Expression of Bruton´s tyrosine kinase in different type of brain lesions of multiple sclerosis patients and during experimental demyelination

BackgroundInhibition of Bruton’s tyrosine kinase (BTK) is an emerging multiple sclerosis (MS) therapy. BTK inhibitors (BTKi) cross the blood-brain barrier and modulate B cells and microglia, major cellular players in active and chronic active lesions.ObjectiveTo assess potential lesional and cellular targets of BTKi, we examined BTK expression in different type of MS white matter (WM) lesions, in unmanipulated CNS resident cells, and in a degenerative MS model associated with microglia activation in vivo.MethodsWe examined BTK expression by next-generation RNA-sequencing in postmortem 25 control WM, 19 NAWM, 6 remyelinating, 18 active, 13 inactive and 17 chronic active lesions. Presence of B cells and microglia were examined by immunohistochemistry. CNS resident cells were isolated from the mouse brain by magnetic sorting. BTK expression was examined by quantitative PCR in isolated cells and dissected corpus callosum from mice treated with cuprizone (CPZ).ResultsBTK expression was significantly increased in active and chronic active lesions with upregulated complement receptors and Fcγ receptors. Active lesions contained high number of perivascular B cells, microglia, and macrophages. Chronic active lesions were characterized by microglia/macrophages in the rim. Microglia expressed BTK at high level (120-fold) in contrast to other CNS cell types (2-4-fold). BTK expression was increasing during CPZ treatment reaching significance after stopping CPZ.ConclusionConsidering BTK expression in MS lesions and resident cells, BTKi may exert effect on B cells, microglia/macrophages in active lesions, and limit microglia activation in chronic active lesions, where tissue damage propagates

Muscle Mitochondrial ATP Synthesis and Glucose Transport/Phosphorylation in Type 2 Diabetes

BACKGROUND: Muscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM). METHODS AND FINDINGS: We measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with (31)P/(1)H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux. CONCLUSIONS: Patients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients

Medical Management of Hyperglycemia in Type 2 Diabetes: A Consensus Algorithm for the Initiation and Adjustment of Therapy: A consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes

The consensus algorithm for the medical management of type 2 diabetes was published in August 2006 with the expectation that it would be updated, based on the availability of new interventions and new evidence to establish their clinical role. The authors continue to endorse the principles used to develop the algorithm and its major features. We are sensitive to the risks of changing the algorithm cavalierly or too frequently, without compelling new information. An update to the consensus algorithm published in January 2008 specifically addressed safety issues surrounding the thiazolidinediones. In this revision, we focus on the new classes of medications that now have more clinical data and experience

Insulin Resistance Is Not Conserved in Myotubes Established from Women with PCOS

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects.Using radiotracer techniques, RT-PCR and enzyme kinetic analysis we examined myotubes established from PCOS subjects with or without pioglitazone treatment, versus healthy control subjects who had been extensively metabolically characterized in vivo. Results. Myotubes established from PCOS and matched control subjects comprehensively expressed all insulin-sensitive biomarkers; glucose uptake and oxidation, glycogen synthesis and lipid uptake. There were no significant differences between groups either at baseline or during acute insulin stimulation, although in vivo skeletal muscle was insulin resistant. In particular, we found no evidence for defects in insulin-stimulated glycogen synthase activity between groups. Myotubes established from PCOS patients with or without pioglitazone treatment also showed no significant differences between groups, neither at baseline nor during acute insulin stimulation, although in vivo pioglitazone treatment significantly improved insulin sensitivity. Consistently, the myotube cultures failed to show differences in mRNA levels of genes previously demonstrated to differ in PCOS patients with or without pioglitazone treatment (PLEK, SLC22A16, and TTBK).These results suggest that the mechanisms governing insulin resistance in skeletal muscle of PCOS patients in vivo are not primary, but rather adaptive.ClinicalTrials.gov NCT00145340

Children overcoming picky eating (COPE) – A cluster randomised controlled trial

Objectives Food neophobia limits dietary variety in children and adults. Interventions to alleviate the impact of neophobia on children's dietary variety have had varying success. The potential effectiveness of mindfulness, a process of bringing awareness to the present moment, has received little attention. This trial aimed to explore the effectiveness of two mindfulness exercises on novel food acceptance for children. Methods A cluster-randomised controlled trial with three trial arms compared the impact of two mindfulness exercises (mindful breathing and mindful raisin-eating) and a non-mindful control task on anticipated liking and intake of a novel fruit. Seventy-one children aged 10–12 years engaged in one of the three tasks at school over five days and were offered a novel fruit at the end of the intervention. Children self-reported mindfulness, food neophobia and anxiety at baseline and follow-up. Results Two mixed-effects models showed that, controlling for school effects and covariates (including mindfulness, food neophobia and anxiety), children in the mindful raisin-eating arm reported greater anticipated liking of a novel fruit and children in both mindfulness arms consumed greater amounts of a novel fruit than children in the control arm. Mixed-design ANOVAs indicated that mindfulness, food neophobia and anxiety did not change over time in each trial arm. Conclusions The results provide promising evidence for the potential effectiveness of mindfulness interventions in encouraging children to try new foods. The mechanisms underlying effectiveness remain unclear and further research, exploring long-term effects and the possibility to generalise these findings to other food groups such as vegetables, is needed

