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Background: HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s).Methods: Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP).Results: Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication.Conclusions: We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS. [ABSTRACT FROM AUTHOR]

HIV entry into the CNS is an early event after peripheral infection, resulting in neurologic dysfunction in a significant number of individuals despite successful anti-retroviral therapy. The mechanisms by which HIV mediates CNS dysfunction are not well understood. Our group recently demonstrated that HIV infection of astrocytes results in survival of HIV infected cells and apoptosis of surrounding uninfected astrocytes by the transmission of toxic intracellular signals through gap junctions. In the current report, we characterize the intracellular signaling responsible for this bystander apoptosis. Here, we demonstrate that HIV infection of astrocytes results in release of cytochrome C from the mitochondria into the cytoplasm, and dysregulation of inositol trisphosphate/intracellular calcium that leads to toxicity to neighboring uninfected astrocytes. Blocking these dysregulated pathways results in protection from bystander apoptosis. These secondary messengers that are toxic in uninfected cells are not toxic in HIV infected cells, suggesting that HIV protects these cells from apoptosis. Thus, our data provide novel mechanisms of HIV mediated toxicity and generation of HIV reservoirs. Our findings provide new potential therapeutic targets to reduce the CNS damage resulting from HIV infection and to eradicate the generation of viral reservoirs. [ABSTRACT FROM AUTHOR]

Macrophages play a significant role in HIV infection, viral rebound, and the development of AIDS. However, the function of host proteins in viral replication is incompletely characterized in macrophages. Purinergic receptors P2X and P2Y are major components of the macrophage immune response to pathogens, inflammation, and cellular damage. We demonstrate that these receptors are necessary for HIV infection of primary human macrophages. Inhibition of purinergic receptors results in a significant reduction in HIV replication in macrophages. This inhibition is independent of viral strain and is dose dependent. We also identify that P2X1, P2X7, and P2Y1 receptors are involved in viral replication. We show that P2X1, but not P2X7 or P2Y1, is necessary for HIV entry into macrophages. We demonstrate that interaction of the HIV surface protein gpl20 with macrophages stimulates an increase in ATP release. Thus, we propose that HIV's binding to macrophages triggers a local release of ATP that stimulates purinergic receptors and facilitates HIV entry and subsequent stages of viral replication. Our data implicate a novel role for a family of host proteins in HIV replication in macrophages and suggest new therapeutic targets to reduce the devastating consequences of HIV infection and AIDS. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) entry into the CNS is an early event after infection, resulting in neurological dysfunction in a significant number of individuals. As people with acquired immunodeficiency syndrome (AIDS) live longer, the prevalence of cognitive impairment is increasing, despite antiretroviral therapy. The mechanisms that mediate CNS dysfunction are still not completely understood, and include inflammation, viral presence, and/or replication. In this report, we characterize a novel role of gap junctions in transmitting and thereby amplifying toxic signals originating from HIV-infected astrocytes that trigger cell death in uninfected astrocytes. HIV-infected astrocytes were resistant to apoptosis; however, uninfected astrocytes forming gap junctions with infected astrocytes were apoptotic. Gap junction blockers abolished apoptosis in uninfected astrocytes, supporting the role of these channels in amplifying cell death. Our findings describe a novel mechanism of toxicity within the brain, triggered by low numbers of HIV-infected astrocytes and amplified by gap junctions, contributing to the pathogenesis of NeuroAIDS. [ABSTRACT FROM AUTHOR]

Leukocyte transmigration across the blood–brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review. [Copyright &y& Elsevier]

The article offers information on the antibody-targeted positron emission tomography (immunoPET), that can visualize replication sites of HIV virus. It mentions that the method allows the noninvasive imaging of simian immunodeficiency virus (SIV) viral reservoirs responsible for SIV persistence in macaques. It also focuses on noninvasive nuclear imaging which is used in diagnosis, prognosis and follow-up of treated patients for clinical oncology and cardiology.

Persistent inflammation contributes to the development of cardiovascular disease (CVD) as an HIV-associated comorbidity. Innate immune cells such as monocytes are major drivers of inflammation in men and women with HIV. The study objectives are to examine the contribution of circulating non-classical monocytes (NCM, CD14dimCD16+) and intermediate monocytes (IM, CD14+CD16+) to the host response to long-term HIV infection and HIV-associated CVD. Women with and without chronic HIV infection (H) were studied. Subclinical CVD (C) was detected as plaques imaged by B-mode carotid artery ultrasound. The study included H-C-, H+C-, H-C+, and H+C+ participants (23 of each, matched on race/ethnicity, age and smoking status), selected from among enrollees in the Women's Interagency HIV Study. We assessed transcriptomic features associated with HIV or CVD alone or comorbid HIV/CVD comparing to healthy (H-C-) participants in IM and NCM isolated from peripheral blood mononuclear cells. IM gene expression was little affected by HIV alone or CVD alone. In IM, coexisting HIV and CVD produced a measurable gene transcription signature, which was abolished by lipid-lowering treatment. In NCM, versus non-HIV controls, women with HIV had altered gene expression, irrespective of whether or not they had comorbid CVD. The largest set of differentially expressed genes was found in NCM among women with both HIV and CVD. Genes upregulated in association with HIV included several potential targets of drug therapies, including LAG3 (CD223). In conclusion, circulating monocytes from patients with well controlled HIV infection demonstrate an extensive gene expression signature which may be consistent with the ability of these cells to serve as potential viral reservoirs. Gene transcriptional changes in HIV patients were further magnified in the presence of subclinical CVD. [ABSTRACT FROM AUTHOR]

HIV-1 enters the CNS soon after peripheral infection and causes chronic neuroinflammation and neuronal damage that leads to cognitive impairment in 40-70% of HIV-infected people. The nonpathogenic cellular isoform of the human prion protein (PrPc) is an adhesion molecule constitutively expressed in the CNS. Previously, our laboratory showed that shed PrPc (sPrPc) is increased in the cerebrospinal fluid of HIV-infected people with cognitive deficits as compared with infected people with no impairment. In this article, we demonstrate that CCL2 and TNF-a, inflammatory mediators that are elevated in the CNS of HIV-infected people, increase shedding of PrPc from human astrocytes by increasing the active form of the metalloprotease ADAM10.We show that the consequence of this shedding can be the production of inflammatory mediators, because treatment of astrocytes with rPrPc increased secretion of CCL2, CXCL-12, and IL-8. Supernatants from rPrPc-treated astrocytes containing factors produced in response to this treatment, but not rPrPc by itself, cause increased chemotaxis of both uninfected and HIV-infected human monocytes, suggesting a role for sPrPc in monocyte recruitment into the brain. Furthermore, we examined whether PrPc participates in glutamate uptake and found that rPrPc decreased uptake of this metabolite in astrocytes, which could lead to neurotoxicity and neuronal loss. Collectively, our data characterize mediators involved in PrPc shedding and the effect of this sPrPc on monocyte chemotaxis and glutamate uptake from astrocytes. We propose that shedding of PrPc could be a potential target for therapeutics to limit the cognitive impairment characteristic of neuroAIDS. [ABSTRACT FROM AUTHOR]

Analyzes whether chemokines can induce their own expression or that of other chemokines using in vitro culture system. Detection of monocyte chemoattractant protein-1 (MCP-1) expression regardless of chemokine treatment; Effect of the treatment with macrophage inflammatory protein (MIP-1) alpha and MPI-1 beta on the expression of endogenous MCP-1.

Background and purpose: People with human immunodeficiency virus (HIV; PWH) present a complex array of immunologic and medical disorders that impact brain structure and metabolism, complicating the interpretation of neuroimaging. This pilot study of well‐characterized multi‐morbid PWH examined how medical and immunologic factors predicted brain characteristics on proton MR spectroscopy (1H‐MRS) and diffusion‐weighted imaging (DWI). Methods: Eighteen individuals on combination antiretroviral therapy (cART), with mean age of 56 years, underwent medical history review, neuroimaging, and on the day of imaging, blood draw for assay of 20 plasma cytokines and flow cytometric characterization of peripheral blood mononuclear cell subsets. Predictors of n‐acetyl aspartate, choline, myoinositol, glutamate/glutamine, fractional anisotropy and mean diffusivity were identified through bivariate correlation; those significant at p <.1000 were advanced to multivariate analysis, with models created for each neuroimaging outcome. Results: Monocyte subsets and diverse cytokines accounted for 16 of 25 (64%) variables predicting 1H‐MRS spectra in frontal gray and white matter and basal ganglia; monocyte subsets did not predict any DWI characteristic. In contrast, age, presence of hypertension, and duration of HIV infection accounted for 13 of 25 (52%) variables predicting diffusion characteristics in the corpus callosum, thalamic radiations, and basal ganglia but only 3 of 25 (12%) predictors of 1H‐MRS features. Conclusions: 1H‐MRS neurometabolites were most often predicted by immunologic factors sensitive to temporal variation, whereas DWI metrics were more often related to longer‐term disease state. In multi‐morbid cART‐era populations, selection and interpretation of neuroimaging modalities should account for complex temporal and pathogenetic influences of immunologic abnormality, disease state, and aging. [ABSTRACT FROM AUTHOR]

