Vaccinia virus-induced smallpox postvaccinal encephalitis in case of blood-brain barrier damage.

Smallpox vaccination is the only currently effective mean to combat the threat of variola virus used as a bioterrorism agent, although it is responsible for a rare but serious complication, the postvaccinal encephalitis (PVE). Development of safer vaccines therefore is a high priority as the PVE physiopathology is not well understood to date. If vaccinia virus (VACV) is responsible for PVE by central nervous system (CNS) dissemination, trans-migration of the VACV across the blood-brain barrier (BBB) would be supposed to be essential. Given the complexity of the pathogenesis of vaccinia neurovirulence, an in vitro BBB model was used to explore the mechanism of VACV to induce BBB permeability. Two VACV strains were studied, the neurovirulent Western Reserve strain (VACV-WR) and the vaccine reference Lister strain (VACV-List). A mouse model was also developed to study the ability of these two viral strains to propagate in the brain from the blood compartment, their neurovirulence and their neuropathogenesis. In vitro, the loss of permeability resulted from the tight-junctions disruption was induced by virus replication. The ability of VACV to release infectious particles at the abluminal side suggests the capacity of both VACV strains to migrate across the BBB from the blood to the CNS. In vivo, the virus replication in mice CNS was strain-dependent. The VACV-WR laboratory strain proved to be neuroinvasive and neurovirulent, whereas the VACV-List strain is safe in physiological conditions. Mice PVE was observed only with VACV-WR in the co-infection model, when BBB opening was obtained by lipopolysaccharide (LPS) treatment. This study suggests that VACV is able to cross the BBB but encephalitis occurs only in the presence of a co-infection by bacteria. So, a model of co-infection, mimicked by LPS treatment, could have important implication towards the assessment of neurovirulence of new vaccines.

Radiation-induced blood-brain barrier damages: an in vitro study.

A radiation-induced blood-brain barrier (BBB) breakdown has been supposed to explain the acute radiation syndrome and the delayed brain radiation injury, but it has been clearly demonstrated only at high doses. In a previous study (Diserbo et al., 2002), we showed that non-lethal total body irradiation produced an early transient increase in BBB permeability in rats but the underlying mechanisms of radiation-induced BBB breakdown remain unclear. In the present work, the effects of ionizing radiation were studied on an in vitro BBB model. Gamma irradiation induced an increase in [(14)C]-sucrose BBB permeability that can be detected 72 h after exposure at doses up to 4 Gy. This increase was more important 8 days after irradiation and could be limited by dexamethasone treatment. An increase in fluorescein and FITC-dextrans (4 kDa/70 kDa) permeability was also observed, which can be related to a substantial opening of endothelial cell tight-junctions but without massive modification of tight-junction protein (ZO-1, ZO-2, claudin-5, occludin) immunolabeling even 8 days after 25 Gy exposure. Formation of actin stress fibers occurred in endothelial cells 8 days after 25 Gy exposure. A progressive decrease in cellular density associated with a simultaneous spreading of the endothelial cells was also observed after irradiation. Anti-γH2AX immunolabeling was used to investigate both DNA double-strand break induction and repair rates in endothelial cells. It revealed long-lasting DNA double-strand breaks after gamma irradiation. A better understanding and awareness of these phenomena are essential for designing appropriate pharmacotherapy in radiation-therapy and treatment of accidental overexposure.

Gulf War illness: Effects of repeated stress and pyridostigmine treatment on blood-brain barrier permeability and cholinesterase activity in rat brain.

