Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

A decade's experience with retrograde open mesenteric stenting for acute mesenteric ischemia.

OBJECTIVE: Retrograde open mesenteric stenting (ROMS) is an alternative to mesenteric bypass in patients with acute mesenteric ischemia (AMI) with variable reported 30-day mortality rates. Large studies evaluating patient outcomes following ROMS are scarce. Our study aims to assess the results of this approach among patients presenting with AMI. METHODS: We reviewed all the patients with AMI who were treated with ROMS (2011-2022). Patient demographics, presentation, operative details, and outcomes were analyzed. Primary end points were in-hospital, 30-day, and 1-year mortality. Kaplan-Meier estimate for 1-year mortality and primary patency loss were generated. Secondary end points included postoperative 30-day complications. RESULTS: Between 2011 and 2022, ROMS was attempted on a total of 42 patients. The median age was 70 ± 15 years and the majority of patients were female. Pain out of proportion to the physical examination was the most common presenting symptom (n = 18, 42.9%) followed by peritonitis (n = 14, 33.4%). All patients underwent preoperative intravenous contrast computed tomography imaging. In situ thrombosis was identified as the etiology of AMI in 36 patients (85.7%). Technical success was achieved in 40 patients (95.2%). Conventional, non-hybrid operating rooms were used for the majority of cases. Revascularization of all 40 patients involved angioplasty and stenting of superior mesenteric artery. A single stent was placed in 35 patients (87.5%) and the reminder had more than one stent. Eighty percent of patients required bowel resection. A second-look laparotomy was required in 34 patients (85.0%). The mean operative time, including both the general surgery and vascular surgery portions of the index procedure, was 192 ± 57 minutes. Sepsis was the most common complication observed within 30 days, occurring in 8 patients (20.0%). In terms of mortality, 13 patients (32.5%) died during their index hospitalization, and 9 died (22.5%) within 30 days. On Kaplan-Meier analysis, the 1-year overall patient survival rate was 58.6%, and the primary patency rate for stents was 51.4%. CONCLUSIONS: ROMS has an excellent technical success rate in management of AMI with lower than traditionally reported mortality rates for AMI. The dual benefits of rapid revascularization and bowel evaluation should make this surgical modality an alternative approach for treatment of AMI.

Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

STUDY QUESTION: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? SUMMARY ANSWER: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. WHAT IS KNOWN ALREADY: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. STUDY DESIGN, SIZE, DURATION: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. MAIN RESULTS AND THE ROLE OF CHANCE: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. LIMITATIONS, REASONS FOR CAUTION: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. WIDER IMPLICATIONS OF THE FINDINGS: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Racial and Ethnic Differences in Initiation of Menthol Tobacco Smoking and Subsequent Tobacco Use in the Population Assessment of Tobacco and Health Study, Waves 1-4 (2013-2018).

INTRODUCTION: Certain subpopulations in the United States are highly vulnerable to tobacco initiation and addiction, and elimination of disparities among those groups is crucial to reducing the burden of tobacco use. AIMS AND METHODS: This study evaluated the racial and ethnic differences in smoking initiation of menthol flavored cigarettes and cigars among never-users, and in subsequent tobacco use among new users of menthol-flavored products, using longitudinal data from waves 1-4 of the Population Assessment of Tobacco and Health Study. The outcomes of interest were new use of menthol-flavored products, and subsequent past 30-day and past 12-month cigarette and cigar smoking, irrespective of flavors, after initiation. RESULTS: The percentages of new users of menthol-flavored cigarettes and cigars at waves 2-4 were disproportionately higher in non-Hispanic black and Hispanic than in non-Hispanic white people. Adjusting for age and sex, black people who first used any menthol cigars had higher risk of past 30-day use of the same cigar category at the subsequent wave (adjusted risk ratio, aRR 1.48; 95% confidence interval [CI] 1.11 to 1.96) and past 12 months (aRR 1.74; 95% CI 1.55 to 2.63) compared to non-Hispanic white smokers. Black people who first used menthol-flavored cigarettes had marginally higher risk of subsequent past 30-day cigarette use (aRR 1.44; 95% CI 0.99 to 2.10) compared with their non-Hispanic white counterparts. CONCLUSIONS: This study shows that racial and ethnic differences exist in both initiation of menthol-flavored tobacco products and product-specific subsequent use after first using menthol-flavored products; black and Hispanic people have higher rates of initiation; black people also have higher rates of subsequent use. IMPLICATIONS: Use of menthol flavors in tobacco products is confirmed to be a contributor to large disparities in tobacco use; black and Hispanic people are more likely to maintain smoking through use of mentholated products than non-Hispanic white people. The findings suggest educational and regulatory actions on menthol-flavored tobacco products including restricting the selective marketing to vulnerable communities and banning characterizing flavors in cigarettes and cigars may reduce tobacco-related disparities and inform the Food And Drug Administration's evidence-based rulemaking process.

Oral Cladribine Impairs Intermediate, but Not Conventional, Monocyte Transmigration in Multiple Sclerosis Patients across a Model Blood-Brain Barrier.

Multiple sclerosis (MS) is a disease in which the immune system damages components of the central nervous system (CNS), leading to the destruction of myelin and the formation of demyelinating plaques. This often occurs in episodic "attacks" precipitated by the transmigration of leukocytes across the blood-brain barrier (BBB), and repeated episodes of demyelination lead to substantial losses of axons within and removed from plaques, ultimately leading to progressive neurological dysfunction. Within leukocyte populations, macrophages and T and B lymphocytes are the predominant effectors. Among current immunotherapies, oral cladribine's impact on lymphocytes is well characterised, but little is known about its impact on other leukocytes such as monocytes and dendritic cells (DCs). The aim of this study was to determine the transmigratory ability of monocyte and DC subsets in healthy subjects and untreated and cladribine-treated relapse-remitting MS (RRMS) patients using a well-characterised model of the BBB. Peripheral blood mononuclear cells from subjects were added to an in vitro transmigration assay to assess cell migration. Our findings show that while prior treatment with oral cladribine inhibits the migration of intermediate monocytes, it has no impact on the transmigration of DC subsets. Overall, our data indicate a previously unrecognised role of cladribine on intermediate monocytes, known to accumulate in the brain active MS lesions.

HNRNPR Variants that Impair Homeobox Gene Expression Drive Developmental Disorders in Humans.

The heterogeneous nuclear ribonucleoprotein (HNRNP) genes code for a set of RNA-binding proteins that function primarily in the spliceosome C complex. Pathogenic variants in these genes can drive neurodegeneration, through a mechanism involving excessive stress-granule formation, or developmental defects, through mechanisms that are not known. Here, we report four unrelated individuals who have truncating or missense variants in the same C-terminal region of hnRNPR and who have multisystem developmental defects including abnormalities of the brain and skeleton, dysmorphic facies, brachydactyly, seizures, and hypoplastic external genitalia. We further identified in the literature a fifth individual with a truncating variant. RNA sequencing of primary fibroblasts reveals that these HNRNPR variants drive significant changes in the expression of several homeobox genes, as well as other transcription factors, such as LHX9, TBX1, and multiple HOX genes, that are considered fundamental regulators of embryonic and gonad development. Higher levels of retained intronic HOX sequences and lost splicing events in the HOX cluster are observed in cells carrying HNRNPR variants, suggesting that impaired splicing is at least partially driving HOX deregulation. At basal levels, stress-granule formation appears normal in primary and transfected cells expressing HNRNPR variants. However, these cells reveal profound recovery defects, where stress granules fail to disassemble properly, after exposure to oxidative stress. This study establishes an essential role for HNRNPR in human development and points to a mechanism that may unify other "spliceosomopathies" linked to variants that drive multi-system congenital defects and are found in hnRNPs.

Peripheral B-cell dysregulation is associated with relapse after long-term quiescence in patients with multiple sclerosis.

B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be identified. We utilized mass cytometry to analyze B cells from the blood of patients with relapse-remitting MS (RRMS) before and after alemtuzumab treatment, and during relapse. A complementary RRMS cohort was analyzed by single-cell RNA sequencing. The R package "Spectre" was used to analyze these data, incorporating FlowSOM clustering, sparse partial least squares-discriminant analysis and permutational multivariate analysis of variance. Immunoglobulin (Ig)A+ and IgG1 + B-cell numbers were altered, including higher IgG1 + B cells during relapse. B-cell linker protein (BLNK), CD40 and CD210 expression by B cells was lower in patients with RRMS compared with non-MS controls, with similar results at the transcriptomic level. Finally, alemtuzumab restored BLNK, CD40 and CD210 expression by IgA+ and IgG1 + B cells, which was altered again during relapse. These data suggest that impairment of IgA+ and IgG1 + B cells may contribute to MS pathogenesis, which can be restored by alemtuzumab.

Female Gender is a Predictor of Lower Iliac Vein Stenting Patency Rates.

BACKGROUND: Iliac venous stenting (IVS) for thrombotic and nonthrombotic venous disease is increasingly used as evidence of the safety, efficacy and durability of these interventions increases. Female gender has been implicated as a predictor of failure in arterial endovascular interventions. We hypothesize that female gender could be predictive of patency rates of iliac vein stenting. METHODS: Consecutive patients who underwent IVS for thrombotic or nonthrombotic venous disease at our institution from 2007 until 2019 were identified and divided into groups based on gender. Operative notes, venograms, and the electronic health record were then queried to obtain operative details, co-morbid conditions, postoperative outcomes and stent patency. Study outcome was long term patency rate. The data was analyzed using chi-square, logistic regression, and Kaplan-Meier analysis as appropriate. RESULTS: A total of 200 consecutive patients (231 limbs) were identified in our retrospective analysis, with a mean age of 48.8 ± 17.3, and BMI of 31.6 ± 8.6. Of those, 119 (59.5%) patients, (131 [56.8%] limbs) were female. Comparisons between the gender groups revealed no difference in age, BMI, or preoperative comorbidities. There was no difference in type of venous disease between male (85% thrombotic, 15% nonthrombotic) and female (84% thrombotic, 16% nonthrombotic), P= 0.830. The male cohort was more likely to present with leg ulceration (17% vs. 4.6%, P = 0.002), and the female cohort was more likely to present with leg edema (98.5% vs. 93.0%, P= 0.03). The male cohort had a higher rate of caval (48% vs. 33.6%, P= 0.027) and infrainguinal stent extension. (11% vs. 6.9%, P= 0.02). Females had a higher rate of left sided stenting (80.9% vs. 66/0%, P= 0.010). There was no difference in the median stent diameter used between the cohorts. Primary patency at 5 years was significantly higher for the male cohort (94.1% vs. 74.4%, P= 0.01) On adjusted multivariable cox regression female gender was a predictor of loss of primary patency within 5 years (HR, 4.04; P= 0.007). CONCLUSIONS: In this single center retrospective analysis of IVS, male patients were found to have better primary stent patency compared to female.

Glycine promotes longevity in Caenorhabditis elegans in a methionine cycle-dependent fashion.

The deregulation of metabolism is a hallmark of aging. As such, changes in the expression of metabolic genes and the profiles of amino acid levels are features associated with aging animals. We previously reported that the levels of most amino acids decline with age in Caenorhabditis elegans (C. elegans). Glycine, in contrast, substantially accumulates in aging C. elegans. In this study we show that this is coupled to a decrease in gene expression of enzymes important for glycine catabolism. We further show that supplementation of glycine significantly prolongs C. elegans lifespan, and early adulthood is important for its salutary effects. Moreover, supplementation of glycine ameliorates specific transcriptional changes that are associated with aging. Glycine feeds into the methionine cycle. We find that mutations in components of this cycle, methionine synthase (metr-1) and S-adenosylmethionine synthetase (sams-1), completely abrogate glycine-induced lifespan extension. Strikingly, the beneficial effects of glycine supplementation are conserved when we supplement with serine, which also feeds into the methionine cycle. RNA-sequencing reveals a similar transcriptional landscape in serine- and glycine-supplemented worms both demarked by widespread gene repression. Taken together, these data uncover a novel role of glycine in the deceleration of aging through its function in the methionine cycle.

Sitting less elicits metabolic responses similar to exercise and enhances insulin sensitivity in postmenopausal women.

AIMS/HYPOTHESIS: In our current society sedentary behaviour predominates in most people and is associated with the risk of developing type 2 diabetes. It has been suggested that replacing sitting time by standing and walking could be beneficial for individuals with type 2 diabetes but the underlying mechanisms are unknown and direct comparisons with exercise are lacking. Our objective was to directly compare metabolic responses of either sitting less or exercising, relative to being sedentary. METHODS: We performed a randomised, crossover intervention study in 12 overweight women who performed three well-controlled 4 day activity regimens: (1) sitting regimen (sitting 14 h/day); (2) exercise regimen (sitting 13 h/day, exercise 1 h/day); and (3) sitting less regimen (sitting 9 h/day, standing 4 h/day and walking 3 h/day). The primary outcome was insulin sensitivity measured by a two-step hyperinsulinaemic-euglycaemic clamp. We additionally performed metabolomics on muscle biopsies taken before the clamp to identify changes at the molecular level. RESULTS: Replacing sitting time by standing and walking over 4 days resulted in improved peripheral insulin sensitivity, comparable with the improvement achieved by moderate-to-vigorous exercise. Specifically, we report a significant improvement in peripheral insulin sensitivity in the sitting less (~13%) and the exercise regimen (~20%), compared with the sitting regimen. Furthermore, sitting less shifted the underlying muscle metabolome towards that seen with moderate-to-vigorous exercise, compared with the sitting regimen. CONCLUSIONS/INTERPRETATIONS: Replacing sitting time by standing and walking is an attractive alternative to moderate-to-vigorous exercise for improving metabolic health. TRIAL REGISTRATION: ClinicalTrials.gov NCT03912922.

Patterns of joint damage in severe haemophilia A treated with prophylaxis.

OBJECTIVE: The primary objective of this study was to assess whether there are different patterns (classes) of joint health in young boys with severe haemophilia A (SHA) prescribed primary tailored prophylaxis. We also assessed whether age at first index joint bleed, blood group, FVIII gene abnormality variant, factor VIII trough level, first-year bleeding rate and adherence to the prescribed prophylaxis regimen significantly predicted joint damage trajectory, and thus class membership. METHODS: Using data collected prospectively as part of the Canadian Hemophilia Primary Prophylaxis Study (CHPS), we implemented a latent class growth mixture model technique to determine how many joint damage classes existed within the cohort. We used a multinomial logistic regression to predict the odds of class membership based on the above predictors. We fitted a survival model to assess whether there were differences in the rate of dose escalation across the groups. RESULTS: We identified three distinct classes of trajectory: persistently low, moderately increasing and rapidly increasing joint scores. By multinomial regression, we found that only age at first index joint bleed predicted rapidly increasing joint scores. The rapidly increasing joint score class group moved through dose escalation significantly faster than the other two groups. CONCLUSIONS: Using tailored prophylaxis, boys with SHA follow one of three joint health trajectories. By using knowledge of disease trajectories, clinicians may be able to adjust treatment according to a subject's predicted long-term joint health and institute cost-effective programmes of prophylaxis targeted at the individual subject level.

An open label pilot study of the safety and tolerability of perampanel in amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Perampanel, a selective noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist, is capable of slowing the progression of the amyotrophic lateral sclerosis (ALS) phenotype and increasing the number of anterior horn cells in transgenic mice. Trials of perampanel in epilepsy showed a favorable tolerability profile. In this study we aimed to determine the tolerability and safety of perampanel in patients with ALS. METHODS: Enrolled subjects were started on 2 mg/day of perampanel and the dose was increased by 2 mg/day every week to a maximum dose of 8 mg/day. Our primary outcome measure was tolerability, which was evaluated by monitoring adverse events. The secondary outcome measure was clinical progression, assessed using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) and spirometry. RESULTS: Six participants were enrolled. All had adverse events, mostly behavioral. Two completed the trial and the other four withdrew due to adverse events. All participants reported resolution of these events after discontinuation of the drug. The trial was halted due to the large number of adverse events. DISCUSSION: The use of perampanel in this study of ALS was limited by its poor tolerability.

Utilization and Outcomes of Radial Artery Access for Lower Extremity Endovascular Intervention.

