Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier

The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Ovarian sertoli-leydig cell tumour with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alphafetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrineinactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood–brain barrier hyperpermeability

Aims: Hyperglycaemia (HG), in stroke patients, is associated with worse neurological outcome by compromising endothelial cell function and the blood–brain barrier (BBB) integrity. We have studied the contribution of HG-mediated generation of oxidative stress to these pathologies and examined whether antioxidants as well as normalization of glucose levels following hyperglycaemic insult reverse these phenomena. Methods: Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures were used to simulate the human BBB. The integrity of the BBB was measured by transendothelial electrical resistance using STX electrodes and an EVOM resistance meter, while enzyme activities were measured by specific spectrophotometric assays. Results: After 5 days of hyperglycaemic insult, there was a significant increase in BBB permeability that was reversed by glucose normalization. Co-treatment of cells with HG and a number of antioxidants including vitamin C, free radical scavengers and antioxidant enzymes including catalase and superoxide dismutase mimetics attenuated the detrimental effects of HG. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C but not phosphoinositide 3 kinase (PI3 kinase) also reversed HG-induced BBB hyperpermeability. In HBMEC, HG enhanced pro-oxidant (NAD(P)H oxidase) enzyme activity and expression that were normalized by reverting to normoglycaemia. Conclusions: HG impairs brain microvascular endothelial function through involvements of oxidative stress and several signal transduction pathways.

Maternal diabetes affects cell proliferation in developing rat placenta

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

Titanium phosphate for Fuel Cell Proton Conduction Membranes

Environmental issues due to increases in emissions of air pollutants and greenhouse gases are driving the development of clean energy delivery technologies such as fuel cells. Low temperature Proton Exchange Membrane Fuel Cells (PEMFC) use hydrogen as a fuel and their only emission is water. While significant advances have been made in recent years, a major limitation of the current technology is the cost and materials limitations of the proton conduction membrane. The proton exchange membrane performs three critical functions in the PEMFC membrane electrode assembly (MEA): (i) conduction of protons with minimal resistance from the anode (where they are generated from hydrogen) to the cathode (where they combine with oxygen and electrons, from the external circuit or load), (ii) providing electrical insulation between the anode and cathode to prevent shorting, and (iii) providing a gas impermeable barrier to prevent mixing of the fuel (hydrogen) and oxidant. The PFSA (perfluorosulphonic acid) family of membranes is currently the best developed proton conduction membrane commercially available, but these materials are limited to operation below 100oC (typically 80oC, or lower) due to the thermochemical limitations of this polymer. For both mobile and stationary applications, fuel cell companies require more durable, cost effective membrane technologies capable of delivering enhanced performance at higher temperatures (typically 120oC, or higher. This is driving research into a wide range of novel organic and inorganic materials with the potential to be good proton conductors and form coherent membranes. There are several research efforts recently reported in the literature employing inorganic nanomaterials. These include functionalised silica phosphates [1,2], fullerene [3] titania phosphates [4], zirconium pyrophosphate [5]. This work addresses the functionalisation of titania particles with phosphoric acid. Proton conductivity measurements are given together with structural properties.

Claudin-7 down-regulation is an important feature in oral squamous cell carcinoma

Aims: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas, especially claudin-7. The aim of this study was to investigate claudin-7 expression and alterations in oral squamous cell carcinoma (OSCC). Methods and results: Expression of claudin-7 was analysed in 132 cases of OSCC organized in a tissue microarray. Claudin-7 mRNA transcript was evaluated using real-time polymerase chain reaction and the methylation status of the promoter was also assessed. Claudin-7 was negative in 58.3% of the cases. Loss of claudin-7 protein expression was associated with recurrence (P = 0.019), tumour size (P = 0.014), clinical stage of OSCC (P = 0.055) and disease-free survival (P = 0.015). Down-regulation of the claudin-7 mRNA transcripts was observed in 78% of the cases, in accordance with immunoexpression. Analysis of the methylation status of the promoter region of claudin-7 revealed that treatment of O28 cells (that did not express claudin-7 mRNA transcripts) with 5-Aza-2`-Deoxycytidine (5-Aza-dC) led to the re-expression of claudin-7 mRNA transcript. Conclusion: Loss of claudin-7 expression is associated with important subcellular processes in OSCC with impact on clinical parameters.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Pediatric glioblastoma cell line shows different patterns of expression of transmembrane ABC transporters after in vitro exposure to vinblastine

Resistance to drug is a major cause of treatment failure in pediatric brain cancer. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histology, is poorly described. To compare the expression profiles of the multidrug resistance genes ABCB1, ABCC1, and ABCG2 in different neuroepithelial pediatric brain tumor cell lines prior and following short-term culture with vinblastine. Immortalized lineages from pilocytic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were exposed to vinblastine sulphate at different schedules (10 and 60 nM for 24 and 72 h). Relative amounts of mRNA expression were analyzed by real-time quantitative polymerase chain reaction. Protein expression was assessed by immunohistochemistry for ABCB1, ABCC1, and ABCG2. mRNA expression of ABCB1 increased together with augmenting concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, ABCB1 levels of expression diminished in SF188. Following chemotherapy, mRNA expression of ABCC1 decreased in all cell lines other than glioblastoma. ABCG2 expression was influenced by vinblastine only for UW3. The mRNA levels showed consistent association to protein expression in the selected sets of cell lines analyzed. The pediatric glioblastoma cell line SF188 shows different pattern of expression of multidrug resistance genes when exposed to vinblastine. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors.