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.

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.

Development and Challenges of Nanotherapeutic Formulations for Targeting Mitochondrial Cell Death Pathways in Lung and Brain Degenerative Diseases.

Mitochondria are among the most dynamic organelles regulating a wide array of cellular processes. They are the cellular hub for oxidative phosphorylation, energy production, and cellular metabolism, and they are important determinants of cell fate, as they control cell death/survival pathways. The mitochondrial network plays a critical role in cellular inflammatory responses, and mitochondria are central in many pathologic conditions such as chronic inflammatory and aging-associated degenerative diseases. Recent advancements in our understanding of the pathogenic pathways and the role of mitochondria therein have identified highly specific therapeutic targets in order to develop personalized nanomedicine approaches for treatment. A wide array of nanoparticle-based formulations has been employed for potential usage in both diagnosing and treating chronic and fatal conditions, with gold nanoparticles and liposomal encapsulation being of particular interest. In this review, we highlight and summarize the advantages and challenges of developing these nanoformulations for targeted and spatiotemporally controlled drug delivery. We discuss the potential of nanotherapy in neoplasms to target the mitochondrial regulated cell death pathways and recent seminal developments in liposomal nanotherapy against chronic inflammatory lung diseases. The need for further development of nanoparticle-based treatment options for neuroinflammatory and neurodegenerative conditions, such as Alzheimer's disease (AD), is also discussed.

Alzheimer's disease: pathogenesis, diagnostics, and therapeutics.

Currently, 47 million people live with dementia globally, and it is estimated to increase more than threefold (~131 million) by 2050. Alzheimer's disease (AD) is one of the major causative factors to induce progressive dementia. AD is a neurodegenerative disease, and its pathogenesis has been attributed to extracellular aggregates of amyloid ß (Aß) plaques and intracellular neurofibrillary tangles made of hyperphosphorylated τ-protein in cortical and limbic areas of the human brain. It is characterized by memory loss and progressive neurocognitive dysfunction. The anomalous processing of APP by ß-secretases and γ-secretases leads to production of Aß40 and Aß42 monomers, which further oligomerize and aggregate into senile plaques. The disease also intensifies through infectious agents like HIV. Additionally, during disease pathogenesis, the presence of high concentrations of Aß peptides in central nervous system initiates microglial infiltration. Upon coming into vicinity of Aß, microglia get activated, endocytose Aß, and contribute toward their clearance via TREM2 surface receptors, simultaneously triggering innate immunoresponse against the aggregation. In addition to a detailed report on causative factors leading to AD, the present review also discusses the current state of the art in AD therapeutics and diagnostics, including labeling and imaging techniques employed as contrast agents for better visualization and sensing of the plaques. The review also points to an urgent need for nanotechnology as an efficient therapeutic strategy to increase the bioavailability of drugs in the central nervous system.

Magnetically guided non-invasive CRISPR-Cas9/gRNA delivery across blood-brain barrier to eradicate latent HIV-1 infection.

CRISPR-Cas9/gRNA exhibits therapeutic efficacy against latent human immunodeficiency virus (HIV) genome but the delivery of this therapeutic cargo to the brain remains as a challenge. In this research, for the first time, we demonstrated magnetically guided non-invasive delivery of a nano-formulation (NF), composed of Cas9/gRNA bound with magneto-electric nanoparticles (MENPs), across the blood-brain barrier (BBB) to inhibit latent HIV-1 infection in microglial (hµglia)/HIV (HC69) cells. An optimized ac-magnetic field of 60 Oe was applied on NF to release Cas9/gRNA from MENPs surface and to facilitate NF cell uptake resulting in intracellular release and inhibition of HIV. The outcomes suggested that developed NF reduced HIV-LTR expression significantly in comparison to unbound Cas9/gRNA in HIV latent hµglia/HIV (HC69) cells. These findings were also validated qualitatively using fluorescence microscopy to assess NF efficacy against latent HIV in the microglia cells. We believe that CNS delivery of NF (CRISPR/Cas9-gRNA-MENPs) across the BBB certainly will have clinical utility as future personalized nanomedicine to manage neuroHIV/AIDS.

