Untersuchung der Zelltodinduktion durch onkolytische Viren bei Glioblastom-Zelllinien

Das Glioblastoma multiforme ist der aggressivste Hirntumor des Menschen. Die derzeitige Therapie sieht eine chirurgische Resektion kombiniert mit einer Radiochemotherapie vor. Dennoch versterben die Patienten im Durchschnitt nach 10 bis 15 Monaten und ein kurativer Therapieansatz, der eine vollständige Tumorregression bewirkt, fehlt. Die nicht humanpathogenen onkolytischen Reo- (RV), Parvo- (PV) und Newcastle-Disease-Viren (NDV) zeichneten sich in der Vergangenheit durch ihre vielversprechende Fähigkeit, selektiv Tumorzellen abtöten zu können, aus. Basierend auf Vorarbeiten und Erkenntnissen aus der Literatur wurde im vorliegenden Dissertationsprojekt die onkolytische Wirksamkeit einer Doppelinfektion im Vergleich zur Einzelinfektion mit onkolytischen Viren (OV) evaluiert, um die Erfolgsaussichten einer kombinierten OV-Therapie abzuschätzen. Dazu wurde die onkolytische Zelltodinduktion charakterisiert. Nach der Produktion der OV und Bestimmung der exakten Virusmenge per real-time polymerase chain reaction (RT-PCR) wurde die Zytotoxizität im 1-(4,5-Dimethylthiazol-2- yl)-3,5-diphenyl-formazan-(MTT)-Assay nachgewiesen. Anschließend wurde die basale Viruskonzentration für eine onkolytische Wirksamkeit erhoben und eine Dosis- Wirkungskurve bestimmt. Daraufhin wurde das Zellsterben mittels Apoptose und Nekrose anhand der zellulären Hoechst- und Propidiumiodid-Bindung und der Auswertung der Zellmorphologie nachgewiesen. In einer durchflusszytometrischen Untersuchung wurde die Apoptose-, Nekroptose- und Nekroserate über die Expression von Phosphatidylserin und die Detektion von Annexin-V bestimmt. Dabei wurde die Einzelinfektion mit der Doppelinfektion von bis zu zwei Glioblastomzelllinien mit verschiedenen Infektionstitern und Inkubationszeiten verglichen. Zusätzlich wurde die Aktivität der Effektorcaspasen-3/7, einer zellulären Protease des Apoptose-Signalweges, zu verschiedenen Zeitpunkten nach Einzel- und Doppelinfektion ermittelt. Nach Ermittlung einer Dosis-Wirkungskurve und Charakterisierung des Zelltods zeigte sich eine zeit- und konzentrationsabhängige onkolytische Wirksamkeit nach Einzel- und Doppelinfektion. 24h und 48h nach kombinierter Gabe von NDV mit einem weiteren Virus in Zusammenfassung 1 den verwendeten niedrigen (1 und 10 cp/c) Viruskonzentrationen waren die ermittelten Zelltod- Aktivitäten gegenüber denen der NDV-Einzelinfektion nicht erhöht. Die Doppelinfektion mit RV und PV mit hoher Viruskonzentration zeigte eine um 12 % erhöhte onkolytische Wirksamkeit im Vergleich zur jeweiligen Einzelinfektion, die sich auch mit geringfügig erhöhten Aktivitäten der untersuchten Zelltode bestätigten. 72h nach Doppelinfektion der U87 Zellen wurde eine zur Einzelinfektion höhere Zytotoxizität nachgewiesen. Dieser Effekt konnte nach Doppelinfektion der sekundären Glioblastomzelllinie U373 nicht bestätigt werden, sodass zelllinienspezifische Ergebnisse vorlagen. Die RV- und NDV-Einzelinfektionen zeigten eine ausgeprägte onkolytische Wirksamkeit. Nach Doppelinfektion unterschieden sich zeitabhängig die induzierten Zelltode von denen nach Einzelinfektion. Je länger die Inkubationszeit, desto höher waren die Aktivitäten der programmierten Zelltodarten Apoptose und Nekroptose. Nach Doppelinfektion wurde im Zeitverlauf hauptsächlich der Zelltod durch Nekroptose eingeleitet. Allerdings wurden in abnehmender Intensität auch Apoptose und Nekrose verzeichnet. Zudem konnte bereits 4h nach Infektion mit RV, NDV, RV+PV, RV+NDV und PV+NDV eine caspase-abhängige Apoptose-Induktion nachgewiesen werden, die je nach untersuchter Inkubationszeit unterschiedlich ausgeprägt war. Die verwendeten Viruskonzentrationen und der untersuchte Zeitraum zur Charakterisierung der Zelltodarten reichten nicht aus, um eine signifikant vermehrte onkolytische Wirksamkeit und höhere Zelltodaktivitäten nach Doppelinfektion nachzuweisen. Daher sollten die vielversprechenden Beobachtungen zur Zelltodinduktion zukünftig mit einer höheren Viruskonzentration und einer längeren Inkubationszeit wiederholt werden. Dadurch