Modelling the Human Blood-Brain Barrier in Huntington Disease.

While blood-brain barrier (BBB) dysfunction has been described in neurological disorders, including Huntington's disease (HD), it is not known if endothelial cells themselves are functionally compromised when promoting BBB dysfunction. Furthermore, the underlying mechanisms of BBB dysfunction remain elusive given the limitations with mouse models and post mortem tissue to identify primary deficits. We established models of BBB and undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived brain-like microvascular endothelial cells (iBMEC) from HD patients or unaffected controls. We demonstrated that HD-iBMECs have abnormalities in barrier properties, as well as in specific BBB functions such as receptor-mediated transcytosis.

Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System.

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).

Structure-Activity Relationship of novel phenylacetic CXCR1 inhibitors.

We reported recently the Structure-Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2.

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis.

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.

IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease.

The lupus-like autoimmune syndrome of MRL/Mp-Tnfrsf6lpr (lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocyte-dependent cytokines, IFN-gamma plays a pivotal role in the abnormal cell activation and the fatal development of the lpr disease. IL-18, an inducer of IFN-gamma in T lymphocytes and natural killer cells, may contribute to the disease because cells from lpr mice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model, in vivo inhibition of IL-18 was attempted. Young lpr mice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-gamma production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome of lpr mice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.