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The contribution of the novel biomarkers, hepatitis B virus (HBV) RNA and HBV core-related antigen (HBcrAg), to characterization of HBV-human immunodeficiency virus (HIV) coinfection is unclear. We evaluated the longitudinal dynamics of HBV RNA and HBcrAg and their association with classical HBV serum biomarkers and liver histology and viral staining.HBV-HIV co-infected adults from 8 North American centers entered a National Institutes of Health-funded prospective cohort study. Demographic, clinical, serological, and virological data were collected at entry and every 24 to 48 weeks for up to 192 weeks. Participants with HBV RNA and HBcrAg measured ≥2 times (N = 95) were evaluated; 56 had paired liver biopsies obtained at study entry and end of follow-up.Participants had a median age of 50 years; 97% were on combination anti-viral therapy. In hepatitis B e antigen (HBeAg)+ participants, there were significant declines in HBV RNA and HBcrAg over 192 weeks that tracked with declines in HBeAg, hepatitis B surface antigen, HBV DNA, and hepatitis B core antigen (HBcAg) hepatocyte staining grade (all P.05). In HBeAg- participants, there were not significant declines in HBV RNA (P = .49) and HBcrAg (P = .63), despite modest reductions in hepatitis B surface antigen (P.01) and HBV DNA (P = .03). HBV serum biomarkers were not significantly related to change in hepatic activity index, Ishak fibrosis score, or hepatocyte HBcAg loss (all P.05).In HBV-HIV coinfected adults on suppressive dually active antiviral therapy, the use of novel HBV markers reveals continued improvement in suppression of HBV transcription and translation over time. The lack of further improvement in HBV serum biomarkers among HBeAg- patients suggests limits to the benefit of combination anti-viral therapy and provide rationale for additional agents with distinct mechanisms of action.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study bydeconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of theexRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of ten exRNA isolation methods across five biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNAseq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle- (EV), ribonucleoprotein-(RNP)-, and high-density lipoprotein- (HDL) specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method, and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

Background and Purpose: Diabetes is recognized to enhance the frequency and severity of atherosclerosis and cardiovascular disease. Recent studies have shown that hyperglycemia is associated with enhanced hematopoiesis and macrophage accumulation in atherosclerotic lesions. We explored whether high glucose concentrations can enhance intercellular communication between mature macrophages and hematopoietic progenitors via exosomes to promote inflammation and diabetic atherosclerosis. Methods: Bone marrow derived macrophages (BMDM) from C57BL/6 mice were cultured with normal (5mM) or high glucose concentrations (25mM). Exosomes were isolated with our cushioned-density gradient ultracentrifugation method followed by nanoparticle tracking and western blot analysis. Pro-inflammatory properties of high glucose exosomes (HGexo) were tested in vitro by exposing them to BMDM cultured in normal low glucose. The capacity for BMDM-derived exosomes to alter systemic and vascular inflammation were next tested by infusing 25-30 weeks-old ApoE -/- mice fed a chow diet with 3 x 10 10 exosomes three times a week, for four weeks. Results: Our data show that HGexo can stimulate the expression of inflammatory cytokines (IL-6, IL-1ß) as well as NADPH oxidases (Nox-1 and Nox-4) in cultured BMDM. Furthermore, our findings show that intraperitoneally injected exosomes distribute to numerous organs and tissues including the bone marrow and the spleen. Lastly, HGexo enhance the expansion of multipotent and lineage committed hematopoietic progenitors. Conclusions: We identify that exosomes derived from cultured BMDM exposed to high glucose have the capacity to exert intercellular communication in vitro , and in vivo. Our findings suggest that exosomes produced by macrophages exposed to hyperglycemia could represent an unsuspected source of inflammation to accelerate atherosclerosis in diabetes.

Background and Purpose: Ultracentrifugation (UC) is recognized as a robust approach for the isolation of exosomes especially when combined with a second step that involves density gradient ultracentrifugation (DGUC). However, recent studies have highlighted limitations associated with the use of UC including low recovery efficiencies and possible aggregation of exosomes. Such effects could subsequently impact on downstream assessments of exosome function in biological systems. Methods: We tested the benefit of using a liquid cushion of iodixanol during the first UC step to improve the yield of exosomes that are concentrated from the conditioned media (CM) of J774.1 murine macrophages in a method we recently termed Cushioned(C)-DGUC. We also compared the yield and purity of exosomes isolated by C-DGUC with those isolated by first subjecting CM to two other forms of concentration that included: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation prior to DGUC. Results: Our data show that the concentration step largely determines the yield and purity of exosomes isolated following the second DGUC step. The use of a high-density iodixanol cushion in cushioned-UC (C-UC) led to a threefold improvement in exosome yield over conventional UC. Although subjecting the CM to UF resulted in a similar exosome recovery efficiency, it retained eight-fold more soluble proteins than C-UC method. Strikingly, PEG precipitation of the CM generated a substantial number of non-exosomal nanoparticles, which could not be efficiently eliminated by the DGUC step. Western blot analysis reproducibly detected exosome markers CD-81, TSG101 and Alix in fractions 6 and 7. Finally, an in vitro assay of exosome-mediated microRNA delivery confirmed that C-DGUC provided the highest yield of functional exosomes. Conclusions: Collectively, our data demonstrate that the use of a high-density liquid cushion of iodixanol during the concentration step of C-DGUC substantially improves the yield and purity of exosomes derived from cell culture media. This approach can therefore facilitate functional studies of cell-derived exosomes in atherosclerosis.