After the first Persian Gulf War, many soldiers have complained of a variety of symptoms designated as "Gulf War Illness". Among several factors, implication of pyridostigmine (PB) in late cognitive dysfunction is highly likely. As a hypothesis to explain these behavioural disorders is a potentiation of the operational stress effects by pyridostigmine. We have previously described that repeated stress combined to pyridostigmine treatment induces learning dysfunction linked to genomic cerebral modifications [Barbier L, Diserbo M, Lamproglou I, Amourette C, Peinnequin A, Fauquette W. Repeated stress in combination with pyridostigmine: part II-changes in selected cerebral genes expression. Behav Brain Res 2009;197:292-300; Lamproglou I, Barbier L, Diserbo M, Fauvelle F, Fauquette W, Amourette C. Repeated stress in combination with pyridostigmine: part I-long-term behavioural consequences. Behav Brain Res 2009;197:301-10]. In the present study, using the same experimental model, we attempted to determine if such modifications are linked to a central passage of pyridostigmine under stress. Indeed it is known that exposure to stress can disrupt blood-brain barrier (BBB) and thereby increase the neurotoxicity induced by chemicals in many cerebral areas. Adult rats were subjected to repeated stress based on a modification of the pole climbing avoidance technique and treated daily by PB (1.5mg/kg/day, oral in water), for two 5-day periods separated by 2-day rest. Just after the last stress session, (3)H-pyridostigmine was administered as a tracer to evaluate BBB breakdown. In brain micro-punches and brain coronal cryosections, we failed to detect any radioactivity in animals chronically stressed and treated by pyridostigmine. Accordingly, no change of ChE activity was noted in any brain area studied. It thus appears that, in our experimental model, pyridostigmine induces effects on central nervous system, but these effects do not seem to be mediated by a central passage of pyridostigmine linked to a BBB opening under stress. These results suggest that pyridostigmine may have central effects, under stress, via indirect mechanisms emerging from a peripheral pathway.

Repeated stress in combination with pyridostigmine Part I: long-term behavioural consequences.

Since their return from the first Persian Gulf War, some veterans have complained of a variety of symptoms that were designated as "Gulf War Illness" (GWI). Among other factors, pyridostigmine, used as a prophylaxis treatment against intoxication by nerve agents, has been proposed by many authors as a cause of late social and/or cognitive dysfunction related to GWI. One of the hypotheses placed to explain these behavioural disorders is that operational stress has modified the side effects of pyridostigmine given to soldiers. In an attempt to establish an experimental model of GWI to evaluate the long-term behavioural effects of pyridostigmine administered in stressful conditions, we have developed a new model of repeated stress based on the pole-climbing avoidance technique. We used it to evaluate the effects of pyridostigmine treatment combined to repeated stress over the months following the end of the treatment. We observed that this stress induces impulsiveness and aggressiveness in adult male rat. Moreover, pyridostigmine treatment administered daily 30 min before each stressful session amplifies these behavioural disorders and induces long-term learning dysfunction and slight but significant decrease in phosphocholine level in hippocampus. This suggests that repeated administration of pyridostigmine combined to pole-climbing avoidance (PCA) stress conditions can induce adverse effects in rat central nervous system.

Repeated stress in combination with pyridostigmine Part II: changes in cerebral gene expression.

Organophosphates (OP) represent a potential threat in terrorism or during military conflicts. Due to its faculty to protect cholinesterase (ChE) activity against irreversible inactivation by OP, pyridostigmine bromide (PB) was used as a prophylaxis treatment during the first Persian Gulf War. To explain dysfunctions reported by Gulf War Veterans (GWV), it was suggested a potentiation of the operational stress effects by PB given to soldiers. Our companion paper (see part 1 in the same journal issue) describes that PB treatment administered in repeated stress conditions results in long-term perturbations of learning and social behaviour. The present paper examines, in adult male Wistar rats, consequences of the association of repeated stress and PB treatment on gene expression in hypothalamus and hippocampus. PB treatment (1.5 mg/kg/day) was orally administered 30 min before each stress session to inhibit 40% of blood ChE as recommended by NATO. 10 days of stress alone induce a decrease in hypothalamic Il-1alpha expression. Treatment with PB alone increases mineralocorticoid receptor expression in hypothalamus which means that PB may thus modify stress perception by animals. Stressed-PB animals showed increase in hippocampal expression of BDNF, TrkB and CamKIIalpha, three genes implicated in memory development. As a supplement to previous studies showing behavioural and biochemical effects of the association of stress with PB, our data reveal that behavioural effects of this association may be linked with genomic changes in hippocampus. Mechanisms underlying these modifications and their link with memory disturbances reported by GWV remain to be further determined.

Soman poisoning induces delayed astrogliotic scar and angiogenesis in damaged mouse brain areas.

