Microfluidic Isolation of Neuronal-Enriched Extracellular Vesicles Shows Distinct and Common Neurological Proteins in Long COVID, HIV Infection and Alzheimer's Disease.

Long COVID (LongC) is associated with a myriad of symptoms including cognitive impairment. We reported at the beginning of the COVID-19 pandemic that neuronal-enriched or L1CAM+ extracellular vesicles (nEVs) from people with LongC contained proteins associated with Alzheimer's disease (AD). Since that time, a subset of people with prior COVID infection continue to report neurological problems more than three months after infection. Blood markers to better characterize LongC are elusive. To further identify neuronal proteins associated with LongC, we maximized the number of nEVs isolated from plasma by developing a hybrid EV Microfluidic Affinity Purification (EV-MAP) technique. We isolated nEVs from people with LongC and neurological complaints, AD, and HIV infection with mild cognitive impairment. Using the OLINK platform that assesses 384 neurological proteins, we identified 11 significant proteins increased in LongC and 2 decreased (BST1, GGT1). Fourteen proteins were increased in AD and forty proteins associated with HIV cognitive impairment were elevated with one decreased (IVD). One common protein (BST1) was decreased in LongC and increased in HIV. Six proteins (MIF, ENO1, MESD, NUDT5, TNFSF14 and FYB1) were expressed in both LongC and AD and no proteins were common to HIV and AD. This study begins to identify differences and similarities in the neuronal response to LongC versus AD and HIV infection.

Blood Markers Show Neural Consequences of LongCOVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists throughout the world with over 65 million registered cases of survivors with post-COVID-19 sequelae, also known as LongCOVID-19 (LongC). LongC survivors exhibit various symptoms that span multiple organ systems, including the nervous system. To search for neurological markers of LongC, we investigated the soluble biomolecules present in the plasma and the proteins associated with plasma neuronal-enriched extracellular vesicles (nEVs) in 33 LongC patients with neurological impairment (nLongC), 12 COVID-19 survivors without any LongC symptoms (Cov), and 28 pre-COVID-19 healthy controls (HC). COVID-19 positive participants were infected between 2020 and 2022, not hospitalized, and were vaccinated or unvaccinated before infection. IL-1ß was significantly increased in both nLongC and Cov and IL-8 was elevated in only nLongC. Both brain-derived neurotrophic factor and cortisol were significantly elevated in nLongC and Cov compared to HC. nEVs from people with nLongC had significantly elevated protein markers of neuronal dysfunction, including amyloid beta 42, pTau181 and TDP-43. This study shows chronic peripheral inflammation with increased stress after COVID-19 infection. Additionally, differentially expressed nEV neurodegenerative proteins were identified in people recovering from COVID-19 regardless of persistent symptoms.

Engineered induced-pluripotent stem cell derived monocyte extracellular vesicles alter inflammation in HIV humanized mice.

Aim: A peripheral inflammatory response can drive neuroinflammation in a number of infections including human immunodeficiency virus (HIV). Monocyte/macrophage (M/Mφ) activation is a hallmark of acute HIV infection and a source of chronic inflammation in a subset of HIV-infected individuals. We sought to decrease peripheral inflammation and M/Mφ transmigration after HIV infection by engineering extracellular vesicles (EV) to antagonize a microRNA (miR) associated with inflammation. We hypothesized that induced pluripotent stem cell (iPSC)-derived monocyte EVs (mEVs), engineered to contain an antagomir to miR-155 (αmiR mEV) would target monocyte inflammation and influence neuroinflammation in an HIV-infected humanized mice. Methods: mEVs were characterized by tetraspanins, nanoparticle tracking analysis, electron microscopy, and their preferential entry into circulating monocytes as well as testing for endogenous selected miRNAs. HIV-infected humanized mice were treated with control or antagomir155 mEVs. Plasma viral load was measured plus activation markers on lymphocytes and monocytes and the number of macrophages in the brain was quantified. Results: mEVs preferentially entered peripheral monocytes. HIV infection increased C-C chemokine receptor type 5 (CCR5) and major histocompatibility complex, class II, DR (HLA-DR) expression on T cells and monocytes. Treatments with mEVs did not decrease plasma HIV viral load; however, mEVs alone resulted in a decrease in %CCR5+ and %HLA-DR+ on T cells and an increase in %CCR5+ monocytes. αmiR mEVs decreased %CCR5 on M/Mφ. The mEV-treated HIV-infected mice did not show an increase in macrophage transmigration to the brain. Conclusion: mEVs alone caused an unexpected decrease in lymphocyte activation and increase in monocyte %CCR5; however, this did not translate to an increase in macrophage transmigration to the brain.