können potenzielle additive Effekte, insbesondere durch den immunogenen Zelltod durch Nekroptose, nach Doppelinfektion aufgedeckt werden.Glioblastoma multiforme is the most aggressive human brain tumor. Even after undergoing standard therapy, which currently consists of surgical resection and radiochemotherapy, patients die after 10 to 15 months on average. There has yet to be discovered a curative therapeutic approach that achieves complete tumor regression. Oncolytic viruses (OV) that are not pathogenic to humans, such as reovirus (RV), parvovirus (PV), and newcastle Disease virus (NDV), have demonstrated the promising ability to selectively kill tumor cells in the past. Based on previous work and findings from literature, this dissertation project evaluates the oncolytic efficacy of a double infection compared to a single infection with OV in order to assess the chances of success of a combined OV therapy. For this purpose, we characterized cell death induction by OV. We performed 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyl-formazan-(MTT)-assays to assess cytotoxicity after producing OV and determining the exact amount of virus concentration using real-time polymerase chain reaction (RT-PCR). Following that, the basal virus concentration for oncolytic concentration was measured, and a dose-response curve was determined. Cell death via apoptosis and necrosis was then detected by using cellular Hoechst and propidium iodide staining and by evaluation of the cell morphology. Then, we performed a flow cytometric assay to determine the activity of apoptosis, necroptosis, and necrosis by the expression of phosphatidylserine and detection of annexin-V. Using different infection titers and incubation times, single infection was compared to double infection of up to two glioblastoma cell lines. Furthermore, the activity of effector caspases-3/7, a cellular protease involved in the apoptosis signaling pathway, was measured at multiple time points after single and double infection. After generating the dose-response curve and characterizing cell death, time- and concentration-dependent oncolytic efficacy for single and double infection could be demonstrated. 24h and 48h after combined administration of NDV with another virus at low (1 and 10 cp/c) virus concentrations, the cell death activity observed was not increased compared with a single NDV infection. RV and PV double infection with high virus Zusammenfassung 3 concentration resulted in a 12% increase in oncolytic efficacy compared to the corresponding single infection, which was also backed by slightly increased activities of the investigated cell death modes. Monotherapy with RV and NDV showed substantial oncolytic efficacy Higher cytotoxicity was detected 72 hours after double infection of U87 cells compared to single infection. This effect could not be reproduced after double infection of the secondary glioblastoma cell line U373, indicating that cell line-specific results were obtained. When compared to a single infection, different cell deaths were induced in a time-dependent manner after double infection. The longer the incubation period, the more active the programmed cell death modes via apoptosis and necroptosis were. As time passed after a double infection, cell death was primarily mediated by necroptosis. Apoptosis and necrosis were also detected in low levels. In addition, caspase-dependent apoptosis induction was detected 4h after infection with RV, NDV, RV+PV, RV+NDV, and PV+NDV. The apoptosis activity varied depending on the incubation period examined. The virus concentrations used, and the incubation period studied to characterize the cell death modes were insufficient to demonstrate significantly increased oncolytic efficacy and higher cell death activities after double infection. Future experiments should reproduce the encouraging findings on cell death induction with a higher virus concentration and a more extended incubation period. This could reveal potential additive effects on tumor cell killing through immunogenic cell death by necroptosis after double infection

