Escherichia coli lipopolysaccharide lps

Before mononuclear cell (MNC) enrichment, the bone marrow aspirate was filtered and washed with sterile PBS. Thereafter, the same procedures were followed for peripheral blood MNCs (PBMCs) and bone marrow MNCs (BM-MNCs). PBMCs/BM-MNCs were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare, Chicago, IL). Cell composition was evaluated by Sysmex analyzer (Sysmex) and with flow cytometry (Table 3, see key resource table (KRT) for RRIDs). PBMCs/BM-MNCs were resuspended in Roswell Park Memorial Institute 1640 Dutch-modified culture medium (RPMI) (Life Technologies/Invitrogen, Waltham, USA) supplemented with 2 mmol/L glutamine (Invitrogen), 10 mg/mL gentamicin (Centrafarm, Etten-Leur, The Netherlands) and 1 mmol/L pyruvate (Invitrogen). Per well, 5 × 10⁵ PBMCs/BM-MNCs were stimulated for 24 hours in duplicate in round-bottom 96-well plates (Corning, NY) with the following stimuli: RPMI, 10 ng/mL Escherichia coli lipopolysaccharide (LPS) (serotype 055:B5 Sigma-Aldrich, St. Louis, MO), and 10 μg/mL Pam3CysK4 (P3C) (EMC Microcollections, Tübingen, Germany). After 24-hour incubation, supernatants were stored after plate centrifugation at −80°C until cytokine assessment.

The micronized form of triamcinolone acetonide (mTA) with a purity of 99.5% was purchased from Pura Quimica^® (Córdoba, Argentina). Poloxamer 188 (P188) was provided by Rupamel S.R.L (Buenos Aires, Argentina), a sales agent of BASF. Ultrapure water was obtained from Water Purification System (HF-Super Easy Series, Heal Force, Shanghai, China). Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma-Aldrich^®. Zirmil^® Yttrium-stabilized zirconium beads of 0.1 mm size (Saint-Gobain ZirPro, Köln, Germany) were used as a collision agent in the milling process. All other chemicals were extra pure grade and used without further purification.

After an overnight incubation at 37°C, monocytes seeded on 24-well plates (1 × 10⁵ cells/well) were infected with viable CT and CP EBs in SPG buffer (infectivity ratio: 5 EBs/cell) for 60 min in the presence of 10 μM Dichlorofluorescein diacetate (DCFDA) or 5 μM Diaminofluorescein (DAF-2), ROS and RNS probes, respectively. Treatments with 0.8 μM Phorbol or 10 μg/ml Escherichia coli Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls to induce ROS or RNS production, respectively [27 (link),28 (link)]. Cells were subsequently washed with phosphate buffered saline (PBS) and each well was filled with 300 μl of PBS and glucose (4,5 mg/ml). The increase in cell fluorescence from each well was measured (λexc = 485 nm; λem = 535 nm) with a spectrofluorometer (Wallac Victor multilabel counter, Perkin-Elmer Inc., Boston, MA, USA) at 3, 6 and 24 hours post infection.

The mononuclear cell fraction was isolated by density centrifugation of EDTA anticoagulated blood, diluted 1 : 1 in pyrogen-free saline, over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Isolated cells were washed twice in saline and resuspended in culture medium (RPMI, Invitrogen, Carlsbad, California, USA) supplemented with 10 μg/mL gentamicin, 10 mM L-glutamine, and 10 mM pyruvate. Cell counts were performed in a Coulter counter (Coulter Electronics). A total of 5 × 10⁵ mononuclear cells in 100 μL were added to round-bottomed 96-well plates (Greiner) with RPMI, sonicated Mycobacterium tuberculosis (MTB) (1 μg/mL end protein concentration, strain H37Rv), Escherichia coli lipopolysaccharide (LPS; 1 ng/mL; Sigma-Aldrich, St. Louis, MO, USA), heat-killed Staphylococcus aureus (1 × 10⁶ microorganisms/mL, clinical isolate), or heat-killed Candida albicans (1 × 10⁶ microorganisms/mL, strain UC820). After 24 h (for determination of TNF-α, IL-1β, and IL-6) or 48 h (for determination of IFN-γ and IL-10) of incubation, plates were centrifuged and supernatants were stored at −20°C until analysis. Cytokines were measured batchwise using commercially available ELISAs (R&D Systems, MN, USA, and Sanquin, Amsterdam, Netherlands) according to the protocols supplied by the manufacturers.

One milliliter of whole blood from T2D and healthy subjects or 0.5 × 10⁶ CD14+ monocytes/well placed in 24-well plates were cultured in HG (30 mM) for 24 h and infected with M. tuberculosis (MOI of 10) or stimulated with 1 ng/ml TLR-2 ligand, synthetic lipoprotein palmitylated N-acyl-S-diacylglyceryl cysteine (Pam₃Cys; EMC Microcollections, Tuebingen, Germany), or 100 ng/ml TLR-4 ligand, Escherichia coli lipopolysaccharide (LPS; Sigma), and were further incubated for 4 h for T2D and healthy subjects or 24 h for monocytes in HG. Supernatants were collected, and interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) levels were measured by a cytometric bead array (BD Bioscience) following the manufacturer's recommendation. CellQuest software was used for sample acquisition, and data were formatted using BD Bioscience CBA software. The results were based on a standard concentration curve, and the cytokine concentration was expressed as pg/ml.

Animals were injected with Escherichia coli lipopolysaccharide (LPS) of serotype O111:B4 (Sigma-Aldrich). In fever experiments, qPCR and immunohistochemistry a dose of 100 μg/kg was used, whereas a dose of 10 μg/kg was used in food intake experiments. The LPS was diluted in 100μl sterile 0.9% NaCl solution and injected intraperitoneally 1.5 h after lights on in fever experiments and 1h before lights off in food intake experiments. A similar volume of vehicle was given to the control group. These doses of LPS are known to induce a strong febrile response without substantial hypothermia and a reproducible anorexia respectively [13 (link), 26 (link), 34 (link)].

RPMI 1640 medium and fetal calf serum (FCS) were obtained from Gibco-BRL/Life Technologies, Italy. HEPES buffer, penicillin G-streptomycin sulfate, Dulbecco's phosphate-buffered saline solution (PBS), Histopaque 1077, and Escherichia coli lipopolysaccharide (LPS) were obtained from Sigma-Aldrich Srl, Milan, Italy. The monoclonal antibody Leu-M3 (anti-CD14) was from Becton Dickinson (San Jose, CA). Enzyme-linked immunosorbent assay (ELISA) kits (human TNF-α DuoSet, human IL-6 DuoSet, human IL-12 p70 DuoSet, human CXCL8/IL-8 DuoSet, and human IL-10 DuoSet) were obtained from R&D Systems (Space Srl, Milan, Italy).