Characterization and Biomarker Analyses of Post-COVID-19 Complications and Neurological Manifestations.

As the SARS-CoV-2 pandemic continues, reports have demonstrated neurologic sequelae following COVID-19 recovery. Mechanisms to explain long-term neurological sequelae are unknown and need to be identified. Plasma from 24 individuals recovering from COVID-19 at 1 to 3 months after initial infection were collected for cytokine and antibody levels and neuronal-enriched extracellular vesicle (nEV) protein cargo analyses. Plasma cytokine IL-4 was increased in all COVID-19 participants. Volunteers with self-reported neurological problems (nCoV, n = 8) had a positive correlation of IL6 with age or severity of the sequalae, at least one co-morbidity and increased SARS-CoV-2 antibody compared to those COVID-19 individuals without neurological issues (CoV, n = 16). Protein markers of neuronal dysfunction including amyloid beta, neurofilament light, neurogranin, total tau, and p-T181-tau were all significantly increased in the nEVs of all participants recovering from COVID-19 compared to historic controls. This study suggests ongoing peripheral and neuroinflammation after COVID-19 infection that may influence neurological sequelae by altering nEV proteins. Individuals recovering from COVID-19 may have occult neural damage while those with demonstrative neurological symptoms additionally had more severe infection. Longitudinal studies to monitor plasma biomarkers and nEV cargo are warranted to assess persistent neurodegeneration and systemic effects.

Inflammatory Mechanisms and Cascades Contributing to Neurocognitive Impairment in HIV/AIDS.

Neurocognitive impairment caused by chronic human immunodeficiency virus (HIV) infection is a growing concern. In this chapter we discuss the inflammatory mechanisms underlying the pathology of asymptomatic and mild neurocognitive impairment in the context of antiretroviral therapy. We discuss the role of HIV, viral proteins, and virally infected cells on the development of neuroinflammation and the effect of viral proteins on the cells of the central nervous system.We examine how these collective factors result in an inflammatory context that triggers the development of neurocognitive impairment in HIV. We assess the contribution of antiretrovirals and drugs of abuse, including methamphetamine, cannabis, and opioids, to the neurotoxic and neuroinflammatory milieu that leads to the development of neurocognitive impairment in HIV-infected individuals. We also examined circulating biomarkers, NF-L, sCD163, and sCD14, pertinent to identifying changes in the CNS that could indicate real-time changes in patient physiology. Lastly, we discuss future studies, such as exosomes and the microbiome, which could play a role in the HIV-induced neuroinflammation that eventually manifests as cognitive impairment.

Using neuronal extracellular vesicles and machine learning to predict cognitive deficits in HIV.

Our objective was to predict HIV-associated neurocognitive disorder (HAND) in HIV-infected people using plasma neuronal extracellular vesicle (nEV) proteins, clinical data, and machine learning. We obtained 60 plasma samples from 38 women and 22 men, all with HIV infection and 40 with HAND. All underwent neuropsychological testing. nEVs were isolated by immunoadsorption with neuron-specific L1CAM antibody. High-mobility group box 1 (HMGB1), neurofilament light (NFL), and phosphorylated tau-181 (p-T181-tau) proteins were quantified by ELISA. Three different computational algorithms were performed to predict cognitive impairment using clinical data and nEV proteins. Of the 3 different algorithms, support vector machines performed the best. Applying 4 different models of clinical data with 3 nEV proteins, we showed that selected clinical data and HMGB1 plus NFL best predicted cognitive impairment with an area under the curve value of 0.82. The most important features included CD4 count, HMGB1, and NFL. Previous published data showed nEV p-T181-tau was elevated in Alzheimer's disease (AD), and in this study, p-T181-tau had no importance in assessing HAND but may actually differentiate it from AD. Machine learning can access data without programming bias. Identifying a few nEV proteins plus key clinical variables can better predict neuronal damage. This approach may differentiate other neurodegenerative diseases and determine recovery after therapies are identified.

Hepatitis C Virus Cure in Human Immunodeficiency Virus Coinfection Dampens Inflammation and Improves Cognition Through Multiple Mechanisms.

BACKGROUND: Chronic inflammation in human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection increases cognitive impairment. With newer, direct-acting antiviral therapies for HCV, our objective was to determine whether chronic inflammation would be decreased and cognition improved with HCV sustained viral response (SVR) in coinfection. METHODS: We studied 4 groups longitudinally: 7 HCV-monoinfected and 12 HIV/HCV-coinfected persons before and after treatment for HCV, 12 HIV-monoinfected persons, and 9 healthy controls. We measured monocyte activation and gene expression, monocyte-derived exosome micro-ribonucleic acid (miRNA) expression, plasma inflammation, and cognitive impairment before and after therapy. RESULTS: Plasma soluble CD163 and neopterin were decreased in HCV mono- and coinfected persons. Blood CD16+ monocytes were decreased in coinfection after HCV treatment. Global deficit score improved 25% in coinfection with the visual learning/memory domain the most improved. Hepatitis C virus SVR decreased monocyte interferon genes MX1, IFI27, and CD169 in coinfection and MX1, LGALS3BP, and TNFAIP6 in HCV monoinfection. Monocyte exosomes from coinfected persons increased in microRNA (miR)-19a, miR-221, and miR-223, all of which were associated with decreasing inflammation and nuclear factor-κB activation. CONCLUSIONS: Hepatitis C virus cure in coinfection brings monocyte activation to levels of HIV alone. Cognitive impairment is significantly improved with cure but not better than HIV infection alone, which strong suggests that cognitive impairment was driven by both HIV and HCV.SummaryHCV cure in HIV coinfection improves monocyte and plasma activation markers and increases cognitive function in the visual learning/memory domain.

Profile of neuronal exosomes in HIV cognitive impairment exposes sex differences.

OBJECTIVE: To use plasma neuron-derived exosomes (NDEs) to detect proteins that diagnose HIV-associated neurocognitive disorders (HAND). To compare NDE cargo from HAND with Alzheimer's disease. DESIGN: Eighty plasma samples were assayed including men (n = 29) and women (n = 51) with and without HAND. METHODS: Plasma NDEs were isolated by immunoadsorption with neuron specific L1 cell adhesion molecule antibody. NDE proteins were quantified by ELISA and proximity extension assays for 184 targets. RESULTS: Neuronal enrichment of NDE was confirmed with elevated synaptophysin and normalized to the exosomal marker, apoptosis-linked gene-2-interacting protein X (ALIX). NDE from men and women had significant divergent results. High mobility group box 1 and neurofilament light were significantly increased in NDE from cognitively impaired men and were unchanged in women. NDE from HIV+ men had decreased p-T181-tau, a marker increased in Alzheimer's disease, compared with no difference in women. NDE amyloid beta was not increased in cognitive impairment. Proximity extension assays analysis showed 25 proteins were differentially expressed in HIV infection alone. Seven proteins identified asymptomatic and mild cognitive impairment in HIV+ women. NDE from women had significantly decreased cathepsin S, total tau, neuronal cell adhesion molecule and contactin 5 in mild impairment. Twelve proteins were increased in NDE from cognitively impaired men, including carboxypeptidase M, cadherin 3, colony stimulating factor 2 receptor alpha subunit and mesencephalic astrocyte-derived neurotropic factor. CONCLUSION: NDE proteins differ in HIV infection alone and cognitive impairment between men and women suggesting mechanistic sex differences associated with HAND. Several NDE targets are different from that reported for Alzheimer's disease.

Plasma neuronal exosomes serve as biomarkers of cognitive impairment in HIV infection and Alzheimer's disease.

