Multi-functional auto-fluorescent nanogels for theranostics.

Here in the present article, the state of art for nanotechnology-enabled nanogel theranostics and the upcoming concepts in nanogel-based therapeutics are summarized. The benefits, innovation, and prospects of nanogel technology are also briefly presented.

Long Noncoding Transcriptome in Chronic Obstructive Pulmonary Disease.

Chronic airway inflammation from recurring exposures to noxious environmental stimuli results in a progressive and irreversible airflow limitation and the lung parenchymal damage that characterizes chronic obstructive pulmonary disease (COPD). The large variability observed in the onset and progression of COPD is primarily driven by complex gene-environment interactions. The transcriptomic and epigenetic memory potential of lung epithelial and innate immune cells drive responses, such as mucus hyperreactivity and airway remodeling, that are tightly regulated by various molecular mechanisms, for which several candidate susceptibility genes have been described. However, the recently described noncoding RNA species, in particular the long noncoding RNAs, may also have an important role in modulating pulmonary responses to chronic inhalation of toxic substances and the development of COPD. This review outlines the features of long noncoding RNAs that have been implicated in regulating the airway inflammatory responses to cigarette smoke exposure and their possible association with COPD pathogenesis. As COPD continues to debilitate the increasingly aging population and contribute to higher morbidity and mortality rates worldwide, the search for better biomarkers and alternative therapeutic options is pivotal.

Targeted Mitochondrial COQ10 Delivery Attenuates Antiretroviral-Drug-Induced Senescence of Neural Progenitor Cells.

HIV infection is associated with symptoms of accelerated or accentuated aging that are likely to be driven not only by HIV itself but also by the toxicity of long-term use of antiretroviral drugs. Therefore, it is crucially important to understand the mechanisms by which antiretroviral drugs may contribute to aging. The aim of this study was to investigate the hypothesis that antiretroviral drugs cause increased reactive oxygen species (ROS) generation that results in mitochondrial dysfunction and culminates in promoting cellular senescence. In addition, we applied targeted nanoparticle (NP)-based delivery to specifically enrich mitochondria with coenzyme Q10 (CoQ10) in order to enhance antioxidant protection. The studies employed neural progenitor cells (NPCs), as differentiation of these cells into mature neurons is affected both during HIV infection and in the aging process. Exposure of cultured NPCs to various combinations of HIV antiretroviral therapy (ART) induced a more than 2-fold increase in mitochondrial ROS generation and mitochondrial membrane potential, a more than 50% decrease in oxygen consumption and ATP levels, a 60% decrease in SIRT3 expression, and a 42% decrease in cell proliferation relative to control levels. These alterations were accompanied by a 37% increase in beta-galactosidase staining and a shortening of the telomere length to more than half of the length of controls as assessed by quantitative telomere-FISH labeling, indicating accelerated NPC senescence in response to ART exposure. Importantly, CoQ10 delivered by targeted nanoparticles effectively attenuated these effects. Overall, these results indicate that ART promotes cellular senescence by causing mitochondrial dysfunction, which can be successfully reversed by supplementation with mitochondria-targeted CoQ10.

Novel nanoformulation to mitigate co-effects of drugs of abuse and HIV-1 infection: towards the treatment of NeuroAIDS.

Drug abuse (e.g., methamphetamine-Meth or cocaine-Coc) is one of the major risk factors for becoming infected with HIV-1, and studies show that in combination, drug abuse and HIV-1 lead to significantly greater damage to CNS. To overcome these issues, we have developed a novel nanoformulation (NF) for drug-abusing population infected with HIV-1. In this work, a novel approach was developed for the co-encapsulation of Nelfinavir (Nel) and Rimcazole (Rico) using layer-by-layer (LbL) assembled magnetic nanoformulation for the cure of neuroAIDS. Developed NF was evaluated for blood-brain barrier (BBB) transmigration, cell uptake, cytotoxicity and efficacy (p24 assay) in HIV-1 infected primary astrocyte (HA) in presence or absence of Coc and Meth. Developed magnetic nanoformulation (NF) fabricated using the LbL approach exhibited higher amounts of drug loading (Nel and Rico) with 100% release of both the therapeutic agents in a sustained manner for 8 days. NF efficacy studies indicated a dose-dependent decrease in p24 levels in HIV-1-infected HA (~55%) compared to Coc + Meth treated (~50%). The results showed that Rico significantly subdued the effect of drugs of abuse on HIV infectivity. NF successfully transmigrated (38.8 ± 6.5%) across in vitro BBB model on the application of an external magnetic field and showed >90% of cell viability with efficient cell uptake. In conclusion, our proof of concept study revealed that sustained and concurrent release of sigma σ1 antagonist and anti-HIV drug from the developed novel sustained release NF can overcome the exacerbated effects of drugs of abuse in HIV infection and may solve the issue of medication adherence in the drug-abusing HIV-1 infected population.

Eicosapentaenoic acid inhibits intestinal ß-carotene absorption by downregulation of lipid transporter expression via PPAR-α dependent mechanism.

The involvement of lipid transporters, the scavenger receptor class B, type I (SR-BI) and Niemann-Pick type C1 Like 1 protein (NPC1L1) in carotenoid absorption is demonstrated in intestinal cells and animal models. Dietary ω-3 fatty acids are known to possess antilipidemic properties, which could be mediated by activation of PPAR family transcription factors. The present study was conducted to determine the effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on intestinal ß-carotene absorption. ß-carotene uptake in Caco-2/TC7 cells was inhibited by EPA (p < 0.01) and PPARα agonist (P < 0.01), but not by DHA, PPARγ or PPARδ agonists. Despite unaltered ß-carotene uptake, both DHA and PPARδ agonists inhibited the NPC1L1 expression. Further, EPA also induced the expression of carnitine palmitoyl transferase 1A (CPT1A) expression, a PPARα target gene. Interestingly, EPA induced inhibition of ß-carotene uptake and SR B1 expression were abrogated by specific PPARα antagonist, but not by PPARδ antagonist. EPA and PPARα agonist also inhibited the basolateral secretion of ß-carotene from Caco-2 cells grown on permeable supports. These results suggest that EPA inhibits intestinal ß-carotene absorption by down regulation of SR B1 expression via PPARα dependent mechanism and provide an evidence for dietary modulation of intestinal ß-carotene absorption.

Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1)-facilitated HIV restriction in astrocytes is regulated by miRNA-181a.

BACKGROUND: Although highly active antiretroviral therapy (HAART) has significantly reduced the morbidity and mortality in HIV patients, virus continues to reside in the central nervous system (CNS) reservoir. Hence, a complete eradication of virus remains a challenge. HIV productively infects microglia/macrophages, but astrocytes are generally restricted to HIV infection. The relative importance of the possible replication blocks in astrocytes, however, is yet to be delineated. A recently identified restriction factor, sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1), restricts HIV infection in resting CD4(+)T cells and in monocyte-derived dendritic cells. However, SAMHD1 expression and HIV-1 restriction activity regulation in the CNS cells are unknown. Though, certain miRNAs have been implicated in HIV restriction in resting CD4(+)T cells, their role in the CNS HIV restriction and their mode of action are not established. We hypothesized that varying SAMHD1 expression would lead to restricted HIV infection and host miRNAs would regulate SAMHD1 expression in astrocytes. RESULTS: We found increased SAMHD1 expression and decreased miRNA expression (miR-181a and miR-155) in the astrocytes compared to microglia. We report for the first time that miR-155 and miR-181a regulated the SAMHD1 expression. Overexpression of these cellular miRNAs increased viral replication in the astrocytes, through SAMHD1 modulation. Reactivation of HIV replication was accompanied by decrease in SAMHD1 expression. CONCLUSIONS: Here, we provide a proof of concept that increased SAMHD1 in human astrocytes is in part responsible for the HIV restriction, silencing of which relieves this restriction. At this time, this concept is of theoretical nature. Further experiments are needed to confirm if HIV replication can be reactivated in the CNS reservoir.