INTRODUCTION: Radial artery access has become popular for cardiac interventions, but its role in lower extremity interventions is not well defined. We aimed to describe current utilization and outcomes of transradial access for lower extremity interventions. METHODS: Peripheral vascular intervention (PVI) from 2016-2020 where transradial access was employed in the Vascular Quality Initiative (VQI) registry were studied. Cases before 2016 were excluded as documentation of transradial access was not possible in earlier years. PVIs involving radial artery access were evaluated with regard to access guidance, access-site complications, target vessels treated and the technical success of these interventions. RESULTS: Of 167,098 PVIs, 1,096 (0.66%) involved radial access. Utilization varied significantly by region (P < 0.01). The left radial artery was used in 66.9% of cases. Ultrasound-guided access was documented in 72.7% of cases. There were no significant differences in age, body mass index, or sex between the transradial group and other PVIs. In 450 procedures, a second access site was utilized, most commonly a retrograde femoral access (60.0%) or retrograde pedal access (16.7%). The largest sheath was 6-Fr in 78.0%. Interventions documenting radial-only access more commonly treated the aortoiliac segment (49.4% vs. 29.5%, P < 0.001) and less commonly treated the tibial segments (7.1% vs. 32.1%, P < 0.001). Technical success was 94.0%, with inability to cross the lesion (3.1%) and residual stenosis after treatment (2.2%) being most common. There were access-site complications in 2.9%, with hematoma (2.4%) being most common. DISCUSSION: Radial access is associated with high technical success rates and low access-site complication rates. Advances in device profile and shaft length may overcome shortcomings of transradial access and lead to further utilization of this access site.

Establishing a point-of-care additive manufacturing workflow for clinical use.

Additive manufacturing, or 3-Dimensional (3-D) Printing, is built with technology that utilizes layering techniques to build 3-D structures. Today, its use in medicine includes tissue and organ engineering, creation of prosthetics, the manufacturing of anatomical models for preoperative planning, education with high-fidelity simulations, and the production of surgical guides. Traditionally, these 3-D prints have been manufactured by commercial vendors. However, there are various limitations in the adaptability of these vendors to program-specific needs. Therefore, the implementation of a point-of-care in-house 3-D modeling and printing workflow that allows for customization of 3-D model production is desired. In this manuscript, we detail the process of additive manufacturing within the scope of medicine, focusing on the individual components to create a centralized in-house point-of-care manufacturing workflow. Finally, we highlight a myriad of clinical examples to demonstrate the impact that additive manufacturing brings to the field of medicine.

Extracellular Vesicles from Mesenchymal Stromal Cells for the Treatment of Inflammation-Related Conditions.

Over the past two decades, mesenchymal stromal cells (MSCs) have demonstrated great potential in the treatment of inflammation-related conditions. Numerous early stage clinical trials have suggested that this treatment strategy has potential to lead to significant improvements in clinical outcomes. While promising, there remain substantial regulatory hurdles, safety concerns, and logistical issues that need to be addressed before cell-based treatments can have widespread clinical impact. These drawbacks, along with research aimed at elucidating the mechanisms by which MSCs exert their therapeutic effects, have inspired the development of extracellular vesicles (EVs) as anti-inflammatory therapeutic agents. The use of MSC-derived EVs for treating inflammation-related conditions has shown therapeutic potential in both in vitro and small animal studies. This review will explore the current research landscape pertaining to the use of MSC-derived EVs as anti-inflammatory and pro-regenerative agents in a range of inflammation-related conditions: osteoarthritis, rheumatoid arthritis, Alzheimer's disease, cardiovascular disease, and preeclampsia. Along with this, the mechanisms by which MSC-derived EVs exert their beneficial effects on the damaged or degenerative tissues will be reviewed, giving insight into their therapeutic potential. Challenges and future perspectives on the use of MSC-derived EVs for the treatment of inflammation-related conditions will be discussed.

Basic insights into Zika virus infection of neuroglial and brain endothelial cells.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain-Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood-brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml-1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Nationwide trends in drug-coated balloon and drug-eluting stent utilization in the femoropopliteal arteries.

