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Background Hepatitis E virus (HEV) is the leading cause of acute hepatitis throughout the world. Increasing blood component transfusion-associated HEV infections highlight the need for reliable virus inactivation procedures for plasma derivatives from pooled plasma donations. Study Design and Methods An animal infection study was conducted to evaluate the efficiency of HEV inactivation by pasteurization during the manufacturing process of the von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate Haemate P/Humate-P (CSL Behring, Marburg, Germany). For this purpose, groups of pigs were inoculated with stabilized VWF/FVIII intermediate spiked with HEV-positive liver homogenate and exposed to increasing incubation times of 0, 3, 6, and 10 h at 60 degrees C. Animals were evaluated for virus replication over 27 days and in a subsequent trial over 92 days. Results Virus replication was detected in animals up to the 6-h pasteurization group. In contrast, pasteurization for 10 h did not reveal virus detection when the observation period was 27 days. In an additional experiment using the 10-h pasteurized material, two individuals started virus excretion and seroconverted when the observation period was extended to 92 days. Based on the total infection rate (2 of 12) of the animals inoculated with the sample pasteurized for 10 h, a virus reduction factor of at least 4.7 log(10) is calculated. Conclusion This study demonstrates that pasteurization at 60 degrees C for 10 h of an HEV-positive plasma derivative leads to the effective reduction of infectivity, resulting in a VWF/FVIII product with an appropriate margin of safety for HEV.

Schwerpunkt dieser Promotionsarbeit war die Identifizierung von genetischen und epigenetischen Markern zur Vorhersage der Gewichtsentwicklung unter Therapie mit Antipsychotika der zweiten Generation. Hierfür wurden neue molekularbiologische Tests entwickelt. Neben dem Polymorphismus rs9939609 des FTO-Gen konnte erstmals rs7185735 im gleichen Gen als vielversprechender Risikomarker identifiziert werden. Im Rahmen der epigenetischen Untersuchungen wurde zudem die Stabilität der Methylierung in Blutproben getestet. The focus of this doctoral thesis was the identification of genetic and epigenetic markers to predict weight gain under therapy with second-generation antipsychotics. Therefore, we developed new molecular biology tests. In addition to the polymorphism rs9939609 of the FTO gene, we identified the rs7185735 of the same gene as a promising risk marker for the first time. As part of the epigenetic studies, we also tested the stability of methylation in blood samples.

Marion Ortner,1 Marion Stange,1 Heike Schneider,1 Charlotte Schröder,2 Katharina Buerger,3 Claudia Müller,3 Felix Müller-Sarnowski,1 Janine Diehl-Schmid,1 Hans Förstl,1 Timo Grimmer1 ,* Werner Steimer2, * 1Department of Psychiatry and Psychotherapy, Technical University of Munich, School of Medicine, Klinikum rechts der Isar, Munich, Germany; 2Institute for Clinical Chemistry and Pathobiochemistry, Technical University of Munich, School of Medicine, Klinikum rechts der Isar, Munich, Germany; 3Institute for Stroke and Dementia Research (ISD), University of Munich, School of Medicine, Klinikum der Universität München, Munich, Germany*These authors contributed equally to this workCorrespondence: Marion Ortner Email marion.ortner@tum.deBackground: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil.Objective: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP 2D6 genotype or gene dose–dependent metabolism of donepezil.Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose.Results: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (β=0.465; p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose.Conclusion: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism.Keywords: Alzheimer’s disease, cytochrome P-450 enzymes, genetic polymorphism, therapeutic drug monitoring, mass spectrometry

Determination of the conversion factor from copies per μl RNA to IU of the WHO standard (DOCX 16 kb)

Calculation of the Infectivity titer of the Liver Used for Inoculation (DOCX 16 kb)

Calculation of the Infectivity titer of the Liver Used for Inoculation (DOCX 16 kb)

Alignment of the hypervariable region. Alignment done with Geneious version 10.2 created by Biomatters. Available from https://www.geneious.com (DOCX 244 kb)

Results of the RT-qPCR from serum and the porcine IgG HEV-Ab ELISA; The graphs display the individual curves of viral RNA in serum and the detection of Anti-HEV-IgG antibodies in correlation to days after infection (DOCX 462 kb)

Results of the RT-qPCR from serum and the porcine IgG HEV-Ab ELISA; The graphs display the individual curves of viral RNA in serum and the detection of Anti-HEV-IgG antibodies in correlation to days after infection (DOCX 462 kb)

Amplification curve of HEV specific qRT-PCR. A) Amplification curve of HEV specific qRT-PCR targeting the HEV standard (red line) and the RNA extracts from the inocula (blue line). B) Standard curves were obtained by Ct values plotted against the log of starting quantity. C) Obtained Ct values and determined copy numbers (DOCX 219 kb)

Determination of the conversion factor from copies per μl RNA to IU of the WHO standard (DOCX 16 kb)

Amplification curve of HEV specific qRT-PCR. A) Amplification curve of HEV specific qRT-PCR targeting the HEV standard (red line) and the RNA extracts from the inocula (blue line). B) Standard curves were obtained by Ct values plotted against the log of starting quantity. C) Obtained Ct values and determined copy numbers (DOCX 219 kb)