Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise

BACKGROUND: Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification. METHODS: Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined. RESULTS: EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events. CONCLUSION: 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.     

Factor analysis of lifestyle-related factors in 12,525 urban Japanese subjects

This study describes the clustering patterns of several lifestyle-related factors in urban Japanese subjects. The effect of aging on these patterns was also investigated. Data of 8 factors that included body mass index (BMI), blood pressure (BP), fasting plasma glucose (FPG), serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLc), gamma-glutamyl-transferase (GGT), and cigarette smoking were analyzed for 12,525 individuals (4,591 men and 7,934 women) aged either 40, 50, or 60 years. Factor analysis showed eight factors clustered into 3 unrelated groups. BMI and BP were excluded in subjects aged 60 years. Our data showed that the effect of obesity on the prevalence of type 2 diabetes mellitus was age dependent. In spite of the established inverse relationship between TG and HDLc, we found that TG had an association with GGT. These results indicated that aging may have a major influence on the expression of multiple risk factors. The influence of BMI on the lifestyle-related factors appeared to be mostly expressed in younger people, while these factors appeared to be independent of BMI at age 60.     

Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism. IDO activity is linked with immunosuppression by its ability to inhibit lymphocyte proliferation, and with neurotoxicity through the generation of quinolinic acid and other toxins. IDO is induced in macrophages by HIV-1 infection, and it is up regulated in macrophages in human brain tissue with HIV-1 encephalitis (HIVE). Using a model of HIVE, we investigated whether IDO inhibitor 1-methyl-d-tryptophan (1-MT) could affect the generation of cytotoxic T lymphocytes (CTLs) and clearance of virus-infected macrophages from the brain. Severe combined immunodeficient mice were reconstituted with human peripheral blood lymphocytes, and encephalitis was induced by intracranial injection of autologous HIV-1-infected monocyte-derived macrophages (MDMs). Animals treated with 1-MT demonstrated increased numbers of human CD3+, CD8+, CD8+/interferon-gamma+ T cells, and HIV-1(gag/pol)-specific CTLs in peripheral blood compared with controls. At week 2 after MDM injection in the basal ganglia, mice treated with 1-MT showed a 2-fold increase in CD8+ T lymphocytes in the areas of the brain containing HIV-1-infected MDMs compared with untreated controls. By week 3, 1-MT-treated mice showed 89% reduction in HIV-infected MDMs in brain as compared with controls. Thus, manipulation of immunosuppressive IDO activity in HIVE may enhance the generation of HIV-1-specific CTLs, leading to elimination of HIV-1-infected macrophages in brain.