Heat-killed E. faecalis (5×107 CFU/mL) were prepared by heating bacteria at 65°C for 30 min. No viability of the bacteria was confirmed by plating an aliquot of the heat-killed bacteria on TSB agar plates. Murine rCCL2 was purchased from BD Biosciences (San Jose, CA, USA), and mAb

directed against CCL2 was obtained from BioLegend (San Diego, CA, USA). rCCL5, rCCL17 and mAbs directed against these chemokines were purchased from R&D Systems Bortezomib solubility dmso (Minneapolis, MN, USA). Biotin-conjugated anti-CD3, anti-F4/80 and anti-CD19 mAbs were obtained from eBioscience (San Jose, CA, USA). Phosphorothioated CCL2 antisense ODNs (5′-AAGCGTGACAGAGACCTGCATAGTGGTGG-3′) and scrambled ODNs (5′-CCACCACTATGCAGGTCTCTGTCACGCTT-3′) were purchased from Sigma-Genosys (The Woodlands, TX, USA). RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin was utilized for the cultivation of various Mϕ preparations. Thermally injured mice were created according to our previously reported protocol 23–25. This procedure consistently produced a third degree burn on approximately 25% of total body surface area (TBSA) for a 26-g

mouse. Immediately after thermal injury, physiologic saline (1 mL per mouse, i.p.) was administered for fluid resuscitation. Deaths within 5 days of 25% TBSA flame burn were not demonstrated Silmitasertib molecular weight after our burn procedure. As controls, mice were anesthetized and shaved but were not exposed to the gas flame. They also received physiologic saline (1 mL per mouse, i.p.). Buprenorphine (2 mg/kg) was given s.c. every 12 h during the postburn period. Sham burn animals also received identical regimens of analgesics (buprenorphine) throughout the study period. Mϕs (F4/80+ cells) were prepared from MLNs of various groups of mice, as previously described 24, 25. F4/80+ cells with

94% or more purity were consistently obtained using this technique. Severely burned mice were subjected to CCL2 antisense ODN gene therapy. Thus, burned mice were treated twice with CCL2 antisense ODNs at 2 and 12 h after burn injury. Based on our Dolichyl-phosphate-mannose-protein mannosyltransferase preliminary studies, CCL2 antisense ODNs were administered s.c. to burned mice at doses ranging from 0.01 to 100 μg/mouse. Weight loss, reduced appetite and abnormal body temperature were not demonstrated in normal mice treated with 100 μg/mouse of CCL2 antisense ODNs twice a day for 7 days. The effect of the gene therapy was confirmed by measuring CCL2 levels in the sera of these mice 24 h after burn injury, because the maximum level of CCL2 in sera of these mice was reached within 24 h of severe burn injury. CCL2 in serum specimens was assayed by ELISA. To determined the efficacy of CCL2 antisense ODNs on the generation of M2Mϕs, MLN-Mϕs were isolated from severely burned mice treated twice with 10 μg/mouse of CCL2 antisense ODNs (2 and 12 h after burn injury) 1–8 days after burn injury.

Because the effective concentration of

Torin 1 price HLA (1–3 nm) used in these assays is below the equilibrium dissociation constant (KD) of most high-affinity peptide–HLA interactions, the peptide concentration leading to half-saturation of the HLA is a reasonable approximation of the affinity of the interaction. Affinity measurements of peptides to recombinant HLA-DRB1*0101, -DRB1*0301, -DRB1*0302, -DRB1*0401, -DRB3*0301, -DRB5*0101 and DPA1*0103/DPB1*0401 molecules were performed according to previous work.32 Briefly, peptides including reference peptides known to bind the used HLA-II alleles [DR-binding peptide HA 306–318 (sequence: YKYVKQNTLKLAT) and DP-binding peptide, Plasm. Falciparum 239–253 (3D7)33 (sequence: YILLKKILSSRFNQM)] were dissolved and titrated in 25% glycerol, 0·1% pluriol (F68) and 150 mm NaCl. An HLA-II stock solution consisting of bacterially expressed and urea-denatured α- and β-chains, at appropriate concentrations

were diluted into refolding buffer: 100 mm Tris/Citrate, 25% glycerol, 0·01% Pluriol F68 containing protease inhibitors (TPCK and Pepstatin both 3·3 μg/ml) at pH 6 (DRB1*0101. DRB5*0101) or pH 7 (remaining HLA-II alleles). The diluted HLA-II stock was subsequently mixed 1 : 1 with peptide titrations and incubated at 18° for 48 hr. Formed HLA-II complexes were detected Selleck Neratinib using a homogeneous proximity assay (Alpha Screen; Perkin Elmer, Waltham, MA, USA); briefly, streptavidin-coated donor Lumacaftor beads and L243 (murine monoclonal anti-DR) coupled acceptor beads, both 5 mg/ml, were diluted 500 times into PBS 0·1% Pluriol (F68). Ten microlitres of bead mix was mixed with 10 μl HLA-II/peptide samples in 384 Optiplates (Perkin Elmer). Following 18 hr of incubation at 18° they were read on an Envision Reader (Perkin Elmer) and analysed accordingly.32 The CD4+ T cells were positively depleted from PBMC according to the manufacturer’s instruction using monoclonal anti-CD4-coated Dynabeads from Dynal Biotech ASA (Oslo, Norway). The PBMC were effectively (>98%) depleted of CD4+ T cells as verified by flow cytometry. The PBMC

were thawed, washed and then used for CD4+ or CD8+ T-cell depletion or cultured directly in RPMI-1640 supplemented with 5% heat-inactivated AB serum (Valley Biomedical, Winchester, VA), 2 mm l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The PBMC (4 × 106 to 6 × 106) or depleted PBMC were cultured in 1 ml culture medium in 24-well plates (Nunc, Roskilde, Denmark) in the presence of individual peptides with a final concentration of 10 μg/ml per well, and incubated for 10 days at 37°, 5% CO2 in humidified air. Recombinant human interleukin-2 (rhIL-2; Proleukin; Chiron, Amsterdam, the Netherlands) 20 U/ml was added on day 1. Cells were harvested on day 10, washed twice in RPMI-1640 and resuspended in complete medium to a final concentration of 1 × 106 to 2 × 106 cells/ml.