stRoke R package

This is an R-toolbox of custom functions for convenient data management and analysis in clinical health research and teaching. The package is mainly collected for personal use, but any use beyond that is encouraged. This package has migrated functions from 'agdamsbo/daDoctoR', and new functions has been added. Version follows months and year. See NEWS/Changelog for release notes. This package includes sampled data from the TALOS trial (Kraglund et al (2018) ). The win_prob() function is based on work by Zou et al (2022) . The age_calc() function is based on work by Becker (2020)

Design of consensus toxins and their use for the discovery of broadly neutralizing antibodies

Snakebite envenoming remains a significant global health challenge, impacting millions of people annually, particularly in regions with limited access to medical care. The primary treatment for snakebite envenoming is antivenom, which consists of polyclonal antibodies derived from the immunization of large animals with snake venom. However, this traditional method poses challenges, including the need to extract venom from snakes, the use of big animals in the manufacturing process, the resulting antivenoms can potentially cause adverse reactions, and these products possess limited cross-reactivity against venoms from species whose venom was not included in the immunization procedure. This thesis explores alternative strategies for developing a fundamentally new type of antivenom, with an emphasis on the utilization of recombinant toxin expression and antibody discovery techniques.The central objective of this research is to innovate antivenom development by eliminating the need to extract venom from snakes and instead enable the heterologous expression of toxins and the discovery of antibodies in vitro. This work focused on α-neurotoxins from the three-finger toxin superfamily. These are crucial components of most elapid venoms and known for their potent neurotoxic effects, which are mediated by the ability of this functional group of toxins to bind and inhibit nicotinic acetylcholine receptors on the postsynaptic side of neuromuscular junctions, ultimately manifesting as paralysis in victims.In the first part of the work behind this thesis, Escherichia coli and Komagataella phaffii (previously known as Pichia pastoris) were employed as hosts for the recombinant expression of α-cobratoxin, which is a well-studied α-neurotoxin. This comparison of different expression systems aimed to evaluate the efficiency and functionality of the recombinantly expressed toxins. The findings demonstrated the potential of both E. coli and K. phaffii in producing α-cobratoxin, presenting promising alternatives to traditional venom extraction methods, although the work also indicated that further optimization is warranted to obtain even higher quality toxins.Afterward, α-cobratoxin was utilized as an antigen to discover single-chain variable fragments (scFvs) as toxin-targeting antibodies using phage display technology. Notably, our process leveraged recombinant α-cobratoxin, which was equally useful as antigen as native α-cobratoxin in the discovery process. This approach represents the first entirely in vitro antibody discovery strategy to discover antibodies targeting snake toxins. The study thus showcased the viability of using recombinant snake toxins for antibody discovery, offering a potentially less hazardous discovery platform with enhanced molecular control for antivenom development, devoid of the need for snake venoms.To broaden the neutralization capacity of toxin-neutralizing antibodies, the use of consensus α-neurotoxins was employed during a phage display-based discovery campaign involving an immune nanobody library. Here, such toxins were designed, expressed, and used as antigens to discover nanobodies with cross-neutralizing properties against snake toxins from different species of elapids from several different genera. These nanobodies demonstrated surprisingly high affinity and broad neutralizing capacities and may therefore be promising leads for the further development of future antivenom therapies.In conclusion, the research presented in this thesis significantly propels antivenom development forward, establishing a more efficient, sustainable, and accessible approach to antivenom development, which I hope will pave the way for improved snakebite envenoming management worldwide. Moreover, the knowledge and insights generated in the work behind this thesis may offer valuable guidance to future researchers in their pursuit of broadly neutralizing antibodies and nanobodies beyond the field of snake envenoming. More specifically, it is my personal belief that the concepts and methodologies developed here hold significant promise in various scientific domains, including the development of consensus proteins and broadly neutralizing antibodies in areas, such as infectious disease research, cancer therapy, drug discovery, and diagnostics, where cross-reactivity may be a key aspect for successful clinical, industrial, and basic research applications