Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile.

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.

Isolation of a serogroup 1-specific antigen from Legionella pneumophila.

Outer membrane material was extracted from a serogroup 1 strain of Legionella pneumophila using ethylenediaminetetraacetate. Ferritin-conjugated antiserum reacted only at the surface of the organism, as seen by electron microscopy. Outer membrane material was fractionated into five peaks by molecular sieve chromatography using a gel that had been equilibrated in a buffer containing sodium deoxycholate. One resolved peak (approximately 4x10(4) daltons) was highly active serologically. When rechromatographed in the absence of sodium deoxycholate, material from this peak reaggregated to approximately 10(6) daltons. Ther serologic activity of this antigen was restricted to L. pneumophila serogroup 2, although minor cross-reactions with strains of serogroups 2 and 4 were detected using an enzyme-linked immunosorbent assay. The antigen was less than 10% carbohydrate, 15% protein, 1.1% phosphate, and the remainder lipid of unknown composition. Neither 2-keto-3-deoxyoctonate nor heptose was detected, and the antigen did not induce a Shwartzman reaction. Only one band was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Legionella pneumophila: growth inhibition by human pharyngeal flora.

Several bacterial isolated from human pharyngeal cultures specifically inhibited the growth of Legionella pneumophila. The inhibitory substance from two strains (Streptococcus species 1-3 and Staphylococcus saprophyticus KC) was isolated from a broth supernatant. The inhibitor was active against all strains of L. pneumophilia tested, including five strains of L. pneumophila serogroup 1 and one strain each of serogroups 2, 3, and 4. The substance did not inhibit growth of 18 fresh clinical and laboratory pathogens (12 general). The substance was dialyzable, was resistant to head and proteolysis, and did not precipitate with ammonium sulfate. Nicotinamide adenine dinucleotide, produced by several upper respiratory tract organisms, did not inhibit L. pneumophila, and L. pneumophila could not be isolated when Streptococcus species 1-3, S. saprophyticus KC, and L. pneumophila were cocultivated. These properties may in part explain the difficulty of isolation and may aid in the identification of L. pneumophila.

The cell envelope of the Legionnaires' disease bacterium. Morphologic and biochemical characteristics.

We studied the cell-envelope structure of the Legionnaires' disease (LD) bacterium by electron microscopy and biochemical assays. There were apparent differences in cell structure by electron microscopy using two different prefixation methods. Organisms prefixed with gluteraldehyde had a single surrounding membrane. The typical two-membrane structure of gram-negative bacteria, however, was observed after prefixation with a combination of gluteraldehyde, formalin, and creosol. The cell wall (peptidoglycan) was seen in electron micrographs of plasmolyzed bacteria and in papain-digested cells. Both cytoplasmic and outer membranes were separated by differential centrifugation of spheroplast sonicates followed by sucrose density gradient ultracentrifugation. We identified each membrane by characteristic enzyme activity (cytoplasmic membrane) and 2-keto-3-deoxyoctonate content (outer membrane).

Characterization of lipopolysaccharide of Haemophilus influenzae.

Lipopolysaccharide from strains of Haemophilus influenzae was extracted and isolated by the hot phenol-water procedure. The preparations were relatively insoluble in water but could be solubilized with surface-active agents. The preparations contained carbohydrate (30%), fatty acid (29%), and phosphate (4.7%); protein content was less than 1%. Thin-layer chromatography, gas-liquid chromatography, and colorimetric assays detected glucose, galactose, glucosamine, heptose, and a 2-keto-3-deoxy-octonate-like molecule (less than 1%). Neither methylpentose nor dideoxyhexose was detected. The lipid portion was composed of fatty acids common to lipopolysaccharide of Salmonella. The preparations provoked positive dermal Shwartzman reactions and biphasic febrile responses in rabbits, responses typical of endotoxic activity. The 50% lethal dose for mice was decreased from 16.5 microgram/g to 0.015 microgram/g by concomitant administration of actinomycin D. The preparations were shown to be polyclonal activators of bone marrow-derived (B) cells. Limulus lysate gelation was seen with 8.0 ng of lipopolysaccharide. Preliminary hemagglutination data suggested at least three different antigenic factors associated with the lipopolysaccharide of H. influenzae type b. The H. influenzae lipopolysaccharide appeared biologically similar to that of enterobacteria but chemically different.