Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Inhibiting tau-induced elevated nSMase2 activity and ceramides is therapeutic in an Alzheimer's disease mouse model.

BACKGROUND: Cognitive decline in Alzheimer's disease (AD) is associated with hyperphosphorylated tau (pTau) propagation between neurons along synaptically connected networks, in part via extracellular vesicles (EVs). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2 (nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that human tau expression elevates brain ceramides and nSMase2 activity. METHODS: To determine the therapeutic benefit of inhibiting this elevation, we evaluated PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor in the transgenic PS19 AD mouse model. Additionally, we directly evaluated the effect of PDDC on tau propagation in a mouse model where an adeno-associated virus (AAV) encoding P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus. The contralateral transfer of the double mutant human tau to the dentate gyrus was monitored. We examined ceramide levels, histopathological changes, and pTau content within EVs isolated from the mouse plasma. RESULTS: Similar to human AD, the PS19 mice exhibited increased brain ceramide levels and nSMase2 activity; both were completely normalized by PDDC treatment. The PS19 mice also exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all of which were pathologic features of human AD. PDDC treatment reduced these changes. The plasma of PDDC-treated PS19 mice had reduced levels of neuronal- and microglial-derived EVs, the former carrying lower pTau levels, compared to untreated mice. In the tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly reduced the amount of tau propagation to the contralateral side. CONCLUSIONS: PDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity, leading to the slowing of tau spread in AD mice.

Inhibition of neutral sphingomyelinase 2 impairs HIV-1 envelope formation and substantially delays or eliminates viral rebound.

Although HIV-1 Gag is known to drive viral assembly and budding, the precise mechanisms by which the lipid composition of the plasma membrane is remodeled during assembly are incompletely understood. Here, we provide evidence that the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) interacts with HIV-1 Gag and through the hydrolysis of sphingomyelin creates ceramide that is necessary for proper formation of the viral envelope and viral maturation. Inhibition or depletion of nSMase2 resulted in the production of noninfectious HIV-1 virions with incomplete Gag lattices lacking condensed conical cores. Inhibition of nSMase2 in HIV-1-infected humanized mouse models with a potent and selective inhibitor of nSMase2 termed PDDC [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] produced a linear reduction in levels of HIV-1 in plasma. If undetectable plasma levels of HIV-1 were achieved with PDDC treatment, viral rebound did not occur for up to 4 wk when PDDC was discontinued. In vivo and tissue culture results suggest that PDDC selectively kills cells with actively replicating HIV-1. Collectively, this work demonstrates that nSMase2 is a critical regulator of HIV-1 replication and suggests that nSMase2 could be an important therapeutic target with the potential to kill HIV-1-infected cells.

Neutral sphingomyelinase 2 is required for HIV-1 maturation.

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Inhibiting tau-induced elevated nSMase2 activity and ceramides is therapeutic in murine Alzheimer's disease.

Background: Cognitive decline in Alzheimer's disease (AD) is associated with prion-like tau propagation between neurons along synaptically connected networks, in part via extracellular vesicles (EV). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2(nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that tau expression triggers an elevation in brain ceramides and nSMase2 activity. Methods: To determine the therapeutic benefit of inhibiting this elevation, we evaluated the efficacy of PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor, in the PS19 tau transgenic AD murine model. Changes in brain ceramide and sphingomyelin levels, Tau content, histopathology, and nSMase2 target engagement were monitored, as well as changes in the number of brain-derived EVs in plasma and their Tau content. Additionally, we evaluated the ability of PDDC to impede tau propagation in a murine model where an adeno-associated virus(AAV) encoding for P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus and the contralateral transfer to the dentate gyrus was monitored. Results: Similar to human AD, PS19 mice exhibited increased brain ceramides and nSMase2 activity; both were completely normalized by PDDC treatment. PS19 mice exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all pathologic features of human AD. PDDC treatment significantly attenuated these aberrant changes. Mouse plasma isolated from PDDC-treated PS19 mice exhibited reduced levels of neuron- and microglia-derived EVs, the former carrying lower phosphorylated Tau(pTau) levels, compared to untreated mice. In the AAV tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly decreased tau spreading to the contralateral side. Conclusions: PDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity leading to the slowing of tau spread in AD mice.