Fluid biomarkers for cognitive impairment have the advantage of being relatively noninvasive and capable of monitoring neuronal and other brain cell health in real time. Biomarkers can predict the onset of dementing illness, but also correlate with cognition in a dynamic way allowing us to follow treatment responses and determine brain recovery. Chronic HIV infection causes cognitive impairment in a subset of individuals suggesting "premature aging." Exosomes are small extracellular vesicles that are shed from all cells. They are important in normal cell-to-cell communication as they contain cellular proteins, mRNA transcripts, and miRNAs. Exosome cargo varies depending on the health of the cell and pathological state; specific proteins/mRNAs and/or miRNAs are present and may serve as biomarkers. Exosomes of variable cellular origin can be isolated from peripheral blood by various methods. Neuron-derived exosomes (NDEs) can be isolated using a precipitation/immunoaffinity approach using antibodies against neuronal cell adhesion molecule L1CAM and the contents queried for central nervous system (CNS) disorders including HIV-associated neurological disorders (HAND) and Alzheimer's disease (AD). As these studies are recent, numerous questions arise including which neuronal proteins are in NDEs and whether their contents differ in different CNS pathologies or with age. In addition, can the NDE cargo predict as well as diagnose cognitive impairment and could exosomal contents be used as therapeutic biomarkers, or theramarkers, of neuronal recovery from effective treatment? This mini-review will show some new data and review recent studies on NDE from individuals with HIV infection and AD. HIV-associated neurocognitive disorders (HAND) are pathologies seen in a subset of individuals with chronic HIV infection. They belong to the spectrum of neurodegenerative diseases that result in death or dysfunction of neurons with similarities to Alzheimer disease (AD) but also distinctive differences (reviewed (Canet et al., Front Cell Neurosci 12: 307, 2018)). Both disorders are difficult to diagnose without neuropsychological testing and both need new biomarkers to judge progression as well as recovery with treatment. Both disorders involve neuroinflammation and several common targets. AD is associated with aging and HIV is thought to initiate premature aging. In HIV infection, amyloid beta (Aß), which is deposited in "plaques" in AD, is soluble and its relevance to HIV-associated cognitive impairment is controversial (Achim et al., J Neuroimmune Pharmacol 4: 190-199, 2009; Rempel and Pulliam, AIDS 19: 127-135, 2005). Aß deposition is required for AD pathological diagnosis, but is not necessarily causative (Barage and Sonawane, Neuropeptides 52: 1-18, 2015; Hardy and Selkoe, Science 297: 353-356, 2002; Morris et al., Acta Neuropathol Commun 2: 135, 2014). Neurofilament light (NF-L) is a surrogate marker in plasma and cerebrospinal fluid (CSF) for neurodegeneration (Abu-Rumeileh et al., Alzheimers Res Ther 10: 3, 2018; Mattsson et al., JAMA Neurol 74: 557-566, 2017) but continues to be a controversial biomarker for both HAND and AD (Gisslen et al., EBioMedicine 3: 135-140, 2016; Kovacs et al., Eur J Neurol 24:1326-e77, 2017; Norgren et al., Brain Res 987: 25-31, 2003; Rolstad et al., J Alzheimers Dis 45: 873-881, 2015; Yilmaz et al., Expert Rev Mol Diagn 17: 761-770, 2017). Blood biomarkers are needed to advance both HAND and AD fields, as blood draws are less costly than neuroimaging and are minimally invasive compared to lumbar punctures required for CSF acquisition. Extracellular vesicles (EVs) are nanoscale membranous vesicles shed from all cells including those of the central nervous system (CNS) and found in all biofluids; they are divided into exosomes (30-150 nm) originating from late endosomes/multivesicular bodies and microvesicles (150-1000 nm) produced through budding of the plasma membrane. Both types of vesicles are implicated in the pathogenesis of neurodegenerative diseases and may provide biomarkers (Bellingham et al., Front Physiol 3: 124, 2012). In this report, we call the vesicles exosomes, since they are the predominant vesicles in our preparations. They are involved in cell-to-cell communication in normal homeostasis and can be carriers of toxic proteins (Aß, tau) (Sardar Sinha et al., Acta Neuropathol 136: 41-56, 2018) shed by cells as waste or actively secreted in a degenerative process (review Gupta and Pulliam, J Neuroinflammation 11: 68, 2014). The idea that exosomes originating from a specific cell can be recovered in the plasma using cellular surface markers of interest is intriguing. Neuron derived exosomes (NDEs) were first described in 2015 and isolated using antibodies against neural cell adhesion molecules NCAM or L1CAM, after total plasma exosome isolation (Fiandaca et al., Alzheimers Dement 11: 600-607 e1, 2015). Characterization of NDEs follows guidelines endorsed by the International Society for Extracellular Vesicles and includes Nanoparticle Tracking Analysis (NTA) to determine EV concentration and average diameter; Western Blots for EV markers; ELISAs for neuronal proteins and transmission EM for visualization (Sun et al., AIDS 31: F9-F17, 2017; Tang et al., FASEB J 30: 3097-106, 2016). This innovative isolation of an exosome sub-population has generated interest in using NDE as biomarkers for neurodegenerative diseases like AD, HAND, traumatic brain injury, posttraumatic stress disorder and more (reviews Agoston et al., Brain Inj 31: 1195-1203, 2017; Gupta and Pulliam, J Neuroinflammation 11: 68, 2014; Hu et al., Cell Death Dis 7: e2481, 2016; Karnati et al., J Neurotrauma, 2018; Osier et al., Mol Neurobiol, 2018). Several biomarkers from plasma NDEs were recently reported by the Pulliam lab to be elevated in general cognitive impairment (Sun et al., AIDS 31: F9-F17, 2017). We review our collective data here on HAND and AD and add to the characterization of plasma NDEs as exciting biomarkers of neurodegeneration.

