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2 incubator with RPMI 1640 and 10% heat-inactivated fetal bovine serum (FBS). Cell-free supernatants were harvested 20 hours later and stored at -20°C until assayed for cytokine concentrations by ELISA as previously described (38, 39). The amount of lipopolysaccharide in all preparations was measured quantitatively with a Limulus Amoebocyte Lysate assay (Whittaker Bioproducts, Walkersville, MD) and found to be <40 pg per microgram of M. tuberculosis fraction or protein, an amount that did not stimulate IL-12 activity by itself.
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0344760091
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note
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The mycolyl arabinogalactan peptidoglycan complex (mAGP) was also derived from the cell wall pellet after SDS detergent extraction. Most of the IL-12 p40-inducing activity was associated with the SCWP fraction compared with mACP (see supplementary material available at www.sciencemag.org/feature/ data/1040444.shl). Because macrophage phagocytosis of protein-adsorbed particulate suspensions has been reported to induce IL-12 release, we filtered both fractions through a 0.2-μm sterile filter. The SCWP fraction retained most of its IL-12 p40-inducing activity after filtration, whereas the mAGP suspension did not induce IL-12 p40.
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14
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0344760089
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note
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3. At each step, fractions were tested for their ability to induce IL-12 p40, and the fractions with potent activity were further purified. Rotofor fraction number 4 was electrophoresed under denaturing conditions on a 4 to 20% trisglycine minigel and transferred to nitrocellulose. Areas containing the four protein bands noted in Fig. 1B were isolated and solubilized as described previously (11) and used to stimulate THP-1 cells.
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All RAW 264.7 cell (ATCC, Manassas, VA) transfections were carried out with the Superfect protocol (Qiagen, Valencia, CA) at a ratio of 1 μg of DNA to 3 μl of Superfect for 2.5 hours. After a PBS wash, cells were divided into two wells and left unactivated or stimulated with LPS or lipoproteins for 24 hours and then harvested for the chloramphenicol acetyltransferase (CAT) assay. In IL-12 p40 and inducible nitric oxide synthase [iNOS) promoter induction studies, RAW 264.7 cells were transiently transfected with either IL-12 p40 (15) or iNOS promoter (36), CAT reporter plasmid (2 μg), and HSP70-β-Gal construct (0.5 μg) as an internal control In the TLR-2 dn1 overexpression experiments, increasing doses of TLR-2 dn1 expression plasmid (100 ng, 500 ng, 1 μg, and 2 μg) (19) were transfected with the IL-12 p40 promoter construct and β-Gal as an internal control. The amount of expression plasmid was normalized to 2 μg with expression plasmid lacking the TLR-2 dn1 coding sequence (pCDNA3). IL-12 p40 promoter transfectants were either left unactivated or stimulated with LPS, 19-kD lipoprotein, or OspA at 50 ng/ml or Tp47 lipopeptide (24) at 10 μM for 24 hours. iNOS promoter transfectants were stimulated with LPS, 19-kD lipoprotein, OspA, or deacylated OspA (d-OspA) from 0.01 to 100 ng/ml, or left unstimulated for 24 hours. The iNOS promoter was also cotransfected with the TLR-2 dn1 construct (1 μg) or vector control (1 μg) and stimulated with LPS at 1 ng/ml, 19-kD lipoprotein and OspA at 5 ng/ml, or Tp47 lipopeptide at 10 μM for 24 hours. IL-12 p40 and iNOS promoter activities were measured according to CAT activity (percent chloramphenicol acetylation) with a phosphorimager. Data were normalized to a cotransfected β-Gal construct for transfection efficiency.
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0345190492
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5 cells/well in six-well plates and transiently transfected them the following day with the NF-κB responsive E-selectin (ELAM) gene enhancer luciferase (pGL3) reporter gene (0.5 μg) and a β-galactosidase reporter plasmid (0.5 μg) as an internal control by the Superfect protocol at a 1:3 ratio of DNA (micrograms to Superfect (microliters). Cells were then incubated with the DNA-Superfect mixture for 2 hours and washed, and multiple transfectants were pooled and divided into separate wells for activation by a titration of LPS or lipoprotein stimuli (19-kD lipoprotein. Tp47, and OspA). Twenty-four hours later cells were stimulated for 6 hours then lysed in 200 μl of reporter lysis buffer (Promega, Madison, Wl), and 20 μl was used in the luciferase assay. HEK 293 control cells and TLR-2 stable clones were also cotransfected with a CD14 expression plasmid (19) (1 μg) or vector control (pCDNA3, 1 μg) with the same protocol.
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Chan, J.1
Xing, Y.2
Magliozzo, R.S.3
Bloom, B.R.4
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J. Chan, Y. Xing, R. S. Magliozzo, B. R. Bloom, J. Exp. Med. 175, 1111 (1992); J. Chan, K. Tanaka, D. Carroll, J. Flynn, B. R. Bloom, Infect. Immun. 63, 736 (1995).
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(1995)
Infect. Immun.
, vol.63
, pp. 736
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Chan, J.1
Tanaka, K.2
Carroll, D.3
Flynn, J.4
Bloom, B.R.5
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59
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0344760071
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note
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2 (Sigma) (40).
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63
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0345190487
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note
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Adherent monocytes were isolated as described previously (14). Cells were treated with no antibody, mouse anti-human TLR-2 neutralizing monoclonal antibody, or an isotype control mouse IgG1 (10 μg/ml) for 30 min before stimulation with LPS or 19-kD lipoprotein (50 ng/ml) and incubated for 16 hours. Supernatants were then harvested and assayed for IL-12 p40 by ELISA. CD40L- and interferon-γ (IFN-γ) - stimulated adherent monocytes were used to control for the anti-TLR-2 blocking of lipoprotein-induced IL-12. Adherent monocytes were treated with human IFN-γ (100 units/ml; Endogen, Cambridge, MA) for 16 hours to up-regulate CD40 expression. Cells were then treated with antibodies as stated above, then stimulated with soluble CD40L trimer (250 ng/ml; Immunex, Seattle, WA) for 16 hours. Neither antibody nor IFN-γ alone induced IL-12 production.
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64
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0344760069
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We thank P. Sieling, G. Cheng, M. Mark, C. Nathan, and H. Herschman for scientific discussion; M. Gately (Hoffman-La Roche) for POD-46D; and C. Nathan (Cornell Medical College, New York, NY) for the iNOS promoter. Supported by the NIH (to R.L.M., P.J.B., B.R.B., M.V.N.), the Howard Hughes Medical Institute (to B.R.B. and S.T.S.), the United Nations Development Programme/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP), and the Dermatology Research Foundation of California, Incorporated.
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