Neuronal deletion of nSMase2 reduces the production of Aß and directly protects neurons.

Extracellular vesicles (EVs) have been proposed to regulate the deposition of Aß. Multiple publications have shown that APP, amyloid processing enzymes and Aß peptides are associated with EVs. However, very little Aß is associated with EVs compared with the total amount Aß present in human plasma, CSF, or supernatants from cultured neurons. The involvement of EVs has largely been inferred by pharmacological inhibition or whole body deletion of the sphingomyelin hydrolase neutral sphingomyelinase-2 (nSMase2) that is a key regulator for the biogenesis of at-least one population of EVs. Here we used a Cre-Lox system to selectively delete nSMase2 from pyramidal neurons in APP/PS1 mice (APP/PS1-SMPD3-Nex1) and found a âˆ¼ 70% reduction in Aß deposition at 6 months of age and âˆ¼ 35% reduction at 12 months of age in both cortex and hippocampus. Brain ceramides were increased in APP/PS1 compared with Wt mice, but were similar to Wt in APP/PS1-SMPD3-Nex1 mice suggesting that elevated brain ceramides in this model involves neuronally expressed nSMase2. Reduced levels of PSD95 and deficits of long-term potentiation in APP/PS1 mice were normalized in APP/PS1-SMPD3-Nex1 mice. In contrast, elevated levels of IL-1ß, IL-8 and TNFα in APP/PS1 mice were not normalized in APP/PS1-SMPD3-Nex1 mice compared with APP/PS1 mice. Mechanistic studies showed that the size of liquid ordered membrane microdomains was increased in APP/PS1 mice, as were the amounts of APP and BACE1 localized to these microdomains. Pharmacological inhibition of nSMase2 activity with PDDC reduced the size of the liquid ordered membrane microdomains, reduced the localization of APP with BACE1 and reduced the production of Aß1-40 and Aß1-42. Although inhibition of nSMase2 reduced the release and increased the size of EVs, very little Aß was associated with EVs in all conditions tested. We also found that nSMase2 directly protected neurons from the toxic effects of oligomerized Aß and preserved neural network connectivity despite considerable Aß deposition. These data demonstrate that nSMase2 plays a role in the production of Aß by stabilizing the interaction of APP with BACE1 in liquid ordered membrane microdomains, and directly protects neurons from the toxic effects of Aß. The effects of inhibiting nSMase2 on EV biogenesis may be independent from effects on Aß production and neuronal protection.

Dendrimer-Conjugated nSMase2 Inhibitor Reduces Tau Propagation in Mice.

Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid-ß and hyperphosphorylated tau (pTau), which can spread throughout the brain via extracellular vesicles (EVs). Membrane ceramide enrichment regulated by the enzyme neutral sphingomyelinase 2 (nSMase2) is a critical component of at least one EV biogenesis pathway. Our group recently identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), the most potent (30 nM) and selective inhibitor of nSMase2 reported to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To enhance its PK properties, we conjugated DPTIP to a hydroxyl-PAMAM dendrimer delivery system, creating dendrimer-DPTIP (D-DPTIP). In an acute brain injury model, orally administered D-DPTIP significantly reduced the intra-striatal IL-1ß-induced increase in plasma EVs up to 72 h post-dose, while oral DPTIP had a limited effect. In a mouse tau propagation model, where a mutant hTau (P301L/S320F) containing adeno-associated virus was unilaterally seeded into the hippocampus, oral D-DPTIP (dosed 3× weekly) significantly inhibited brain nSMase2 activity and blocked the spread of pTau to the contralateral hippocampus. These data demonstrate that dendrimer conjugation of DPTIP improves its PK properties, resulting in significant inhibition of EV propagation of pTau in mice. Dendrimer-based delivery of DPTIP has the potential to be an exciting new therapeutic for AD.

