In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood-Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope = 0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/ reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1b) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.

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The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 Å resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNAprimed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 Å away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.Human immunodeficiency virus, type 1 (HIV-1), reverse transcriptase (RT) is essential for HIV replication. RT converts the single-stranded viral genomic RNA into a linear doublestranded DNA that can be integrated into the host chromosomes (reviewed in ref 1). The enzyme has two activities, (i) a DNA polymerase that can use either RNA or DNA as a template and (ii) an RNase H (RNH) that selectively degrades the RNA strand of an RNA-DNA heteroduplex. The RNH activity of RT is required for virus replication; cellular RNH cannot substitute for the retroviral enzyme (2). The RNH activity degrades the genomic RNA during first-strand ("minus-strand") DNA synthesis, which allows the newly synthesized DNA to be used as a template for second-strand ("plus-strand") DNA synthesis.HIV-1 RT is a heterodimer consisting of 66 kDa (p66) and 51 kDa (p51) subunits. The two polypeptide chains have 440 N-terminal amino acid residues in common. These comprise four polymerase subdomains: the thumb, palm, fingers, and connection (3,4). The C-terminus of p66 contains an additional 120 amino acid residues that form the bulk of the RNH domain. Despite having identical N-terminal sequences, the arrangement of the subdomains in the two subunits differs dramatically. The p66 subunit contains a large cleft formed by the fingers, palm, and thumb subdo-mains that can accommodate double-stranded nucleic acid templateprimers (3-6). Although the p51 subunit contains the same four subdomains, it does not form a nucleic acid binding cleft.Because of its pivotal role in the HIV life cycle, HIV RT is a primary target for antiretroviral agents. All RT inhibitors currently approved for the treatment of acquired immune deficiency syndrome (AIDS) inhibit...

SUMMARYPurpose: P450 enzymes (CYPs) play a major role in hepatic drug metabolism. It is unclear whether these enzymes are functionally expressed by the diseased human bloodbrain barrier (BBB) and are involved in local drug metabolism or response. We have evaluated the cerebrovascular CYP expression and function, hypothesizing possible implication in drug-resistant epilepsy. Methods: CYP P450 transcript levels were assessed by cDNA microarray in primary endothelial cultures established from a cohort of brain resections (n = 12, drug-resistant epilepsy EPI-EC and aneurism domes ANE-EC). A human brain endothelial cell line (HBMEC) and non-brain endothelial cell line (HUVEC) were used as controls. The effect of exposure to shear stress on CYP expression was evaluated. Results were confirmed by Western blot and immunohistochemistry on brain specimens. Endothelial drug metabolism was assessed by high performance liquid chromatography (HPLC-UV). Results: cDNA microarray showed the presence of CYP enzymes in isolated human primary brain endothelial cells. Using EPI-EC and HBMEC we found that CYP mRNA levels were significantly affected by exposure to shear stress. CYP3A4 protein was overexpressed in EPI-EC (290 ± 30%) compared to HBMEC and further upregulated by shear stress exposure. CYP3A4 was increased in the vascular compartment at regions of reactive gliosis in the drugresistant epileptic brain. Metabolism of carbamazepine was significantly elevated in EPI-EC compared to HBMEC. Discussion: These results support the hypothesis of local drug metabolism at the diseased BBB. The direct association between BBB CYP enzymes and the drug-resistant phenotype needs to be further investigated.