Abstract: Macrophages are crucial to host defense, functioning in innate and cell-mediated immunity. MicroRNAs (miRNAs) are small non-coding RNA molecules that repress transcription and protein production. Little is known about miRNA expression in primary human macrophages, or about how macrophage miRNAs contribute to both normal macrophage function and to the pathogenesis of disease in humans. Using Western blot analyses, we demonstrated the production of miRNA machinery proteins by human primary macrophages. Using two different miRNA array techniques, we identified 119 miRNAs expressed by human primary macrophages, including hsa-let-7a, miR-16, -23a, 30b, -103, -146a, -212, and -378 and validated them by quantitative RT-PCR. Our findings provide a knowledge base to which macrophage miRNA expression in organ-specific macrophages or disease processes may be compared in humans. [Copyright &y& Elsevier]

HIV enters the CNS early after peripheral infection, establishing reservoirs in perivascular macrophages that contribute to development of HIV-associated neurocognitive disorders (HAND) in 15–40% of people with HIV (PWH) despite effective antiretroviral therapy (ART). Opioid use may contribute to dysregulated macrophage functions resulting in more severe neurocognitive symptoms in PWH taking opioids. Macroautophagy helps maintain quality control in long-lived cell types, such as macrophages, and has been shown to regulate, in part, some macrophage functions in the CNS that contribute to HAND. Using Western blotting and confocal immunofluorescence in primary human macrophages, we demonstrated that morphine and a commonly prescribed ART regimen induce bulk autophagy. Morphine and ART also inhibited completion of autophagy. HIV infection increased these inhibitory effects. We also examined two types of selective autophagy that degrade aggregated proteins (aggrephagy) and dysfunctional mitochondria (mitophagy). Morphine and ART inhibited selective autophagy mediated by p62 regardless of HIV infection, and morphine inhibited mitophagic flux in HIV-infected cells demonstrating potential mitotoxicity. These results indicate that inhibition of autophagy, both in bulk and selective, in CNS macrophages may mediate neurocognitive dysfunction in PWH using opioids. Increasing autophagic activity in the context of HIV may represent a novel therapeutic strategy for reducing HAND in these individuals. [ABSTRACT FROM AUTHOR]

Chemokines are important mediators of inflammation. It has been demonstrated that there is an increase in chemokine expression in both the sera and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1). The HIV-1 viral protein, Tat, a transcriptional regulator, has been detected in the central nervous system (CNS) of infected individuals, and has been demonstrated to induce chemokines from various cells within the brain. The authors now show that the interaction of human microglia, the resident phagocytes of the brain, with Tat leads to dramatic increases in the secretion of the chemokines CCL2, CXCL8, CXCL10, CCL3, CCL4, and CCL5. Treatment of microglia with Tat plus specific inhibitors of signal transduction pathways demonstrated that the induction of each chemokine is regulated differently. Tat-induced expression of CCL2 and CCL4 was mediated by the activation of the extracellular regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the induction of CXCL8 and CCL3 was mediated only by the p38 MAPK pathway. Tat-induced CXCL10 expression was mediated, to some extent, by activation of the ERK1/2 MAPK pathway, phosphatidylinositol 3-kinase pathway, and the p38 MAPK pathway, whereas CCL5 expression was not mediated by any pathway tested. Western blot analysis demonstrated phosphorylation of ERK 1/2 and Akt upon stimulation of microglia with Tat. These data suggest that a soluble HIV-1 viral protein can alter the chemokine balance in the brain, which can then lead to an influx of inflammatory cells and contribute to the neuropathogenesis of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Nestin expression in the developing human brain was examined with the use of unique human specific anti-nestin antibodies. Double immunostaining of cell cultures and tissue sections derived from first and second trimester human fetal brain (HFB) examined the co-expression of nestin with other cell type specific phenotypic markers. The immunocytochemical analysis shows that from first to second trimester, the majority of developing glial cells exhibited a transitional state marked by co-expression of nestin and GFAP. However, the corresponding transitional state for developing neuronal cells, co-expressing nestin and MAP-2, was rarely detected. These results imply different temporal patterns of nestin expression in cells of glial and neuronal lineages. Confocal microscopy of HFB tissue section staining also revealed a similar pattern of nestin co-expression with glial and neuronal markers. Our results suggest that nestin expression alone may not identify an undifferentiated stem cell, and that progenitor cells in glial and neuronal lineages express nestin in different temporal patterns. [Copyright &y& Elsevier]

Antiretroviral drugs have dramatically improved the morbidity and mortality of people living with HIV (PLWH). While current antiretroviral therapy (ART) regimens are generally well-tolerated, risks for side effects and toxicity remain as PLWH must take life-long medications. Antiretroviral drugs impact autophagy, an intracellular proteolytic process that eliminates debris and foreign material, provides nutrients for metabolism, and performs quality control to maintain cell homeostasis. Toxicity and adverse events associated with antiretrovirals may be due, in part, to their impacts on autophagy. A more complete understanding of the effects on autophagy is essential for developing antiretroviral drugs with decreased off target effects, meaning those unrelated to viral suppression, to minimize toxicity for PLWH. This review summarizes the findings and highlights the gaps in our knowledge of the impacts of antiretroviral drugs on autophagy. [ABSTRACT FROM AUTHOR]

• Purified Hemozoid is internalized into human primary cells. • Internalization triggers toxicity in neurons and astrocytes. • Hemozoid does not need additional host and parasite factors to be toxic. Malaria, caused by an intracellular protozoan parasite of the genus Plasmodium , is one of the most important infectious diseases worldwide. In 2017, a total of 219 millions cases were reported with 435,000 deaths related to malaria. A major complication of malaria infection is cerebral malaria (CM), characterized by enhanced blood-brain barrier permeability, leukocyte infiltration and/or activation, and neuronal dropout resulting in coma and death in significant numbers of individuals, especially children. Despite the high incidence and mortality, the pathogenesis of cerebral malaria is not well characterized. Hemozoin (HMZ) or "malaria pigment," a by-product of intraerythrocytic parasite-mediated hemoglobin catabolism, is released into the bloodstream after lysis of the host infected erythrocyte. The effects of HMZ on brain cells has not been studied due to the contamination/adhesion/aggregation of the HMZ with host and toxic parasitic factors. We now demonstrate that extracellular purified HMZ is taken up by human neurons and astrocytes, resulting in cellular dysfunction and toxicity. These findings contribute to a better understanding of the neuropathogenesis of CM and provide evidence that HMZ accumulation in the bloodstream could result in CNS compromise. Thus, alternative approaches to reducing circulating HMZ could serve as a potential treatment. [ABSTRACT FROM AUTHOR]

cART has significantly improved the life expectancy of people living with HIV (PLWH). However, it fails to eliminate the long-lived reservoir of latent HIV-infected cells. Radioimmunotherapy (RIT) relies on antigen-specific monoclonal antibodies (mAbs) for targeted delivery of lethal doses of ionizing radiation to cells. Previously, we have demonstrated that human mAb 2556 against HIV gp41 conjugated with 213Bismuth radioisotope (t 1/2 = 46 min, alpha-emitter) selectively killed HIV-infected cells. 225Actinium (t 1/2 = 9.92 d, alpha-emitter) and 177Lutetium (t 1/2 = 6.7 d, beta-emitter) are two long-lived clinically proven radioisotopes for cancer treatment which might be more effective in killing infected cells systemically and in CNS. In this study we have conjugated 2556 mAb with 213Bi, 225Ac and 177Lu, and compared their ability to kill HIV-infected human peripheral blood mononuclear cells (PBMCs) and monocytes. PBMCs and monocytes from healthy donors were infected with HIV p49.5 and treated in vitro with increasing concentrations of 213Bi (4–20 μCi)-, 225Ac (20–100 nCi)- and 177Lu (4–50 μCi)-2556 mAb. After three days post-treatment of infected PBMCs and monocytes, 213Bi- and 177Lu-conjugated 2556 mAb reduced virus production measured by p24 level in a dose-dependent manner, whereas, 225Ac-2556 showed minimal effect. However, seven days post-treatment all three radioisotopes showed significantly more pronounced reduction of virus replication as compared to control labeled mAb with 225Ac-2556 showing the least non-specific killing. These results indicate that RIT holds promise as a novel treatment option for the eradication of HIV-infected cells that merits further study in combination with cART and reactivation drugs. [ABSTRACT FROM AUTHOR]

Despite the success of cART, greater than 50% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND). Macrophages are the major cell type infected in the CNS. Unlike for T cells, the virus does not kill macrophages and these long-lived cells may become HIV reservoirs in the brain. They produce cytokines/chemokines and viral proteins that promote inflammation and neuronal damage, playing a key role in HIV neuropathogenesis. HIV Tat is the transactivator of transcription that is essential for replication and transcriptional regulation of the virus and is the first protein to be produced after HIV infection. Even with successful cART, Tat is produced by infected cells. In this study we examined the role of the HIV Tat protein in the regulation of gene expression in human macrophages. Using THP-1 cells, a human monocyte/macrophage cell line, and their infection with lentivirus, we generated stable cell lines that express Tat-Flag. We performed ChIP-seq analysis of these cells and found 66 association sites of Tat in promoter or coding regions. Among these are C5, CRLF2/TSLPR, BDNF, and APBA1/Mint1, genes associated with inflammation/damage. We confirmed the association of Tat with these sequences by ChIP assay and expression of these genes in our THP-1 cell lines by qRT-PCR. We found that HIV Tat increased expression of C5, APBA1, and BDNF, and decreased CRLF2. The K50A Tat-mutation dysregulated expression of these genes without affecting the binding of the Tat complex to their gene sequences. Our data suggest that HIV Tat, produced by macrophage HIV reservoirs in the brain despite successful cART, contributes to neuropathogenesis in HIV-infected people. [ABSTRACT FROM AUTHOR]

Background: Cerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies. Methods: To examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume. Results: The metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability. Conclusions: Taken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM. [ABSTRACT FROM AUTHOR]

Despite successful combined antiretroviral therapy, ~60% of HIV-infected people exhibit HIV-associated neurocognitive disorders (HAND). CCL2 is elevated in the CNS of infected people with HAND and mediates monocyte influx into the CNS, which is critical in neuroAIDS. Many HIV-infected opiate abusers have increased neuroinflammation that may augment HAND. Buprenorphine is used to treat opiate addiction. However, there are few studies that examine its impact on HIV neuropathogenesis. We show that buprenorphine reduces the chemotactic phenotype of monocytes. Buprenorphine decreases the formation of membrane projections in response to CCL2. It also decreases CCL2-induced chemotaxis and mediates a delay in reinsertion of the CCL2 receptor, CCR2, into the cell membrane after CCL2-mediated receptor internalization, suggesting a mechanism of action of buprenorphine. Signaling pathways in CCL2-induced migration include increased phosphorylation of p38 MAPK and of the junctional protein JAM-A. We show that buprenorphine decreases these phosphorylations in CCL2-treated monocytes. Using DAMGO, CTAP, and Nor-BNI, we demonstrate that the effect of buprenorphine on CCL2 signaling is opioid receptor mediated. To identify additional potential mechanisms by which buprenorphine inhibits CCL2-induced monocyte migration, we performed proteomic analyses to characterize additional proteins in monocytes whose phosphorylation after CCL2 treatment was inhibited by buprenorphine. Leukosialin and S100A9 were identified and had not been shown previously to be involved in monocyte migration. We propose that buprenorphine limits CCL2-mediated monocyte transmigration into the CNS, thereby reducing neuroinflammation characteristic of HAND. Our findings underscore the use of buprenorphine as a therapeutic for neuroinflammation as well as for addiction. [ABSTRACT FROM AUTHOR]

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions. [ABSTRACT FROM AUTHOR]

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. [ABSTRACT FROM AUTHOR]

Background: Maternal thyroid hormones play a fundamental role in appropriate fetal development during gestation. Offspring that have been gestated under maternal hypothyroidism suffer cognitive impairment. Thyroid hormone deficiency during gestation can significantly impact the central nervous system by altering the migration, differentiation, and function of neurons, oligodendrocytes, and astrocytes. Given that gestational hypothyroidism alters the immune cell ratio in offspring, it is possible that this condition could result in higher sensitivity for the development of autoimmune diseases. Methods: Adult mice gestated under hypothyroidism were induced with experimental autoimmune encephalomyelitis (EAE). Twenty-one days after EAE induction, the disease score, myelin content, immune cell infiltration, and oligodendrocyte death were evaluated. Results: We observed that mice gestated under hypothyroidism showed higher EAE scores after disease induction during adulthood compared to mice gestated in euthyroidism. In addition, spinal cord sections of mice gestated under hypothyroidism that suffered EAE in adulthood showed higher demyelination, CD4+ and CD8+ infiltration, and increased oligodendrocyte death. Conclusions: These results show for the first time that a deficiency in maternal thyroid hormones during gestation can influence the outcome of a central nervous system inflammatory disease, such as EAE, in their offspring. These data strongly support evaluating thyroid hormones in pregnant women and treating hypothyroidism during pregnancy to prevent increased susceptibility to inflammatory diseases in the central nervous system of offspring. [ABSTRACT FROM AUTHOR]