Inhibition of Amyloid-Beta Production, Associated Neuroinflammation, and Histone Deacetylase 2-Mediated Epigenetic Modifications Prevent Neuropathology in Alzheimer's Disease in vitro Model.

Alzheimer's disease (AD) is a growing global threat to healthcare in the aging population. In the USA alone, it is estimated that one in nine persons over the age of 65 years is living with AD. The pathology is marked by the accumulation of amyloid-beta (Aß) deposition in the brain, which is further enhanced by the neuroinflammatory process. Nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are the major neuroinflammatory pathways that intensify AD pathogenesis. Histone deacetylase 2 (HDAC2)-mediated epigenetic mechanisms play a major role in the genesis and neuropathology of AD. Therefore, therapeutic drugs, which can target Aß production, NLRP3 activation, and HDAC2 levels, may play a major role in reducing Aß levels and the prevention of associated neuropathology of AD. In this study, we demonstrate that withaferin A (WA), an extract from Withania somnifera plant, significantly inhibits the Aß production and NF-κB associated neuroinflammatory molecules' gene expression. Furthermore, we demonstrate that cytokine release inhibitory drug 3 (CRID3), an inhibitor of NLRP3, significantly prevents inflammasome-mediated gene expression in our in vitro AD model system. We have also observed that mithramycin A (MTM), an HDAC2 inhibitor, significantly upregulated the synaptic plasticity gene expression and downregulated HDAC2 in SH-SY5Y cells overexpressing amyloid precursor protein (SH-APP cells). Therefore, the introduction of these agents targeting Aß production, NLRP3-mediated neuroinflammation, and HDAC2 levels will have a translational significance in the prevention of neuroinflammation and associated neurodegeneration in AD patients.

Multifunctional Nanotherapeutics for the Treatment of neuroAIDS in Drug Abusers.

HIV and substance abuse plays an important role in infection and disease progression. Further, the presence of persistent viral CNS reservoirs makes the complete eradication difficult. Thus, neutralizing the drug of abuse effect on HIV-1 infectivity and elimination of latently infected cells is a priority. The development of a multi-component [antiretroviral drugs (ARV), latency reactivating agents (LRA) and drug abuse antagonist (AT)] sustained release nanoformulation targeting the CNS can overcome the issues of HIV-1 cure and will help in improving the drug adherence. The novel magneto-liposomal nanoformulation (NF) was developed to load different types of drugs (LRAs, ARVs, and Meth AT) and evaluated for in-vitro and in-vivo BBB transmigration and antiviral efficacy in primary CNS cells. We established the HIV-1 latency model using human astrocyte cells (HA) and optimized the dose of LRA for latency reversal, Meth AT in in-vitro cell culture system. Further, PEGylated magneto-liposomal NF was developed, characterized for size, shape, drug loading and BBB transport in-vitro. Results showed that drug released in a sustained manner up to 10 days and able to reduce the HIV-1 infectivity up to ~40-50% (>200 pg/mL to <100 pg/mL) continuously using single NF treatment ± Meth treatment in-vitro. The magnetic treatment (0.8 T) was able to transport (15.8% ± 5.5%) NF effectively without inducing any toxic effects due to NF presence in the brain. Thus, our approach and result showed a way to eradicate HIV-1 reservoirs from the CNS and possibility to improve the therapeutic adherence to drugs in drug abusing (Meth) population. In conclusion, the developed NF can provide a better approach for the HIV-1 cure and a foundation for future HIV-1 purging strategies from the CNS using nanotechnology platform.

A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein.