Towards nanomedicines for neuroAIDS.

Although highly active antiretroviral therapy (HAART) has resulted in remarkable decline in the morbidity and mortality in AIDS patients, controlling HIV infections still remain a global health priority. HIV access to the CNS serves as the natural viral preserve because most antiretroviral (ARV) drugs possess inadequate or zero delivery across the brain barriers. Thus, development of target-specific, effective, safe, and controllable drug-delivery approach is an important health priority for global elimination of AIDS progression. Emergence of nanotechnology in medicine has shown exciting prospect for development of novel drug delivery systems to administer the desired therapeutic levels of ARV drugs in the CNS. Neuron-resuscitating and/or antidependence agents may also be delivered in the brain through nanocarriers to countercheck the rate of neuronal degradation during HIV infection. Several nanovehicles such as liposomes, dendrimers, polymeric nanoparticles, micelles, and solid lipid nanoparticles have been intensively explored. Recently, magnetic nanoparticles and monocytes/macrophages have also been used as carrier to improve the delivery of nanoformulated ARV drugs across the blood-brain barrier. Nevertheless, more rigorous research homework has to be elucidated to sort out the shortcomings that affect the target specificity, delivery, release, and/or bioavailability of desired amount of drugs for treatment of neuroAIDS.

Synaptic Plasticity and Neurological Disorders in Neurotropic Viral Infections.

Based on the type of cells or tissues they tend to harbor or attack, many of the viruses are characterized. But, in case of neurotropic viruses, it is not possible to classify them based on their tropism because many of them are not primarily neurotropic. While rabies and poliovirus are considered as strictly neurotropic, other neurotropic viruses involve nervous tissue only secondarily. Since the AIDS pandemic, the interest in neurotropic viral infections has become essential for all clinical neurologists. Although these neurotropic viruses are able to be harbored in or infect the nervous system, not all the neurotropic viruses have been reported to cause disrupted synaptic plasticity and impaired cognitive functions. In this review, we have discussed the neurotropic viruses, which play a major role in altered synaptic plasticity and neurological disorders.

Commentary on the interactions of HIV and alcohol in the central nervous system.

This commentary is to highlight the relevance and public interest of the review published by Silverstein and Kumar, which focuses on the mechanisms by which alcohol and HIV-1 infection cause increased in central nervous system (CNS) damage. The overall review is based on previous literature with cell culture systems and animal models that have demonstrated that exposure to alcohol and HIV infection or HIV viral proteins result in synergistic up-regulation of pro-inflammatory cytokines and oxidative stress. The authors discuss the effects of alcohol on cells in the CNS, followed by a brief discussion on the impact of HIV-1 and HIV proteins on the CNS, and the final section focuses on the combined effects of HIV and alcohol on the CNS as determined by in vitro, in vivo, and clinical studies.

Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation.

The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ∼10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ∼130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

LRRC25 expression during physiological aging and in mouse models of Alzheimer's disease and iPSC-derived neurons.

The leucine-rich repeat-containing protein 25 (LRRC25) is relatively a novel protein with no information on its role in neuronal or brain function. A recent study suggested LRRC25 is a potential risk factor for Alzheimer's disease (AD). As a first step to understanding LRRC25's role in the brain and AD, we found LRRC25 is expressed in both cell membranes and cytoplasm in a punctuate appearance in astrocytes, microglia, and neurons in cell lines as well as mouse brain. We also found that LRRC25 expression is both age- and brain region-dependent and that 1-day-old (1D) pups expressed the least amount of LRRC25 protein compared to adult ages. In the APΔE9 mice, immunoblot quantified LRRC25 protein levels were increased by 166% (**p < 0.01) in the cortex (CX) and by 215% (***p < 0.001) in the hippocampus (HP) relative to wild-type (WT) controls. Both the brainstem (BS) and cerebellum (CB) showed no significant alterations. In the 3xTg mice, only CX showed an increase of LRRC25 protein by 91% (*p < 0.05) when compared to WT controls although the increased trend was noted in the other brain regions. In the AD patient brains also LRRC25 protein levels were increased by 153% (***p < 0.001) when compared to normal control (NC) subjects. Finally, LRRC25 expression in the iPSC-derived neurons quantified by immunofluorescence was increased by 181% (**p < 0.01) in AD-derived neurons when compared to NC-derived neurons. Thus increased LRRC25 protein in multiple models of AD suggests that LRRC25 may play a pathogenic role in either Aß or tau pathology in AD. The mechanism for the increased levels of LRRC25 in AD is unknown at present, but a previous study showed that LRRC25 levels also increase during neonatal hypoxic-ischemia neuronal damage. Based on the evidence that autophagy is highly dysregulated in AD, the increased LRRC25 levels may be due to decreased autophagic degradation of LRRC25. Increased LRRC25 in turn may regulate the stability or activity of key enzymes involved in either Aß or hyperphosphorylated tau generation and thus may contribute to increased plaques and neurofibrillary tangles.

Pathological Responses in Asian House Shrews (Suncus murinus) to the Naturally Acquired Orientia tsutsugamushi Infection.

Scrub typhus is a re-emerging disease caused by Orientia tsutsugamushi, transmitted by mites belonging to the family Trombiculidae. Humans and rodents acquire the infection by the bite of larval mites/chiggers. Suncus murinus, the Asian house shrew, has been reported to harbor the vector mites and has been naturally infected with O. tsutsugamushi. The present study aimed to localize and record O. tsutsugamushi in the tissues and the host response in shrews naturally infected with O. tsutsugamushi. Sheehan's modified May-Grunwald Giemsa staining was carried out in 365 tissues from 87 animals, and rickettsiae were documented in 87 tissues from 20 animals. Immunohistochemical (IHC) staining, using polyclonal antibodies raised against selected epitopes of the 56-kDa antigen, was carried out, and 81/87 tissue sections were tested positive for O. tsutsugamushi. By IHC, in addition to the endothelium, the pathogen was also demonstrated by IHC in cardiomyocytes, the bronchiolar epithelium, stroma of the lungs, hepatocytes, the bile duct epithelium, the epithelium and goblet cells of intestine, the tubular epithelium of the kidney, and splenic macrophages. Furthermore, the pathogen was confirmed by real-time PCR using blood (n = 20) and tissues (n = 81) of the IHC-positive animals. None of the blood samples and only 22 out of 81 IHC-positive tissues were tested positive by PCR. By nucleotide sequencing of the 56-kDa gene, Gilliam and Karp strains were found circulating among these animals. Although these bacterial strains are highly virulent and cause a wide range of pathological alterations, hence exploring their adaptive mechanisms of survival in shrews will be of significance. Given that the pathogen localizes in various organs following a transient bacteremia, we recommend the inclusion of tissues from the heart, lung, intestine, and kidney of reservoir animals, in addition to blood samples, for future molecular surveillance of scrub typhus.

HIV infection and drugs of abuse: role of acute phase proteins.

BACKGROUND: HIV infection and drugs of abuse such as methamphetamine (METH), cocaine, and alcohol use have been identified as risk factors for triggering inflammation. Acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) are the biomarkers of inflammation. Hence, the interactive effect of drugs of abuse with acute phase proteins in HIV-positive subjects was investigated. METHODS: Plasma samples were utilized from 75 subjects with METH use, cocaine use, alcohol use, and HIV-positive alone and HIV-positive METH, cocaine, and alcohol users, and age-matched control subjects. The plasma CRP and SAA levels were measured by ELISA and western blot respectively and the CD4 counts were also measured. RESULTS: Observed results indicated that the CRP and SAA levels in HIV-positive subjects who are METH, cocaine and alcohol users were significantly higher when compared with either drugs of abuse or HIV-positive alone. The CD4 counts were also dramatically reduced in HIV-positive with drugs of abuse subjects compared with only HIV-positive subjects. CONCLUSIONS: These results suggest that, in HIV-positive subjects, drugs of abuse increase the levels of CRP and SAA, which may impact on the HIV infection and disease progression.