Oxysterol misbalance critically contributes to Wilson disease pathogenesis.

Wilson disease (WD) is a metabolic disorder caused by inactivation of the copper-transporting ATPase 2 (ATP7B) and copper (Cu) overload in tissues. Excess Cu causes oxidative stress and pathologic changes with poorly understood mechanistic connections. In Atp7b-/- mice with established liver disease, Cu overload activates the stress-sensitive transcription factor Nrf2 (nuclear factor erythroid-derived 2-like 2). Nrf2 targets, especially sulfotransferase 1e1 (Sult1e1), are strongly induced and cause elevation of sulfated sterols, whereas oxysterols are decreased. This sterol misbalance results in inhibition of the liver X receptor (LXR) and up-regulation of LXR targets associated with inflammatory responses. Pharmacological inhibition of Sult1e1 partially reverses oxysterol misbalance and LXR inhibition. Contribution of this pathway to advanced hepatic WD was demonstrated by treating mice with an LXR agonist. Treatment decreased inflammation by reducing expression of proinflammatory molecules, diminished fibrosis by down-regulating the noncanonical transforming growth factor-ß signaling pathway, and improved liver morphology and function. Thus, the identified pathway is an important driver of WD.

Immunometabolic Reprogramming in Response to HIV Infection Is Not Fully Normalized by Suppressive Antiretroviral Therapy.

BACKGROUND: HIV infection results in immunometabolic reprogramming. While we are beginning to understand how this metabolic reprogramming regulates the immune response to HIV infection, we do not currently understand the impact of ART on immunometabolism in people with HIV (PWH). METHODS: Serum obtained from HIV-infected (n = 278) and geographically matched HIV seronegative control subjects (n = 300) from Rakai Uganda were used in this study. Serum was obtained before and ~2 years following the initiation of ART from HIV-infected individuals. We conducted metabolomics profiling of the serum and focused our analysis on metabolic substrates and pathways assocaited with immunometabolism. RESULTS: HIV infection was associated with metabolic adaptations that implicated hyperactive glycolysis, enhanced formation of lactate, increased activity of the pentose phosphate pathway (PPP), decreased ß-oxidation of long-chain fatty acids, increased utilization of medium-chain fatty acids, and enhanced amino acid catabolism. Following ART, serum levels of ketone bodies, carnitine, and amino acid metabolism were normalized, however glycolysis, PPP, lactate production, and ß-oxidation of long-chain fatty acids remained abnormal. CONCLUSION: Our findings suggest that HIV infection is associated with an increased immunometabolic demand that is satisfied through the utilization of alternative energetic substrates, including fatty acids and amino acids. ART alone was insufficient to completely restore this metabolic reprogramming to HIV infection, suggesting that a sustained impairment of immunometabolism may contribute to chronic immune activation and comorbid conditions in virally suppressed PWH.

Neutral sphingomyelinase 2 inhibition attenuates extracellular vesicle release and improves neurobehavioral deficits in murine HIV.

People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PLH, CI, and MDD. Here we evaluated EcoHIV-infected mice for behavioral abnormalities relevant to depression and cognition deficits, and assessed the behavioral and biochemical effects of nSMase2 inhibition. Mice were infected with EcoHIV and daily treatment with either vehicle or the nSMase2 inhibitor (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) began 3 weeks post-infection. After 2 weeks of treatment, mice were subjected to behavior tests. EcoHIV-infected mice exhibited behavioral abnormalities relevant to MDD and CI that were reversed by PDDC treatment. EcoHIV infection significantly increased cortical brain nSMase2 activity, resulting in trend changes in sphingomyelin and ceramide levels that were normalized by PDDC treatment. EcoHIV-infected mice also exhibited increased levels of brain-derived EVs and altered microRNA cargo, including miR-183-5p, miR-200c-3p, miR-200b-3p, and miR-429-3p, known to be associated with MDD and CI; all were normalized by PDDC. In conclusion, inhibition of nSMase2 represents a possible new therapeutic strategy for the treatment of HIV-associated CI and MDD.

Fibroblast growth factor-21 improves insulin action in nonlactating ewes.

During metabolically demanding physiological states, ruminants and other mammals coordinate nutrient use among tissues by varying the set point of insulin action. This set point is regulated in part by metabolic hormones with some antagonizing (e.g., growth hormone and TNFα) and others potentiating (e.g., adiponectin) insulin action. Fibroblast growth factor-21 (FGF21) was recently identified as a sensitizing hormone in rodent and primate models of defective insulin action. FGF21 administration, however, failed to improve insulin action in dairy cows during the naturally occurring insulin resistance of lactation, raising the possibility that ruminants as a class of animals or lactation as a physiological state are unresponsive to FGF21. To start addressing this question, we asked whether FGF21 could improve insulin action in nonlactating ewes. Gene expression studies showed that the ovine FGF21 system resembles that of other species, with liver as the major site of FGF21 expression and adipose tissue as a target tissue based on high expression of the FGF21 receptor complex and activation of p44/42 extracellular signal-regulated kinase (ERK1/2) following exogenous FGF21 administration. FGF21 treatment for 13 days reduced plasma glucose and insulin over the entire treatment period and improved glucose disposal during a glucose tolerance test. FGF21 increased plasma adiponectin by day 3 of treatment but had no effect on the plasma concentrations of total, C16:0-, or C18:0-ceramide. Overall, these data confirm that the insulin-sensitizing effects of FGF21 are conserved in ruminants and raise the possibility that lactation is an FGF21-resistant state.

Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1ß injection and two hours later plasma and brain were collected. IL-1ß injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1ß-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1ß or PDDC. IL-1ß injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1ß-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

Effects of serine palmitoyltransferase inhibition by myriocin in ad libitum-fed and nutrient-restricted ewes.

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, 12 non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from day 1 to 14 and were nutrient-restricted (straw only) from day 15 to 17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on day 17. Plasma was analyzed for free fatty acids (FFAs), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by day 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in the liver and omental and mesenteric adipose, VLC ceramide and DHCer in the pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.

Characterization of the Plasma Lipidome in Dairy Cattle Transitioning from Gestation to Lactation: Identifying Novel Biomarkers of Metabolic Impairment.

The discovery of novel biomarkers for peripartal diseases in dairy cows can improve our understanding of normal and dysfunctional metabolism, and lead to nutritional interventions that improve health and milk production. Our objectives were to characterize the plasma lipidome and identify metabolites associated with common markers of metabolic disease in peripartal dairy cattle. Multiparous Holstein cows (n = 27) were enrolled 30 d prior to expected parturition. Blood and liver samples were routinely collected through to d 14 postpartum. Untargeted lipidomics was performed using quadrupole time-of-flight mass spectrometry. Based on postpartum measures, cows were categorized into low or high total fatty acid area under the curve (total FAAUC; d 1-14 postpartum; 4915 ± 1369 vs. 12,501 ± 2761 (µmol/L × 14 d); n = 18), ß-hydroxybutyrate AUC (BHBAAUC; d 1-14 postpartum; 4583 ± 459 vs. 7901 ± 1206 (µmol/L × 14 d); n = 18), or liver lipid content (d 5 and 14 postpartum; 5 ± 1 vs. 12 ± 2% of wet weight; n = 18). Cows displayed decreases in plasma triacylglycerols and monoalkyl-diacylglycerols, and the majority of phospholipids reached a nadir at parturition. Phosphatidylcholines (PC) 32:3, 35:5, and 37:5 were specific for high total FAAUC, PC 31:3, 32:3, 35:5, and 37:5 were specific for high BHBAAUC, and PC 31:2, 31:3, and 32:3 were specific for high liver lipid content. PC 32:3 was specific for elevated total FA, BHBA, and liver lipid content. Lipidomics revealed a dynamic peripartal lipidome remodeling, and lipid markers associated with elevated total FA, BHBA, and liver lipid content. The effectiveness of nutrition to impact these lipid biomarkers for preventing excess lipolysis and fatty liver warrants evaluation.

Nipping disease in the bud: nSMase2 inhibitors as therapeutics in extracellular vesicle-mediated diseases.

Extracellular vesicles (EVs) are indispensable mediators of intercellular communication, but they can also assume a nefarious role by ferrying pathological cargo that contributes to neurological, oncological, inflammatory, and infectious diseases. The canonical pathway for generating EVs involves the endosomal sorting complexes required for transport (ESCRT) machinery, but an alternative pathway is induced by the enrichment of lipid membrane ceramides generated by neutral sphingomyelinase 2 (nSMase2). Inhibition of nSMase2 has become an attractive therapeutic strategy for inhibiting EV biogenesis, and a growing number of small-molecule nSMase2 inhibitors have shown promising therapeutic activity in preclinical disease models. This review outlines the function of EVs, their potential role in disease, the discovery and efficacy of nSMase2 inhibitors, and the path to translate these findings into therapeutics.

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Patterns and Predictors of Cognitive Function Among Virally Suppressed Women With HIV.

Cognitive impairment remains frequent and heterogeneous in presentation and severity among virally suppressed (VS) women with HIV (WWH). We identified cognitive profiles among 929 VS-WWH and 717 HIV-uninfected women from 11 Women's Interagency HIV Study sites at their first neuropsychological (NP) test battery completion comprised of: Hopkins Verbal Learning Test-Revised, Trail Making, Symbol Digit Modalities, Grooved Pegboard, Stroop, Letter/Animal Fluency, and Letter-Number Sequencing. Using 17 NP performance metrics (T-scores), we used Kohonen self-organizing maps to identify patterns of high-dimensional data by mapping participants to similar nodes based on T-scores and clustering those nodes. Among VS-WWH, nine clusters were identified (entropy = 0.990) with four having average T-scores ≥45 for all metrics and thus combined into an "unimpaired" profile (n = 311). Impaired profiles consisted of weaknesses in: (1) sequencing (Profile-1; n = 129), (2) speed (Profile-2; n = 144), (3) learning + recognition (Profile-3; n = 137), (4) learning + memory (Profile-4; n = 86), and (5) learning + processing speed + attention + executive function (Profile-5; n = 122). Sociodemographic, behavioral, and clinical variables differentiated profile membership using Random Forest models. The top 10 variables distinguishing the combined impaired vs. unimpaired profiles were: clinic site, age, education, race, illicit substance use, current and nadir CD4 count, duration of effective antiretrovirals, and protease inhibitor use. Additional variables differentiating each impaired from unimpaired profile included: depression, stress-symptoms, income (Profile-1); depression, employment (Profile 2); depression, integrase inhibitor (INSTI) use (Profile-3); employment, INSTI use, income, atazanavir use, non-ART medications with anticholinergic properties (Profile-4); and marijuana use (Profile-5). Findings highlight consideration of NP profile heterogeneity and potential modifiable factors contributing to impaired profiles.

Associations between Antiretrovirals and Cognitive Function in Women with HIV.

