Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood-brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1( MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture condition

TRANSPORT OF THE ␤-LACTAM ANTIBIOTIC BENZYLPENICILLIN AND THE DIPEPTIDE GLYCYLSARCOSINE BY BRAIN CAPILLARY ENDOTHELIAL CELLS IN VITRO

This paper is available online at http://www.dmd.org ABSTRACT: Peripherally administered ␤-lactam antibiotics, which are structural analogs of tripeptides, may cause neurotoxic reactions or induce seizures. Previous in vivo studies provided evidence for brain uptake of these antibiotics. In the present work, we studied the extent and mechanism of the uptake of benzylpenicillin and glycylsarcosine by brain microvessel endothelial cells in vitro, using freshly isolated and cultured porcine brain capillary endothelial cells. Characterization of the cell culture model demonstrated the functional expression of the system transporting the neutral amino acids leucine and phenylalanine. The initial rate of uptake of benzylpenicillin was >3-fold greater than the rate of uptake of the extracellular marker sucrose (ratio, 3.29 ؎ 0.37), whereas uptake of glycylsarcosine did not differ from that of sucrose. The differences in cellular uptake correlated with the octanol/buffer partition coefficients for glycylsarcosine and benzylpenicillin (1.16 ؋ 10 ؊3 for glycylsarcosine and 6.83 ؋ 10 ؊2 for benzylpenicillin). The concentration-dependent uptake of benzylpenicillin (1-2000 M) was not saturable and was not sensitive to shifts in pH or temperature. The permeability-surface area product for the uptake of benzylpenicillin at pH 7.4 was determined from these experiments and was found to be 8.1 ؋ 10 ؊5 ml/sec/g of brain. This value was very close to the value determined in in vivo studies. Uptake of benzylpenicillin and glycylsarcosine was not reduced in the presence of 1 mM ceftibuten or 100 M probenecid. The findings with cultured cell monolayers were confirmed using freshly isolated endothelial cells. These in vitro data are compatible with benzylpenicillin, but not glycylsarcosine, being able to penetrate endothelial cells. Uptake of benzylpenicillin by brain capillary endothelial cells occurs by a slow nonsaturable process, with no evidence for carriermediated transport

Increased Expression and Activity of Brain Cortical cPLA2 Due to Chronic Lipopolysaccharide Administration in Mouse Model of Familial Alzheimer’s Disease

Cytosolic phospholipase A2 (cPLA2) is an enzyme regulating membrane phospholipid homeostasis and the release of arachidonic acid utilized in inflammatory responses. It represents an attractive target for the treatment of Alzheimer’s disease (AD). Previously, we showed that lipopolysaccharide (LPS)-induced systemic inflammation caused abnormal lipid metabolism in the brain of a transgenic AD mouse model (APdE9), which might be associated with potential changes in cPLA2 activity. Here, we investigated changes in cPLA2 expression and activity, as well as the molecular mechanisms underlying these alterations due to chronic LPS administration in the cerebral cortex of female APdE9 mice as compared to saline- and LPS-treated female wild-type mice and saline-treated APdE9 mice. The study revealed the significant effects of genotype LPS treatment on cortical cPLA2 protein expression and activity in APdE9 mice. LPS treatment resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in the cortex of APdE9 mice. The gene expressions of inflammation markers Il1b and Tnfa were significantly elevated in the cortex of both APdE9 groups compared to the wild-type groups. The study provides evidence of the elevated expression and activity of cPLA2 in the brain cortex of APdE9 mice after chronic LPS treatment, which could be associated with NFkB activation

Hepatocellular effects of cyclosporine A and its derivative

ABSTRACT The novel immunosuppressive drug O-hydroxyethyl-D(Ser) 8 -cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na ϩ and Ca ϩϩ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na ϩ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure

Correction to: Current research into brain barriers and the delivery of therapeutics for neurological diseases: a report on CNS barrier congress London, UK, 2017

After publication of the article [1], it has been brought to our attention that there are some errors in the formatting of names in the final version of the article

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions