Entwicklung siRNA basierter Therapieansätze im Mausmodell am Beispiel viraler Hepatitiden

Die RNAi Technologie bietet einen vielversprechenden Ansatz zur Behandlung von hepatischen Viruserkrankungen. Um einen sicheren Einsatz von siRNAs gewährleisten zu können, ist es notwendig, die hepatischen Wirkmechanismen von siRNA zu kennen. Im ersten Teil dieser Arbeit wurde die Beteiligung von endosomalen-MyD88-abhängigen Toll-like Rezeptoren an der siRNA-vermittelten Immunantwort in vivo nachgewiesen. Diese Immunantwort wurde durch die nicht-parenchymalen Leberzellen vermittelt und resultierte in der Expression verschiedener Zytokine. Auch in weiteren Organen wie Milz, Niere und Herz war eine Immunreaktion detektierbar. Durch chemische Modifikationen am Riboserückgrat der siRNA wie 2’O-Methyl oder 2’Fluor konnte diese immunologische Nebenwirkung unterbunden werden. Des Weiteren konnte im zweiten Abschnitt mittels der Isg15-siRNA gezeigt werden, dass der Einsatz von LNP01-formulierten siRNAs im Mausmodell zu einem Hepatozyten-spezifischen Knockdown führte. Die verschiedenen Ribose-Modifikationen hatten hierbei keinen signifikanten Einfluss auf die Effizienz der siRNAs. Zusätzlich konnte gezeigt werden, dass die LNP01-Formulierung auch in einem humanisierten Mausmodell zur spezifischen Gensuppression in den humanen Hepatozyten führte, sowie zur Suppression der HCV-Replikation in vivo. Der etablierte, siRNA-vermittelte Knockdown des Wirtsfaktors Isg15 in vivo, ist eine Option in der HCV Therapie, da er die HCV-Replikation direkt supprimiert und zusätzlich zu einer verstärkten hepatischen Reaktion auf endogenes und exogenes Interferon alpha führte. In vitro zeigte sich die Kombination aus siISG15 und einem Protease-Inhibitor (PI) als effiziente Therapieoption in der HCV Behandlung, mit dem Vorteil, dass die Entstehung von Resistenzen gegenüber des PIs den Therapieerfolg weniger beeinflusst. Der dritte Teil dieser Arbeit nutzte den Isg15 Knockdown in vivo sowie in vitro im HCV-Replikon Modell, um die Bedeutung von Isg15 im Rahmen der HCV-Replikation aufzuklären. Die Proteomanalyse identifizierte verschiedene Proteine, welche von der Isg15 Expression abhängig waren, welche einen direkten Effekt auf die HCV-Replikation hatten (Hnrnp K und Hmgcs1) oder in den Lipid-Metabolismus und in den Zusammenbau der Viruspartikel (assembly) involviert waren. Psma6 wurde als negativ Regulator der Isg15 Expression identifiziert und scheint damit indirekt die HCV-Replikation zu beeinflussen. Die Anwendung von siRNA im HBV transgenen Mausmodell im letzten Abschnitt dieser Arbeit zeigte zum einen, dass siRNAs effiziente Werkzeuge sind, um die virale Replikation auch direkt zu hemmen. Zum anderen diente der Einsatz der siRNA der Untersuchung der HBV-induzierten Immunantwort. Es zeigte sich, dass in den HBVtg Tieren ohne HBsAg Expression eine konsistente TLR3-induzierte IFN/ISG Antwort vorlag. Auch die Expression von pro-viralen Wirtsfaktoren wie Hnrnp K und Hmgcs1 war erhöht. Die Tiere wiesen ein funktionelles TLR-System auf, jedoch war die TLR3- und TLR7-abhängige Immunantwort in den Tieren reduziert, aber nicht komplett inhibiert. Diese Arbeit zeigt das Anwendungsspektrum von siRNAs in vivo, sie können der Untersuchung von Virus-Wirts-Interaktionen, ebenso wie der therapeutischen Anwendung gegen virale Hepatitiden, dienen.RNAi technology is a promising approach for the treatment of viral hepatitis. To ensure safety and efficacy of this technology, it is necessary to determine the hepatic off-target responses induced by siRNA. This study identified the involvement of endosomal MyD88-dependent Toll-like receptors in siRNA-induced immune responses in vivo. These immune responses were orchestrated by the non-parenchymal liver cells (NPC) and resulted in the expression of different cytokines. In other organs like spleen, kidney and heart these immune reactions were detectable as well. These off-target effects could be avoided by chemical modifications of the ribose backbone of the siRNA, like 2’O-methyl or 2’ fluor modifications. Furthermore, it was shown that LNP01-formulated Isg15-specific siRNAs led to a hepatocyte-restricted knockdown in mice. The knockdown efficiencies were not impaired by the different ribose-modifications. Additionally, suppression of ISG15 in humanized, HCV-infected mice led to the suppression of HCV replication. The knockdown of this pro-viral host factor is a promising option for the future HCV therapy strategies, because it directly suppresses HCV replication and enhanced hepatic response to endogenous and exogenous Interferon alpha. The combination of siISG15 and a HCV protease inhibitor (PI) exhibits the advantage to prevent resistance development against the PI in vitro. The investigation of the Isg15 knockdown in vitro and in vivo revealed the enigmatic link between Isg15 and HCV. Proteome analysis identified different proteins which depend on the Isg15 expression, directly influenced the HCV replication (Hnrnp K and Hmgcs1) or were likely be involved in lipid metabolism and virus assembly. Psma6 was identified as a negative regulator of the Isg15 expression and may therefore influence the HCV replication indirectly. The application of siRNA in a HBV transgenic mouse model identified siRNAs as efficient tools to directly block viral replication. Additionally, the siRNAs were used to investigate immune response in these animals. It was shown, that the HBVtg mice lacking the HBsAg, exhibited consistent TLR3-dependent IFN/ISG responses. The expression of pro-viral host factors like Hnrnp K and Hmgcs1 was elevated as well. Furthermore, the animals showed a functional TLR system, here TLR3- and TLR7-dependent immune responses were reduced but not completely abrogated. In conclusion, this study showed a broad range of siRNA applications in vivo including the analysis of host virus interactions and direct therapeutic approaches against viral hepatitis

A culture-sensitive semi-quantitative FFQ for use among the adult population in Nairobi, Kenya: development, validity and reproducibility

Objective: To develop a semi-quantitative FFQ and to evaluate its validity and reproducibility for the assessment of total dietary intake of Kenyan urban adult population, given its non-existence in Kenya. Design: The current study adopted a cross-sectional design. A culture-sensitive semi-quantitative FFQ was developed and its validity was tested relative to thre non-consecutive 24-h recalls (24hR). Reproducibility was tested by the test–retest method, with a 3-week interval. Spearman’s correlation coefficients and intra-class correlation coefficients were calculated for several macro- and micronutrients. Cross-classification into quartiles and Bland and Altman plots were analysed. Setting: Nairobi county (Dagoreti South and Starehe constituencies). Participants: A convenient sample was recruited in three different clusters in Nairobi. Results: A culture-sensitive 123-food-item semi-quantitative FFQ showed higher nutrient intakes compared with the 24hR (total energy median 12543·632 v. 8501·888 kJ, P < 0·001). Energy-adjusted and deattenuated Spearman’s correlations for macronutrients ranged between 0·21 (total fat) and 0·47 (protein). The agreement in the same quartile varied from 28 % (protein) to 41 % (carbohydrates). Including adjacent quartiles, the range increased: 76 % (protein and fat) to 81 % (carbohydrates). The extreme disagreement was low. The first FFQ application resulted in higher mean values for all nutrients compared with the second FFQ (total energy median 12459·952 v. 10485·104 kJ, P < 0·001). Energy-adjusted correlations for macronutrients ranged from 0·28 (carbohydrates) to 0·61 (protein). Intra-class correlation coefficients for macronutrients were moderate, between 0·6 and 0·7. Conclusions: The developed semi-quantitative FFQ was shown to be a valid and reproducible tool for ranking urban adult Kenyans according to their dietary intake.N/

Detailed plan for the COMET WP3 initial research activity - list of research projects and goals, participants and timing

Detailed plan for the COMET Work Package (WP) 3

Nutritional intake and food sources in an adult urban Kenyan population

Publisher Copyright: © 2022 The Authors. Nutrition Bulletin published by John Wiley & Sons Ltd on behalf of British Nutrition Foundation.Urbanisation is hastening the transition from traditional food habits to less healthy diets, which are becoming more common among Kenyans. No up-to-date studies on usual dietary intake and the main food sources of adult Kenyans are available. The aim of the present study was to identify the main food sources of nutrients in the diet of urban adult Kenyans and explore potential associations with demographic variables including age, sex, level of education, occupation and body mass index. The study adopted a cross-sectional design. The dietary intake of 486 adult Kenyans from Nairobi was assessed using a validated, culture-sensitive, semi-quantitative food frequency questionnaire. Binary logistic regression models were used to evaluate associations between food sources and demographic variables. Macronutrient intakes as a proportion of total energy intake (TEI) were within international dietary guidelines. Cereals and grain products (34.0%), sugar, syrups, sweets and snacks (9.8%), fruits (9.7%) and meat and eggs (8.8%) were the major contributors to TEI. Cereals and grain products contributed 42.5% to carbohydrates, followed by fruits (12.4%) and sugar, syrups, sweets and snacks (10.6%). The most important sources of protein and total fat were cereals and grain products (23.3% and 19.7%, respectively) and meat and eggs (22.0% and 18.7%, respectively). Sex, age and level of education were associated with the choice of food groups. Although macronutrient intakes were within guidelines, the Kenyan diet was revealed to be high in sugars, salt and fibre, with differences in food sources according to demographic variables. These results can act as an incentive to national authorities to implement nutritional strategies aiming to raise awareness of healthier dietary patterns among Kenyans.Peer reviewe

Liver-First Approach for Synchronous Colorectal Metastases : Analysis of 7360 Patients from the LiverMetSurvey Registry

Background The liver-first approach in patients with synchronous colorectal liver metastases (CRLM) has gained wide consensus but its role is still to be clarified. We aimed to elucidate the outcome of the liver-first approach and to identify patients who benefit at most from this approach. Methods Patients with synchronous CRLM included in the LiverMetSurvey registry between 2000 and 2017 were considered. Three strategies were analyzed, i.e. liver-first approach, colorectal resection followed by liver resection (primary-first), and simultaneous resection, and three groups of patients were analyzed, i.e. solitary metastasis, multiple unilobar CRLM, and multiple bilobar CRLM. In each group, patients from the three strategy groups were matched by propensity score analysis. Results Overall, 7360 patients were analyzed: 4415 primary-first, 552 liver-first, and 2393 simultaneous resections. Compared with the other groups, the liver-first group had more rectal tumors (58.0% vs. 31.2%) and higher hepatic tumor burden (more than three CRLMs: 34.8% vs. 24.0%; size > 50 mm: 35.6% vs. 22.8%; p < 0.001). In patients with solitary and multiple unilobar CRLM, survival was similar regardless of treatment strategy, whereas in patients with multiple bilobar metastases, the liver-first approach was an independent positive prognostic factor, both in unmatched patients (3-year survival 65.9% vs. primary-first 60.4%: hazard ratio [HR] 1.321, p = 0.031; vs. simultaneous resections 54.4%: HR 1.624, p < 0.001) and after propensity score matching (vs. primary-first: HR 1.667, p = 0.017; vs. simultaneous resections: HR 2.278, p = 0.003). Conclusion In patients with synchronous CRLM, the surgical strategy should be decided according to the hepatic tumor burden. In the presence of multiple bilobar CRLM, the liver-first approach is associated with longer survival than the alternative approaches and should be evaluated as standard.Peer reviewe