This work, as a proof of principle, presents a sensitive and selective electrochemical immunosensor for Zika-virus (ZIKV)-protein detection using a functionalized interdigitated micro-electrode of gold (IDE-Au) array. A miniaturized IDE-Au immunosensing chip was prepared via immobilization of ZIKV specific envelop protein antibody (Zev-Abs) onto dithiobis(succinimidyl propionate) i.e., (DTSP) functionalized IDE-Au (electrode gap/width of 10 µm). Electrochemical impedance spectroscopy (EIS) was performed to measure the electrical response of developed sensing chip as a function of ZIKV-protein concentrations. The results of EIS studies confirmed that sensing chip detected ZIKV-protein selectively and exhibited a detection range from 10 pM to 1 nM and a detection limit of 10 pM along with a high sensitivity of 12 kΩM-1. Such developed ZIKV immune-sensing chip can be integrated with a miniaturized potentiostat (MP)-interfaced with a smartphone for rapid ZIKV-infection detection required for early stage diagnostics at point-of-care application.

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.

Investigation of ac-magnetic field stimulated nanoelectroporation of magneto-electric nano-drug-carrier inside CNS cells.

In this research, we demonstrate cell uptake of magneto-electric nanoparticles (MENPs) through nanoelectroporation (NEP) using alternating current (ac)-magnetic field stimulation. Uptake of MENPs was confirmed using focused-ion-beam assisted transmission electron microscopy (FIB-TEM) and validated by a numerical simulation model. The NEP was performed in microglial (MG) brain cells, which are highly sensitive for neuro-viral infection and were selected as target for nano-neuro-therapeutics. When the ac-magnetic field optimized (60 Oe at 1 kHz), MENPs were taken up by MG cells without affecting cell health (viability > 92%). FIB-TEM analysis of porated MG cells confirmed the non-agglomerated distribution of MENPs inside the cell and no loss of their elemental and crystalline characteristics. The presented NEP method can be adopted as a part of future nanotherapeutics and nanoneurosurgery strategies where a high uptake of a nanomedicine is required for effective and timely treatment of brain diseases.

Development of magneto-plasmonic nanoparticles for multimodal image-guided therapy to the brain.

Magneto-plasmonic nanoparticles are one of the emerging multi-functional materials in the field of nanomedicine. Their potential for targeting and multi-modal imaging is highly attractive. In this study, magnetic core/gold shell (MNP@Au) magneto-plasmonic nanoparticles were synthesized by citrate reduction of Au ions on magnetic nanoparticle seeds. Hydrodynamic size and optical properties of magneto-plasmonic nanoparticles synthesized with the variation of Au ions and reducing agent concentrations were evaluated. The synthesized magneto-plasmonic nanoparticles exhibited superparamagnetic properties, and their magnetic properties contributed to the concentration-dependent contrast in magnetic resonance imaging (MRI). The imaging contrast from the gold shell part of the magneto-plasmonic nanoparticles was also confirmed by X-ray computed tomography (CT). The transmigration study of the magneto-plasmonic nanoparticles using an in vitro blood-brain barrier (BBB) model proved enhanced transmigration efficiency without disrupting the integrity of the BBB, and showed potential to be used for brain diseases and neurological disorders.

Development of TIMP1 magnetic nanoformulation for regulation of synaptic plasticity in HIV-1 infection.

Although the introduction of antiretroviral therapy has reduced the prevalence of severe forms of neurocognitive disorders, human immunodeficiency virus (HIV)-1-associated neurocognitive disorders were observed in 50% of HIV-infected patients globally. The blood-brain barrier is known to be impermeable to most of antiretroviral drugs. Successful delivery of antiretroviral drugs into the brain may induce an inflammatory response, which may further induce neurotoxicity. Therefore, alternate options to antiretroviral drugs for decreasing the HIV infection and neurotoxicity may help in reducing neurocognitive impairments observed in HIV-infected patients. In this study, we explored the role of magnetic nanoparticle (MNP)-bound tissue inhibitor of metalloproteinase-1 (TIMP1) protein in reducing HIV infection levels, oxidative stress, and recovering spine density in HIV-infected SK-N-MC neuroblastoma cells. We did not observe any neuronal cytotoxicity with either the free TIMP1 or MNP-bound TIMP1 used in our study. We observed significantly reduced HIV infection in both solution phase and in MNP-bound TIMP1-exposed neuronal cells. Furthermore, we also observed significantly reduced reactive oxygen species production in both the test groups compared to the neuronal cells infected with HIV alone. To observe the effect of both soluble-phase TIMP1 and MNP-bound TIMP1 on spine density in HIV-infected neuronal cells, confocal microscopy was used. We observed significant recovery of spine density in both the test groups when compared to the cells infected with HIV alone, indicting the neuroprotective effect of TIMP1. Therefore, our results suggest that the MNP-bound TIMP1 delivery method across the blood-brain barrier can be used for reducing HIV infectivity in brain tissue and neuronal toxicity in HIV-infected patients.

