Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

Application of infrared matrix-assisted laser desorption electrospray ionization mass spectrometry for morphine imaging in brain tissue.

Here, we present a method developed for the analysis of spatial distributions of morphine in mouse brain tissue using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer. The method is also capable of evaluating spatial distributions of the antiretroviral drug abacavir. To maximize sensitivity to morphine, we analyze various Orbitrap mass spectrometry acquisition modes utilizing signal abundance and frequency of detection as evaluation criteria. We demonstrate detection of morphine in mouse brain and establish that the selected ion monitoring mode provides 2.5 times higher sensitivity than the full-scan mode. We find that distributions of morphine and abacavir are highly correlated with the Pearson correlation coefficient R = 0.87. Calibration showed that instrument response is linear up to 40 pg/mm2 (3.8 µg/g of tissue).

Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia.

SUMMARY STATEMENT: HIV/HIV-1 Tat and morphine independently increase pathologic phosphorylation of TAR DNA binding protein 43 in the striatum. HIV- and opioid-induced pathologic phosphorylation of TAR DNA binding protein 43 may involve enhanced CK2 activity and protein levels.

Independent actions by HIV-1 Tat and morphine to increase recruitment of monocyte-derived macrophages into the brain in a region-specific manner.

Despite advances in the treatment of human immunodeficiency virus (HIV), approximately one-half of people infected with HIV (PWH) experience neurocognitive impairment. Opioid use disorder (OUD) can exacerbate the cognitive and pathological changes seen in PWH. HIV increases inflammation and immune cell trafficking into the brain; however, less is known about how opioid use disorder affects the recruitment of immune cells. Accordingly, we examined the temporal consequences of HIV-1 Tat and/or morphine on the recruitment of endocytic cells (predominantly perivascular macrophages and microglia) in the dorsal striatum and hippocampus by infusing multi-colored, fluorescently labeled dextrans before and after exposure. To address this question, transgenic mice that conditionally expressed HIV-1 Tat (Tat+), or their control counterparts (Tat-), received three sequential intracerebroventricular (i.c.v.) infusions of Cascade Blue-, Alexa Fluor 488-, and Alexa Fluor 594-labeled dextrans, respectively infused 1 day before, 1-day after, or 13-days after morphine and/or Tat exposure. At the end of the study, the number of cells labeled with each fluorescent dextran were counted. The data demonstrated a significantly higher influx of newly-labeled cells into the perivascular space than into the parenchyma. In the striatum, Tat or morphine exposure increased the number of endocytic cells in the perivascular space, while only morphine increased the recruitment of endocytic cells into the parenchyma. In the hippocampus, morphine (but not Tat) increased the influx of dextran-labeled cells into the perivascular space, but there were too few labeled cells within the hippocampal parenchyma to analyze. Collectively, these data suggest that HIV-1 Tat and morphine act independently to increase the recruitment of endocytic cells into the brain in a region-specific manner.

Histone acetylation at the sulfotransferase 1a1 gene is associated with its hepatic expression in normal aging.

OBJECTIVES: Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects. METHODS: We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months. RESULTS: Our sample shows a significant loss of 5mC at Sult1a1 (ß = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (ß = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6. CONCLUSIONS: Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults.

Treating viruses in the brain: Perspectives from NeuroAIDS.

Aggressive use of antiretroviral therapy has led to excellent viral suppression within the systemic circulation. However, despite these advances, HIV reservoirs still persist. The persistence of HIV within the brain can lead to the development of HIV-associated neurocognitive disorders (HAND). Although the causes of the development of neurocognitive disorders is likely multifactorial, the inability of antiretroviral therapy to achieve adequate concentrations within the brain is likely a major contributing factor. Information about antiretroviral drug exposure within the brain is limited. Clinically, drug concentrations within the cerebrospinal fluid (CSF) are used as markers for central nervous system (CNS) drug exposure. However, significant differences exist; CSF concentration is often a poor predictor of drug exposure within the brain. This article reviews the current information regarding antiretroviral exposure within the brain in humans as well as preclinical animals and discusses the impact of co-morbidities on antiretroviral efficacy within the brain. A more thorough understanding of antiretroviral penetration into the brain is an essential component to the development of better therapeutic strategies for neuroAIDS.