Proceedings of the 2017 ISEV symposium on "HIV, NeuroHIV, drug abuse, & EVs".

Despite the success of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-1-infected individuals on cART, which poses a major health care challenge. Adding further complexity to this long-term antiretroviral use is the comorbidity with drugs of abuse such as morphine, cocaine, and methamphetamine, which can in turn, exacerbate neurologic and cognitive deficits associated with HAND. Furthermore, HIV proteins, such as the transactivator of transcription (Tat) and the envelope protein (gp120), as well as antiretrovirals themselves can also contribute to the progression of neurodegeneration underlying HAND. In the field of NeuroHIV and drug addiction, EVs hold the potential to serve as biomarkers of cognitive dysfunction, targets of therapy, and as vehicles for therapeutic delivery of agents that can ameliorate disease pathogenesis. Based on the success of a previous Satellite Symposium in 2015 at the ISEV meeting in Washington, experts again expanded on their latest research findings in the field, shedding light on the emerging trends in the field of Extracellular Vesicle (EV) biology in NeuroHIV and drug abuse. The satellite symposium sought to align experts in the fields of NeuroHIV and drug abuse to share their latest insights on the role of EVs in regulating neuroinflammation, neurodegeneration, peripheral immune response, and HIV latency in HIV-infected individuals with or without the comorbidity of drug abuse.

Immune activated monocyte exosomes alter microRNAs in brain endothelial cells and initiate an inflammatory response through the TLR4/MyD88 pathway.

The host immune response is critical for homeostasis; however, when chronic low level activation of the immune response with or without the driver continues, a cascade of events can trigger immunological dysfunction. Monocytes are key peripheral sensors of the immune response and their activation is instrumental in the development of cognitive impairment. Here, we show that monocytes activated by interferon alpha, lipopolysaccharide or a combination of both generate exosomes carrying significantly altered microRNA profiles compared to non-activated monocytes. These exosomes alone can activate human brain microvascular endothelial cells to stimulate adhesion molecules, CCL2, ICAM1, VCAM1 and cytokines, IL1ß and IL6. This activation is through the toll like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) pathway that activates nuclear factor-κB and increases monocyte chemotaxis. Inhibition of monocyte exosome release reverses endothelial cell activation and monocyte chemotaxis. Our study suggests that activated monocytes have an impact on brain vascular function through intercellular exosome signaling.

Proceedings of the ISEV symposium on "HIV, NeuroAIDS, drug abuse & EVs".

Extracellular vesicles (EVs) are globular, membrane bound nanovesicles (30-100 nm range) that are shed both during normal cellular functioning and under pathological conditions by most cell types. In recent years, there has been significant interest in the study of these vesicles as conduits for the delivery of information between cells from both analogous and disparate tissues. Their ability to carry specialised cargo including signalling mediators, proteins, messenger RNA and miRNAs characterises these vesicles as primary facilitators of cell-to-cell communication and regulation. EVs have also been demonstrated to play important roles in the field of cancer biology and metastasis. However, significant knowledge gaps exist in the role these vesicles play in the context of HIV infection and drug abuse. To foster discussion in this area a satellite symposium on "HIV, NeuroAIDS, Drug Abuse & EVs", was held in conjunction with the annual meeting of the International Society for Extracellular Vesicles (ISEV) in Bethesda, in April 2015. Experts in HIV and drug abuse fields were invited to share their findings on the role of EVs in HIV-1 infection and drug addiction. Additional discussion included current areas of research in EV biology in HIV infection and drug abuse.

Blood neuron-derived exosomes as biomarkers of cognitive impairment in HIV.