HIV Latency and Nanomedicine Strategies for Anti-HIV Treatment and Eradication.

Antiretrovirals (ARVs) reduce Human Immunodeficiency Virus (HIV) loads to undetectable levels in infected patients. However, HIV can persist throughout the body in cellular reservoirs partly due to the inability of some ARVs to cross anatomical barriers and the capacity of HIV-1 to establish latent infection in resting CD4+ T cells and monocytes/macrophages. A cure for HIV is not likely unless latency is addressed and delivery of ARVs to cellular reservoir sites is improved. Nanomedicine has been used in ARV formulations to improve delivery and efficacy. More specifically, researchers are exploring the benefit of using nanoparticles to improve ARVs and nanomedicine in HIV eradication strategies such as shock and kill, block and lock, and others. This review will focus on mechanisms of HIV-1 latency and nanomedicine-based approaches to treat HIV.

Machine learning assisted-nanomedicine using magnetic nanoparticles for central nervous system diseases.

Magnetic nanoparticles possess unique properties distinct from other types of nanoparticles developed for biomedical applications. Their unique magnetic properties and multifunctionalities are especially beneficial for central nervous system (CNS) disease therapy and diagnostics, as well as targeted and personalized applications using image-guided therapy and theranostics. This review discusses the recent development of magnetic nanoparticles for CNS applications, including Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, and drug addiction. Machine learning (ML) methods are increasingly applied towards the processing, optimization and development of nanomaterials. By using data-driven approach, ML has the potential to bridge the gap between basic research and clinical research. We review ML approaches used within the various stages of nanomedicine development, from nanoparticle synthesis and characterization to performance prediction and disease diagnosis.

Emerging trends in point-of-care biosensing strategies for molecular architectures and antibodies of SARS-CoV-2.

COVID-19, a highly contagious viral infection caused by the occurrence of severe acute respiratory syndrome coronavirus (SARS-CoV-2), has turned out to be a viral pandemic then ravaged many countries worldwide. In the recent years, point-of-care (POC) biosensors combined with state-of-the-art bioreceptors, and transducing systems enabled the development of novel diagnostic tools for rapid and reliable detection of biomarkers associated with SARS-CoV-2. The present review thoroughly summarises and discusses various biosensing strategies developed for probing SARS-CoV-2 molecular architectures (viral genome, S Protein, M protein, E protein, N protein and non-structural proteins) and antibodies as a potential diagnostic tool for COVID-19. This review discusses the various structural components of SARS-CoV-2, their binding regions and the bioreceptors used for recognizing the structural components. The various types of clinical specimens investigated for rapid and POC detection of SARS-CoV-2 is also highlighted. The importance of nanotechnology and artificial intelligence (AI) approaches in improving the biosensor performance for real-time and reagent-free monitoring the biomarkers of SARS-CoV-2 is also summarized. This review also encompasses existing practical challenges and prospects for developing new POC biosensors for clinical monitoring of COVID-19.

Anti-inflammatory effects of CBD in human microglial cell line infected with HIV-1.

Human immunodeficiency virus (HIV) infection is associated with a chronic inflammatory stage and continuous activation of inflammasome pathway. We studied the anti-inflammatory effects of the compound cannabidiol (CBD) in comparison with Δ (9)-tetrahydrocannabinol [Δ(9)-THC] in human microglial cells (HC69.5) infected with HIV. Our results showed that CBD reduced the production of various inflammatory cytokines and chemokines such as MIF, SERPIN E1, IL-6, IL-8, GM-CSF, MCP-1, CXCL1, CXCL10, and IL-1 ß compared to Δ(9)-THC treatment. In addition, CBD led to the deactivation of caspase 1, reduced NLRP3 gene expression which play a crucial role in the inflammasome cascade. Furthermore, CBD significantly reduced the expression of HIV. Our study demonstrated that CBD has anti-inflammatory properties and exhibits significant therapeutic potential against HIV-1 infections and neuroinflammation.

