Missense-depleted regions in population exomes implicate ras superfamily nucleotide-binding protein alteration in patients with brain malformation.

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss

Gender differences in the evaluation of care for patients with type 2 diabetes:a cross-sectional study (ZODIAC-52)

BACKGROUND: Little is known about the association between patient-related factors and patients' evaluation of care. Aim was to investigate which patient-related factors are associated with patients' evaluation of care in men and women with type 2 diabetes (T2D) in primary care. METHODS: This cross-sectional study included 1102 patients with T2D from 52 general practices. We measured patients' evaluation with the EUROPEP questionnaire and collected demographic, clinical and psychological data from questionnaires and health records. Stepwise linear regression analyses were used. RESULTS: The location where the questionnaire was completed (at home or at the general practice) was associated with all outcomes in men and women. Next to this, in men, explanatory factors for the care provider EUROPEP subscale were use of insulin, having some problems with T2D self-care and coffee consumption (R2 8.4%); coffee consumption was associated with the general practice subscale (R2 4.0%). In women, well-being, quality of life, following a general diet, and use of oral glucose-lowering drugs were associated with the care provider subscale (R2 16.8%). For the general practice subscale, well-being and age were explanatory factors (R2 9.4%). CONCLUSIONS: Only a few factors were found to be associated with patients' evaluation of care for men and women with T2D. Taken together, these factors explained only a small part of the variance of the EUROPEP scores. This explained variance was largely attributable to the location where the questionnaire was completed. We therefore advise to be aware of the possible consequences of filing-out questionnaires about patients' evaluation of care at the general practice. TRIAL REGISTRATION: NCT01570140 (Clinicaltrials.gov). Registered 29 March 2012

DEVELOPMENT OF INACTIVATED POLIO VACCINE FROM ATTENUATED SABIN STRAINS FOR CLINICAL STUDIES AND TECHNOLOGY-TRANSFER PURPOSES

Recently, responding to WHO’s call for new polio vaccines, the development of Sabin-IPV (injectable, formalin-Inactivated Polio Vaccine, based on attenuated ‘Sabin’ polio virus strains) was initated at NVI. This activity plays an important role in the WHO polio eradication strategy. The use of Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Initially, the Sabin-IPV production process will be based on the scale-down model of the current, and well-established, Salk-IPV process. In parallel, process development, optimization and formulation research is being carried out to further modernize the process and reduce cost per dose. The lab-scale accelerated process development, product characterization, clinical lot production, and preparations for technology transfer will be discussed. Multivariate data analysis (MVDA) was applied on data from current IPV production (more than 60 Vero cell culture based runs) to extract relevant information, like operating ranges. Subsequently, based on the MVDA analysis, a 3-L scale-down model of the current twin 750-L bioreactors has been setup. Currently, in this lab-scale process, cell and virus culture approximate the large-scale and process improvement studies are in progress. This includes the application of increased cell densities, animal component free media, and DOE optimization in multiple parallel bioreactors. Also, results will be shown from large-scale (to prepare for future technology transfer) generation and testing of Master- and Working virus seedlots, and clinical lot (for phase I studies) production under cGMP conditions. The obtained product was used for immunogenicity studies in rats. It was shown that Sabin-IPV induces a good immune response, and a comparison will be made to regular Salk-IPV. Finally, technology transfer to vaccine manufacturers in low and middle–income countries will take place. For that, an international Sabin-IPV manufacturing course, including practical training at pilot-scale, is being setup

Design of the e-Vita diabetes mellitus study: effects and use of an interactive online care platform in patients with type 2 diabetes (e-VitaDM-1/ZODIAC-40)

Background Due to ongoing rise in need for care for people with chronic diseases and lagging increase in number of care providers, alternative forms of care provision and self-management support are needed. Empowering patients through an online care platform could help to improve patients’ self-management and reduce the burden on the healthcare system. Methods Access to laboratory results and educational modules on diabetes will be offered through a platform for subjects with type 2 diabetes mellitus treated in primary care. Differences in socio-demographic and clinical characteristics between subjects expressing interest vs. disinterest to use the platform will be explored. Platform usage will be tracked and compared. Patient satisfaction and quality of life will be measured by validated questionnaires and economic analyses will be performed. Discussion This study is designed to assess the feasibility of use of an online platform in routine primary healthcare for subjects with type 2 diabetes mellitus in the Netherlands, and to study effects of use of the platform on treatment satisfaction, quality of life and clinical parameters. Although providing access to a online platform is not a novel intervention, usage and effects have not yet been studied in this patient population

Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK

Introduction: Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. Methods: We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. Results: We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Discussion: Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.</p

Duration of rectal colonization with extended-spectrum beta-lactamase-producing Escherichia coli: results of an open, dynamic cohort study in Dutch nursing home residents (2013–2019)

Background: In 2016, a study in a Dutch nursing home showed prolonged colonization duration of extended-spectrum β-lactamase-producing (ESBL)-ST131 compared to ESBL-non-ST131. In this study, we assessed the duration of rectal ESBL-producing E. coli (ESBL-EC) colonization in residents in the same nursing home for an extended period of six years. We aimed to estimate the influence of a possible bias when follow up is started during an outbreak. Methods: Between 2013 and 2019, repetitive point prevalence surveys were performed by culturing rectal or faecal swabs from all residents. Kaplan–Meier survival analysis was performed to calculate the median time to clearance of ESBL-EC with a log-rank analysis to test for differences between ESBL-ST131 and ESBL-non-ST131. Results: The study showed a median time to clearance of 13.0 months (95% CI 0.0–27.9) for ESBL-ST131 compared to 11.2 months (95% CI 4.8–17.6) for ESBL-non-ST131 (p = 0.044). In the subgroup analysis of residents who were ESBL-EC positive in their first survey, the median time to clearance for ST131 was 59.7 months (95% CI 23.7–95.6) compared to 16.2 months (95% CI 2.1–30.4) for ESBL-non-ST131 (p = 0.036). In the subgroup analysis of residents who acquired ESBL-EC, the median time to clearance for ST131 was 7.2 months (95% CI 2.1–12.2) compared to 7.9 months (95% CI 0.0–18.3) for ESBL-non-ST131 (p = 0.718). The median time to clearance in the ESBL-ST131 group was significantly longer in residents who were ESBL-ST131 colonised upon entering the study than in residents who acquired ESBL-ST131 during the study (p = 0.001). Conclusion: A prolonged colonization with ESBL-ST131 was only found in the subgroup who was ESBL-EC positive upon entering the study. The prolonged duration with ESBL-ST131 in the previous study was probably biased by factors that occured during (the start of) the outbreak