BLyS: Member of the tumor necrosis factor family and B lymphocyte stimulator

Science

Volumn 285, Issue 5425, 1999, Pages 260-263

  Moore, Paul A ^a   Belvedere, Ornella ^a   Orr, Amy ^a   Pieri, Krystyna ^a   LaFleur, David W ^a   Feng, Ping ^a   Soppet, Daniel ^a   Charters, Meghan ^a   Gentz, Reiner ^a   Parmelee, David ^a   Li, Yuling ^a   Galperina, Olga ^a   Giri, Judith ^a   Roschke, Viktor ^a   Nardelli, Bernardetta ^a   Carrell, Jeffrey ^a   Sosnovtseva, Svetlana ^a   Greenfield, Wilbert ^a   Ruben, Steven M ^a   Olsen, Henrik S ^a   Fikes, James ^a   Hilbert, David M ^a   more..

^a GLAXOSMITHKLINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN; TUMOR NECROSIS FACTOR;

AMINO ACID SEQUENCE; ANIMAL EXPERIMENT; ARTICLE; B LYMPHOCYTE; B LYMPHOCYTE ACTIVATION; CONTROLLED STUDY; FLOW CYTOMETRY; HUMAN; HUMAN CELL; IMMUNOGLOBULIN BLOOD LEVEL; IMMUNOGLOBULIN PRODUCTION; LYMPHOCYTE PROLIFERATION; MOUSE; NONHUMAN; PRIORITY JOURNAL;

AMINO ACID SEQUENCE; ANIMALS; B-CELL ACTIVATING FACTOR; B-CELL ACTIVATION FACTOR RECEPTOR; B-LYMPHOCYTE SUBSETS; B-LYMPHOCYTES; CELL LINE; CELLS, CULTURED; HUMANS; IMMUNOGLOBULINS; INTERFERON TYPE II; LYMPHOCYTE ACTIVATION; MEMBRANE PROTEINS; MICE; MICE, INBRED BALB C; MOLECULAR SEQUENCE DATA; MONOCYTES; RECEPTORS, CYTOKINE; RECEPTORS, TUMOR NECROSIS FACTOR; RECOMBINANT PROTEINS; SEQUENCE ALIGNMENT; SPECIES SPECIFICITY; TUMOR NECROSIS FACTOR-ALPHA; UP-REGULATION;

ANIMALIA;

EID: 0033538468     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5425.260     Document Type: Article

References (35)

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28. A full-length BLyS clone was Identified, sequenced, and submitted to GenBank (accession number AF132600). BLyS was expressed in baculovirus and purified from Sf9 cell supernatant using a combination of ion exchange (poros HQ-50, poros HS-50, poros PI-50; PE Biosystems, Framingham, MA), size exclusion (Sephacryl S100 HR; Amersham Pharmacia Biotech, Piscataway, NJ), and hydrophobic interaction (Toyopearl Hexyl 650C; Tosohass, Montgomeryville, PA) columns. Purification of BLyS was monitored by SDS-PAGE analysis and the B cell costimulation assay. BLyS was formulated in 0.15 M NaCl and 50 mM sodium acetate at pH 6. Endotoxin concentrations were below the detection limit in the LAL assay (Associates of Cape Cod, Falmouth, MA). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
29. BLyS expression was assessed on the indicated cell types using BLyS-specific mAb 2E5 (IgG1) followed by phycoerythrin (PE)-conjugated F(ab′)2 goat antibody to mouse IgG (CALTAG Laboratories, Burlingate, CA). Purified monocytes were cultured in tissue culture-treated plastic wells (Falcon #3043; Becton-Dickinson, Lincoln Park, NJ) for 3 days in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM 1-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in the presence or absence of IFN-γ (100 U/ml). Comparable results were obtained with monocytes purified from three different donors in three independent experiments. BLyS binding was assessed using rBLyS biotinylated with a N-hydroxy-succinimidobiotin reagent (Pierce, Rockford, IL) and PE-conjugated streptavidin (Dako Corp., Glostrup, Denmark).
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32. BALB/cAnNCR mice (6 to 8 weeks old) from Charles River Laboratories were maintained according to recommended standards in microisolator cages with recycled paper bedding (Harlan Sprague-Dawley, Indianapolis, IN) and were provided with pelleted rodent diet (Harlan Sprague-Dawley) and bottled drinking water on an ad libitum basis. The animal protocols used in this study were reviewed and approved by the Human Genome Sciences Institutional Animal Care and Use Committee.
33. 3H]thymidine (6.7 Ci/mM) beginning 72 hours after factor addition. Analyses of both human and mouse B cell cultures indicated that the proliferative response was evident 24 hours after BLyS addition and progressively increased with a maximal response observed between 72 and 92 hours after culture initiation. The positive and negative controls were IL-2 and medium, respectively. SAC alone yielded background counts of 1427 ± 316. Values are reported as mean ± standard deviation of triplicate wells. Anti-IgM costimulation was performed as described for SAC with the exception that individual wells were precoated with 50 μl of a 10 μg/ml solution of anti-human IgM mAb (IgG1) for 12 hours at 4°C, after which wells were washed before addition of cells. An isotype-matched (IgG1) control antibody was included as a control for nonspecific Ig effects. Similar results were obtained using rBLyS purified from stable CHO transfectants and transiently transfected HEK-293T cells.
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35. We thank D. Russell, R. Wager, N. Nguyen, C. Lincoln, N. Madary, D. Morahan, E. Cochrane, E. Hayden, and K. Florence for their work and review of the manuscript.