OBJECTIVE: Drug-coated balloons (DCB) and drug-eluting stents (DES) have significantly altered treatment paradigms for femoropopliteal lesions. We aimed to describe changes in practice patterns as a result of the infusion of these technologies into the treatment of peripheral arterial disease. METHODS: We queried the Vascular Quality Initiative registry from 2010 to 2017 for all peripheral vascular interventions involving the superficial femoral artery and/or the popliteal artery. Cases were divided into a PRE and a POST era with a cutoff of September 2016, when specific device identity was first recorded in Vascular Quality Initiative. For each artery, a primary treatment was identified as either plain balloon angioplasty, atherectomy, DCB, bare-metal stent, or DES. The relative distribution of primary treatments between the PRE and POST eras was evaluated, as were lesion characteristics associated with DCB and DES use and regional variability in the adoption of these new technologies. RESULTS: Of 210,666 arteries in the dataset, 91,864 femoropopliteal arteries (across 74,842 procedures in 55,437 patients) were included. Each artery received 1.5 ± 0.6 treatments. Primary treatment use changed from 40% balloon angioplasty, 45% stenting, and 15% atherectomy in the PRE era to 22% plain balloon angioplasty, 26% bare-metal stent, 8% atherectomy, 37% DCB, and 8% DES in the POST era (P < .001). Forty-three percent of arteries received a drug-containing device as a primary or adjunctive therapy and 1.3% received both a DCB and DES in the POST era. DCB use as the primary treatment was highest in lesions with length 10.0 to 19.9 cm (42%), TransAtlantic InterSociety A, B, or C lesions (38%), and lesions with mild to no calcification (38%). DES use was highest in lesions with a length of 20 cm or more (12%), TransAtlantic InterSociety D lesions (13%), and lesions with moderate to severe calcification (9%). The range of use across 18 regions was 125 to 40% for DCB and 1% to 14% for DES. Regional variability was greater for DES (SD 4% vs mean 8%) than for DCB (SD 7% vs mean 29%). CONCLUSIONS: There has been a rapid dissemination of DCB and DES technology in the femoropopliteal vessels, with nearly one-half of arteries receiving a drug-containing therapy in modern practice. DCBs are most used in medium length, minimally calcified lesions and DESs are most used in longer, more heavily calcified lesions. There is significant regional variability in adoption, especially with DES.

Identification of longevity compounds with minimized probabilities of side effects.

It is hypothesized that treating the general aging population with compounds that slow aging, geroprotectors, could provide many benefits to society, including a reduction of age-related diseases. It is intuitive that such compounds should cause minimal side effects, since they would be distributed to otherwise healthy individuals for extended periods of time. The question therefore emerges of how we should prioritize geroprotectors discovered in model organisms for clinical testing in humans. In other words, which compounds are least likely to cause harm, while still potentially providing benefit? To systematically answer this question we queried the DrugAge database-containing hundreds of known geroprotectors-and cross-referenced this with a recently published repository of compound side effect predictions. In total, 124 geroprotectors were associated to 800 unique side effects. Geroprotectors with high risks of side effects, some even with risk for death, included lamotrigine and minocycline, while compounds with low side effect risks included spermidine and D-glucosamine. Despite their popularity as top geroprotector candidates for humans, sirolimus and metformin harbored greater risks of side effects than many other candidate geroprotectors, sirolimus being the more severe of the two. Furthermore, we found that a correlation existed between maximum lifespan extension in worms and the likelihood of causing a side effect, suggesting that extreme lifespan extension in model organisms should not necessarily be the priority when screening for novel geroprotectors. We discuss the implications of our findings for prioritizing geroprotectors, suggesting spermidine and D-glucosamine for clinical trials in humans.

Extracellular vesicles and microvascular pathology: Decoding the active dialogue.

Extracellular vesicles (EV) are a heterogeneous collection of membrane-surrounded structures released from all studied cells, under both physiological and pathological conditions. These nano-size vesicles carry complex cargoes including different classes of proteins, lipids and nucleic acids and are known to act as a communication and signalling vesicles in various cellular process. In addition to their role in development and progression of pathological disorders which make them potentially great biomarkers, EV have beneficial effects, as they take part in homeostasis. In this review we have analysed the evidence for the role of microvesicles and exosomes secreted from other cells on microvascular endothelium (EV uptake) as well as the role of endothelial-derived vesicles on their neighbouring and distant cells (EV release).

The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis.

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C. neoformans was present in the perivascular space (PVS) of post-capillary venules. This was associated with a massive influx of blood-derived monocytes, neutrophils, and T lymphocytes into the PVS and a predominantly proinflammatory cytokine/chemokine response. Phagocytes containing cryptococci were present only in the lumen and corresponding PVS of post-capillary venules. Free cryptococci were observed breaching the glia limitans, the protective barrier between the PVS and the cerebral parenchyma. Parenchymal cryptococcomas were typically in direct contact with post-capillary venules and lacked surrounding immune cell infiltrates. Phagocyte depletion abrogated cryptococcoma formation and PVS infiltrates. Together, these observations suggest that cryptococcomas can originate via phagocyte-dependent transport across post-capillary venular endothelium into the PVS and thence via passage of free cryptococci into the brain. In conclusion, we demonstrate for the first time that the PVS of cortical post-capillary venules is the major site of the early innate immune response to, and phagocyte-dependent entry of, C. neoformans.