OBJECTIVE: To investigate proteins associated with neuronal damage in plasma neuron-derived exosomes (NDE) of HIV-infected study participants as a liquid biomarker for cognitive impairment. METHODS: Plasma NDE were isolated using precipitation and immunoadsorption with antibody to a cell surface-specific neuronal marker. Total exosomes and NDE were enumerated, characterized, and proteins extracted and targets quantified by ELISA. RESULTS: Plasma NDE from 23 HIV seropositive individuals of which 11 had mild cognitive impairment, and 12 HIV seronegative controls of which three had cognitive impairment were isolated. NDE were enriched for the neuronal markers neurofilament light (NF-L) and synaptophysin (SYP). Neuropsychologically impaired individuals had fewer NDE compared with neuropsychologically normal study participants. NDE from neuropsychologically impaired study participants had significantly higher levels of high-mobility group box 1 (HMGB1), NF-L, and amyloid ß proteins compared with neuropsychologically normal individuals. NDE HMGB1 protein significantly decreased with age in HIV-infected individuals. CONCLUSION: Plasma NDE were altered in several ways in HIV infection. Elevated HMGB1, NF-L, and amyloid ß proteins could distinguish cognitive impairment. NDE contents reflect neuronal health in 'real time' and may be useful for following cognitive impairment and response to therapy in HIV infection.

Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-κB in endothelial cells.

HIV-infected individuals have activated monocytes with an IFNα phenotype and elevated levels of circulating LPS. These individuals also have a risk of premature cardiovascular disease. The effect of activated monocyte exosomes (Exos) on endothelial cells is unknown. To determine whether Exos from immune-activated monocytes could alter endothelial cell expression and contribute to monocyte/macrophage transmigration and adhesion, we isolated Exos from monocytes stimulated with IFNα, LPS, or both (I/L). We show that monocyte Exos contain different inflammatory microRNA cargo depending on stimulation. When LPS Exos or I/L Exos were added to HUVECs, we found a significant increase in adhesion molecule ICAM-1, chemokine ligand (CCL)-2, and cytokine IL-6 mRNAs and proteins compared with cells treated with IFNα Exos or Exos derived from unstimulated monocytes. Inhibition of transcription factor NF-κB, a common inflammatory cytokine pathway, prevented induction of CCL2, IL6, and ICAM1 Inhibition of TLR4 resulted in differential blockage of the targets. Our results demonstrate for the first time that primary human monocyte Exos enter endothelial cells and cause dysfunction via the TLR4 and NF-κB pathways, which may contribute to heart disease in HIV infection and other diseases involving chronic immune activation.-Tang, N., Sun, B., Gupta, A., Rempel, H., Pulliam, L. Monocyte exosomes induce adhesion molecules and cytokines via activation of NF-κB in endothelial cells.

Human plasma platelet-derived exosomes: effects of aspirin.

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin.

Modulation of cellular function through immune-activated exosomes.

Extracellular vesicles classified as exosomes, microvesicles, or apoptotic bodies based on size are shed from most cells under normal as well as pathological conditions. They are released into the surrounding milieu, including plasma, urine, saliva, and tissues. Exosomes are highly enriched in microRNAs (miRs), which function in recipient cells by regulating posttranscriptional processing of targeted genes. Interaction of a miR with its mRNA target typically results in suppression of its gene expression. Peripheral inflammatory conditions can modulate miR expression in immune cells such as circulating monocytes that can influence their migration and differentiation. Changes within monocyte-derived macrophage miR expression can influence exosome content and further affect end-organ target cells.

Cognitive consequences of a sustained monocyte type 1 IFN response in HIV-1 infection.

With successful antiretroviral therapy, HIV-1-infected subjects can achieve undetectable peripheral viral loads and immune homeostasis. However, in a subset of individuals on therapy, peripheral monocytes have a gene expression profile characteristic of a type 1 interferon α (IFN) response. This type 1 IFN response correlates with a number of pathogenic conditions including neural cell injury and in combination with HCV infection, cognitive impairment. Lessons from the non-human primate models of pathogenic and nonpathogenic SIV suggest that returning the initial IFN spike in acute SIV infection to normal allows the immune system to control infection and return to homeostasis. An IFN "alarm" signature, defined as monocyte activation with overexpression of the type1 IFN genes IFI27 and CD169, would be useful for identifying a subset of subjects with HIV-1 infection that could progress to a number of pathologies associated with immune activation including cognitive dysfunction. This strategy is being actively pursued for autoimmune diseases that are characterized by an IFN signature. Therapies to block the IFN signature are under investigation as a means to reset the immune system and in a subset of HIV-1-infected subjects may be an adjuvant to standard antiviral therapy to return cognitive function.

Exosomes as mediators of neuroinflammation.

Exosomes are membrane-bound nanovesicles that are shed by cells of various lineages under normal as well as pathological conditions. Previously thought to be 'extracellular debris', exosomes have recently generated immense interest following their discovery as mediators of intercellular communication by delivering functional proteins, mRNA transcripts as well as miRNAs to recipient cells. Although suggested to primarily serve as signaling organelles which also remove unwanted cellular components in the brain, accumulating evidence suggests that exosomes can also significantly contribute to the development of several neuropathologies. Toxic forms of aggregated proteins such as α-synuclein, amyloid ß and prions, that are responsible for the development of Parkinson's disease, Alzheimer's disease and Creutzfeldt-Jacob disease (CJD) respectively, have been shown to get effectively packaged into exosomes and spread from one cell to another, initiating an inflammatory cascade. In addition, exosomes secreted by resident brain cells in response to pathogenic stimuli such as viral proteins can also influence bystander cells by the transfer of dysregulated miRNAs that suppress the expression of essential genes in the recipient cells. Given the relevance of exosomes in brain communication and neuropathogenesis, novel therapeutic strategies are now being developed that exploit the biology of these vesicles to deliver anti-inflammatory molecules to the CNS. Exosomes may alter the way we think about brain disorders and their treatments.

Monocyte activation from interferon-α in HIV infection increases acetylated LDL uptake and ROS production.

Atherosclerosis is an inflammatory disease that is accelerated in human immunodeficiency virus (HIV) infection. Individuals with HIV infection have an activated type I interferon (IFN) monocyte phenotype, which may enhance uptake of modified low-density lipoprotein (LDL) thereby initiating a prefoam cell pathology and recruitment into atherosclerotic plaques. In a sampling of HIV-infected subjects, an increase in monocyte activation genes, MX1 and CXCL10, correlated with monocyte expression of the scavenger receptor A (SR-A), a major receptor for lipid uptake and foam cell formation. Monocytes from HIV-infected subjects accumulated more lipid than control uninfected subjects. We modeled increased activation in HIV infection by priming human monocytes with IFNα followed by exposure to acetylated LDL (acLDL). Exposure to IFNα increased acLDL uptake, which generated increased cellular reactive oxygen species (ROS). We posit that HIV infection augments formation of arterial plaques by triggering monocyte activation with a type I IFN profile, which induces SR-A expression, lipid uptake, and subsequent ROS production. These findings may explain in part why HIV-infected individuals with chronic immune activation have an increased risk of atherosclerosis.

Monocyte activation in HIV/HCV coinfection correlates with cognitive impairment.

Coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) challenges the immune system with two viruses that elicit distinct immune responses. Chronic immune activation is a hallmark of HIV infection and an accurate indicator of disease progression. Suppressing HIV viremia by antiretroviral therapy (ART) effectively prolongs life and significantly improves immune function. HIV/HCV coinfected individuals have peripheral immune activation despite effective ART control of HIV viral load. Here we examined freshly isolated CD14 monocytes for gene expression using high-density cDNA microarrays and analyzed T cell subsets, CD4 and CD8, by flow cytometry to characterize immune activation in monoinfected HCV and HIV, and HIV-suppressed coinfected subjects. To determine the impact of coinfection on cognition, subjects were evaluated in 7 domains for neuropsychological performance, which were summarized as a global deficit score (GDS). Monocyte gene expression analysis in HIV-suppressed coinfected subjects identified 43 genes that were elevated greater than 2.5 fold. Correlative analysis of subjects' GDS and gene expression found eight genes with significance after adjusting for multiple comparisons. Correlative expression of six genes was confirmed by qPCR, five of which were categorized as type 1 IFN response genes. Global deficit scores were not related to plasma lipopolysaccharide levels. In the T cell compartment, coinfection significantly increased expression of activation markers CD38 and HLADR on both CD4 and CD8 T cells but did not correlate with GDS. These findings indicate that coinfection is associated with a type 1 IFN monocyte activation profile which was further found to correlate with cognitive impairment, even in subjects with controlled HIV infection. HIV-suppressed coinfected subjects with controlled HIV viral load experiencing immune activation could benefit significantly from successful anti-HCV therapy and may be considered as preferential candidates.