The Radioecology Exchange

The Radioecology Exchange (www.radioecology-exchange.org) was created in 2011 under the EU FP7 STAR (STrategy for Allied Radioecology, www.star-radioecology.org) Network of Excellence; (2011-2015). This project aims to integrate radioecological research efforts of European organisations into a sustainable network. In 2013, the EU FP7 COMET (COordination and iMplementation of a pan-European instrumenT for radioecology (2013- 2017); www.comet-radioecology.org) project commenced; COMET will build upon the work initiated under STAR. The Radioecology Exchange has therefore become the web resource for activities from both projects which will ultimately be maintained by the European Radioecology Alliance (ALLIANCE; www.er-alliance.org). The Radioecology Exchange is intended to become a ‘gateway’ for information related to European (and wider) radioecological research

Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation

BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation

Why “intergenerational feminist media studies”?

Feminism and generation are live and ideologically freighted issues that are subject to a substantial amount of media engagement. The figure of the millennial and the baby boomer, for example, regularly circulate in mainstream media, often accompanied by hyperbolic and vitriolic discourses and affects of intergenerational feminist conflict. In addition, theories of feminist generation and waves have been and continue to be extensively critiqued within feminist theory. Given the compelling criticisms directed at these categories, we ask: why bother examining and foregrounding issues of generation, intergeneration, and transgeneration in feminist media studies? Whilst remaining sceptical of linearity and familial metaphors and of repeating reductive, heteronormative, and racist versions of feminist movements, we believe that the concept of generation does have critical purchase for feminist media scholars. Indeed, precisely because of the problematic ways that is it used, and the prevalence of it as a volatile, yet only too palpable, organizing category, generation is both in need of continual critical analysis, and is an important tool to be used—with care and nuance—when examining the multiple routes through which power functions in order to marginalize, reward, and oppress. Exploring both diachronic and synchronic understandings of generation, this article emphasizes the use of conjunctural analysis to excavate the specific historical conditions that impact upon and create generation. This special issue of Feminist Media Studies covers a range of media forms—film, games, digital media, television, print media, as well as practices of media production, intervention, and representation. The articles also explore how figures at particular lifestages—particularly the girl and the aging woman—are constructed relationally, and circulate, within media, with particular attention to sexuality. Throughout the issue there is an emphasis on exploring the ways in which the category of generation is mobilized in order to gloss sexism, racism, ageism, class oppression, and the effects of neoliberalism

Genome-wide determinants of mortality and motor progression in Parkinson’s disease

There are 90 independent genome-wide significant genetic risk variants for Parkinson’s disease (PD) but currently only five nominated loci for PD progression. The biology of PD progression is likely to be of central importance in defining mechanisms that can be used to develop new treatments. We studied 6766 PD patients, over 15,340 visits with a mean follow-up of between 4.2 and 15.7 years and carried out genome-wide survival studies for time to a motor progression endpoint, defined by reaching Hoehn and Yahr stage 3 or greater, and death (mortality). There was a robust effect of the APOE ε4 allele on mortality in PD. We also identified a locus within the TBXAS1 gene encoding thromboxane A synthase 1 associated with mortality in PD. We also report 4 independent loci associated with motor progression in or near MORN1, ASNS, PDE5A, and XPO1. Only the non-Gaucher disease causing GBA1 PD risk variant E326K, of the known PD risk variants, was associated with mortality in PD. Further work is needed to understand the links between these genomic variants and the underlying disease biology. However, these may represent new candidates for disease modification in PD