Toxicity in Neuronal Cells Caused by Cerebrospinal Fluid from Pneumococcaland Gram-Negative Meningitis

To identify neurotoxic factors in meningitis, a neuronal cell line (HN33.1) was exposed to cerebrospinal fluid (CSF) obtained from rabbits with pneumococcal meningitis or Escherichia coli meningitis or 2 h and 6 h after meningitis was induced by proinflammatory bacterial products (pneumococcal cell walls, endotoxin). CSF from all types of meningitis induced similar degrees of cytotoxicity. When a soluble tumor necrosis factor (TNF) receptor that completely blocked TNF-mediated toxicity at 10-7 M was used, all toxicity in meningitis caused by E. coli, endotoxin, or pneumococcal cell wall administration (2 h afterwards) was mediated by TNF. In contrast, CSF from animals with meningitis caused by live pneumococci or pneumococcal cell wall injection (6 h afterwards) retained cytotoxicity in the presence of the TNF receptor. Thus, in established pneumococcal meningitis, but not in the other forms of meningitis, TNF is not the only component toxic in this neuronal cell lin

55-kd Tumor Necrosis Factor Receptor Is Expressed by Human Keratinocytes and Plays a Pivotal Role in Regulation of Human Keratinocyte ICAM-1 Expression

Tumor necrosis factor α (TNFα) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNFα is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNFα induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37°C for 24h increased ICAM-1 expression in a TNFα-like fashion. Moreover, the well known synergistic effect of IFNγ plus TNFα on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFNγ plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFNγ-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression

Pretreatment with a 55-kDa Tumor Necrosis Factor Receptor-Immunoglobulin Fusion Protein Attenuates Activation of Coagulation, but not of Fibrinolysis, during Lethal Bacteremia in Baboons

Baboons (Papio anubis) receiving a lethal intravenous infusion with live Escherichia coli were pretreated with either a 55-kDa tumor necrosis factor (TNF) receptor-IgG fusion protein (TNFR55:IgG) (n = 4, 4.6 mg/kg) or placebo (n = 4). Neutralization of TNF activity in TNFR55:IgG-treated animals was associated with a complete prevention of mortality and a strong attenuation of coagulation activation as reflected by the plasma concentrations of thrombin-antithrombin III complexes (P < .05). Activation of fibrinolysis was not influenced by TNFR55:IgG (plasma tissue-type plasminogen activator and plasmin-a2-antiplasmin complexes), whereas TNFR55:IgG did inhibit the release of plasminogen activator inhibitor type I (P < .05). Furthermore, TNFR55:IgG inhibited neutrophil degranulation (plasma levels of elastase-α1-antitrypsin complexes, P < .05) and modestly reduced release of secretory phospholipase A2. These data suggest that endogenous TNF contributes to activation of coagulation, but not to stimulation of fibrinolysis, during severe bacteremi

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte./macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. ln the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells

Expression and characterisation of Plasmepsin I from Plasmodium falciparum

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5–10–fold lower Kcat/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite