, compared with control (no exercise), exercise-based rehabilitation may increase exercise capacity (SMD -0.47, 95% CI -0.81 to -0.13; participants = 140; studies = 2, quality of evidence: moderate). There was insufficient evidence at 12 months follow-up for the return to work outcome (RR 0.55 (95% CI 0.19 to 1.56); participants = 44; studies = 1; quality of evidence: low). Due to limited information, trial sequential analysis could not be performed as planned.Our findings suggest that exercise-based rehabilitation for adults after heart valve surgery, compared with no exercise, may improve exercise capacity. Due to a lack of evidence, we cannot evaluate the impact on other outcomes. Further high-quality randomised clinical trials are needed in order to assess the impact of exercise-based rehabilitation on patient-relevant outcomes, including mortality and quality of life

Rationale and design of ARTS: a randomized, double‐blind study of BAY 94‐8862 in patients with chronic heart failure and mild or moderate chronic kidney disease

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106085/1/ejhfhfs061.pd