Diagnostic potential of exosomal extracellular vesicles in oncology.

Liquid biopsy can detect circulating cancer cells or tumor cell-derived DNA at various stages of cancer. The fluid from these biopsies contains extracellular vesicles (EVs), such as apoptotic bodies, microvesicles, exomeres, and exosomes. Exosomes contain proteins and nucleic acids (DNA/RNA) that can modify the microenvironment and promote cancer progression, playing significant roles in cancer pathology. Clinically, the proteins and nucleic acids within the exosomes from liquid biopsies can be biomarkers for the detection and prognosis of cancer. We review EVs protein and miRNA biomarkers identified for select cancers, specifically melanoma, glioma, breast, pancreatic, hepatic, cervical, prostate colon, and some hematological malignancies. Overall, this review demonstrates that EV biomolecules have great potential to expand the diagnostic and prognostic biomarkers used in Oncology; ultimately, EVs could lead to earlier detection and novel therapeutic targets. Clinical implicationsEVs represent a new paradigm in cancer diagnostics and therapeutics. The potential use of exosomal contents as biomarkers for diagnostic and prognostic indicators may facilitate cancer management. Non-invasive liquid biopsy is helpful, especially when the tumor is difficult to reach, such as in pancreatic adenocarcinoma. Moreover, another advantage of using minimally invasive liquid biopsy is that monitoring becomes more manageable. Identifying tumor-derived exosomal proteins and microRNAs would allow a more personalized approach to detecting cancer and improving treatment.

HIV-1 and opiates modulate miRNA profiles in extracellular vesicles.

Opiate abuse increases the risk of HIV transmission and exacerbates HIV neuropathology by increasing inflammation and modulating immune cell function. Exosomal EVs(xEV) contain miRNAs that may be differentially expressed due to HIV infection or opiate abuse. Here we develop a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use. PBMCs infected with HIV were treated with increasing dosages of morphine for 72 hours, the culture supernatants were collected, and the exosomes isolated using differential centrifugation. Exosomal miRNAs were extracted, expression levels determined via Nanostring multiplexed microRNA arrays, and analyzed with Webgestalt. The effect of the exosomes on neuronal function was determined by measuring calcium. Preliminary findings show that HIV-1 infection altered the miRNA profile of PBMC-derived EVs concurrently with opiate exposure. MicroRNA, hsa-miR-1246 was up-regulated 12-fold in the presence of morphine, relative to uninfected control. PBMCs infected with HIV-1 MN, an X4-tropic HIV-1 strain and exposed to morphine, displayed a trend which suggests potential synergistic effects between HIV-1 infection and morphine exposure promoting an increase in viral replication. Dose-dependent differences were observed in miRNA expression as a result of opiate exposure. The xEVs derived from PBMCs exposed to morphine or HIV modulated neuronal cell function. SH-SY5Y cells, treated with xEVs derived from ART-treated PBMCs, exhibited increased viability while for SH-SY5Ys exposed to xEVs derived from HIV-1 infected PBMCs viability was decreased compared to the untreated control. Exposing SH-SY5Y to xEVs derived from HIV-infected PBMCs resulted in significant decrease in calcium signaling, relative to treatment with xEVs derived from uninfected PBMCs. Overall, HIV-1 and morphine induced differential miRNA expression in PBMC-derived exosomes, potentially identifying mechanisms of action or novel therapeutic targets involved in opiate use disorder, HIV neuropathology, TNF signaling pathway, NF-κB signaling pathway, autophagy, and apoptosis in context of HIV infection.

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.

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.

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.

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.

Extracellular Vesicles in the Pathogenesis of Viral Infections in Humans.

Most cells can release extracellular vesicles (EVs), membrane vesicles containing various proteins, nucleic acids, enzymes, and signaling molecules. The exchange of EVs between cells facilitates intercellular communication, amplification of cellular responses, immune response modulation, and perhaps alterations in viral pathogenicity. EVs serve a dual role in inhibiting or enhancing viral infection and pathogenesis. This review examines the current literature on EVs to explore the complex role of EVs in the enhancement, inhibition, and potential use as a nanotherapeutic against clinically relevant viruses, focusing on neurotropic viruses: Zika virus (ZIKV) and human immunodeficiency virus (HIV). Overall, this review's scope will elaborate on EV-based mechanisms, which impact viral pathogenicity, facilitate viral spread, and modulate antiviral immune responses.

Bioengineered Exosomal Extracellular Vesicles in Cancer Therapeutics.

Liquid or blood-based biopsy is a less invasive and more efficient method in which to clinicians can identify diagnostic, prognostic, and therapeutic responsive biomarkers in cancer patients. Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), RNAs, proteins, metabolites, and extracellular vesicles (EVs) are all potential biomarkers found in liquid biopsies. All nucleated cells including healthy, virally infected, and cancer cells release EVs. Since the early 1980s, evidence has mounted to support the pathophysiological role of EVs in cancer. Here we focus on the smallest of the EV, the exosome, and their clinical relevance as nanotherapeutics for cancers. Exosomes obtained from tumors have been reported to promote and/or facilitate malignancy of cancers especially in terms of metastatic potential. Exosomal EVs have also contributed to the development of therapeutic resistance. Recent studies demonstrate that intrinsic and bioengineered exosomes can serve as effective therapeutic agents that disrupt cancer progression. Here we review the current literature regarding the utilization of bioengineered exosomes for therapeutics to treat prevalent cancers such as melanoma, glioma, breast, pancreatic, hepatic, cervical, prostate, and colon cancers. Overall, studies reviewed show that bioengineered exosomes are effective and promising for targeted cancer therapy.

Magnetic Nanoparticle and Exosomal Therapeutic (M-NEXT) Effects on HIV-Associated Neurotoxicity.

The human immunodeficiency virus (HIV) envelope glycoprotein protein 120 (gp120) induces neurotoxicity associated with HIV-associated neurocognitive disorders (HAND). Mechanism of Gp120-mediated neurotoxicity is primarily apoptosis. Currently, there are no therapeutics that address gp120 neurotoxicity. A biocompatible, efficacious therapeutic that easily crosses the blood-brain barrier (BBB) is needed to treat neuronal toxicity observed in HIV-infected individuals. Magnetic nanoparticles (MNPs) have successfully delivered anti-HIV agents across in vitro BBB transwell model. However, MNPs at high doses may damage cells. Exosomal extracellular vesicles (xEVs) are endogenous nanocarriers capable of crossing the BBB. Unlike MNPs, xEVs interact with cells in a paracrine or juxtracrine manner, lacking long-range site specificity. Here we investigated the efficacy of an MNP and xEV-coupled therapeutic (M-NEXT) as a nanocarrier for targeted delivery of anti-HIV fusion agent across the BBB to inhibit HIV-gp120 associated neuropathology. M-NEXT consisting of MNPs encapsulated within xEV carrying T20 peptide on the surface was synthesized and characterized via zeta potential, dynamic light scattering, and TEM imaging. Preliminary efficacy studies using SH-SY5Y cocultured with the in vitro BBB model showed that the M-NEXT-T20-fusion peptide protected neurons from HIV gp120-mediated neurotoxicity. Additionally, BBB integrity and permeability assessed via trans-endothelial resistance (TEER) and a Dextran-FITC transport assay was unaffected. SH-SY5Y viability measured by XTT assay was not significantly modulated by M-NEXT. In summary, preliminary findings support M-NEXT as effective nanocarriers for delivery of anti-HIV gp120 associated neurotoxicity agents.

Preparation and characterization of anti-HIV nanodrug targeted to microfold cell of gut-associated lymphoid tissue.

The human immunodeficiency virus 1 (HIV-1) still remains one of the leading life-threatening diseases in the world. The introduction of highly active antiretroviral therapy has significantly reduced disease morbidity and mortality. However, most of the drugs have variable penetrance into viral reservoir sites, including gut-associated lymphoid tissue (GALT). Being the largest lymphoid organ, GALT plays a key role in early HIV infection and host-pathogen interaction. Many different treatment options have been proposed to eradicate the virus from GALT. However, it becomes difficult to deliver traditional drugs to the GALT because of its complex physiology. In this regard, we developed a polymer-based Pluronic nanocarrier containing anti-HIV drug called efavirenz (EFV) targeting Microfold cells (M-cells) in the GALT. M-cells are specialized epithelial cells that are predominantly present in the GALT. In this work, we have exploited this paracellular transport property of M-cells for targeted delivery of Pluronic nanocarrier tagged EFV, bioconjugated with anti-M-cell-specific antibodies to the GALT (nanodrug). Preliminary characterization showed that the nanodrug (EFV-F12-COOH) is of 140 nm size with 0.3 polydispersion index, and the zeta potential of the particles was -19.38±2.2 mV. Further, drug dissolution study has shown a significantly improved sustained release over free drugs. Binding potential of nanodrug with M-cell was also confirmed with fluorescence microscopy and in vitro uptake and release studies. The anti-HIV activity of the nanodrug was also significantly higher compared to that of free drug. This novel formulation was able to show sustained release of EFV and inhibit the HIV-1 infection in the GALT compared to the free drug. The present study has potential for our in vivo targeted nanodrug delivery system by combining traditional enteric-coated capsule technique via oral administration.