Background: Perivascular macrophages and microglia are critical to CNS function. Drugs of abuse increase extracellular dopamine in the CNS, exposing these cells to elevated levels of dopamine. In rodent macrophages and human T-cells, dopamine was shown to modulate cellular functions through activation of dopamine receptors and other dopaminergic proteins. The expression of these proteins and the effects of dopamine on human macrophage functions had not been studied. Methods: To study dopaminergic gene expression, qRT-PCR was performed on mRNA from primary human monocyte derived macrophages (MDM). Expression and localization of dopaminergic proteins was examined by immunoblotting isolated plasma membrane, total membrane and cytosolic proteins from MDM. To characterize dopamine-mediated changes in cytokine production in basal and inflammatory conditions, macrophages were treated with different concentrations of dopamine in the presence or absence of LPS and cytokine production was assayed by ELISA. Statistical significance was determined using two-tailed Students’ T-tests or Wilcoxen Signed Rank tests. Results: These data show that MDM express mRNA for all five subtypes of dopamine receptors, and that dopamine receptors 3 and 4 are expressed on the plasma membrane. MDM also express mRNA for the dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC). DAT is expressed on the plasma membrane, VMAT2 on cellular membranes and TH and AADC are in the cytosol. Dopamine also alters macrophage cytokine production in both untreated and LPS-treated cells. Untreated macrophages show dopamine mediated increases IL-6 and CCL2. Macrophages treated with LPS show increased IL-6, CCL2, CXCL8 and IL-10 and decreased TNF-α. Conclusions: Monocyte derived macrophages express dopamine receptors and other dopaminergic proteins through which dopamine may modulate macrophage functions. Thus, increased CNS dopamine levels due to drug abuse may exacerbate the development of neurological diseases including Alzheimer’s disease and HIV associated neurological disorders. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To characterize acute (postprandial) and chronic (after a 6-month period of weight loss) effects of a low-carbohydrate vs. a low-fat diet on subclinical markers of cardiovascular disease (CVD) in adults with type 2 diabetes. Design: At baseline and 6 months, measures of C-reactive protein (CRP), interleukin-6 (IL-6), soluble intercellular adhesion molecule (sICAM) and soluble E-selectin were obtained from archived samples (n=51) of participants randomized in a clinical trial comparing a low-carbohydrate and a low-fat diet. In a subset of participants (n=27), postprandial measures of these markers were obtained 3 h after a low-carbohydrate or low-fat liquid meal. Endothelial function was also measured by reactive hyperemic peripheral arterial tonometry during the meal test. Paired t tests and unpaired t tests compared within- and between-group changes. Results: There were no significant differences observed in postprandial measures of inflammation or endothelial function. After 6 months, CRP (mean±S.E.) decreased in the low-fat arm from 4.0±0.77 to 3.0±0.77 (P=.01). In the low-carbohydrate arm, sICAM decreased from 234±22 to 199±23 (P=.001), and soluble E-selectin decreased from 93±10 to 82±10 (P=.05.) A significant correlation between change in high-density lipoprotein and change in soluble E-selectin (r=−0.33, P=.04) and with the change in ICAM (r=−0.43, P=.01) was observed. Conclusions: Low-carbohydrate and low-fat diets both have beneficial effects on CVD markers. There may be different mechanisms through which weight loss with these diets potentially reduces CVD risk. [Copyright &y& Elsevier]

HIV infection of the CNS is an early event after primary infection, resulting in neurological complications in a significant number of individuals despite antiretroviral therapy (ART). The main cells infected with HIV within the CNS are macrophages/microglia and a small fraction of astrocytes. The role of these few infected astrocytes in the pathogenesis of neuroAIDS has not been examined extensively. Here, we demonstrate that few HIV-infected astrocytes (4.7±2.8% in vitro and 8.2±3.9% in vivo) compromise blood- brain barrier (BBB) integrity. This BBB disruption is due to endothelial apoptosis, misguided astrocyte end feet, and dysregulation of lipoxygenase/cyclooxygenase, BKCa channels, and ATP receptor activation within astrocytes. All of these alterations in BBB integrity induced by a few HIV-infected astrocytes were gap junction dependent, as blocking these channels protected the BBB from HIV-infected astrocytemediated compromise. We also demonstrated apoptosis in vivo of BBB cells in contact with infected astrocytes using brain tissue sections from simian immunodeficiency virus-infected macaques as a model of neuroAIDS, suggesting an important role for these few infected astrocytes in the CNS damage seen with HIV infection. Our findings describe a novel mechanism of bystander BBB toxicity mediated by low numbers of HIV-infected astrocytes and amplified by gap junctions. This mechanism of toxicity contributes to understanding how CNS damage is spread even in the current ART era and how minimal or controlled HIV infection still results in cognitive impairment in a large population of infected individuals. [ABSTRACT FROM AUTHOR]

Abstract: The prevalence of human immunodeficiency virus 1 (HIV) associated neurocognitive disorders resulting from infection of the central nervous system (CNS) by HIV continues to increase despite the success of combination antiretroviral therapy. Although monocytes are known to transport HIV across the blood–brain barrier (BBB) into the CNS, there are few specific markers that identify monocyte subpopulations susceptible to HIV infection and/or capable of infiltrating the CNS. We cultured human peripheral blood monocytes and characterized the expression of the phenotypic markers CD14, CD16, CD11b, Mac387, CD163, CD44v6 and CD166 during monocyte/macrophage (Mo/Mac) maturation/differentiation. We determined that a CD14+CD16+CD11b+Mac387+ Mo/Mac subpopulation preferentially transmigrates across our in vitro BBB model in response to CCL2. Genes associated with Mo/Mac subpopulations that transmigrate across the BBB and/or are infected by HIV were identified by cDNA microarray analyses. Our findings contribute to the understanding of monocyte maturation, infection and transmigration into the brain during the pathogenesis of NeuroAIDS. [Copyright &y& Elsevier]

