The effect of combined ß-lactoglobulin supplementation and resistance exercise training prior to limb immobilisation on muscle protein synthesis rates in healthy young adults: study protocol for a randomised controlled trial.

BACKGROUND: The decline in skeletal muscle mass experienced following a short-term period (days to weeks) of muscle disuse is mediated by impaired rates of muscle protein synthesis (MPS). Previous RCTs of exercise or nutrition prehabilitation interventions designed to mitigate disuse-induced muscle atrophy have reported limited efficacy. Hence, the aim of this study is to investigate the impact of a complex prehabilitation intervention that combines ß-lactoglobulin (a novel milk protein with a high leucine content) supplementation with resistance exercise training on disuse-induced changes in free-living integrated rates of MPS in healthy, young adults. METHODS/DESIGN: To address this aim, we will recruit 24 healthy young (18-45 years) males and females to conduct a parallel, double-blind, 2-arm, randomised placebo-controlled trial. The intervention group will combine a 7-day structured resistance exercise training programme with thrice daily dietary supplementation with 23 g of ß-lactoglobulin. The placebo group will combine the same training programme with an energy-matched carbohydrate (dextrose) control. The study protocol will last 16 days for each participant. Day 1 will be a familiarisation session and days 2-4 will be the baseline period. Days 5-11 represent the 'prehabilitation period' whereby participants will combine resistance training with their assigned dietary supplementation regimen. Days 12-16 represent the muscle disuse-induced 'immobilisation period' whereby participants will have a single leg immobilised in a brace and continue their assigned dietary supplementation regimen only (i.e. no resistance training). The primary endpoint of this study is the measurement of free-living integrated rates of MPS using deuterium oxide tracer methodology. Measurements of MPS will be calculated at baseline, over the 7-day prehabilitation period and over the 5-day immobilisation period separately. Secondary endpoints include measurements of muscle mass and strength that will be collected on days 4 (baseline), 11 (end of prehabilitation) and 16 (end of immobilisation). DISCUSSION: This novel study will establish the impact of a bimodal prehabilitation strategy that combines ß-lactoglobulin supplementation and resistance exercise training in modulating MPS following a short-term period of muscle disuse. If successful, this complex intervention may be translated to clinical practice with application to patients scheduled to undergo, for example, hip or knee replacement surgery. TRIAL REGISTRATION: NCT05496452. Registered on August 10, 2022. PROTOCOL VERSION: 16-12-2022/1.

Onset of androgen deprivation therapy leads to rapid deterioration of body composition, physical performance, cardiometabolic health and quality-of-life in prostate cancer patients.

OBJECTIVES: To assess the adverse impact of the first 5 months of androgen deprivation therapy on body composition, physical performance, cardiometabolic health and health-related quality-of-life in prostate cancer patients. MATERIALS AND METHODS: Thirty-four prostate cancer patients (70 ± 7 years) were assessed shortly after initiation of androgen deprivation therapy and again 5 months thereafter. Measurements consisted of whole-body dual-energy x-ray absorptiometry (body composition), computed tomography scanning of the upper leg (muscle mass), one-repetition maximum leg press (muscle strength), cardiopulmonary exercise testing (aerobic capacity), blood draws (metabolic parameters), accelerometry (habitual physical activity) and questionnaires (health-related quality-of-life). Data were analyzed with Student's paired t-tests. RESULTS: Over time, whole-body fat mass (from 26.2 ± 7.7 to 28.4 ± 8.3 kg, p < 0.001) and fasting insulin (from 9.5 ± 5.8 to 11.3 ± 6.9 mU/L, p < 0.001) increased. Declines were observed for quadriceps cross-sectional area (from 66.3 ± 9.1 to 65.0 ± 8.5 cm2, p < 0.01), one-repetition maximum leg press (from 107 ± 27 to 100 ± 27 kg, p < 0.01), peak oxygen uptake (from 23.2 ± 3.7 to 20.3 ± 3.4 mL/min/kg body weight, p < 0.001), step count (from 7,048 ± 2,277 to 5,842 ± 1,749 steps/day, p < 0.01) and health-related quality-of-life (from 84.6 ± 13.5 to 77.0 ± 14.6, p < 0.001). CONCLUSIONS: Androgen deprivation therapy induces adverse changes in body composition, muscle strength, cardiometabolic health and health-related quality-of-life already within 5 months after the start of treatment, possibly largely contributed by diminished habitual physical activity. Prostate cancer patients should, therefore, be stimulated to increase their habitual physical activity immediately after initiation of androgen deprivation therapy, to limit adverse side-effects and to improve health-related quality-of-life.

Declining prevalence of cerebral palsy in children born at term in Denmark.

