Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation.

Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

Characteristics of the low density corneal endothelial monolayer.

Corneal endothelial cells form a leaky barrier on the posterior surface of the cornea, allowing influx of nutrient-carrying aqueous humor through the paracellular space and efflux of excess fluid. Corneal edema arises when the density of these non-proliferative endothelial cells declines from endothelial disease or intraocular surgery. The cellular changes occurring at low densities are ill-defined. We therefore investigated the paracellular pathway of corneal endothelial cell monolayers of varying density to determine alterations occurring in paracellular permeability and monolayer morphology. Primary cultures of bovine corneal endothelial cells (BCECs) were passaged onto permeable supports under varying culture conditions to obtain confluent monolayers of <1000, 1000-1999 and >2000 cells/mm(2). Culture growth was monitored by transendothelial electrical resistance measurements. Diffusional permeability to sodium fluorescein, FITC-dextran MW 4000 or FITC-dextran MW 20,000 was measured. Confluent cultures were also analyzed by immunofluorescence localization of the tight junction protein ZO-1 and by transmission electron microscopy. For comparison, we evaluated ZO-1 for low and high density human corneal endothelium. Our results showed that all BCEC cultures grew to the same final transendothelial electrical resistance regardless of final density. In the diffusional permeability assay, permeability increased significantly only for the smallest tracer molecule (sodium fluorescein) in the lowest density monolayers (<1000 cells/mm(2)). ZO-1 immunofluorescence distinctly localized to intercellular junctions in high density BCEC cultures but had more diffuse localization at lower densities. Transmission electron microscopy imaging revealed cells with thinner cross-sectional profiles and longer overlapping intercellular processes at low density relative to high density cultures. Low density human corneal endothelium lacked the diffuse ZO-1 distribution seen in BCECs. Our data supports the hypothesis that barrier integrity is the primary function disrupted in low density corneal endothelial monolayers and contradicts the idea of a linear decline in barrier function with decreasing cell density.

Characterisation of Listeria monocytogenes isolates from cattle using a bovine caruncular epithelial cell model.

Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.

Biomacromolecules as carriers in drug delivery and tissue engineering.

Natural biomacromolecules have attracted increased attention as carriers in biomedicine in recent years because of their inherent biochemical and biophysical properties including renewability, nontoxicity, biocompatibility, biodegradability, long blood circulation time and targeting ability. Recent advances in our understanding of the biological functions of natural-origin biomacromolecules and the progress in the study of biological drug carriers indicate that such carriers may have advantages over synthetic material-based carriers in terms of half-life, stability, safety and ease of manufacture. In this review, we give a brief introduction to the biochemical properties of the widely used biomacromolecule-based carriers such as albumin, lipoproteins and polysaccharides. Then examples from the clinic and in recent laboratory development are summarized. Finally the current challenges and future prospects of present biological carriers are discussed.

Study of the transcytosis of an anti-transferrin receptor antibody with a Fab' cargo across the blood-brain barrier in mice.

One strategy used to transport pharmacologically active substances across the blood-brain barrier (BBB) is to link the substance to a molecule capable of crossing the BBB using a receptor-mediated transcellular transport system in brain capillary endothelial cells. The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 antibody, directed against mouse TfR, is able to induce a receptor response. In this work, the potential of 8D3 to carry molecules across the BBB was investigated. This antibody (either unlabeled or FITC-labeled) was intravenously administered to ICR-CD1 mice. Immunocomplexes (ICs) consisting of 8D3 antibody (carrier) and Fab' fragments (simulated cargo), in some cases directly fluorolabeled with FITC, were also administered to ICR-CD1 mice. At different time-points of recirculation, the IC components were studied using immunohistochemical procedures and fluorescence resonance energy transfer (FRET). The results suggested that 8D3 alone or in an IC with Fab' fragments entered the endothelial cells. FRET analysis indicated that the colocalization of their fluorescent signals inside the endothelial cells decreased with time, indicating that ICs can be processed and Fab' fragments probably separated from 8D3. However, neither 8D3 alone nor the IC components crossed the BBB.

Endothelial calcium dynamics, connexin channels and blood-brain barrier function.

Situated between the circulation and the brain, the blood-brain barrier (BBB) protects the brain from circulating toxins while securing a specialized environment for neuro-glial signaling. BBB capillary endothelial cells exhibit low transcytotic activity and a tight, junctional network that, aided by the cytoskeleton, restricts paracellular permeability. The latter is subject of extensive research as it relates to neuropathology, edema and inflammation. A key determinant in regulating paracellular permeability is the endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)]i) that affects junctional and cytoskeletal proteins. Ca(2+) signals are not one-time events restricted to a single cell but often appear as oscillatory [Ca(2+)]i changes that may propagate between cells as intercellular Ca(2+) waves. The effect of Ca(2+) oscillations/waves on BBB function is largely unknown and we here review current evidence on how [Ca(2+)]i dynamics influence BBB permeability.

Carbon nanotubes for delivery of small molecule drugs.

In the realm of drug delivery, carbon nanotubes (CNTs) have gained tremendous attention as promising nanocarriers, owing to their distinct characteristics, such as high surface area, enhanced cellular uptake and the possibility to be easily conjugated with many therapeutics, including both small molecules and biologics, displaying superior efficacy, enhanced specificity and diminished side effects. While most CNT-based drug delivery system (DDS) had been engineered to combat cancers, there are also emerging reports that employ CNTs as either the main carrier or adjunct material for the delivery of various non-anticancer drugs. In this review, the delivery of small molecule drugs is expounded, with special attention paid to the current progress of in vitro and in vivo research involving CNT-based DDSs, before finally concluding with some consideration on inevitable complications that hamper successful disease intervention with CNTs.