HIV Subtypes B and C gp120 and Methamphetamine Interaction: Dopaminergic System Implicates Differential Neuronal Toxicity.

HIV subtypes or clades differentially induce HIV-associated neurocognitive disorders (HAND) and substance abuse is known to accelerate HIV disease progression. The HIV-1 envelope protein gp120 plays a major role in binding and budding in the central nervous system (CNS) and impacts dopaminergic functions. However, the mechanisms utilized by HIV-1 clades to exert differential effects and the methamphetamine (METH)-associated dopaminergic dysfunction are poorly understood. We hypothesized that clade B and C gp120 structural sequences, modeling based analysis, dopaminergic effect, and METH potentiate neuronal toxicity in astrocytes. We evaluated the effect of clade B and C gp120 and/or METH on the DRD-2, DAT, CaMKs and CREBP transcription. Both the structural sequence and modeling studies demonstrated that clade B gp120 in V1-V4, α -2 and N-glycosylated sites are distinct from clade C gp120. The distinct structure and sequence variation of clade B gp120 differentially impact DRD-2, DAT, CaMK II and CaMK IV mRNA, protein and intracellular expression compared to clade C gp120. However, CREB transcription is upregulated by both clade B and C gp120, and METH co-treatment potentiated these effects. In conclusion, distinct structural sequences of HIV-1 clade B and C gp120 differentially regulate the dopaminergic pathway and METH potentiates neurotoxicity.

Immunopathogenesis of HIV infection in cocaine users: role of arachidonic acid.

Arachidonic acid (AA) is known to be increased in HIV infected patients and illicit drug users are linked with severity of viral replication, disease progression, and impaired immune functions. Studies have shown that cocaine accelerates HIV infection and disease progression mediated by immune cells. Dendritic cells (DC) are the first line of antigen presentation and defense against immune dysfunction. However, the role of cocaine use in HIV associated acceleration of AA secretion and its metabolites on immature dendritic cells (IDC) has not been elucidated yet. The aim of this study is to elucidate the mechanism of AA metabolites cyclooxygenase-2 (COX-2), prostaglandin E2 synthetase (PGE2), thromboxane A2 receptor (TBXA2R), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), 14-3-3 ζ/δ and 5-lipoxygenase (5-LOX) mediated induction of IDC immune dysfunctions in cocaine using HIV positive patients. The plasma levels of AA, PGE2, 15d-PGJ2, 14-3-3 ζ/δ and IDC intracellular COX-2 and 5-LOX expression were assessed in cocaine users, HIV positive patients, HIV positive cocaine users and normal subjects. Results showed that plasma concentration levels of AA, PGE2 and COX-2, TBXA2R and 5-LOX in IDCs of HIV positive cocaine users were significantly higher whereas 15d-PGJ2 and 14-3-3 ζ/δ were significantly reduced compared to either HIV positive subjects or cocaine users alone. This report demonstrates that AA metabolites are capable of mediating the accelerative effects of cocaine on HIV infection and disease progression.

Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study.

We have previously described an antiapoptotic steady-state gene expression profile in circulating human monocytes from asymptomatic viremic HIV(+) donors, but the mechanism associated with this apoptosis resistance remains to be fully elucidated. Here, we show that Rb1 activation is a dominant feature of apoptosis resistance in monocytes exposed to HIV-1 in vivo (as measured ex vivo) and in vitro. Monocytes from asymptomatic viremic HIV(+) individuals show a positive correlation between levels of hypophosphorylated (active) Rb1 and VL in conjunction with increases in other p53-inducible proteins associated with antiapoptosis regulation, such as p21 and PAI-1 (SERPINE1), when compared with circulating monocytes from uninfected donors. Monocytes exposed in vitro to HIV-1 R5 isolates but not X4 isolates showed lower caspase-3 activation after apoptosis induction, indicating a role for the CCR5 signaling pathway. Moreover, monocytes exposed to R5 HIV-1 or MIP-1 ß induced Rb1 and p21 expression and an accumulation of autophagy markers, LC3 and Beclin. The inhibition of Rb1 activity in HIV-1 R5 viral-exposed monocytes using siRNA led to increased apoptosis sensitivity, thereby confirming a central role for Rb1 in the antiapoptotic phenotype. Our data identify Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes in association with protective autophagy and contributing to monocyte survival during immune activation and/or HIV-1 viremia.

Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia.

Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4(+)T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia.

Herpesvirus saimiri terminal membrane proteins modulate HIV-1 replication by altering Nef and Tat functions.

Herpesvirus saimiri (HVS)-transformed human T cells expressing terminal membrane proteins (TMPs) tyrosine kinase interacting protein (Tip) and saimiri transformation associated protein strain C (StpC) are highly permissive for R5 and X4 strains of HIV-1. StpC expression enhances replication of R5 and X4 strains of HIV-1 and induces latent reservoirs of replication competent HIV-1 in cell lines derived from T cells or monocytes. Paradoxically Tip expression restricts replication and cytopathic effects of R5 and X4 strains of HIV-1 in T cells and monocytes post-retrotransposition. Understanding the canonical pathways whereby Tip and StpC alter HIV-1 replication may uncover novel therapeutic approaches to HIV-1 infection. Here we show Tip inhibits Tat-mediated transcriptional activation of the long terminal repeat (LTR). Tip mediated inhibition of Tat transactivation is reversed by Nef. Tip also mediates restriction of late-stage replication of HIV-1 by disrupting Nef interaction with lymphocyte-specific protein-tyrosine kinase (Lck) in lipid rafts. Specifically, in the presence of Tip, Lck does not localize to lipid rafts reducing Nef interaction with Lck within the lipid rafts. Finally, the permissive phenotype conferred by StpC is the result of synergy with Tat during transcriptional activation of the HIV-1 LTR. This transcriptional synergy between StpC and Tat requires Lck and NF-kappaB consensus binding sequences. These findings demonstrate that the HVS TMPs influence transcriptional and post-transcriptional stages in HIV-1 replication. We propose that HVS-encoded TMPs associated with T cell transformation have evolved ability to modulate the replication of competing retroviruses. Gene based approaches utilizing Tip and StpC may provide therapeutic models for treating acute and latent HIV-1 infections, respectively.

H. saimiri tyrosine-kinase interacting protein inhibits Tat function: a prototypic strategy for restricting HIV-1-induced cytopathic effects in immune cells.

Herpesvirus saimiri (HVS)-transformed human T cells become permissive for X4 and R5 strains of human immunodeficiency virus type 1 (HIV-1), evidence that HVS-encoded proteins associated with T cell transformation enhance HIV-1 replication. Analyzing the contribution of transformation-associated bicistronic HVS open reading frames (ORF) to HIV-1 replication revealed expression of the second ORF saimiri transformation-associated protein type C (StpC) conferred the permissive phenotype to T cells. In contrast, expression of the first HVS ORF tyrosine-kinase interacting protein (Tip) in the absence of StpC enhanced restriction of HIV-1 replication in T cell lines and peripheral blood mononuclear cells. Understanding the mechanism whereby Tip enhanced restriction of HIV-1 replication may uncover unique pathways that could be targeted therapeutically. Here we report that Tip restricts HIV-1 replication in a monocyte-derived cell line and restricts reactivation of replication of HIV-1 in a T cell line harboring provirus. In this report, we begin to unravel the molecular underpinnings of Tip-mediated restriction. Tip mediates both lymphocyte-cell-specific kinase (Lck)-dependent and -independent effects on HIV-1 replication. We also provide evidence that Tip-mediated restriction is in part due to inhibition of Tat transactivation of the HIV-1 long terminal repeat (LTR). Expression of Tip in T cells increased activation of Stat1 and Stat3, as well as activation of protein kinase RNA-dependent (PKR/p68) and interferon-gamma production. Taken together, these results provide evidence that Tip restricts HIV-1 replication and reactivation by inhibiting HIV-1 transcription while inducing an intercellular antiviral state. We propose that genetically engineered vectors driving Tip expression could provide a prototypic strategy for restricting HIV-1 replication and reactivation in diverse cell lineages.

Herpesvirus saimiri-encoded proteins Tip and StpC modulate human immunodeficiency virus type 1 replication in T-cell lines and lymphocytes independently of viral tropism.

Herpesvirus saimiri (HVS)-transformed T-lymphocytes are permissive for both X4 and R5 strains of human immunodeficiency virus type 1 (HIV-1). HVS-encoded proteins tyrosine-kinase interacting protein (Tip) and saimiri transformation-associated protein subgroup C (StpC) were previously implicated in altering HIV permissiveness. MOLT4 cells expressing StpC or StpC and Tip are permissive for X4 strains of HIV-1. In contrast, HIV-1 was restricted in MOLT4 cells expressing Tip alone. Here we show that MOLT4 cells and primary lymphocytes expressing StpC are permissive for R5 strains of HIV-1 while Tip expression restricted R5 strains. These results suggest that intracellular immunization with Tip and StpC could be developed as models for therapeutic strategies targeting both X4 and R5 strains of HIV-1.