Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells.

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.

Listeria monocytogenes infection of HeLa cells results in listeriolysin O-mediated transient activation of the Raf-MEK-MAP kinase pathway.

In this study we investigated the effect of Listeria monocytogenes infection on the activation of the Raf-MEK-MAP kinase pathway in eukaryotic host cells. HeLa cell infection with L. monocytogenes EGD resulted in a rapid, but transient, phosphorylation of the MAP kinases erk-1 and erk-2, a transient phosphorylation of the MAP kinase kinase MEK-1, and a transient activation of the MAP kinase kinase kinase Raf. In parallel to the transient phosphorylation of the MAP kinases, we detected induced expression of the MAP kinase phosphatase MKP-1. Additionally we present evidence that listeriolysin O is the inducing agent for activation of the Raf-MEK-MAP kinase pathway.

HAB-1, a new heteromyeloma for continuous production of human monoclonal antibodies.

To obtain suitable cell lines for the immortalisation of human lymphocytes, we constructed a heteromyeloma between the murine myeloma Ag8 and human lymphocytes from a highly malignant polymorphic, centroblastic B-cell lymphoma. The thioguanine-resistant and HAT-sensitive heteromyeloma HAB-1 neither secretes nor contains cytoplasmatic immunoglobulins, the cells being EBV negative but positively stained for HLA-BC and the human proliferation marker Ki-67. The karyotype consists of about 50 murine and 20 human chromosomes. The HAB-1 cells grow in suspension and have a doubling rate of about 25-30 h. In fusion experiments with spleen cells from stomach carcinoma patients HAB-1 cells show a 5-7 times higher fusion efficiency than murine Ag8 cells or another heteromyeloma SPM4-0 and give stable antibody producing products. The cell line will be made available to interested scientists.