The beta-amyloid (Abeta) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Abeta responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Abeta peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Abeta were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Abeta. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Abeta; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Abeta on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC 4 (3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC 4 (3) provided a robust flow cytometric indicator for bacterial cell viability.

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5-3-O-(thio)-triphosphate (GTP␥S) binding assays, the potency of this compound was 2.6 -53.6 nM, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTP␥S binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe 111 at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC 50 . In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.The Epstein-Barr virus (EBV) induced receptor 2 (EBI2; also known as GPR183) is an orphan member of the 7TM 2 receptor family A. EBI2 is up-regulated up to 200-fold in B cells following EBV infection (1). In agreement with an expression pattern primarily restricted to immunological cell types (1, 2), it was recently demonstrated that EBI2 orchestrates the positioning of B cells in the follicle (3, 4). Specifically, expression of EBI2 allowed activated B cells to translocate to the outer follicular regions; in contrast, the absence of EBI2 permitted the cells to enter the germinal centers. Thus, EBI2 mediates the correct localization of B cells during humoral immune responses. To date, no endogenous ligand for EBI2 has been identified. Given that this receptor does not have a close homolog, the chemical nature of such a ligand is difficult to infer. Accordingly, EBI2 has been placed in varying 7TM receptor subgroups by different phylogenetic analyses as being a target of peptide (1, 5) or lipid ligands (6). Nevertheless, many aspects of EBI2 signaling have been revealed. Thus, we have previously demonstrated that EBI2 is constitutively active through G␣ i (but not G␣ q or G␣ s ) and serum response element (but not NFAT or NFB...

Two strains of Legionella pneumophila (LP) serogroup I, of differing virulence, were examined in terms of numbers of viable organisms in tissues, pyrexia and mortality following aerosol infection. The Corby strain was the more virulent, with pyrexia and deaths of guinea-pigs 3 to 6 days after infection. This strain multiplied very rapidly in the lungs to reach a peak of 5 X 10(11) viable organisms/lung. Organisms were present in the blood, liver, spleen and kidney. The Philadelphia-1 strain (NCTC 11192) was unable to replicate in the lung and was cleared between 14 and 21 days after infection. Pyrexia was not observed. No guinea-pigs died and viable LP was not found in any organ other than the lung. Lung lavages on aerosol infected animals were performed and the virulent Corby strain was found to be mainly intracellular. The avirulent Philadelphia-1 strain was found predominantly in the extracellular location. There were approximately 10 times the number of viable virulent LP in the lung macrophage fraction than in the lung PMNL fraction. In comparison, there were approximately equal numbers of the viable avirulent strain in the macrophages and the PMNL. Experimental evidence suggests that the macrophage preferentially supports the growth of the virulent Corby strain compared with the PMNL. The avirulent strain on the other hand appears to be destroyed by both the macrophages and the PMNL.

The Epstein–Barr virus induced gene 2 (EBI2) was recently identified as the first oxysterol-activated 7TM receptor. EBI2 is essential for B cell trafficking within lymphoid tissues and thus the humoral immune response in general. Here we characterize the antagonism of the non-peptide molecule GSK682753A, which blocks oxysterol-induced G-protein activation, β-arrestin recruitment and B-cell chemotaxis. We furthermore demonstrate that activation triggers pertussis toxin-sensitive MAP kinase phosphorylation, which is also inhibited by GSK682753A. Thus, EBI2 signalling in B cells mediates key phenotypic functions via signalling pathways amenable to manipulation providing additional therapeutic options for inhibiting EBI2 activity.