Background and Purpose: Apolipoprotein (apo) E suppresses atherosclerosis by exerting a profound control over cells of the innate immune system including monocytes and macrophages. In mice with hyperlipidemia, apoE also prevents an exaggerated proliferation of hematopoietic stem and progenitor cells (HSPC) to further limit the expansion of innate immune cells. However, apoE’s control over hematopoiesis and its subsequent shaping of adaptive immunity remains unexplored. Methods: We explored this question by studying HypoE mice deficient in the LDL receptor ( Apoe h/h Ldlr –/– mice) that display plasma lipid levels similar to those of Apoe -/- Ldlr -/- mice when fed a chow diet, but accumulate apoE in plasma and display reduced atherosclerosis. Populations of HSPC were examined in the bone marrow (BM) and spleen, while populations of mature immune cells and their levels of cellular activation were examined in the blood, spleen, lymph nodes and aorta of twenty-week-old mice. Results: Our findings show that apoE suppressed the expansion of HSPC in both the spleen and BM of Apoe h/h Ldlr -/- mice, which remained similar to levels seen in normal wildtype mice. Specifically, in the spleens of Apoe h/h Ldlr -/- mice, apoE reduced the expansion of a population of multi-potent progenitors termed MPP4 responsible for lineages of lymphoid cells. Accordingly, Apoe h/h Ldlr -/- mice displayed smaller spleens and lymph nodes that contained fewer myeloid- and lymphoid-derived leukocytes compared to Apoe -/- Ldlr -/- mice. The spleens and lymph nodes of Apoe h/h Ldlr -/- mice had a higher percentage of T cells that displayed a naïve phenotype, while the percentage of effector T cells was decreased and produced 50% less IFN-γ. Dendritic cells isolated from spleens of Apoe h/h Ldlr -/- mice showed reduced levels of CD86 that plays a key role in T cell activation. Digested aortas of Apoe h/h Ldlr -/- mice revealed a decrease in both T cell and myeloid cell number that also displayed a lower percentage of the pro-inflammatory CD11b + subtype but a higher percentage of the anti-inflammatory CD103 + subtype. Conclusion: Collectively, our findings show that the control of hematopoiesis exerted by apoE results in reduced adaptive immunity to suppress atherosclerosis in hyperlipidemic mice.

AIMS:We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(–/–) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(–/–) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS:HypoE/SR-BI(–/–) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS:Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

Plasma lipoproteins are essential vehicles of lipid distribution for cellular energy and structural requirements as well as for excretion of lipid excess. Imbalances in lipoprotein metabolism are known to contribute to metabolic diseases ranging from vascular inflammation and atherosclerosis to obesity and diabetes. The lipid and protein cargo carried by lipoprotein subclasses have long been the focus of studies exploring the contribution of plasma lipoproteins in health and in metabolic disorders. More recent studies have revealed the presence of non-coding RNA as a new form of cargo carried by plasma lipoproteins. Lipoprotein-associated microRNAs have been identified to distribute differentially among plasma lipoprotein subclasses and contribute to cellular signaling. These findings highlight plasma lipoprotein-associated RNA as a potential source of biological signaling and warrants a renewed interest in the study of plasma lipoprotein biology. This chapter describes principles and methods based on density ultracentrifugation and size exclusion chromatography for the isolation of plasma lipoproteins as a source of extracellular RNA.

Exosomes represent one class of extracellular vesicles that are thought to be shed by all cell types. Although the exact nature of exosome biogenesis and function remains incompletely understood, they are increasingly recognized as a source of intercellular communication in health and disease. Recent observations of RNA exchange via donor cell-derived exosomes that exert genetic regulation in recipient cells have led to a boon into exosome research. The excitement and promise of exosomes as a new therapeutic avenue for human pathologies remain limited by challenges associated with their isolation from culture media and bio-fluids. The introduction of new methodologies to facilitate the isolation of exosomes has simultaneously raised concerns related to the reproducibility of studies describing exosome effector functions. Even high speed ultracentrifugation, the first and long considered gold standard approach for exosome isolation has recently been noted to be subject to uncontrolled variables that could impact functional readouts of exosome preparations. This chapter describes principles and methods that attempt to overcome such limitations by first concentrating exosomes in a liquid cushion and subsequently resolving them using density gradient ultracentrifugation. Our approach avoids possible complications associated with direct pelleting onto plastic tubes and allows for further purification of exosomes from dense protein aggregates.