Drug Delivery to the Brain: Recent Advances and Unmet Challenges.

Brain cancers and neurodegenerative diseases are on the rise, treatments for central nervous system (CNS) diseases remain limited. Despite the significant advancement in drug development technology with emerging biopharmaceuticals like gene therapy or recombinant protein, the clinical translational rate of such biopharmaceuticals to treat CNS disease is extremely poor. The blood-brain barrier (BBB), which separates the brain from blood and protects the CNS microenvironment to maintain essential neuronal functions, poses the greatest challenge for CNS drug delivery. Many strategies have been developed over the years which include local disruption of BBB via physical and chemical methods, and drug transport across BBB via transcytosis by targeting some endogenous proteins expressed on brain-capillary. Drug delivery to brain is an ever-evolving topic, although there were multiple review articles in literature, an update is warranted due to continued growth and new innovations of research on this topic. Thus, this review is an attempt to highlight the recent strategies employed to overcome challenges of CNS drug delivery while emphasizing the necessity of investing more efforts in CNS drug delivery technologies parallel to drug development.

Recent Advances in Nanotherapeutics for Neurological Disorders.

Neurological disorders remain a significant health and economic burden worldwide. Addressing the challenges imposed by existing drugs, associated side- effects, and immune responses in neurodegenerative diseases is essential for developing better therapies. The immune activation in a diseased state has complex treatment protocols and results in hurdles for clinical translation. There is an immense need for the development of multifunctional nanotherapeutics with various properties to address the different limitations and immune interactions exhibited by the existing therapeutics. Nanotechnology has proven its potential to improve therapeutic delivery and enhance efficacy. Promising advancements have been made in developing nanotherapies that can be combined with CRISPR/Cas9 or siRNA for a targeted approach with unique potential for clinical translation. Engineering natural exosomes derived from mesenchymal stem cells (MSCs), dendritic cells (DCs), or macrophages to both deliver therapeutics and modulate the immune responses to tumors or in neurodegenerative disease (ND) can allow for targeted personalized therapeutic approaches. In the present review, we summarize and overview the recent advances in nanotherapeutics in addressing the existing treatment limitations and neuroimmune interactions for developing ND therapies and provide insights into the upcoming advancements in nanotechnology-based nanocarriers.

Protein cargo of Nef-containing exosomal extracellular vesicles may predict HIV-associated Neurocognitive Impairment status.

Exosomal extracellular vesicles (xEVs) in plasma and cerebrospinal fluid (CSF) of aviremic people living with HIV/AIDS (PLWHA) contain the HIV Negative factor (Nef) protein. However, the role of xEVs and Nef-containing-xEVs(xEV-Nef) in HIV-associated neuropathology is unknown. Here we performed a cross-sectional analysis of the content of xEVs derived from matched serum and CSF samples of PLWHAs diagnosed with either asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), or HIV-associated dementia (HAD). The overall objective was to determine whether the content of the matched xEVs derived plasma or CSF correlated with the neurocognitive impairment (NCI) status. The size and protein content of the xEVs were characterized via dynamic light scattering (DLS) and LC-MS/MS, respectively. xEV size was not significantly different between ANI, MND, or HAD groups. CSF of PLWHAs with NCI contained significantly more xEVs than matched plasma. xEV-Nef CSF concentration was elevated in PLWHAs with NCI and correlated with CD4 T-cell count. Plasma-derived xEV protein profiles from PLWHAs with ANI or MND differed from PLWHAs without NCI. Over-representation analysis using Reactome and KEGG databases show proteins involved in pathways associated with heme scavenging, signaling(MAP kinase and integrin-alpha),Toll-like receptor regulation, clot formation, complement, and cytosolic calcium level were elevated in MND. Pathways upregulated within the ANI group involved high-density lipid (HDL) remodeling, post-translational protein phosphorylation, and platelet activation. Overall, the data shows that xEV protein profiles of ANI and MND differ, suggesting protein profiles of peripheral xEVs, xEV-Nef, and CD4 T-cell count may discern NCI status.