Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR.

Group B Streptococcus (GBS) remains the most common Gram-positive bacterium causing neonatal meningitis and GBS meningitis continues to be an important cause of mortality and morbidity. In this study, we showed that GBS penetration into the brain occurred initially in the meningeal and cortex capillaries, and exploits a defined host cell signaling network comprised of S1P2 , EGFR, and CysLT1. GBS exploitation of such network in penetration of the blood-brain barrier was demonstrated by targeting S1P2 , EGFR, and CysLT1 using pharmacological inhibition, gene knockout and knockdown cells, and gene knockout animals, as well as interrogation of the network (up- and downstream of each other). More importantly, counteracting such targets as a therapeutic adjunct to antibiotic therapy was beneficial in improving the outcome of animals with GBS meningitis. These findings indicate that investigating GBS penetration of the blood-brain barrier provides a novel approach for therapeutic development of GBS meningitis.

The response to brain tumor-derived growth factors is altered in radioresistant human brain endothelial cells.

INTRODUCTION: Radioresistant brain tumor vasculature is thought to hamper the efficiency of adjuvant cancer therapies. However, little is known regarding the signalling pathways involved in the angiogenic response to brain tumor-derived growth factors in irradiated human brain microvascular endothelial cells (HBMEC). The goal of this study is to assess the effect of ionizing radiation (IR) on HBMEC survival, migration and tubulogenesis. METHODS: HBMEC were cultured and irradiated at sublethal single doses. Cell survival was assessed by nuclear cell counting and flow cytometry. HBMEC migration in response to brain tumor-derived growth factors (U-87 GF) and tubulogenesis were assayed using modified Boyden chambers and Matrigel, respectively. RESULTS: We observed that single administration of 3-10 Gy IR doses only reduced cell survival by 30%. Radioresistant HBMEC overexpressed RhoA, a small GTPase protein regulating cellular adhesion and migration, and Rho-kinase (ROK), a serine-threonine protein kinase and one of RhoA's major targets. HBMEC migration was induced by vascular endothelial growth factor (VEGF), but even more so in response to sphingo-sine-1-phosphate (S1P) and to U-87 GF. Following IR exposure, HBMEC basal migration increased more than two-fold, whereas the response to S1P and to U-87 GF was significantly diminished. Similarly, the inhibitor of ROK Y-27632 decreased HBMEC migration in response to S1P and U-87 GF. Overexpression of RhoA decreased tubulogenesis, an effect also observed in irradiated HBMEC. CONCLUSION: Our results suggest that radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis are altered following IR. The RhoA/ROK signalling pathway is involved in the IR-altered angiogenic functions and may represent a potential molecular target for enhancing the impact of radiotherapy on tumor-associated endothelial cells.

Meningitis-Associated Escherichia coli.

Escherichia coliis the most common Gram-negative organism causing neonatal meningitis. Neonatal E. colimeningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis and pathophysiology contributes to such mortality and morbidity. Recent reports of neonatal meningitis caused by E. coli strains producing CTX-M-type or TEM-type extended-spectrum ß-lactamases create a challenge. E. colipenetration into the brain, the essential step in the development of E. coli meningitis, requires a high-degree of bacteremia and penetration of the blood-brain barrier as live bacteria, but the underlying mechanisms remain incompletely understood. Recent functional genomic approaches of meningitis-causing E. coli in both in vitro and in vivo models of the blood-brain barrier (e.g., human brain microvascular endothelial cells and animal models of experimental hematogenousE. colimeningitis, respectively) have identified several E. coli factors contributing to a high-degree of bacteremia, as well as specific microbial factors contributing to E. coli invasion of the blood-brain barrier. In addition, E. coli penetration of the blood-brain barrier involves specific host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors and host cell signaling molecules is efficient in preventing E. coli penetration into the brain. Continued investigation of the microbial and host factors contributing to E. colibacteremia andinvasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis, thereby limiting the exposure to emerging antimicrobial-resistant E. coli.

Role of human brain microvascular endothelial cells during central nervous system infection. Significance of indoleamine 2,3-dioxygenase in antimicrobial defence and immunoregulation.

The cerebral endothelium is involved both in regulating the influx of immune cells into the brain and in modifying immunological reactions within the CNS. A number of human pathogens may cause encephalitis or meningitis when this important protective barrier is impaired. We have previously shown that interferon-gamma activated human brain microvascular endothelial cells (HBMEC) restrict the growth of bacteria and parasites. We now provide evidence that HBMEC are also capable of inhibiting viral replication after stimulation with IFN-gamma, an effect further augmented by costimulation with IL-1. This antiviral effect was completely blocked in the presence of L-tryptophan, indicating the induction of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) to be responsible for the observed antiviral effect. Apart from exerting antimicrobial effects tryptophan depletetion has also been described as a regulatory mechanism in T cell responses to both allo- and autoantigens. We were able to demonstrate that IDO mediated degradation of L-tryptohan in HBMEC is responsible for a significant reduction in T lymphocyte proliferation. Resupplementation of L-tryptophan and restoration of initial T cell responses demonstrated the central role of this essential amino acid in the reduction of T-cell proliferation. Brain endothelial cells appear to limit microbial expansion in the CNS by local degradation of tryptophan, thus acting in concert with other IDO-positive cell populations on the parenchymal side of the blood-brain barrier such as astrocytes, microglia and neurons. Since all dietary tryptophan must cross the blood-brain barrier, the microvascular endothelial cells may play a key role in restricting tryptophan influx from the bloodstream into the brain. As deleterious effects of brain infections can often be attributed to subsequently invading immune cells, an IDO-mediated reduction of lymphocyte proliferation may be beneficial for preventing collateral brain damage.

Induction of intercellular adhesion molecule-1 on human brain endothelial cells by HIV-1 gp120: role of CD4 and chemokine coreceptors.

Central nervous system dysfunction is commonly observed in children with HIV-1 infection, but the mechanisms whereby HIV-1 causes encephalopathy are not completely understood. We have previously shown that human brain microvascular endothelial cells (HBMEC) from children are responsive to gp120 derived from X4 HIV-1 by increasing expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1. However, the mechanisms involved in gp120-mediated up-regulation of cell adhesion molecule expression is unclear. In the present study, we found that gp120 derived from both X4 and R5 HIV-1 induced increased expression of ICAM-1 on HBMEC, but the degree of this up-regulation differed among the various HBMEC isolates. The up-regulation of ICAM-1 was inhibited by anti-CD4 antibodies as well as by specific antibodies directed against chemokine receptors and small-molecule coreceptor inhibitors. Anti-CD4 antibodies inhibited the increase in ICAM-1 expression mediated by gp120 derived from X4 and R5 HIV-1, whereas antibodies against chemokine receptors displayed a differential inhibition depending on the source of gp120. Both X4 and R5 gp120-induced ICAM-1 expression was sensitive to pertussis toxin and involved the nuclear factor-kB pathway. These findings indicate a direct involvement of CD4 and a differential involvement of chemokine receptors in the activation of pediatric HBMEC by X4 and R5 gp120. The activation of brain endothelium of children by HIV-1 protein gp120 by way of CD4 and chemokine receptors may have implications for the pathogenesis of HIV-1 encephalopathy in the pediatric population.