Gliotic scar formation and angiogenesis are two biological events involved in the tissue reparative process generally occurring in the brain after mechanically induced injury, ischemia or cerebral tumor development. For the first time, in this study, neo-vascularization and glial scar formation were investigated in the brain of soman-poisoned mice over a 3-month period after nerve agent exposure (1.2 LD50 of soman). Using anti-claudin-5 and anti-vascular endothelial growth factor (VEGF) immunostaining techniques on brain sections, blood vessels were quantified and VEGF expression was verified to appraise the level of neo-angiogenesis induced in damaged brain areas. Furthermore, glial scar formation and neuropathology were estimated over time in the same injured brain regions by anti-glial fibrillary acidic protein (GFAP) immunohistochemistry and hemalun-phloxin (H&P) dye staining, respectively. VEGF over-expression was noticed on post-soman day 3 in lesioned areas such as the hippocampal CA1 field and amygdala. This was followed by an increase in the quantity of mature blood vessels, 3 months after soman poisoning, in the same brain areas. On the other hand, massive astroglial cell activation was demonstrated on post-soman day 8. Reactive astroglial cells were located only in damaged cerebral regions where H&P-stained eosinophilic neurons were found. For longer experimental times, astroglial response slowly decreased overtime but remained detectable on post-soman day 90 in some discrete brain regions (i.e. CA1 field and amygdala) evidencing the formation of a glial scar. In this study, we discuss the key role of VEGF in the angiogenic process and in the glial or neuronal response induced by soman poisoning.

Normal breast epithelial cells induce p53-dependent apoptosis and p53-independent cell cycle arrest of breast cancer cells.

Cancer development depends not only on the nature of cancerous cells themselves, but also on the regulatory effects of various normal cells. The present study was performed to investigate the effect of normal breast epithelial cells (NBEC) on the growth of breast cancer cells under various conditions. We demonstrated that NBEC-conditioned medium (NBEC-CM) inhibited growth of breast cancer cell lines in monolayer culture and three-dimensional collagen gel culture, as well as in soft agar. In MCF-7 and T-47D cells which have a functional p53, NBEC-CM induced apoptosis without modifying cell cycle progression. In MDA-MB-231 and BT-20 cells that have a non-functional p53, NBEC-CM did not induce apoptosis, although a slight G1 blokage was observed in MDA-MB-231 cells. Transient transfections of MCF-7 and T-47D cells demonstrated that NBEC-triggered apoptosis was mediated by endogenous p53. Moreover, pifithrin-alpha which specifically inhibits the transcriptional activity of p53, completely abolished NBEC-induced apoptosis in both MCF-7 and T-47D cells, indicating that p53 mediated apoptosis via its transcriptional activity. Finally, orthovanadate, a protein tyrosine phosphatase inhibitor, completely inhibited NBEC-triggered apoptosis, indicating that NBEC-triggered apoptosis was regulated by tyrosine phosphatases.

Cross-talk between mesenchyme and epithelium increases H19 gene expression during scattering and morphogenesis of epithelial cells.

The H19 gene is an imprinted gene expressed from the maternal allele. It is known to function as an RNA molecule. We previously reported that in breast adenocarcinoma, H19 is often overexpressed in stromal cells and preferentially located at the epithelium/stroma boundary, suggesting that epithelial/mesenchymal interactions can control H19 RNA expression. In some cases of breast adenocarcinoma with poor prognosis, H19 is overexpressed in epithelial cells. Therefore we examined whether mesenchymal factors can induce H19 expression in epithelial cells. Using quantitative RT-PCR and in situ hybridization, we found that when mammary epithelial cells were cultured in collagen gels, H19 expression was strongly up-regulated compared to when cells were cultured on plastic. Collagen gels allow three-dimensional growth of epithelial cells and morphogenetic responses to soluble factors. A conditioned medium from MRC-5 fibroblasts caused branching morphogenesis of HBL-100 cells and invasive growth of MDA-MB-231 cells, whereas MCF-7 cells were unresponsive. Induction of H19 expression correlated with morphological changes in HBL-100 and in MDA-MB-231 cells, whereas H19 expression was not induced in MCF-7 cells. Using a blocking antibody, HGF/SF was identified as the fibroblast-derived growth factor capable of inducing H19 expression and cell morphogenesis. We further demonstrated that H19 promoter activity was stimulated by various growth factors using transient transfection in MDCK epithelial cells. HGF/SF was more efficient than EGF or FGF-2 in transactivating the H19 promoter, whereas IGF-2, TGFbeta-1, and TNF-alpha were ineffective. This activation by HGF/SF was prevented by pharmacological inhibition of MAP kinase or of phospholipase C. We conclude that H19 is a target gene for HGF/SF, a known regulator of epithelial/mesenchymal interactions, and suggest that the up-regulation of H19 may be implicated in morphogenesis and/or migration of epithelial cells.