The blood-brain barrier (BBB) is the specialized system of brain microvascular endothelial cells (BMVEC) that shields the brain from toxic substances in the blood, supplies brain tissues with nutrients, and filters harmful compounds from the brain back to the bloodstream. The close interaction between BMVEC and other components of the neurovascular unit (astrocytes, pericytes, neurons, and basement membrane) ensures proper function of the central nervous system (CNS). Transport across the BBB is strictly limited through both physical (tight junctions) and metabolic barriers (enzymes, diverse transport systems). A functional polarity exists between the luminal and abluminal membrane surfaces of the BMVEC. As a result of restricted permeability, the BBB is a limiting factor for the delivery of therapeutic agents into the CNS. BBB breakdown or alterations in transport systems play an important role in the pathogenesis of many CNS diseases (HIV-1 encephalitis, Alzheimer's disease, ischemia, tumors, multiple sclerosis, and Parkinson's disease). Proinflammatory substances and specific disease-associated proteins often mediate such BBB dysfunction. Despite seemingly diverse underlying causes of BBB dysfunction, common intracellular pathways emerge for the regulation of the BBB structural and functional integrity. Better understanding of tight junction regulation and factors affecting transport systems will allow the development of therapeutics to improve the BBB function in health and disease.

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.

Background Factors limiting the efficacy of conventional antiretroviral therapy for HIV-1 infection include treatment adherence, pharmacokinetics and penetration into viral sanctuaries. These affect the rate of viral mutation and drug resistance. In attempts to bypass such limitations, nanoparticles containing ritonavir, indinavir and efavirenz (described as nanoART) were manufactured to assess macrophage-based drug delivery. Methods NanoART were made by high-pressure homogenization of crystalline drug with various surfactants. Size, charge and shape of the nanoparticles were assessed. Monocyte-derived macrophage nanoART uptake, drug release, migration and cytotoxicity were determined. Drug levels were measured by reverse-phase high-performance liquid chromatography. Results Efficient monocyte-derived macrophage cytoplasmic vesicle uptake in less than 30 min based on size, charge and coating was observed. Antiretroviral drugs were released over 14 days and showed dose-dependent reduction in progeny virion production and HIV-1 p24 antigen. Cytotoxicities resulting from nanoART carriage were limited. Conclusion These results support the continued development of macrophage-mediated nanoART carriage for HIV-1 disease.

The relationship among neuroinflammation, blood-brain barrier (BBB) dysfunction, and progressive HIV-1 infection as they affect the onset and development of neuroAIDS is incompletely understood. IntroductionHuman immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) commonly results in behavioral, motor, and cognitive impairments. [1][2][3] Although disease severity and progression has slowed, in part, as a result of antiretroviral therapy, 76% to 83% of brain autopsies continue to show observable neuropathological abnormalities. [4][5][6] Disease pathology ranges from mild brain atrophy and gliosis to robust viral replication, multinucleated giant cell formation, astrogliosis and microgliosis, myelin pallor, and neuronal loss. 7,8 These pathological findings are collectively termed HIV-1 encephalitis (HIVE). HIVE is a common correlate to the later stages of behavioral, motor, neuropsychiatric, and neurologic consequences of disease termed HIV-1-associated dementia (HAD) (for recent reviews, see McArthur 1 ; Grant et al 2 ; Ghafouri 3 ). HIVE is fueled by viral infection and immune activation of brain mononuclear phagocytes (MPs: blood-derived perivascular macrophages and microglia). 9 Such MP-and virus-associated neuroinflammation promotes monocyte trafficking across the bloodbrain barrier (BBB), MP infiltration into the CNS, and neurodegeneration. [10][11][12] Thus, dysfunction of the BBB is one critical feature of HIV-1 neuropathogenesis.Brain microvascular endothelial cells, a major component of BBB function and integrity, are connected by tight junctions (TJs) that limit paracellular flux and restrict permeability. 13 Indeed, under normal physiologic conditions, the brain endothelium functions as an interface between the blood and the brain parenchyma, strictly regulating influx of ions, molecules, and leukocytes into the CNS. Nonetheless, in disease, a variety of environmental, toxic, degenerative, and microbial insults could cause BBB breakdown. 12,14 Such a breakdown occurs during progressive HIV-1 infection 12,[15][16][17] and was documented in laboratory, animal models, human clinical observations, and autopsy studies. 10,[16][17][18][19][20] Underlying mechanisms of BBB dysfunction and how it affects ongoing disease are incompletely understood. 1,12,14 Dysfunction of the BBB enhances penetration of cell-free virus, ingress of activated HIV-1-infected monocytes across the BBB, accumulation of MP in the CNS, and spread of the virus to neighboring microglia and astrocytes. 15,21 Thus, BBB breech is commonly associated with accelerated disease and the development of behavioral and cognitive deficits that are signatures of HAD. 1,16 Based on these observations, the elucidation of the signaling pathways mediating BBB compromise can prove important for understanding disease mechanisms and development of new therapies.Signal transducers and activators of transcription (STATs) proteins are latent cytoplasmic transcription factors that are phosphorylated by Janus kinases (JAKs) in...

Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration90 of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.