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.