Profile of Class I Histone Deacetylases (HDAC) by Human Dendritic Cells after Alcohol Consumption and In Vitro Alcohol Treatment and Their Implication in Oxidative Stress: Role of HDAC Inhibitors Trichostatin A and Mocetinostat.

Epigenetic mechanisms have been shown to play a role in alcohol use disorders (AUDs) and may prove to be valuable therapeutic targets. However, the involvement of histone deacetylases (HDACs) on alcohol-induced oxidative stress of human primary monocyte-derived dendritic cells (MDDCs) has not been elucidated. In the current study, we took a novel approach combining ex vivo, in vitro and in silico analyses to elucidate the mechanisms of alcohol-induced oxidative stress and role of HDACs in the periphery. ex vivo and in vitro analyses of alcohol-modulation of class I HDACs and activity by MDDCs from self-reported alcohol users and non-alcohol users was performed. Additionally, MDDCs treated with alcohol were assessed using qRT-PCR, western blot, and fluorometric assay. The functional effects of alcohol-induce oxidative stress were measured in vitro using PCR array and in silico using gene expression network analysis. Our findings show, for the first time, that MDDCs from self-reported alcohol users have higher levels of class I HDACs compare to controls and alcohol treatment in vitro differentially modulates HDACs expression. Further, HDAC inhibitors (HDACi) blocked alcohol-induction of class I HDACs and modulated alcohol-induced oxidative stress related genes expressed by MDDCs. In silico analysis revealed new target genes and pathways on the mode of action of alcohol and HDACi. Findings elucidating the ability of alcohol to modulate class I HDACs may be useful for the treatment of alcohol-induced oxidative damage and may delineate new potential immune-modulatory mechanisms.

Effect of Cocaine on HIV Infection and Inflammasome Gene Expression Profile in HIV Infected Macrophages.

We have observed significantly increased HIV infection in HIV infected macrophages in the presence of cocaine that could be due to the downregulation of BST2 restriction factor in these cells. In human inflammasome PCR array, among different involved in inflammasome formation, in HIV infected macrophages in the presence of cocaine, we have observed significant upregulation of NLRP3, AIM2 genes and downstream genes IL-1ß and PTGS2. Whereas negative regulatory gene MEFV was upregulated, CD40LG and PYDC1 were significantly downregulated. Among various NOD like receptors, NOD2 was significantly upregulated in both HIV alone and HIV plus cocaine treated cells. In the downstream genes, chemokine (C-C motif) ligand 2 (CCL2), CCL7 and IL-6 were significantly up regulated in HIV plus cocaine treated macrophages. We have also observed significant ROS production (in HIV and/or cocaine treated cells) which is one of the indirect-activators of inflammasomes formation. Further, we have observed early apoptosis in HIV alone and HIV plus cocaine treated macrophages which may be resultant of inflammasome formation and cspase-1 activation. These results indicate that in case of HIV infected macrophages exposed to cocaine, increased ROS production and IL-1ß transcription serve as an activators for the formation of NLRP3 and AIM2 mediated inflammasomes that leads to caspase 1 mediated apoptosis.

Magnetically guided central nervous system delivery and toxicity evaluation of magneto-electric nanocarriers.

Least component-based delivery of drug-tagged-nanocarriers across blood-brain-barriers (BBB) will allow site-specific and on-demand release of therapeutics to prevent CNS diseases. We developed a non-invasive magnetically guided delivery of magneto-electric nanocarriers (MENCs), ~20 nm, 10 mg/kg, across BBB in C57Bl/J mice. Delivered MENCs were uniformly distributed inside the brain, and were non-toxic to brain and other major organs, such as kidney, lung, liver, and spleen, and did not affect hepatic, kidney and neurobehavioral functioning.

Platelets Contribute to BBB Disruption Induced by HIV and Alcohol.

The role of platelets in the neurological diseases that underlie cognitive impairment has attracted increasing attention in recent years. Multiple pathways in platelets contribute to host defenses, as well as to CNS function. In the current study, we hypothesize that the Blood Brain Barrier (BBB) is disrupted when exposed to platelets from patients with triple Co-morbidity (hazardous alcohol users+ HIV+ thrombocytopenia), compared to those with dual, single or no morbidity (HIV only, alcohol only or healthy controls).

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.

DJ1 expression downregulates in neuroblastoma cells (SK-N-MC) chronically exposed to HIV-1 and cocaine.

BACKGROUND: HIV-associated neurological disorder (HAND) has long been recognized as a consequence of human immunodeficiency virus (HIV) infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson's disease) in regulating dopamine (DA) transmission and reactive oxygen species (ROS) production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder. METHODS: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC). Gene and protein expression analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry. Effect of DJ1 dysregulation on oxidative stress was analyzed by measuring ROS production in these cells. RESULTS: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine which is inversely proportional to ROS production. CONCLUSION: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1 and/or cocaine indicating oxidative stress level of DA neurons.

Investigation of Neuropathogenesis in HIV-1 Clade B and C Infection Associated with IL-33 and ST2 Regulation.

In present research work, for the first time, we demonstrate that neuropathogenesis in HIV-1 clade B and C infection is associated with IL-33 and ST2 dysregulation, that is, implication toward neuropathogenesis. It is known that neuropathogenesis of HIV infected individuals is clade dependent. Proinflammatory cytokines and related receptors play a significant role in the complex regulatory mechanisms of neuropathogenesis in HIV-1 infection. Among them, IL-33 is an inflammatory cytokine expressed in the central nervous system (CNS) and activates microglia cells and may affect neuroimmune inflammatory processes involved in HIV neuropathogenesis. Beside this, IL-33 receptor (ST2) plays a role in neuroinflammatory processes through the modulation of the biological action of IL-33. quantitative real time PCR (qRT-PCR), ELISA, Western blot (WB), and flow cytometry experiments were performed to elucidate the role of IL-33/ST2 in HIV neuropathogenesis in CNS cells. Apoptosis and mechanisms of IL-33 in neuronal cells were studied using caspase-3 assay and RT-PCR. Results of the studies suggest that the infection in CNS cells with HIV-1 clade B resulted in higher levels of IL-33/ST2L expression compared to HIV-1 clade C infection. Furthermore, higher concentrations of IL-33 were associated with a decrease in myocyte enhancer factor 2C (MEF2C) expression, a transcription factor that regulates synaptic function, and an increase in apoptosis, NOD2, and SLC11A1 in clade B infection. This led to neuroinflammation which dysregulates synaptic function and apoptosis. These parameters are common in neuroAIDS provoked by HIV infection.

Electrochemical sensing method for point-of-care cortisol detection in human immunodeficiency virus-infected patients.

A novel electrochemical sensing method was devised for the first time to detect plasma cortisol, a potential psychological stress biomarker, in human immunodeficiency virus (HIV)-positive subjects. A miniaturized potentiostat (reconfigured LMP91000 chip) interfaced with a microfluidic manifold containing a cortisol immunosensor was employed to demonstrate electrochemical cortisol sensing. This fully integrated and optimized electrochemical sensing device exhibited a wide cortisol-detection range from 10 pg/mL to 500 ng/mL, a low detection limit of 10 pg/mL, and sensitivity of 5.8 µA (pg mL)(-1), with a regression coefficient of 0.995. This cortisol-selective sensing system was employed to estimate plasma cortisol in ten samples from HIV patients. The electrochemical cortisol-sensing performance was validated using an enzyme-linked immunosorbent assay technique. The results obtained using both methodologies were comparable within 2%-5% variation. The information related to psychological stress of HIV patients can be correlated with disease-progression parameters to optimize diagnosis, therapeutic, and personalized health monitoring.