Abstract: Members of the TNF-ligand and receptor superfamilies are important in the pathogenesis of lupus nephritis, a major cause of mortality and morbidity in SLE. TWEAK, a member of the TNF-ligand superfamily, is markedly increased in urine from patients with active lupus nephritis, and urinary TWEAK levels significantly correlate with renal disease activity. To support a possible role of TWEAK in the pathogenesis of lupus nephritis and other inflammatory nephritides, we examined the effects of TWEAK in human kidney mesangial cells, podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. We found that TWEAK induces human kidney cells to express multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1α, ICAM-1, and VCAM-1. Cytokine production is mediated through NF-κB activation, and is inhibited by anti-TWEAK monoclonal antibodies. TWEAK stimulated chemokines induced migration of human PBMC, particularly monocytes/macrophages. Furthermore, we found that TWEAK promotes kidney infiltration of inflammatory cells, and stimulates proliferation of kidney cells in vitro and in vivo. Thus, TWEAK may play an important pathogenic role in the development of glomerulonephritis by promoting a local inflammatory environment and inducing kidney cell proliferation. Blocking TWEAK/Fn14 interactions may be a promising therapeutic target in immune-mediated renal diseases. [Copyright &y& Elsevier]

Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1ADA) or clade C (HIV-1Indie-C1) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1Indie-C1-infected macrophages recruited monocytes poorly in vitro compared with HIV-1ADA-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1. [ABSTRACT FROM AUTHOR]

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAlDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-D-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis. suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAlDS. [ABSTRACT FROM AUTHOR]

Abstract: The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1α), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1β and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1β. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS. [Copyright &y& Elsevier]

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood--brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1α, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS. [ABSTRACT FROM AUTHOR]

Abstract: Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of Gαi signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms. [Copyright &y& Elsevier]

: PurposeTo determine whether endovascular radiation can inhibit intimal hyperplasia in a swine model of hemodialysis access.: Materials and methodsPolytetrafluoroethylene arteriovenous grafts (6 mm in diameter) were placed between the common carotid artery and the external jugular vein bilaterally in 8 pigs. Two days after the surgery, fistulography was performed. Gamma radiation (12 Gy) was delivered endovascularly to one of the grafts at the venous anastomosis by using an iridium192 source. Thus, the other graft in each pig served as an untreated control. Fistulas were evaluated with fistulography or venography 6 week after radiation. All grafts were then harvested for histological and immunohistochemical examination.: ResultsSeven grafts on the treated side and 5 grafts on the control side remained patent for at least 6 weeks. Angiography demonstrated that the percentage stenosis at the venous anastomosis was significantly lower for the treated group (15.9 ± 14.1 versus 32.6 ± 16.7%, P = 0.045). Histopathologic analyses revealed that the mean intimal area and maximal intimal thickness were significantly lower with reduced smooth muscle cell proliferation at the venous anastomosis on the treated side compared to the control side (0.68 ± 0.30 versus 1.06 ± 0.29 mm2, P = 0.017, and 0.18 ± 0.08 versus 0.26 ± 0.07 mm, P = 0.004, respectively). The residual lumen was significantly greater for the treated group (1.59 ± 0.42 versus 1.06 ± 0.37 mm2, P = 0.031). No significant differences were found in the area, nor maximal thickness in the vein either proximal or distal to the anastomosis between the two groups.: ConclusionsIn an animal model of hemodialysis access, brachytherapy with iridium192 delivered 2 days after graft implantation reduces intimal hyperplasia and stenosis at the venous anastomosis. The reduced smooth-muscle cells found in the radiated veins suggests that brachytherapy may exert its effect on neointimal formation by inhibition of smooth muscle cell proliferation. [Copyright &y& Elsevier]

Hepatocyte growth factor (HGF) has multiple effects on target cells upon activation of its receptor, c-Met. In endothelial cells, HGF induces migration, proliferation, and angiogenesis. HGF can also act as an anti-apoptotic factor for several cell types. The signal transduction pathways involved in mediating its anti-apoptotic effects have not been fully clarified. We demonstrated that HGF is anti-apoptotic for human endothelial cells, and identified the signaling pathways by which it mediates its effects. Human umbilical vein endothelial cells (HUVEC) exhibited significant levels of apoptosis after serum deprivation. HGF inhibited apoptosis in a dose dependent manner in serum-deprived cultures. HGF induced the phosphorylation of Akt and Erk1/2, cell survival factors, in a time dependent manner in serum deprived HUVEC. Inhibition of Akt and Erk1/2 activation abolished the anti-apoptotic effects of HGF. The transcription factor, NF-κB, can also play a role in promoting cell survival. However, NF-κB does not appear to contribute to the anti-apoptotic properties of HGF, as nuclear translocation of NF-κB was not detected in HGF-treated cultures. Endothelial cell migration, proliferation, and apoptosis contribute to the pathogenesis of atherosclerosis, and HGF may play a role in the development and progression of vascular lesions. Immunohistochemical analysis of human carotid artery sections demonstrated HGF protein localization within atherosclerotic lesions but not in normal vessels, suggesting that HGF may participate in atherogenesis. [Copyright &y& Elsevier]

Chemokines are small molecular weight proteins that play important roles in inflammation. Originally described as chemotactic cytokines, chemokines stimulate the influx of leukocytes into specific tissue compartments. These molecules also modulate gene expression in both infiltrating and resident cells to mediate a vast array of cellular functions, and their importance in disease processes has been well documented. This study examined the expression of chemokines during myocardial ischemia and established a pathway by which two, MIP-2 and JE/MCP-1, modulate cardiac myocyte viability during this process. To focus on the direct effects of chemokines on these cells, a mouse model of ischemia without reperfusion was used. The expression of chemokines and chemokine receptors was induced in the left ventricular free wall as early as 1 h post-ischemia, with the most significant increases in MIP-2 (CXCL2) and JE/MCP-1 (CCL2). Expression of their respective receptors, CXCR2 and CCR2, was also induced. Similar changes in gene expression occurred at the mRNA and protein levels in isolated neonatal mouse cardiac myocytes stimulated by hypoxia. Antibody to MIP-2 inhibited hypoxia-induced JE/MCP-1 expression, demonstrating that MIP-2 is critical for this event. Moreover, in vivo intramyocardial injection of either an adenovirus expressing MIP-2 or the recombinant protein itself was sufficient to upregulate JE/MCP-1 production even in the absence of ischemia. Thus, MIP-2 regulates JE/MCP-1 expression both in cell culture and in vivo. Furthermore, JE/MCP-1 markedly decreased hypoxia-induced cell death in cultured cardiac myocytes. Thus, JE/MCP-1 appears to mediate an unanticipated survival pathway in target cardiac myocytes themselves. These findings indicate an important role for MIP-2 and JE/MCP-1 in regulating the response of cardiac myocytes to myocardial ischemia. [ABSTRACT FROM AUTHOR]

Examines HIV envelope protein for the activation of human arterial smooth muscle cells. Expression of tissue factor; Induction of tissue factor by the HIV envelope protein; Generation of reactive oxygen species.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain. [ABSTRACT FROM AUTHOR]

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND. [ABSTRACT FROM AUTHOR]

• We describe studies demonstrating that substances of abuse exacerbate HIV neuropathogenesis. • We suggest areas for research. • We discuss potential therapies. Approximately 37 million people worldwide are infected with human immunodeficiency virus (HIV). One highly significant complication of HIV infection is the development of HIV-associated neurocognitive disorders (HAND) in 15–55% of people living with HIV (PLWH), that persists even in the antiretroviral therapy (ART) era. The entry of HIV into the central nervous system (CNS) occurs within 4–8 days after peripheral infection. This establishes viral reservoirs that may persist even in the presence of ART. Once in the CNS, HIV infects resident macrophages, microglia, and at low levels, astrocytes. In response to chronic infection and cell activation within the CNS, viral proteins, inflammatory mediators, and host and viral neurotoxic factors produced over extended periods of time result in neuronal injury and loss, cognitive deficits and HAND. Substance abuse is a common comorbidity in PLWH and has been shown to increase neuroinflammation and cognitive disorders. Additionally, it has been associated with poor ART adherence, and increased viral load in the cerebrospinal fluid (CSF), that may also contribute to increased neuroinflammation and neuronal injury. Studies have examined mechanisms that contribute to neuroinflammation and neuronal damage in PLWH, and how substances of abuse exacerbate these effects. This review will focus on how substances of abuse, with an emphasis on methamphetamine (meth), cocaine, and opioids, impact blood brain barrier (BBB) integrity and transmigration of HIV-infected and uninfected monocytes across the BBB, as well as their effects on monocytes/macrophages, microglia, and astrocytes within the CNS. We will also address how these substances of abuse may contribute to HIV-mediated neuropathogenesis in the context of suppressive ART. Additionally, we will review the effects of extracellular dopamine, a neurotransmitter that is increased in the CNS by substances of abuse, on HIV neuropathogenesis and how this may contribute to neuroinflammation, neuronal insult, and HAND in PLWH with active substance use. Lastly, we will discuss some potential therapies to limit CNS inflammation and damage in HIV-infected substance abusers. [ABSTRACT FROM AUTHOR]

As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14+CD16+ monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14+CD16+ monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14+CD16+ monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14+ cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14+CD16+ monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14+CD16+ monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14+CD16+ monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis. [ABSTRACT FROM AUTHOR]