A scalable adenovirus production process, from cell culture to purified bulk

Adenovirus (AdV) vectors are commonly used in cancer gene therapy trials, evaluated in gene therapy and used as vaccines for various diseases. AdV vectors are well studied and are suitable as vaccine vectors due to their ability to infect different cell types, remain episomal and produce stable high titer material. Manufacturing of safe and efficacious clinical-grade virus relies on a scalable and cost-effective production process. In this study, we have combined experimental work and process economy calculations, from AdV production in cell culture to purified bulk product up to 10L scale. An efficient and scalable process for AdV production was developed by evaluation of each process step. The most studied vector is serotype 5, making this a suitable system for process development of AdV vectors. Human AdV5 expressing the green fluorescent protein (GFP) was used for process development. First, suspension HEK 293 cells adapted to serum-free cell culture medium were optimized for AdV production and evaluated in different single use bioreactor systems. Tween 20 was used for cell lysis as a replacement for the traditionally used Triton X-100 (now on the Authorization list (Annex XIV) of REACH, the regulation on Registration, Evaluation, Authorization and restriction of Chemicals). A residual Tween 20 assay with low detection limit was set-up. Filters and conditions for clarification, concentration and buffer exchange by tangential flow filtration were optimized. Anion exchange based capture step alternatives were compared, including different chromatography resins and membrane formats. Finally, core bead technology was evaluated as an alternative to size exclusion chromatography for the polishing step before the final formulation. Analytical methods for virus titer are challenging and depend on purity and quality of the sample. For total virus titer, qPCR and HPLC methods were used. Furthermore, a method based on surface plasmon resonance (Biacore) was developed for analysis of adenovirus titer. For infectious virus titer, we have used a cell based assay with automatic image analysis. Based on analytical data different downstream process alternatives were compared regarding load capacity, recovery and purity and we propose a robust and scalable process with a favorable process economy. Please click Additional Files below to see the full abstract

Relations between lipoprotein(a) concentrations, LPA genetic variants, and the risk of mortality in patients with established coronary heart disease: a molecular and genetic association study

Background: Lipoprotein(a) concentrations in plasma are associated with cardiovascular risk in the general population. Whether lipoprotein(a) concentrations or LPA genetic variants predict long-term mortality in patients with established coronary heart disease remains less clear. Methods: We obtained data from 3313 patients with established coronary heart disease in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. We tested associations of tertiles of lipoprotein(a) concentration in plasma and two LPA single-nucleotide polymorphisms ([SNPs] rs10455872 and rs3798220) with all-cause mortality and cardiovascular mortality by Cox regression analysis and with severity of disease by generalised linear modelling, with and without adjustment for age, sex, diabetes diagnosis, systolic blood pressure, BMI, smoking status, estimated glomerular filtration rate, LDL-cholesterol concentration, and use of lipid-lowering therapy. Results for plasma lipoprotein(a) concentrations were validated in five independent studies involving 10 195 patients with established coronary heart disease. Results for genetic associations were replicated through large-scale collaborative analysis in the GENIUS-CHD consortium, comprising 106 353 patients with established coronary heart disease and 19 332 deaths in 22 studies or cohorts. Findings: The median follow-up was 9·9 years. Increased severity of coronary heart disease was associated with lipoprotein(a) concentrations in plasma in the highest tertile (adjusted hazard radio [HR] 1·44, 95% CI 1·14–1·83) and the presence of either LPA SNP (1·88, 1·40–2·53). No associations were found in LURIC with all-cause mortality (highest tertile of lipoprotein(a) concentration in plasma 0·95, 0·81–1·11 and either LPA SNP 1·10, 0·92–1·31) or cardiovascular mortality (0·99, 0·81–1·2 and 1·13, 0·90–1·40, respectively) or in the validation studies. Interpretation: In patients with prevalent coronary heart disease, lipoprotein(a) concentrations and genetic variants showed no associations with mortality. We conclude that these variables are not useful risk factors to measure to predict progression to death after coronary heart disease is established. Funding: Seventh Framework Programme for Research and Technical Development (AtheroRemo and RiskyCAD), INTERREG IV Oberrhein Programme, Deutsche Nierenstiftung, Else-Kroener Fresenius Foundation, Deutsche Stiftung für Herzforschung, Deutsche Forschungsgemeinschaft, Saarland University, German Federal Ministry of Education and Research, Willy Robert Pitzer Foundation, and Waldburg-Zeil Clinics Isny

Association of Factor V Leiden with Subsequent Atherothrombotic Events:A GENIUS-CHD Study of Individual Participant Data