Opioid and neuroHIV Comorbidity - Current and Future Perspectives.

With the current national opioid crisis, it is critical to examine the mechanisms underlying pathophysiologic interactions between human immunodeficiency virus (HIV) and opioids in the central nervous system (CNS). Recent advances in experimental models, methodology, and our understanding of disease processes at the molecular and cellular levels reveal opioid-HIV interactions with increasing clarity. However, despite the substantial new insight, the unique impact of opioids on the severity, progression, and prognosis of neuroHIV and HIV-associated neurocognitive disorders (HAND) are not fully understood. In this review, we explore, in detail, what is currently known about mechanisms underlying opioid interactions with HIV, with emphasis on individual HIV-1-expressed gene products at the molecular, cellular and systems levels. Furthermore, we review preclinical and clinical studies with a focus on key considerations when addressing questions of whether opioid-HIV interactive pathogenesis results in unique structural or functional deficits not seen with either disease alone. These considerations include, understanding the combined consequences of HIV-1 genetic variants, host variants, and µ-opioid receptor (MOR) and HIV chemokine co-receptor interactions on the comorbidity. Lastly, we present topics that need to be considered in the future to better understand the unique contributions of opioids to the pathophysiology of neuroHIV. Graphical Abstract Blood-brain barrier and the neurovascular unit. With HIV and opiate co-exposure (represented below the dotted line), there is breakdown of tight junction proteins and increased leakage of paracellular compounds into the brain. Despite this, opiate exposure selectively increases the expression of some efflux transporters, thereby restricting brain penetration of specific drugs.

DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver.

Aging is associated with reduced liver function that may increase the risk for adverse drug reactions in older adults. We hypothesized that age-related changes to epigenetic regulation of genes involved in drug metabolism may contribute to this effect. We reviewed published epigenome-wide studies of human blood and identified the cytochrome P450 2E1 (CYP2E1) gene as a top locus exhibiting epigenetic changes with age. To investigate potential functional changes with age in the liver, the primary organ of drug metabolism, we obtained liver tissue from mice aged 4-32 months from the National Institute on Aging. We assayed global DNA methylation (5-methylcytosine, 5mC), hydroxymethylation (5-hydroxymethylcytosine, 5hmC), and locus-specific 5mC and histone acetylation changes around mouse Cyp2e1. The mouse livers exhibit significant global decreases in 5mC and 5hmC with age. Furthermore, 5mC significantly increased with age at two regulatory regions of Cyp2e1 in tandem with decreases in its gene and protein expressions. H3K9ac levels also changed with age at both regulatory regions of Cyp2e1 investigated, while H3K27ac did not. To test if these epigenetic changes are associated with varying rates of drug metabolism, we assayed clearance of the CYP2E1-specific probe drug chlorzoxazone in microsome extracts from the same livers. CYP2E1 intrinsic clearance is associated with DNA methylation and H3K9ac levels at the Cyp2e1 locus but not with chronological age. This suggests that age-related epigenetic changes may influence rates of hepatic drug metabolism. In the future, epigenetic biomarkers could prove useful to guide dosing regimens in older adults.

Educational Outcomes Resulting From Restructuring a Scholarship Course for Doctor of Pharmacy Students.

Objective. To compare educational outcomes between two iterations of a scholarship and research course for Doctor of Pharmacy (PharmD) students at Virginia Commonwealth University's School of Pharmacy. Methods. The first iteration of a course intended to teach pharmacy students the knowledge and skills necessary to design and conduct research involved lectures and application exercises, including limited guided questions about different aspects of the research process. In the fall of 2015, multiple structured activities and accompanying grading rubrics, each designed around the structure and content of a section of a research proposal, were introduced to the course to supplement lectures. Both iterations of the course culminated with students submitting a research proposal. After establishing interrater reliability, faculty members graded a random sample of 20 research proposals, 10 from each version of the course, and section-specific and overall proposal scores were compared. Results. In the proposals submitted after the course revisions, significant improvements in three areas were identified: the overall score, the section-specific scores for research hypothesis/specific aims, and institutional review board (IRB) discussion/informed consent. Nominal, though not statistically significant, improvements were observed in other sections. Conclusion. Additional research is needed regarding the best instructional strategies to reinforce data analysis and statistical testing knowledge and skills in PharmD students. Overall, our findings support the hypothesis that a more formalized, guided approach for teaching research methods improves learning outcomes for PharmD students.

Discovery of Second-Generation NLRP3 Inflammasome Inhibitors: Design, Synthesis, and Biological Characterization.

NLRP3 inflammasomes have recently emerged as an attractive drug target for neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as selective inhibitors of NLRP3 inflammasomes. Initial characterization of the lead HL16 demonstrated improved, however, nonselective inhibition on the NLRP3 inflammasome. Structure-activity relationship studies of HL16 identified a new lead, 17 (YQ128), with an IC50 of 0.30 ± 0.01 µM. Further studies from in vitro and in vivo models confirmed its selective inhibition on the NLRP3 inflammasome and its brain penetration. Furthermore, pharmacokinetic studies in rats at 20 mg/kg indicated extensive systemic clearance and tissue distribution, leading to a half-life of 6.6 h. However, the oral bioavailability is estimated to be only 10%, which may reflect limited GI permeability and possibly high first-pass effects. Collectively, these findings strongly encourage development of more potent analogues with improved pharmacokinetic properties from this new chemical scaffold.

Cell-type specific differences in antiretroviral penetration and the effects of HIV-1 Tat and morphine among primary human brain endothelial cells, astrocytes, pericytes, and microglia.

The inability to achieve adequate intracellular antiretroviral concentrations may contribute to HIV persistence within the brain and to neurocognitive deficits in opioid abusers. To investigate, intracellular antiretroviral concentrations were measured in primary human astrocytes, microglia, pericytes, and brain microvascular endothelial cells (BMECs), and in an immortalized brain endothelial cell line (hCMEC/D3). HIV-1 Tat and morphine effects on intracellular antiretroviral concentrations also were evaluated. After pretreatment for 24 h with vehicle, HIV-1 Tat, morphine, or combined Tat and morphine, cells were incubated for 1 h with equal concentrations of a mixture of tenofovir, emtricitabine, and dolutegravir at one of two concentrations (5 µM or 10 µM). Intracellular drug accumulation was measured using LC-MS/MS. Drug penetration differed depending on the drug, the extracellular concentration used for dosing, and cell type. Significant findings included: 1) Dolutegravir (at 5 µM or 10 µM) accumulated more in HBMECs than other cell types. 2) At 5 µM, intracellular emtricitabine levels were higher in microglia than other cell types; while at 10 µM, emtricitabine accumulation was greatest in HBMECs. 3) Tenofovir (5 or 10 µM extracellular dosing) displayed greater accumulation inside HBMECs than in other cell types. 4) After Tat and/or morphine pretreatment, the relative accumulation of antiretroviral drugs was greater in morphine-exposed HBMECs compared to other treatments. The opposite effect was observed in astrocytes in which morphine exposure decreased drug accumulation. In summary, the intracellular accumulation of antiretroviral drugs differed depending on the particular drug involved, the concentration of the applied antiretroviral drug, and the cell type targeted. Moreover, morphine, and to a lesser extent Tat, exposure also had differential effects on antiretroviral accumulation. These data highlight the complexity of optimizing brain-targeted HIV therapeutics, especially in the setting of chronic opioid use or misuse.

HIV-1 Tat and opioids act independently to limit antiretroviral brain concentrations and reduce blood-brain barrier integrity.

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

Identifying Components of Success Within Health Sciences-Focused Mentoring Programs Through a Review of the Literature.

Objective. To identify programmatic components and structural features associated with success of mentoring programs within the health sciences. Findings. Thirty-eight manuscripts representing 34 individual programs were reviewed. Of the institutions represented, 68% were public. Sixty-eight percent of programs included single disciplines only, with four focused in pharmacy, 13 in medicine, and six in nursing. Of the 34 individual programs, all programs reporting participant confidence and self-efficacy reported success in that domain. Eighteen programs reported outcomes related to scholarly activity that included publications or funding/grantsmanship; 16 reported success. Eleven of 16 programs reporting promotion/tenure and/or faculty retention rates reported success. Program components associated with successful programs included frequent meetings (at least monthly) and delivering content within formal curricula. Content categories common within programs reporting success were content related to research, funding/grantsmanship and networking/collaboration. In addition, specific for the promotion/retention domain, content related to curriculum/teaching was commonly found within successful programs. Summary. Although somewhat dependent on the program's specific goals, curriculum most commonly associated with success contained content on research, grantsmanship/funding, curriculum/teaching, and networking/collaboration. Among many programs, the reporting lacked objective, standardized metrics and often included only generalized descriptions/categorization of course content. The incomplete and inconsistent reporting limited our ability to draw conclusions regarding individual topics important for each program component. Proper planning, execution, and assessment of faculty mentoring programs is critical to the identification of additional program characteristics for optimal faculty success.

Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1-100 ng mL-1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.

Building a schizophrenia genetic network: transcription factor 4 regulates genes involved in neuronal development and schizophrenia risk.

The transcription factor 4 (TCF4) locus is a robust association finding with schizophrenia (SCZ), but little is known about the genes regulated by the encoded transcription factor. Therefore, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 in neural-derived (SH-SY5Y) cells to identify genome-wide TCF4 binding sites, followed by data integration with SCZ association findings. We identified 11 322 TCF4 binding sites overlapping in two ChIP-seq experiments. These sites are significantly enriched for the TCF4 Ebox binding motif (>85% having ≥1 Ebox) and implicate a gene set enriched for genes downregulated in TCF4 small-interfering RNA (siRNA) knockdown experiments, indicating the validity of our findings. The TCF4 gene set was also enriched among (1) gene ontology categories such as axon/neuronal development, (2) genes preferentially expressed in brain, in particular pyramidal neurons of the somatosensory cortex and (3) genes downregulated in postmortem brain tissue from SCZ patients (odds ratio, OR = 2.8, permutation P < 4x10-5). Considering genomic alignments, TCF4 binding sites significantly overlapped those for neural DNA-binding proteins such as FOXP2 and the SCZ-associated EP300. TCF4 binding sites were modestly enriched among SCZ risk loci from the Psychiatric Genomic Consortium (OR = 1.56, P = 0.03). In total, 130 TCF4 binding sites occurred in 39 of the 108 regions published in 2014. Thirteen genes within the 108 loci had both a TCF4 binding site ±10kb and were differentially expressed in siRNA knockdown experiments of TCF4, suggesting direct TCF4 regulation. These findings confirm TCF4 as an important regulator of neural genes and point toward functional interactions with potential relevance for SCZ.

Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier.

A principal function of endothelial cells is the formation of a barrier between the blood and tissues.  This barrier arises from the physical connections at cell-cell junctions, which includes cytoskeletal tight junction and adherens junction proteins. Methods that alter barrier function must therefore affect these cell-cell connections. The blood brain barrier (BBB) represents perhaps the most selective endothelial barrier, which arises from endothelial cell interactions with astrocytes and pericytes. Even in non-central nervous system (CNS) endothelial cells, barrier properties can be enhanced, mimicking the BBB, through induction of intercellular junctions, by either direct co-culture with astrocytes, supplementation with astrocyte conditioned medium (ACM) and/or pharmacologic enhancement of cAMP. To understand how cell-cell junctions change during endothelial barrier enhancement, we examined the effects of ACM and/or cAMP donors added to standard media on human umbilical vein endothelial cells (HUVEC). HUVEC cultured with cAMP-elevating agents had the most enhanced barrier function as measured by Electric Cell-substrate Impedance Sensing (ECIS®), a real-time, label-free, impedance based method of studying cell barrier properties. However, subtle differences in actin and cell-cell junction proteins were seen across all four culture conditions. cAMP-elevating agents also triggered the redistribution of ZO-1 and VE-cadherin to cell-cell junctions, and intensified the actin microfilament network at the cell cortex.  Using a VE-cadherin FRET-force sensor, we observed a decrease in VE-cadherin force in HUVEC cultured with ACM with cAMP donors. Our data indicate cAMP elevation induces both junctional strengthening and reduced VE-cadherin forces. Additionally, treatment with an inhibitor of formin, which reduced actin stress fibers, enhanced barrier function. These data suggest that barrier function is modulated both through the trafficking of proteins to cell-cell junctions, and through the modulation and a relaxation of mechanical force through adherens junctions as intercellular junctional complexes become established.

New Drugs in the Pipeline for the Treatment of HIV: a Review.

