Plasmacytoid dendritic cells are present in lymphoid and nonlymphoid tissue and contribute substantially to both innate and adaptive immunity. Recently, we have described several monoclonal antibodies that recognize a plasmacytoid dendritic cell-specific antigen, which we have termed BDCA-2. Molecular cloning of BDCA-2 revealed that BDCA-2 is a novel type II C-type lectin, which shows 50.7% sequence identity at the amino acid level to its putative murine ortholog, the murine dendritic cell–associated C-type lectin 2. Anti–BDCA-2 monoclonal antibodies are rapidly internalized and efficiently presented to T cells, indicating that BDCA-2 could play a role in ligand internalization and presentation. Furthermore, ligation of BDCA-2 potently suppresses induction of interferon α/β production in plasmacytoid dendritic cells, presumably by a mechanism dependent on calcium mobilization and protein-tyrosine phosphorylation by src-family protein-tyrosine kinases. Inasmuch as production of interferon α/β by plasmacytoid dendritic cells is considered to be a major pathophysiological factor in systemic lupus erythematosus, triggering of BDCA-2 should be evaluated as therapeutic strategy for blocking production of interferon α/β in systemic lupus erythematosus patients.

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During prolonged recombinant interferon (rIFN)-alpha 2 therapy, a minority of patients develop high-titer neutralizing IFN-alpha antibodies. Sera from nine IFN-alpha antibody-positive patients were studied to characterize the specificity of anti-IFN-alpha neutralizing antibodies by their ability to inhibit the antiviral and antiproliferative activity of different rIFN-alpha subtypes and rIFN-alpha 1/alpha 2 hybrids. These therapy-induced antibodies (Tab) were compared with IFN-alpha-specific autoantibodies (Aab) from two patients with systemic lupus erythematosus who had never received any exogenous IFN-alpha. Although IFN-alpha subtypes are closely related in structure, Tab inhibited the antiviral activity of only recombinant (r)IFN-alpha 2 and rIFN-alpha 6, but not or slightly that of rIFN-alpha 1, -alpha 7, -alpha 8 and -alpha 14. Furthermore, of four different rIFN-alpha 1/alpha 2 hybrids tested, Tab inhibited only those which contained the N-terminal residues 17-64 of rIFN-alpha 2. Comparison of the primary sequences of neutralized and not neutralized subtypes suggests an epitope involving the residues 22-31 of IFN-alpha 2 is recognized. Thus, Tab block rIFN-alpha 2 by reacting with only one of two functional domains. In contrast, Aab possessed a broad specificity and neutralized both the antiviral and antiproliferative activity of rIFN-alpha 2, -alpha 6, -alpha 7, -alpha 8, and -alpha 14. They also neutralized all four rIFN-alpha 1/alpha 2 hybrids tested. These data demonstrate that Tab are highly specific for the therapeutic IFN-alpha subtype and specifically neutralize rIFN-alpha 2 by binding to its N-terminal functional domain.

Immunoprecipitation is an often used technique to enrich target protein(s) from a cell lysate for subsequent detection or functional studies. Immunoprecipitation is usually performed by forming an immune complex between the protein(s) of interest, the specific antibody and Protein A sepharose beads and subsequently precipitating the complex by centrifugation. Though efficient, this method usually requires lengthy incubation steps. Here we introduce a novel way of immunoprecipitating proteins by replacing the centrifugation step by magnetic separation. Magnetic labeling was performed using Protein A or G conjugated to super-paramagnetic MicroBeads and the labeled immune complex was separated over a (IMACS magnet and column. The extremely small size of the MicroBeads allowed a very rapid magnetic labeling which was completed in 30 minutes. Various intracellular and surface proteins were successfully immunoprecipitated as analyzed by SDS-PAGE and Western blotting. Pre-absorption steps proved to be not necessary as the magnetic separation procedure which uses a combination of a column and a magnet allowed very effective washing steps. A future automation of the magnetic technique may allow highthroughput immunoprecipitations which may be of special interest for upcoming proteomics projects.paramagnetic MicroBeads 687 A protein disulfide isomerase gene fusion expression proteins.-a. C. Ohto2). M. Murarnatsu2). S. Obata2).system that suppresses the aggregation of heterologousWe have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. The fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G 8-fold; and geranylgeranyl pyrophosphate synthase: 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intra-disulfide. Whereas aggregation of the protein without intra-disulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. 688 Induction of lactose catabolism in plasmid-free Lactococcus ladis IL1403 strain.Lactic Acid Bacteria LAB are widely used in various food production processes. The ability to hydrolyse lactose is a very important trait, especially in the dairy industry. The homofermentation pathway of lactose utilisation in LAB leads to the acidification of milk and reduction of lactose concentration. Lactose catabolism is encoded in Lactococcus lactis strains by plasmid genes organised in lactose operon. In this work we show the induction of chromosomal genes for lacto...