BACKGROUND: Studies examining the role of factor V Leiden among patients at higher risk of atherothrombotic events, such as those with established coronary heart disease (CHD), are lacking. Given that coagulation is involved in the thrombus formation stage on atherosclerotic plaque rupture, we hypothesized that factor V Leiden may be a stronger risk factor for atherothrombotic events in patients with established CHD. METHODS: We performed an individual-level meta-analysis including 25 prospective studies (18 cohorts, 3 case-cohorts, 4 randomized trials) from the GENIUS-CHD (Genetics of Subsequent Coronary Heart Disease) consortium involving patients with established CHD at baseline. Participating studies genotyped factor V Leiden status and shared risk estimates for the outcomes of interest using a centrally developed statistical code with harmonized definitions across studies. Cox proportional hazards regression models were used to obtain age- and sex-adjusted estimates. The obtained estimates were pooled using fixed-effect meta-analysis. The primary outcome was composite of myocardial infarction and CHD death. Secondary outcomes included any stroke, ischemic stroke, coronary revascularization, cardiovascular mortality, and all-cause mortality. RESULTS: The studies included 69 681 individuals of whom 3190 (4.6%) were either heterozygous or homozygous (n=47) carriers of factor V Leiden. Median follow-up per study ranged from 1.0 to 10.6 years. A total of 20 studies with 61 147 participants and 6849 events contributed to analyses of the primary outcome. Factor V Leiden was not associated with the combined outcome of myocardial infarction and CHD death (hazard ratio, 1.03 [95% CI, 0.92-1.16]; I2=28%; P-heterogeneity=0.12). Subgroup analysis according to baseline characteristics or strata of traditional cardiovascular risk factors did not show relevant differences. Similarly, risk estimates for the secondary outcomes including stroke, coronary revascularization, cardiovascular mortality, and all-cause mortality were also close to identity. CONCLUSIONS: Factor V Leiden was not associated with increased risk of subsequent atherothrombotic events and mortality in high-risk participants with established and treated CHD. Routine assessment of factor V Leiden status is unlikely to improve atherothrombotic events risk stratification in this population

Subsequent Event Risk in Individuals with Established Coronary Heart Disease:Design and Rationale of the GENIUS-CHD Consortium

BACKGROUND: The "GENetIcs of sUbSequent Coronary Heart Disease" (GENIUS-CHD) consortium was established to facilitate discovery and validation of genetic variants and biomarkers for risk of subsequent CHD events, in individuals with established CHD. METHODS: The consortium currently includes 57 studies from 18 countries, recruiting 185,614 participants with either acute coronary syndrome, stable CHD or a mixture of both at baseline. All studies collected biological samples and followed-up study participants prospectively for subsequent events. RESULTS: Enrollment into the individual studies took place between 1985 to present day with duration of follow up ranging from 9 months to 15 years. Within each study, participants with CHD are predominantly of self-reported European descent (38%-100%), mostly male (44%-91%) with mean ages at recruitment ranging from 40 to 75 years. Initial feasibility analyses, using a federated analysis approach, yielded expected associations between age (HR 1.15 95% CI 1.14-1.16) per 5-year increase, male sex (HR 1.17, 95% CI 1.13-1.21) and smoking (HR 1.43, 95% CI 1.35-1.51) with risk of subsequent CHD death or myocardial infarction, and differing associations with other individual and composite cardiovascular endpoints. CONCLUSIONS: GENIUS-CHD is a global collaboration seeking to elucidate genetic and non-genetic determinants of subsequent event risk in individuals with established CHD, in order to improve residual risk prediction and identify novel drug targets for secondary prevention. Initial analyses demonstrate the feasibility and reliability of a federated analysis approach. The consortium now plans to initiate and test novel hypotheses as well as supporting replication and validation analyses for other investigators