PURPOSE OF REVIEW: The purpose of this paper is to review therapies with new mechanisms of action for the treatment of HIV that are at least in phase 2 clinical trials. RECENT FINDINGS: There are several new mechanisms of action being represented within clinical development, including histone deacetylase (HDAC) inhibitors, gene therapies, broadly neutralizing anti-HIV antibodies, immune modulation, and drugs with new mechanisms to block HIV entry. The new therapies are being developed for both as add-on therapy to existing combination antiretroviral therapy and as agents to be used during treatment interruption. The current drugs in development have had varying degrees of success in the early trials. Each of these new drugs may potentially fill a void in current antiretroviral therapy (ART) therapies, which will ultimately lead to improved outcomes in HIV-infected individuals.

Effects of HIV-1 Tat and Methamphetamine on Blood-Brain Barrier Integrity and Function In Vitro.

Human immunodeficiency (HIV) infection results in neurocognitive deficits in about one half of infected individuals. Despite systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting central nervous system (CNS)-localized infection. Drug abuse exacerbates HIV-induced cognitive and pathological CNS changes. This study's purpose was to investigate the effects of the HIV-1 protein Tat and methamphetamine on factors affecting drug penetration across an in vitro BBB model. Factors affecting paracellular and transcellular flux in the presence of Tat and methamphetamine were examined. Transendothelial electrical resistance, ZO-1 expression, and lucifer yellow (a paracellular tracer) flux were aspects of paracellular processes that were examined. Additionally, effects on P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP-1) mRNA (via quantitative PCR [qPCR]) and protein (via immunoblotting) expression were measured; Pgp and MRP-1 are drug efflux proteins. Transporter function was examined after exposure of Tat with or without methamphetamine using the P-gp substrate rhodamine 123 and also using the dual P-gp/MRP-1 substrate and protease inhibitor atazanavir. Tat and methamphetamine elicit complex changes affecting transcellular and paracellular transport processes. Neither Tat nor methamphetamine significantly altered P-gp expression. However, Tat plus methamphetamine exposure significantly increased rhodamine 123 accumulation within brain endothelial cells, suggesting that treatment inhibited or impaired P-gp function. Intracellular accumulation of atazanavir was not significantly altered after Tat or methamphetamine exposure. Atazanavir accumulation was, however, significantly increased by simultaneous inhibition of P-gp and MRP. Collectively, our investigations indicate that Tat and methamphetamine alter aspects of BBB integrity without affecting net flux of paracellular compounds. Tat and methamphetamine may also affect several aspects of transcellular transport.

HIV-1 Tat disrupts blood-brain barrier integrity and increases phagocytic perivascular macrophages and microglia in the dorsal striatum of transgenic mice.

HIV-1 infection results in blood-brain barrier (BBB) disruption, which acts as a rate-limiting step for HIV-1 entry into the CNS and for subsequent neuroinflammatory/neurotoxic actions. One mechanism by which HIV may destabilize the BBB involves actions of the HIV-1 regulatory protein, trans-activator of transcription (Tat). We utilized a conditional, Tat-expressing transgenic murine model to examine the influence of Tat1-86 expression on BBB integrity and to assess the relative numbers of phagocytic perivascular macrophages and microglia within the CNS in vivo. The effects of Tat exposure on sodium-fluorescein (Na-F; 0.376kDa), horseradish peroxidase (HRP; 44kDa), and Texas Red-labeled dextran (70kDa) leakage into the brain were assessed in Tat-exposed (Tat+) and control (Tat-) mice. Exposure to HIV-1 Tat significantly increased both Na-F and HRP, but not the larger sized Texas Red-labeled dextran, confirming BBB breakdown and also suggesting the breach was limited to molecules <70kDa. Additionally, at 5 d after Tat induction, Alexa Fluor® 488-labeled dextran was bilaterally infused into the lateral ventricles 5 d before the termination of the experiment. Within the caudate/putamen, Tat induction increased the proportion of dextran-labeled Iba-1+ phagocytic perivascular macrophages (∼5-fold) and microglia (∼3-fold) compared to Tat- mice. These data suggest that HIV-1 Tat exposure is sufficient to destabilize BBB integrity and to increase the presence of activated, phagocytic, perivascular macrophages and microglia in an in vivo model of neuroAIDS.