Background. Critically ill, extremely premature infants develop anemia because of intensive laboratory blood testing and undergo multiple red blood cell (RBC) transfusions in the early weeks of life. To date, researchers have had only limited success in finding ways to reduce transfusions significantly in this patient population.Objective. To reduce RBC transfusions for these infants by using a point-of-care bedside monitor that returns analyzed blood to the patient.Design, Setting, and Patients. This was a prospective, 2-center, randomized, open, controlled, clinical trial with a 1:1 assignment of extremely low birth weight infants (weighing 500–1000 g at birth) to control or monitor groups and analysis with the intention-to-treat approach. Predefined RBC transfusion criteria were applied uniformly in the 2 groups.Interventions. Clinical treatment of study subjects with an in-line, ex vivo, bedside monitor that withdraws blood through an umbilical artery catheter, analyzes blood gases and sodium, potassium, and hematocrit levels, and returns the sample to the patient.Main Outcome Measures. The total volume and number of RBC transfusions during the first 2 weeks of life and the total volume of blood removed for laboratory testing.Results. The trial was terminated prematurely when one center's NICU changed its standard method of laboratory testing. In the first 2 weeks of life, there was a nonsignificant 17% lower cumulative RBC transfusion volume in the monitor group (n = 46), compared with the control group (n = 47). However, data from the first week only (the period of greater catheter use) demonstrated a significant 33% lower cumulative RBC transfusion volume in the monitor group. Cumulative phlebotomy loss was ∼25% less in the monitor group throughout the 2-week study period. There was no difference between groups in neonatal mortality, morbidity, and neurodevelopmental outcome rates at 18 to 24 months. This is the first randomized trial documenting that RBC transfusions administered to neonates can by reduced by decreasing laboratory phlebotomy loss.Conclusions. As long as an umbilical artery catheter is available for blood sampling with an in-line blood gas and chemistry monitor, significant reductions in neonatal RBC transfusions can be achieved. The patients most likely to benefit from monitor use are the smallest, most critically ill newborns.

Background & Aims T cells play a critical role in viral infection. We examined whether T-cell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). Methods We enrolled 200 adults with CHB who participated in the National Institutes of Health−supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon gamma and interleukin 10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1, and cytotoxic T lymphocyte-associated antigen-4 was examined by flow cytometry. Immune measures were compared with clinical parameters, including physician-defined immune-active, immune-tolerant, or inactive CHB phenotypes, in a blinded fashion. Results Compared with controls, patients with CHB had weak T-cell proliferative, interferon gamma, and interleukin 10 responses to HBV, with increased frequency of circulating FOXP3 + CD127 − regulatory T cells and CD4 + T-cell expression of programmed death-1 and cytotoxic T lymphocyte-associated antigen-4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in hepatitis B e antigen (HBeAg) + than HBeAg − patients (percent responders: 3% vs 23%; P = .00008). Although in vitro blockade of programmed death-1 or cytotoxic T lymphocyte-associated antigen-4 increased T-cell responses to HBV, the effect was weaker in HBeAg + than HBeAg − patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls. Conclusions HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms, including circulating HBeAg, but without distinct T-cell−based immune signatures for clinical phenotypes. These findings suggest additional T-cell−independent or regulatory mechanisms of CHB pathogenesis that warrant further investigation.

In a previous report, it was shown that a dimension of personality entitled dependence/independence could be used to distinguish the cardiovascular and catecholaminergic response to stress in type A individuals. This study is a continuation of the previous work. It examines the relative effects of beta-blockade on stress-induced changes in heart rate, blood pressure, and catecholamines in two groups of type A individuals who differ in reactivity. Sixty-six healthy, middle-aged men with type A behavior were classified as dependent (high reactivity) or independent (low reactivity). The effects of beta-blockade on the two groups were tested in a randomized, double-blind, crossover trial utilizing a mental stressor, a physical stressor, and work day monitoring. Results showed that, in comparison with the independent type As, those who had been classified as dependent had higher heart rates in all three stress conditions and had higher epinephrine levels during both the mental and physical stressors. For both groups, beta-blockade lowered the heart rate and systolic blood pressure but increased norepinephrine with the mental stressor and epinephrine and norepinephrine with the physical stressor. It also reduced the mean work day heart rate. For the dependent type As, the drug had a greater effect on increasing epinephrine during the physical stressor and by reducing the percent of the work day spent in high heart rate zones. The results are discussed from the point of view of identifying type As who may be particularly coronary prone and the potential effects of beta-blockade on the reactivity associated with type A behavior.