Summary:Purpose: A biotechnologic breakthrough for the study of drug permeability across the blood-brain barrier (BBB) would be the use of a reproducible in vitro model that recapitulates the functional, structural, and pathologic properties of the BBB in situ. We developed a humanized dynamic in vitro BBB model (DIV-BBB) based on cocultures of human microvascular endothelial cells (HBMECs) from "normal" and drug-resistant epileptic brain tissue with human brain astrocytes (HAs) from epilepsy patients or controls.Methods: HBMECs and HAs were cocultured for 28 days in polypropylene capillaries. HBMECs were exposed to physiologic levels of shear stress generated by intraluminal flow. Permeability to [3 H]sucrose, [ 14 C]phenytoin, and [ 14 C]diazepam was measured in control and drug-resistant DIV-BBB with and without pretreatment with the MDR1 inhibitor XR9576. BBB integrity was monitored by transendothelial electrical resistance measurements (TEERs). Cell growth and viability were assessed by measurement of glucose consumption and lactate production.Results: P Sucrose and TEER values did not depend on the origin of the endothelium used (epileptic or normal). P Phenytoin was 10-fold less (1.54 × 10 −6 cm/s) in drug-resistant BBB models than in controls (1.74 × 10 −5 cm/s). MDR1 blockade with XR9576 was effective (3.5-fold increase) only in drug-resistant cultures. P Diazepam in control and drug-resistant DIV-BBB was not affected by XR9576 and did not depend on the epileptic or control origin of endothelia. The overall contribution of epileptic glia to pharmacoresistance was negligible.Conclusions: These results show that, for the substances used, the humanized DIV-BBB recapitulates the physiologic permeability properties of the BBB in vivo and is also capable of mimicking a drug-resistant BBB phenotype.

SUMMARYPurpose: Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug-resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co-localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival. Methods: Brain specimens were obtained from eight patients undergoing resection to relieve drug-resistant seizures or to remove a cavernous angioma. Each specimen was partitioned for either immunostaining or primary culture of human endothelial cells and astrocytes. Immunostaining was performed using anti-CYP3A4, MDR1, GFAP, or NeuN antibodies. High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was used to quantify carbamazepine (CBZ) metabolism by these cells.CYP3A4 expression was correlated to DAPI) condensation, a marker of cell viability. Human embryonic kidney (HEK) cells were transfected with 4¢,6-diamidino-2-phenylindole (CYP3A4 to further evaluate the link between CYP3A4 levels, CBZ metabolism, and cell viability. Key Findings: CYP3A4 was expressed by blood-brain barrier (BBB) endothelial cells and by the majority of neurons (75 ± 10%). Fluorescent immunostaining showed coexpression of CYP3A4 and MDR1 in endothelial cells and neurons. CYP3A4 expression inversely correlated with DAPI nuclear condensation. CYP3A4 overexpression in HEK cells conferred resistance to cytotoxic levels of carbamazepine. CYP3A4 levels positively correlated with the amount of CBZ metabolized. Significance: CYP3A4 brain expression is not only associated with drug metabolism but may also represent a cytoprotective mechanism. Coexpression of CYP3A4 and MDR1 may be involved in cell survival in the diseased brain.

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Abstractobjective To determine the impact of environmental nudges on handwashing behaviours among primary school children as compared to a high-intensity hygiene education intervention.methods In a cluster-randomised trial (CRT), we compared the rates of handwashing with soap (HWWS) after a toileting event among primary school students in rural Bangladesh. Eligible schools (government run, on-site sanitation and water, no hygiene interventions in last year, fewer than 450 students) were identified, and 20 schools were randomly selected and allocated without blinding to one of four interventions, five schools per group: simultaneous handwashing infrastructure and nudge construction, sequential infrastructure then nudge construction, simultaneous infrastructure and highintensity hygiene education (HE) and sequential handwashing infrastructure and HE. The primary outcome, incidence of HWWS after a toileting event, was compared between the intervention groups at different data collection points with robust-Poisson regression analysis with generalised estimating equations, adjusting for school-level clustering of outcomes.results The nudge intervention and the HE intervention were found to be equally effective at sustained impact over 5 months post-intervention (adjusted IRR 0.81, 95% CI 0.61-1.09). When comparing intervention delivery timing, the simultaneous delivery of the HE intervention significantly outperformed the sequential HE delivery (adjusted IRR 1.58 CI 1.20-2.08), whereas no significant difference was observed between sequential and simultaneous nudge intervention delivery (adjusted IRR 0.75, 95% CI 0.48-1.17).conclusion Our trial demonstrates sustained improved handwashing behaviour 5 months after the nudge intervention. The nudge intervention's comparable performance to a high-intensity hygiene education intervention is encouraging.