Motiverande samtals inverkan på diabetespatienter

Bakgrund: Antalet personer med diabetes i världen ökar ständigt. Dåliga levnadsvanor ökar risken för diabeteskomplikationer. Vården har ett ansvar att stärka patientens motivation och förmåga till egenvård.  Syfte: Undersöka effekten av MI som behandlingsmetod för att hjälpa diabetespatienter till förbättrad egenvård.    Metod: Litteraturstudie. Artikelsökningar gjordes i de medicinska databaserna Pubmed, Cinahl och Scopus. I studien inkluderades 10 originalartiklar varav fem var av medelkvalitet och fem av hög kvalitet. Dessa delades in i två grupper vilka var effekt på HbA1c och blodfetter samt effekt på egenvård.    Resultat: Utifrån inkluderade studier kunde ingen signifikant effekt påvisas i HbA1c och blodfetter eller gällande kost, fysisk aktivitet eller medicinsk följsamhet. Däremot sågs positiv effekt vid mätningar av upplevd kompetens, kunskap och egenkontroll. En studie visade positiv effekt på HbA1c av MI i kombination med KBT.  De effekter som kunde påvisas avtog kort tid efter behandlingssessionerna upphört. Studiernas utformning och mätmetoder ifrågasattes.    Slutsats: I nuläget finns få studier inom området därför är det för tidigt att rekommendera MI som metod inom diabetesvården. Mer forskning behövs om MI med tydligare fokus på personalens relation med patienten och personalens MI-färdigheter om man syftar till att mäta effekten av MI-samtalet.Background: The number of people with diabetes in the world is constantly increasing. Bad food habits increase the risk of diabetes complications. Healthcare has a responsibility to enhance the patient's motivation and ability for self-care. Objective: Investigate the impact of MI as a treatment to help patients with diabetes to better self-care. Method: Literature review. The search for articles were made in the medical databases Pubmed , Cinahl and Scopus. The study included 10 original articles of which five were medium and five high quality. These were divided into two groups, effect on HbA1c and blood lipids and effect on self-care. Results: Based on the included studies, no significant effect is demonstrated in HbA1c and blood lipids or current diet, physical activity or medical adherence. There was however a positive effect in measurements of perceived competence, knowledge and self-control. One study demonstrates a positive effect on HbA1c of MI in combination with CBT. The effects found subsided shortly after the treatment sessions ended. Study design and measurement methods were questioned. Conclusion: Few studies exist in the field, therefore, it is too early to yet recommend the MI-method in diabetes care. More research is needed on MI with a clearer focus on the interviewers relationship with the patient and the MI skills of the interviewers if one aims to measure the effect of the MI-sessions

Early English Instruction : A study of teachers' views on the instruction in English in the early years

Vårt syfte med denna studie är att undersöka hur åtta lärare i förskoleklass till och med år 3 på tre skolor i Mellansverige, fördelat på en kommunal skola och två friskolor, ser på undervisning i det engelska språket, hur de väljer att arbeta med detta samt vilken effekt de tror att tidig engelskundervisning har. Vi vill även ta reda på hur lärarna arbetar med inlärning av engelska samt om de anser att deras egna kunskaper är tillräckliga för att undervisa i ämnet. Vi valde att genomföra studien med en kvalitativ metod som grund för enkäter och intervjuer. Fördelen med att komplettera enkätfrågor med intervjuer har gett oss möjlighet att få en djupare förståelse för svaren som informanterna gett samt ta del av deras tankar, resonemang och handlingar. En kvalitativ metod var för denna studie rimlig för att uppnå det syfte vi har med undersökningen. Svaren vi erhållit i vår undersökning är spridda men informanterna är i stort sett överens om att beslut som berör i vilken årskurs engelskundervisning ska börja tas av kommunen där skolan finns och att informanterna rättar sig efter detta. De är inte heller av åsikten att de skulle vilja börja vare sig tidigare eller senare med undervisningen om de själva fick välja. Informanterna använder sig mest av material som de själva tagit fram, då färdiga material inte förekommer för låga åldrar. Rim, ramsor och sånger är en stor del av dessa material. De är dessutom överens om att engelskundervisning i tidiga åldrar ska syfta till att eleverna ska kunna tala engelska i enkla former. I förlängningen leder en sådan tidig start av undervisning till att eleverna känner sig trygga i att använda språket både i skolmiljö och på sin fritid. Resultatet visar även på att informanterna inte har något intresse i kompetensutveckling i det engelska språket utan hellre lägger vikt vid andra ämnen

Wastewater treatment in Sweden 2018

Development over the last 200 years has taken Swedish society from dugout latrines, to underground sewers flowing into lakes or coastal waters, to advanced wastewater treatment plants. Wastewater issues have changed from resolving local sanitary problems to a global environmental issue. This paper ’Wastewater Treatment in Sweden’ is published by the Swedish Environmental Protection Agency (Swedish EPA) to provide an historical overview of development of urban wastewater treatment from 1900s to the present. The paper is published biannually and includes the most recent statistical data from 2016 for releases and sludge from wastewater treatment plants. This information is published in accordance with Article 16 of the Urban Wastewater Treatment Directive (91/271/EEC). The Directive applies to all wastewater collected in sewage systems, but specific requirements apply only to treatment plants serving more than 2,000 persons. For Sweden, this corresponds to over 400 wastewater treatment plants