Cognitive complications persist in antiretroviral therapy(ART)-treated people with HIV. However, the pattern and severity of domain-specific cognitive performance is variable and may be exacerbated by ART-mediated neurotoxicity. 929 women with HIV(WWH) from the Women's Interagency HIV Study who were classified into subgroups based on sociodemographic and longitudinal behavioral and clinical data using semi-parametric latent class trajectory modelling. Five subgroups were comprised of: 1) well-controlled HIV with vascular comorbidities(n = 116); 2) profound HIV legacy effects(CD4 nadir <250 cells/µL; n = 275); 3) primarily <45 year olds with hepatitis C(n = 165); 4) primarily 35-55 year olds(n = 244), and 5) poorly-controlled HIV/substance use(n = 129). Within each subgroup, we fitted a constrained continuation ratio model via penalized maximum likelihood to examine adjusted associations between recent ART agents and cognition. Most drugs were not associated with cognition. However, among the few drugs, non-nucleoside reverse transcriptase inhibitor (NNRTIs) and protease inhibitors(PIs) were most commonly associated with cognition, followed by nucleoside reverse transcriptase inhibitors(NRTIs) and integrase inhibitors(IIs). Directionality of ART-cognition associations varied by subgroup. Better psychomotor speed and fluency were associated with ART for women with well-controlled HIV with vascular comorbidities. This pattern contrasts women with profound HIV legacy effects for whom poorer executive function and fluency were associated with ART. Motor function was associated with ART for younger WWH and primarily 35-55 year olds. Memory was associated with ART only for women with poorly-controlled HIV/substance abuse. Findings demonstrate interindividual variability in ART-cognition associations among WWH and highlight the importance of considering sociodemographic, clinical, and behavioral factors as an underlying contributors to cognition. Are antiretroviral agents a risk factor for cognitive complications in women with HIV? We examind associations between ART-agents and cognitive function among similar subgroups of women with HIV from the Women's Interagency HIV study. The patterns of associations depended on sociodemographic, clinical, and behavioral characteristics of women.

White Matter Injury Is Associated with Reduced Manual Dexterity and Elevated Serum Ceramides in Subjects with Cerebral Small Vessel Disease.

INTRODUCTION: We have demonstrated that asymptomatic cerebral small vessel disease (cSVD) measured by white matter hyperintensity volume is associated with reduced manipulative manual dexterity on the Grooved Peg Board Test (GPBT) in middle-aged healthy individuals with a family history of early coronary artery disease. In this current study, we aim to identify the association of subcortical white matter microstructural impairment measured by diffusion tensor imaging, manual dexterity measured by GPBT and circulating serums ceramide, another marker for white matter injury. We hypothesize that lower regional fractional anisotropy (rFA) is associated with worse performance on GPBT and elevated serum ceramides in the same study population. METHODS: rFA of 48 regions representing the subcortical white matters were analyzed in GeneSTAR participants in addition to serum ceramides and GPBT scores. Unadjusted univariable analyses with Bonferroni correction for multiple comparisons were completed using Spearman correlation for testing the associations between ceramides, rFA of subcortical white matter, and GPBT performance. Subsequently, sensitivity analyses were performed after excluding the participants that had any physical limitation that may influence their performance on GPBT. Finally, in the adjusted analysis using generalized estimating equation, linear regression models were performed for the areas that met significance threshold in the unadjusted analyses. RESULTS: 112 subjects (age [49 ± 11], 51% female, 39.3% African American) were included. Adjusted analyses for the significant correlations that met the Bonferroni correction threshold in the unadjusted univariable analyses identified significant negative associations between rFA of the right fornix (RF) and log-GPBT score (ß = -0.497, p = 0.037). In addition, rFA of RF negatively correlated with log serum ceramide levels (C18: ß = -0.03, p = 0.003, C20: ß = -0.0002, p = 0.004) and rFA of left genu of corpus callosum negatively correlated with log C18 level (ß = -0.0103, p = 0.027). CONCLUSIONS: These results demonstrate that subcortical microstructural white matter disruption is associated with elevated serum ceramides and reduced manual dexterity in a population with cSVD. These findings suggest that injury to white matter tracts undermines neural networks, with functional consequences in a middle-aged population with cardiovascular risk factors.