A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions

The aim of this study was to develop a three-dimensional (3D) model of the human blood-brain barrier in vitro, which mimics the cellular architecture of the CNS and could be used to analyse the delivery of nanoparticles to cells of the CNS. The model includes human astrocytes set in a collagen gel, which is overlaid by a monolayer of human brain endothelium (hCMEC/D3 cell line). The model was characterised by transmission electron microscopy (TEM), immunofluorescence microscopy and flow cytometry. A collagenase digestion method could recover the two cell types separately at 92-96% purity. Astrocytes grown in the gel matrix do not divide and they have reduced expression of aquaporin-4 and the endothelin receptor, type B compared to two-dimensional cultures, but maintain their expression of glial fibrillary acidic protein. The effects of conditioned media from these astrocytes on the barrier phenotype of the endothelium was compared with media from astrocytes grown conventionally on a two-dimensional (2D) substratum. Both induce the expression of tight junction proteins zonula occludens-1 and claudin-5 in hCMEC/D3 cells, but there was no difference between the induced expression levels by the two media. The model has been used to assess the transport of glucose-coated 4nm gold nanoparticles and for leukocyte migration. TEM was used to trace and quantitate the movement of the nanoparticles across the endothelium and into the astrocytes. This blood-brain barrier model is very suitable for assessing delivery of nanoparticles and larger biomolecules to cells of the CNS, following transport across the endothelium

Multiple lumbar transverse process stress fractures as a cause of chronic low back ache in a young fast bowler - a case report

A rare case of multilevel transverse process stress fractures as a cause of low back ache in a professional cricket player has been presented. The report discusses the possible mechanism of such an injury in a cricket player and also highlights the preventive and therapeutic aspects of management in such patients. The report also stresses upon the need for early identification of such sports related injuries to prevent long term morbidity in the athletes

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

Lack of efficacy of Doxil® in TNF-α-based isolated limb perfusion in sarcoma-bearing rats

textabstractHere we show that Doxil® has minimal antitumour activity in the isolated limb perfusion (ILP) setting and its activity was not enhanced by the addition of tumour necrosis factor (TNF). Doxil® accumulation in tumour tissue was low and also not augmented by TNF. In contrast, activity of free conventional doxorubicin was enhanced by TNF. We conclude that application of Doxil® in a TNF-based ILP is not a useful alternative to free conventional doxorubicin or melphalan

In vivo isolated kidney perfusion with tumour necrosis factor α (TNF-α) in tumour-bearing rats

Isolated perfusion of the extremities with high-dose tumour necrosis factor α (TNF-α) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-α and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 μg TNF-α. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-α and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-α alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-α, the minimal threshold concentration of TNF-α to exert its anti-tumour effects was not reached. The applicability of TNF-α in isolated kidney perfusion for human tumours seems, therefore, questionable. © 1999 Cancer Research Campaig

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria