Polymyxin b pmb

Cryopreserved primary ascTAM derived from different ovarian cancer patients were cultured in ascites (pool of 10 different patients) for 6 days followed by overnight starvation in RPMI1680 medium supplemented with 1 mM sodium pyruvate (Sigma Aldrich). Cells were treated with 1 μg/ml recombinant human Hsp70 protein (rhHsp70, low endotoxin; Enzo Life Sciences, Lörrach, Germany). A control (Ctrl^low) containing 0.005 ng/ml LPS from E. coli (Sigma Aldrich) corresponding to the endotoxin level of the rhHSP70 (indicated by the manufacturer) was included. To further address the potential effect of endotoxin contaminations of rhHSP70, TAM were pre‐incubated with 10 μg/ml polymyxin B (PMB, Sigma Aldrich) for 2 h prior to stimulation.

For heat-denaturation, LPS or Rv2145c were incubated at 100°C for 1 h. For digestion of proteinase K (PK), LPS or Rv2145c were incubated along with 50 μg/ml soluble PK followed by heating for 20 min at 100°C to deactivate the enzyme, and then prepared stimulant were treated in BMDMs. For pretreatment with polymyxin B (PMB; Sigma), LPS and Rv2145c were incubated in medium containing 50 μg/ml of PMB for 1 h at room temperature. After 24 h, cytokine levels in the supernatant were analyzed by sandwich ELISA.

Cell proliferation was analyzed by CFSE dilution assay or BrdU incorporation. For CFSE dilution assay, B cells were incubated with 1 µM CFSE (eBioscience) for 10 min at room temperature, and then washed with medium containing 10% FCS. CFSE-labeled B cells were cultured with ALD-DNA, LPS, E. coli DNA, ALD-DNA plus LPS, or E. coli DNA plus LPS for 3 days. The cells were analyzed using a CyAn ADP analyzer (Beckman-Coulter) and the Modfit software (Verity Software House, Topsham, ME).
For BrdU incorporation, ALD-DNA, anti-CD40 antibody, LPS, or a combination of these stimuli were added to the cultures for 3 days. In some cases, cells were pretreated with 20 µg/ml polymyxin B (PMB, Sigma-Aldrich) for 20 min at 37°C. Cells were harvested after 24-hour incubation with 10 µM BrdU (Roche Diagnostics, Mannheim, Germany). BrdU incorporation was analyzed using Cell proliferation ELISA Kit (Roche Diagnostics) according to the manufacturer's instruction.

Lipooligosaccharides (LOS) was extracted from E.coli B according to the procedure described by Galanos [7 (link),12 ]. The specific method for the extraction of LOS requires mixture of aqueous phenol, chloroform and petroleum ether. Polymyxin B (PmB) (Sigma-Aldrich Chemie GmbH, Germany) was dissolved in drinking water and given to mice in dose of 29.5 mg/l.Both compounds were added in proper concentrations to in vitro cultures.

Antibodies against proIL-1β, iNOS, COX-2, NF-κB SN50 cell permeable inhibitory peptide (sc-3060) and pyrrolidine dithiocarbamic acid ammonium salt (PDTC) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against actin, LPS, pharmaceutical inhibitors, N-acetyl cysteine (NAC), polymyxin B (PMB) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059, SB203580, SP600125 and antibodies against phospho-proteins were obtained from Cell Signaling Technology (Beverly, MA, USA). The NF-κB reporter reagent (QUANTI-Blue) and ATP were obtained from InvivoGen (San Diego, CA, USA). ELISA kits were obtained from Affymetrix eBioscience (San Diego, CA, USA). The intracellular ROS indicator was obtained from Molecular Probes (Eugene, OR, USA). The florescent E. coli particles and Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). M-CSF was obtained from Peprotech (London, UK). Lipid IVa was obtained from MyBioSource (San Diego, CA, USA).

The natural boswellic acids are commercially available as Bioswellix (BOSW) (Interpharma, Prague, Czech Republic), containing acetyl-β-boswellic and acetyl-11-keto-β-boswellic acid (min. 35% w/w). The representatives of currently used antibiotics, erythromycin (ERM) and polymyxin B (PMB), were purchased from Sigma-Aldrich, Prague, Czech Republic. BOSW was dissolved in 100% dimethylsulfoxide (DMSO, Penta, Prague, Czech Republic) to a 1% maximum final concentration of DMSO in culture medium (control samples with 1% DMSO were included in all assays). Each of the antibiotics were dissolved in the appropriate growth medium.

The antibiotics chosen for this in vitro study were based on those commonly used in United Kingdom clinical practice, namely the bactericidal antibiotics ciprofloxacin (CIP, Sigma–Aldrich), cefuroxime 0.5% (w/v) (CXM, Hampshire Hospitals NHS), polymyxin B (PMB, Sigma–Aldrich), gentamicin (GEN, Sigma–Aldrich), ofloxacin (0.3% w/v) (OFX, Allergan), levofloxacin (0.5% w/v) (LVX, Santen) and the bacteriostatic antibiotic chloramphenicol 0.5% (w/v) (CHL, Bausch and Lomb). A broth dilution assay was done to determine PAO1 susceptibility to each antibiotic. Various concentrations of antibiotic (0.01, 0.1, 1, 10, 50, 100, and 200 μg/mL) were added to equal volumes of LB broth medium with various concentrations of bacteria (∼10⁵, ∼10⁶ and ∼10⁷ Colony Forming Units (CFU)/mL). Absorbance (Optical Density (OD) λ₆₀₀ nm) as a measure of bacterial growth was assessed hourly up to 9 h and then at 24 h of incubation at 37°C with shaking (200 rpm). Comparison of the 24 h OD readings for bacterial growth with and without antibiotics was used to calculate the lowest concentration of antibiotic that completely reduced OD growth by 100% and the concentration of antibiotic that reduced OD growth by 50%.