AIM: To investigate reasons for the declining prevalence of cerebral palsy (CP) in children born at term in Denmark by evaluating obstetric and neonatal factors associated with CP, and their changes over time. METHOD: In this cohort study, we included 987 495 children (504 600 [51.1%] males and 482 895 [48.9%] females) born after 37 completed gestational weeks during birth years 1997 to 2013. Risk ratios of CP for each factor were calculated with log-binominal regression analyses. Significant factors were evaluated concerning their development in prevalence over time. RESULTS: In the antenatal period, there were significant associations with an increased risk of CP and high maternal body mass index (BMI), smoking during pregnancy, nulliparity, male sex, gestational age, and low birthweight. In the study period, fewer females smoked during pregnancy and fewer children were born post-term, dropping from 22.6% to 11.4% and 9.4% to 2.5% respectively. Conversely, the proportion of females with high BMI increased. Most significant risk factors were found in the neonatal period, with an increase in children with diagnosed birth defects and children admitted to neonatal care. INTERPRETATION: Reasons for the declining prevalence of CP appear to be multifactorial and likely include the decline in maternal smoking and children born post-term along with centralization and advances in neonatal treatment.

Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia.

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

SFPQ rescues F508del-CFTR expression and function in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.

Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages.

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase.

A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance.

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-ß-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation.

OBJECTIVES: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody-mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease. METHODS: We performed an observational case-control study including infants hospitalised for RSV infection, hernia surgery or RSV-negative respiratory viral infections. We determined RSV antigen-specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry. RESULTS: We found that RSV-specific maternal antibodies activate NK cells in vitro. While concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon-γ production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls. CONCLUSION: Our results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines.

Benefits and Harm of Treatment with P2Y12 Inhibitors beyond 12 Months in Patients with Coronary Artery Disease.

The trade-off between the benefits and harm of long-term (> 12 months) treatment with P2Y12 inhibitors in patients with coronary artery disease (CAD) after percutaneous coronary intervention (PCI) remains controversial. This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. PubMed and Embase were searched without time restrictions to identify randomized controlled trials comparing > 12-month P2Y12 inhibition versus ≤ 12-month treatment in patients with acute coronary syndrome (ACS) or stable CAD undergoing PCI. A qualitative assessment was performed using the assessment tool from the National Heart, Lung, and Blood Institute of the National Institutes of Health. We performed a meta-analysis of the following endpoints: primary outcome (primarily major cardiovascular events), all-cause death, and major bleeding. Eight trials, comprising 40,218 patients, were included. Five studies were rated "good," two studies "fair," and one study "poor." The meta-analysis showed that > 12-month P2Y12 inhibition significantly reduced the primary outcomes compared with ≤ 12-month treatment (hazard ratio [HR]: 0.85; 95% confidence interval (CI): 0.75-0.97; p = 0.01). No significant difference was demonstrated between groups in all-cause death (HR: 1.02; 95% CI: 0.76-1.36; p = 0.91) or major bleedings (HR: 1.26; 95% CI: 0.93-1.70; p = 0.14). I 2 test showed low to moderate heterogeneity among the included studies (21.6-62.3%). This systematic review and meta-analysis therefore demonstrates a reduction in major cardiovascular events during extended P2Y12-inhibitor treatment beyond 12 months compared with ≤ 12 months in patients with ACS or stable CAD undergoing PCI. There was no significant difference in all-cause death or major bleedings.

Platelet microRNA expression and association with platelet maturity and function in patients with essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by persistently elevated platelet counts and an increased risk of thromboembolic events. Dysregulated expression of small noncoding microRNAs (miRNAs) have been shown in ET and may influence platelet maturity and function in ET patients. In this study, we included 22 ET patients and 19 healthy controls to investigate the expression of 12 platelet miRNAs previously reported to be dysregulated in ET. Further, we investigated the correlation between the expression of selected miRNAs and platelet maturity and platelet function. Total RNA was isolated from platelets, and expression analyses were performed using TaqMan quantitative PCR (qPCR). Mean platelet volume (MPV) and immature platelet count and -fraction (IPC and IPF) were measured using the Sysmex XE-5000 automated haematology system. Platelet function was investigated by multiple electrode aggregometry (agonists: arachidonic acid (AA), thrombin-receptor-activating-peptide (TRAP) and adenosine diphosphate (ADP)), while platelet activation was determined by multi-colour flow cytometry (antibodies: bound-fibrinogen, CD63 and P-selectin (CD62p), agonists: AA, TRAP and ADP). We showed that miR-9 and miR-490 were significantly upregulated in ET patients compared with healthy controls (p-values < 0.01), while miR-10a, miR-28, miR-126, miR-155, miR-221, miR-222, miR-223 and miR-431 were significantly downregulated in ET patients (all p-values < 0.001). A significant positive correlation was observed between miR-431 and MPV, IPC and IPF (all p-values < 0.05). The expression of miR-126 was negatively correlated with platelet aggregation induced by AA and TRAP (p < 0.05). In addition, we found the expression of miR-9 and miR-490 to be negatively correlated with the percentage of fibrinogen-, CD63- and P-selectin- positive platelets using TRAP as agonist (p < 0.05). In conclusion, our data indicate that platelet microRNAs may play a role in ET and that specific microRNAs are correlated with platelet maturity and platelet function.

Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis.

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation.

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

Once- versus twice-daily aspirin treatment in patients with essential thrombocytosis.

Insufficient platelet inhibition has been reported in up to 40% of aspirin-treated patients, including patients with essential thrombocytosis. To maintain sufficient platelet inhibition, a shorter dosing interval with aspirin has been suggested. We aimed to investigate the antiplatelet effect of low-dose aspirin given twice-daily compared to standard once-daily dosing in patients with essential thrombocytosis. We included 22 patients, who were treated for 7 days with standard once-daily aspirin (75 mg once-daily) followed by 7 days treatment of twice-daily aspirin (37.5 mg twice-daily). The two regimens were separated by 14 days aspirin washout. Blood samples were obtained 1h and 24h/12h after the last pill intake in each regimen. The effect of aspirin was evaluated by: (1) platelet aggregation measured by whole blood impedance aggregometry (Multiplate® Analyser) using arachidonic acid (ASPItest 0.5 mM) as agonist and (2) serum thromboxane B2 levels determined using an enzyme-linked immunosorbent assay. The difference in platelet aggregation from 1h to the end of the dosing interval (24h/12h) was used to compare the two regimens. We demonstrated a significantly smaller difference in platelet aggregation in the twice-daily regimen compared to the once-daily: mean of difference = 228 AU*min (95% confidence interval (CI): 92-363, p < 0.01). In addition, a significantly smaller difference in thromboxane B2 was demonstrated in the twice-daily regimen compared to the once-daily regimen: mean of difference = 16.3 ng/mL (95% CI: 9.9-22.7, p < 0.01). In conclusion, twice-daily dosing with low-dose aspirin provides a more consistent platelet inhibition compared with standard once-daily dosing in patients with essential thrombocytosis.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy.

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.

Platelet characteristics in patients with essential thrombocytosis.

BACKGROUND: Essential thrombocytosis (ET) is a myeloproliferative disorder characterized by an increased platelet count. ET is associated with an increased risk of thrombosis, and procoagulant features of the disease may include an increased number of reactive reticulated platelets and an increased aggregation potential. We aimed to explore the association between platelet count, platelet turnover, and platelet aggregation in patients with ET. METHODS: We included 24 ET patients who discontinued antiplatelet therapy prior to blood sampling. Reticulated platelets were assessed as immature platelet count (IPC) and immature platelet fraction by automated flow cytometry (Sysmex XE-5000). Platelet aggregation was investigated by impedance aggregometry (Multiplate® Analyzer) and aggregation potential by flow cytometry (NAVIOS). RESULTS: Our results showed that ET patients had increased IPC compared to healthy individuals (median 12.3 vs. median 6.9, P < 0.0001). Furthermore, a positive correlation between platelet count and impedance aggregation was demonstrated using arachidonic acid (r = 0.48, P = 0.02), thrombin-receptor-activating-peptide (r = 0.46, P = 0.03) and adenosine diphosphate (r = 0.56, P = 0.007) as agonists. Finally, an increased aggregation potential was demonstrated in ET patients compared to healthy individuals. CONCLUSIONS: The study showed that ET patients compared to healthy individuals have an increased amount of reticulated platelets and increased aggregation potential. These findings might in part explain the increased thromboembolic risk in patients with ET. © 2018 International Clinical Cytometry Society.

Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.

Recurrent Cardiovascular Events Despite Antiplatelet Therapy in a Patient with Polycythemia Vera and Accelerated Platelet Turnover.

BACKGROUND Clopidogrel is commonly used in the prevention and treatment of cardiovascular events. However, despite clopidogrel treatment, some patients experience recurrent ischemic events. CASE REPORT We present the case of a 58-year-old man with polycythemia vera and concomitant thrombocytosis who suffered 6 episodes of cerebral infarctions and 1 myocardial infarction, despite treatment with clopidogrel. Following his last ischemic event, the antiplatelet therapy was intensified from initially clopidogrel monotherapy to dual antiplatelet therapy with aspirin 75 mg once daily and ticagrelor 90 mg twice daily. Since then, no cardiovascular event has been reported. CONCLUSIONS This case report illustrates that insufficient platelet inhibition with clopidogrel monotherapy in a patient with thrombocytosis may be associated with recurrent arterial thrombosis. The exact reasons for the insufficient platelet inhibition are not known, but a plausible explanation may be an accelerated platelet turnover reflected by an increased number of immature platelets in this patient.

Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.