Renal denervation reduces atrial remodeling in hypertensive rats with metabolic syndrome

Atrial fibrillation (AF) is highly prevalent in hypertensive patients with metabolic syndrome and is related to inflammation and activation of the sympathoadrenergic system. The multi-ligand Receptor-for-Advanced-Glycation-End-products (RAGE) activates inflammation-associated tissue remodeling and is regulated by the sympathetic nervous system. Its counterpart, soluble RAGE (sRAGE), serves as anti-inflammatory decoy receptor with protective properties. We investigated the effect of sympathetic modulation by renal denervation (RDN) on atrial remodeling, RAGE/sRAGE and RAGE ligands in metabolic syndrome. RDN was performed in spontaneously hypertensive obese rats (SHRob) with metabolic syndrome compared with lean spontaneously hypertensive rats (SHR) and with normotensive non-obese control rats. Blood pressure and heart rate were measured by telemetry. The animals were killed 12 weeks after RDN. Left atrial (LA) and right atrial (RA) remodeling was assessed by histological analysis and collagen types. Sympathetic innervation was measured by tyrosine hydroxylase staining of atrial nerve fibers, RAGE/sRAGE, RAGE ligands, cytokine expressions and inflammatory infiltrates were analyzed by Western blot and immunofluorescence staining. LA sympathetic nerve fiber density was higher in SHRob (+44%) versus controls and reduced after RDN (-64% versus SHRob). RAGE was increased (+718%) and sRAGE decreased (− 62%) in SHRob as compared with controls. RDN reduced RAGE expression (− 61% versus SHRob), significantly increased sRAGE levels (+162%) and induced a significant decrease in RAGE ligand levels in SHRob (− 57% CML and − 51% HMGB1) with reduced pro-inflammatory NFkB activation (− 96%), IL-6 production (− 55%) and reduced inflammatory infiltrates. This led to a reduction in atrial fibrosis (− 33%), collagen type I content (− 72%), accompanied by reduced LA myocyte hypertrophy (− 21%). Transfection experiments on H9C2 cardiomyoblasts demonstrated that RAGE is directly involved in fibrosis formation by influencing cellular production of collagen type I. In conclusion, suppression of renal sympathetic nerve activity by RDN prevents atrial remodeling in metabolic syndrome by reducing atrial sympathetic innervation and by modulating RAGE/sRAGE balance and reducing pro-inflammatory and pro-fibrotic RAGE ligands, which provides a potential therapeutic mechanism to reduce the development of AF

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Twelve-Month Outcomes of the AFFINITY Trial of Fluoxetine for Functional Recovery After Acute Stroke: AFFINITY Trial Steering Committee on Behalf of the AFFINITY Trial Collaboration

Background and Purpose: The AFFINITY trial (Assessment of Fluoxetine in Stroke Recovery) reported that oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and seizures. After trial medication was ceased at 6 months, survivors were followed to 12 months post-randomization. This preplanned secondary analysis aimed to determine any sustained or delayed effects of fluoxetine at 12 months post-randomization. Methods: AFFINITY was a randomized, parallel-group, double-blind, placebo-controlled trial in adults (n=1280) with a clinical diagnosis of stroke in the previous 2 to 15 days and persisting neurological deficit who were recruited at 43 hospital stroke units in Australia (n=29), New Zealand (4), and Vietnam (10) between 2013 and 2019. Participants were randomized to oral fluoxetine 20 mg once daily (n=642) or matching placebo (n=638) for 6 months and followed until 12 months after randomization. The primary outcome was function, measured by the modified Rankin Scale, at 6 months. Secondary outcomes for these analyses included measures of the modified Rankin Scale, mood, cognition, overall health status, fatigue, health-related quality of life, and safety at 12 months. Results: Adherence to trial medication was for a mean 167 (SD 48) days and similar between randomized groups. At 12 months, the distribution of modified Rankin Scale categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio, 0.93 [95% CI, 0.76–1.14]; P =0.46). Compared with placebo, patients allocated fluoxetine had fewer recurrent ischemic strokes (14 [2.18%] versus 29 [4.55%]; P =0.02), and no longer had significantly more falls (27 [4.21%] versus 15 [2.35%]; P =0.08), bone fractures (23 [3.58%] versus 11 [1.72%]; P =0.05), or seizures (11 [1.71%] versus 8 [1.25%]; P =0.64) at 12 months. Conclusions: Fluoxetine 20 mg daily for 6 months after acute stroke had no delayed or sustained effect on functional outcome, falls, bone fractures, or seizures at 12 months poststroke. The lower rate of recurrent ischemic stroke in the fluoxetine group is most likely a chance finding. REGISTRATION: URL: http://www.anzctr.org.au/ ; Unique identifier: ACTRN12611000774921