Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.

Human immunodeficiency virus-1 (HIV-1) encephalitis is characterized by brain infiltration of virus-infected monocytes and macrophages. Cellular products and viral proteins secreted by infected cells likely play an important role in blood-brain barrier (BBB) impairment and the development of HIV-1-associated dementia (HAD). We previously demonstrated that HIV-1 envelope glycoprotein gp120 induces toxicity and alters expression of tight junction proteins in human brain microvascular endothelial cells (HBMECs). Here, we delineate the mechanisms of gp120-induced BBB dysfunction. Human brain microvascular endothelial cells expressed HIV-1 co-receptors (CCR5 and CXCR4). Exposure of HBMECs to gp120 derived from macrophage (CCR5) or lymphocyte (CXCR4)-tropic viruses decreased BBB tightness, increased permeability, and enhanced monocyte migration across in vitro BBB models. Blood-brain barrier integrity was restored after gp120 removal. CCR5 antibodies and inhibitors of myosin light chain kinase or protein kinase C (PKC) blocked gp120-enhanced monocyte migration and permeability of BBB in vitro. Exposure of HBMECs to gp120 induced release of intracellular calcium ([Ca(2+)](i)) that was prevented by CCR5 antibody and partially blocked by CXCR4 antagonist. Human immunodeficiency virus-1 gp120 activated three PKC isoforms in HBMECs [PKC-alpha/betaII, PKC(pan)-betaII and PKC-zeta/lambda]. Furthermore, specific PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biologic significance of these results. Thus, gp120 can cause dysfunction of BBB via PKC pathways and receptor mediated [Ca(2+)](i) release leading to cytoskeletal alterations and increased monocyte migration.

These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.

IntroductionThe pathogenesis of HIV-1 infection is linked to dysfunction and depletion of CD4 ϩ T lymphocytes. [1][2][3] The virus persists and disseminates over years, despite an apparently intact host immune response. The inability to eliminate HIV-1 suggests that negativeregulatory (tolerogenic) signals may shield HIV-1 from adaptive immune clearance. 4,5 However, the specific mechanisms by which the virus might protect itself from clearance remain unresolved.HIV-1 is known to persist at low levels within the central nervous system (CNS) during most of the disease course. 6 Significant productive HIV-1 replication occurs in brain mononuclear phagocytes (MPs; perivascular macrophages and microglia) during late stages of infection only in a subset of individuals with severe immune suppression and high peripheral viral loads. Secretory viral and cellular products from HIV-1-infected and immune competent brain MPs are known to induce neuronal dysfunction and injury. [7][8][9] These include virotoxins such as Tat, Nef, and gp120 and cellular toxins such as proinflammatory cytokines, chemokines, arachidonic acid and its metabolites, platelet activating factor, nitric oxide, and quinolinic acid. Indoleamine 2,3-dioxygenase (IDO) is the first and the rate-limiting enzyme in the generation of quinolinic acid from tryptophan via the kynurenine pathway. 10 An increase of functional IDO enzymatic activity in the brain could lead to enhanced production of neurotoxins, resulting in neurocognitive dysfunction and HIV-1-associated dementia (HAD). 11-13 Signs of increased IDO activity correlate with tryptophan depletion, progression of systemic and brain HIV-1 infection, and HAD. 14,15 Accumulating evidence suggests that IDO serves immunoregulatory and tolerogenic functions. [16][17][18][19][20][21] It appears that certain antigen presenting cells (APCs) may regulate T-cell responses through the expression of IDO. 22 A number of studies indicate that IDO overexpression by APCs may result in immune suppression and reduced T-cell responses. 17,18,20,[23][24][25] Therefore, HIV-induced IDO activity in the brain may participate not only in local neurotoxicity, but also in the failure of the immune system to clear HIV from this reservoir.A strong association between HAD and profound immunodeficiency supports the notion that a lack of effective adaptive immune responses is associated with ongoing viral replication in the brain. One plausible explanation is that circulating HIV-1-specific CD8 ϩ cells could be partially anergic and may be unable to eliminate HIV-1-infected cells in vivo in the setting of functionally impaired helper CD4 ϩ T cells during late stages of infection. 26,27 Based on these observations, we hypothesize that IDOexpressing APCs (specifically, HIV-1-infected macrophages) might help to create a protected reservoir for HIV-1 persistence in the brain. To test this idea we used a mouse model of HIV-1 For personal use only. on June 19, 2019. by guest www.bloodjournal.org From encephalitis (HIVE) in which nonobese di...

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive.

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Objective Poor penetration of anti-retroviral therapy across the blood brain barrier (BBB) poses an impediment on control of HIV-1 infection in brain macrophages. PPARγ, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. Since PPARγ agonists are rapidly absorbed by oral administration and efficiently permeate the BBB we hypothesized that PPARγ stimulation may suppress HIV-1 replication. Design & Methods We investigated the effect of PPARγ ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages (MDM) and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis (HIVE). Results Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARγ stimulation inhibited virus replication by modulating NF-κB activation in a receptor-depended manner, leading to down-regulation of HIV-1 LTR promoter activity and suppression of HIV-1 replication. These effects were PPARγ specific as PPARγ silencing or addition of PPARγ antagonist abolished effects of PPARγ stimulation on HIV-1 LTR and virus replication. Using a murine model for HIVE, we demonstrated that PPARγ ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. Conclusion In vitro data delineated the novel mechanism by which PPARγ activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARγ agonists to reduce HIV-1 replication in lymphocytes and brain macrophages offering new therapeutic intervention in brain and systemic infection.

Oxidative damage of the endothelium disrupts the integrity of blood brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS, superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of anti-oxidant systems like reduction of catalase and superoxide dismutase (SOD) activity in ethanol exposure may elevate the levels of ROS/NO in endothelium resulting in BBB damage. The present study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and activity of anti-oxidative enzymes. SOD activity was increased for a much longer period of time as compared to catalase activity. Decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the mitochondria protecting agent, acetyl-L-carnitine (ALC), and anti-inflammatory agent, rosiglitazone, suppressed ROS levels with marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and anti-oxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS neutralizing anti-oxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.