Abstract. Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosome acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cationindependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluidphase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Bar), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-conraining endocytic compartments in living cells revealed elevated endosomal pH values (pH >7.0) within 2 min after addition of Bar. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe 3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +_ 0.08 and 1.57 + 0.25, respectively. Consequently, little if any change in TfR expression at the cell surface was measured during Bar treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine crosslinking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Bar, sorting was initiated after 20 min of uptake, reaching ~40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.URING receptor-mediated endocytosis, ligand-receptor complexes are internalized and transported via clathrin-coated vesicles to endosomes (for reviews see Van Deurs et al., 1989;Courtoy, 1991). In endosomes, ligands and receptors may dissociate from each other, after which receptors and ligands are transported to their specific destination in the cell. Since endocytosed proteins are transported from endosomes to lysosomes, the trans-Golgi network (TGN) 1, or to either apical or ba-

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Background Blood donors are at risk for reduced iron stores, because of which donor iron monitoring received increased attention in the last decade. Despite the importance for donor health, international consensus on an appropriate policy for iron monitoring is lacking. Therefore, we conduct a trial to evaluate to what extent ferritin-guided donation intervals are effective in increasing haemoglobin and ferritin levels, decreasing low-haemoglobin deferral, increasing donor return and improving the health of whole blood donors in the Netherlands. Methods Sanquin Blood Bank is implementing ferritin-guided donation intervals to prevent donors from increasing iron loss at repeated donations. Using a stepped wedge cluster randomised trial approach, the design involves a random crossover of 29 clusters of blood collection centres from the existing policy without ferritin measurements to a ferritin-guided donation interval policy. This new policy includes ferritin measurements for all new donors and at every 5th whole blood donation, extending donation intervals to 6 months if ferritin is 15–≤ 30 ng/mL and to 12 months if ferritin is < 15 ng/mL. We measure ferritin levels of whole blood donors from stored plasma samples and collect haemoglobin levels and information on low-haemoglobin deferral and donor return from the donor database before, during and after the implementation period. We measure donor health during and after the implementation period using questionnaires, assessing physical and mental wellbeing and iron deficiency- and donation-related symptoms. We use multilevel analyses to investigate differences in ferritin and haemoglobin levels, low-haemoglobin deferral rates, donor return and donor health from whole blood donors, between blood collection centres that have versus those that have not yet implemented the ferritin-guided donation interval policy. Discussion This stepped wedge cluster randomised trial will provide insight into the effectiveness of ferritin-guided donation intervals in lowering iron deficiency, decreasing donor deferrals due to low haemoglobin and improving donor health. We will evaluate a policy that is implemented nationwide in a real-life setting. Our study is therefore not limited to a small experimental setting and the results will guide policymakers seeking an appropriate policy for iron monitoring. Trial registration The Dutch trial registry NTR6738. Registered on 29 September 2017. Retrospectively registered.

Cardiovascular disease (CVD) risk is associated with prenatal and infancy growth. However, the relative importance of these time periods for the CVD risk is uncertain. To elucidate this, we tested in a very preterm cohort the effects of birth weight for gestational age and weight gain between birth and 3 mo post-term (early postnatal weight gain) and between 3 mo and 1 y post-term (late infancy weight gain) on the lipid profile and carotid intimamedia thickness (CIMT) at age 19 y. A less favorable lipid profile was strongly associated with higher current body mass index (BMI), greater waist circumference, and greater absolute fat mass. CIMT was positively associated with current height, and with low-density lipoprotein (LDL) cholesterol and apolipoprotein B (ApoB) levels, and LDL/high-density lipoprotein (HDL) cholesterol and ApoB/ apolipoprotein AI (ApoAI) ratios. Lipid profile and CIMT were unrelated to gestational age, birth weight standard deviation score (SDS) and early postnatal weight gain. CIMT was positively associated with late infancy weight gain, but the relationship disappeared after correction for current height. Our findings in 19 y olds born very preterm argue for an effect of current body composition, rather than of early growth, on the CVD risk. Attempts to reduce the CVD risk in this specific population should focus on weight reduction in young adulthood rather than on optimizing the early growth pattern.

To assess the magnitude of differences in QCA outcomes between two cooperating core laboratories in a single trial, we have carried out an inter-core laboratory variability study. Two QCA experts at the Montreal Heart Institute and Heart Core Leiden both analyzed 32 lesions (pre- and post-intervention) in accordance with previously agreed upon standard operating procedures. One of the experts analyzed the whole image set twice to determine the intraobserver variability. The inter-core laboratory differences in the acute gain (n = 31 pairs) are non-significant. The systematic errors of the individual measurements (n = 63 analyses) show an excellent intraclass correlation coefficient of reliability (>75%), except for the stent length (67.7%). The corresponding random errors are small. In general, the intra-observer systematic and random errors are both slightly smaller than those for the inter-core laboratory study. QCA analyses in clinical trials can be carried out in core laboratories at two different locations if and only if highly standardized conditions are maintained.