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Objectives Our aim was to develop and validate a non-invasive imaging tool to visualize HDL’s in vivo behavior by positron emission tomography (PET), with an emphasis on its plaque targeting abilities. Background High-density lipoprotein (HDL) is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. Methods Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (APOA1) and the phospholipid DMPC. For radiolabeling with Zirconium-89 (89Zr), the chelator DFO was introduced by conjugation to APOA1 or as a phospholipid-chelator (DSPE-DFO). Radiolabeled HDL’s biodistribution and plaque targeting was studied in established murine, rabbit and porcine atherosclerosis models by PET combined with computed tomography (PET/CT) or with magnetic resonance imaging (PET/MRI). Ex vivo validation was conducted by radioactivity counting, autoradiography and near infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. Results We observed distinct pharmacokinetic profiles for the two 89Zr-HDL nanoparticles. Both APOA1- and phospholipid-labeled HDL mainly accumulated in kidneys, liver and spleen with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3–4-fold higher than in controls at 5 days p.i. for both 89Zr-HDL nanoparticles. In the porcine model, we observed increased accumulation of radioactivity in lesions by in vivo PET imaging. Irrespective of the radiolabel’s location we found HDL nanoparticles to preferentially target plaque macrophages and monocytes. Conclusions 89Zr labeling of HDL allows studying its in vivo behavior by non-invasive PET imaging, including visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL’s main components, i.e. phospholipids and APOA1.

The intestinal microbiota has been linked to the development and prevalence of steatohepatitis in humans. Interestingly, steatohepatitis is significantly lower in individuals taking a plant‐based, low‐animal‐protein diet, which is thought to be mediated by gut microbiota. However, data on causality between these observations in humans is scarce. In this regard, fecal microbiota transplantation (FMT) using healthy donors is safe and is capable of changing microbial composition in human disease. We therefore performed a double‐blind randomized controlled proof‐of‐principle study in which individuals with hepatic steatosis on ultrasound were randomized to two study arms: lean vegan donor (allogenic n = 10) or own (autologous n = 11) FMT. Both were performed three times at 8‐week intervals. A liver biopsy was performed at baseline and after 24 weeks in every subject to determine histopathology (Nonalcoholic Steatohepatitis Clinical Research Network) classification and changes in hepatic gene expression based on RNA sequencing. Secondary outcome parameters were changes in intestinal microbiota composition and fasting plasma metabolomics. We observed a trend toward improved necro‐inflammatory histology, and found significant changes in expression of hepatic genes involved in inflammation and lipid metabolism following allogenic FMT. Intestinal microbial community structure changed following allogenic FMT, which was associated with changes in plasma metabolites as well as markers of . Conclusion: Allogenic FMT using lean vegan donors in individuals with hepatic steatosis shows an effect on intestinal microbiota composition, which is associated with beneficial changes in plasma metabolites and markers of steatohepatitis.

An altered intestinal microbiota composition has been implicated in the pathogenesis of metabolic disease including obesity and type 2 diabetes mellitus (T2DM). Low grade inflammation, potentially initiated by the intestinal microbiota, has been suggested to be a driving force in the development of insulin resistance in obesity. Here, we report that bacterial DNA is present in mesenteric adipose tissue of obese but otherwise healthy human subjects. Pyrosequencing of bacterial 16S rRNA genes revealed that DNA from the Gram-negative species Ralstonia was most prevalent. Interestingly, fecal abundance of Ralstonia pickettii was increased in obese subjects with pre-diabetes and T2DM. To assess if R. pickettii was causally involved in development of obesity and T2DM, we performed a proof-of-concept study in diet-induced obese (DIO) mice. Compared to vehicle-treated control mice, R. pickettii-treated DIO mice had reduced glucose tolerance. In addition, circulating levels of endotoxin were increased in R. pickettii-treated mice. In conclusion, this study suggests that intestinal Ralstonia is increased in obese human subjects with T2DM and reciprocally worsens glucose tolerance in DIO mice.