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21 protocols using Anakinra

Tumour cell lines were seeded at 1 × 107 cells in 75cm2 flasks for 24 h in serum‐containing medium, washed three times with phosphate‐buffered saline (PBS) then incubated with 6 mL serum‐free medium for 24 h. Conditioned medium was collected, centrifuged at 400g for 5 min to remove cell debris and stored at −80 °C. NTF were grown to 80% confluence in 75cm2 flasks, washed three times with PBS and then incubated with either cancer cell line conditioned media or serum‐free medium for 24 h. Cancer cell+NTF or NTF alone conditioned medium was then retrieved, centrifuged at 400g for 5 min and stored at −80 °C for later analysis. For IL‐1R blocking experiments, 6 × 105 NTF were incubated with 10 μg/mL anakinra or placebo control (Amgen, Thousand Oaks, CA) for 2 h before stimulation with conditioned medium from SCC89 OPC cells or medium containing 5 ng/mL recombinant IL‐1β as a positive control for 4 h. Levels of anakinra or placebo were maintained throughout the experiment. A cytotoxicity assay using the tetrazolium dye MTT was performed on anakinra treated NTF as previously described.25
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PLGA microspheres were fabricated as described previously, using the water/oil/water double emulsion technique [8 (link),9 (link)]. As in our previous work, we used the pharmaceutical form of IL-1ra, anakinra (Amgen, Thousand Oaks, CA). The initial water/oil emulsion was formed by combining 225μL of IL-1ra (0.15 g/ml) with 1 ml of 75:25 PLGA (0.1 g/ml) (Durect Corporation, Pelham, AL). For fluorescent microsphere studies, the PLGA was supplemented with 3 mg/ml coumarin-6 (Sigma Aldrich; λemission =505 nm; λabsorption =444 nm), and saline was substituted for IL-1ra. Fabricated microspheres were lyophilized until required for surgeries, and sterilized via exposure to ultraviolet light for 10 min prior to injection. The release profile of IL-1ra from these microspheres was described previously, and is characterized by an initial burst release, decreasing to an approximately linear and sustained release over the initial 10 days. In vitro, bioactive IL-1ra continues to be released for up to 20 days [8 (link)].
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Primary human umbilical vein endothelial cells (HUVECs) were isolated as described.19 (link) The experiment was approved by the Ethics Committee of Second Affiliated Hospital of Medical College, Xi’an Jiaotong University. HUVECs were cultured in M199 medium (Gibco, Invitrogen) supplemented with 15% fetal bovine serum (Gibco, Invitrogen), 3 ng/mL endothelial cells growth factor (Sigma) and 100 U/mL penicillin-streptomycin at 37°C. HUVECs were treated with IL-1β (Sino Biological Inc., North Wales, PA, USA) at 5–20 ng/mL with or without IL-1β receptor antagonist Anakinra (Kineret, Amgen, USA) at 5  μg/mL for 12–48 hrs. Cell viability was assessed by trypan blue exclusion. HUVECs at approximately 50–70% confluence were transfected with NLRP3 shRNA, scrambled shRNA or copGFP control lentiviral particles (Santa Cruz Biotechnology) in 5 μg/mL polybrene/complete medium for 12 hrs.
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All treatments were administered via intraperitoneal injection at a volume of 10 ml kg−1. Morphine hydrochloride (McFarlan Smith, Sydney, NSW, Australia) in 0.9% saline was administered at 20 mg kg−1 (base-corrected). Codeine phosphate (GlaxoSmithKline, Melbourne, VIC, Australia) in 0.9% saline, was administered at 21 mg kg−1 (base-corrected equimolar dose to morphine). Anakinra (Kineret, Amgen, Thousand Oaks, CA, USA), a recombinant, nonglycosylated form of the human interleukin-1 receptor antagonist (IL-1RA) in 0.9% saline was administered at 100 mg kg−1. Ibudilast (Medicinova, San Diego, CA, USA) in 35% polyethelene glycol 400 (BDH Laboratory Supplies, Poole, England) in 0.9% saline was administered at 15 mg kg−1. As ibudilast was administered in a single intraperitoneal injection with concomitant codeine or morphine, the total dose volume for both drugs together was adjusted to 10 ml kg−1 for consistency between experiments. An equal volume of saline 0.9% was administered to control animals in Experiments 1, 2, 3 and 4, whereas control animals in Experiment 5 received 35% polyethylene glycol in 0.9% saline.
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The soluble TNF receptor, Etanercept (Enbrel; Wyeth Europa), which is known to fully inhibit the biological function of murine TNF (Fei et al., 2011 (link); Ali et al., 2015b (link)), was used in the experiment as anti-TNF treatment. Subcutaneous (s.c.) injections of Etanercept (0.2 mg/mouse in 0.1 ml of PBS) were performed. The administration began 2 days prior to exposing the knee joints with Lpl1(+sp), and continued on the day the knee joints where i.a. injected with Lpl1(+sp), and were followed by every other day until the termination date. PBS was s.c. injected as control treatment on days when Etanercept was not administered.
To block the function of IL-1 in the murine model, the IL-1 receptor antagonist Anakinra (Kineret; Amgen) was used as anti-IL-1 treatment (Sgroi et al., 2011 (link); Ali et al., 2015a (link)). Anakinra (1 mg/mouse in 0.1 ml of PBS) was s.c. administered daily, starting 2 days prior to exposing the knee joints with Lpl1(+sp), and continued until the termination date.
Phosphate-buffered saline was administered s.c. daily in the control treatment group, starting 2 days prior to exposing the knee joints with Lpl1(+sp), and continued until termination day.
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LEW1.WR1 rats were bred and housed in our specific pathogen-free facility. This study was carried out in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Colorado Denver (permit number: B-79715(10)1E). To test the hypothesis that therapy with Anakinra and ITF-2357 modulate the gut microbiota, rats at the age of 21–25 days were divided into 6 control and experimental groups. Group 1 was left untreated (control uninfected). Group 2 was injected i.p. with 1 x 107 PFU of KRV only, whereas Group 3 and Group 4 were i.p. injected with Anakinra on 5 consecutive days (50 μg/g body weight, obtained from Amgen, Thousand Oaks, CA) and were uninfected or infected with KRV, respectively (Fig 1). Group 5 and Group 6 were gavaged with ITF-2357 on 5 consecutive days (30 μg/g body weight, obtained from Italfarmaco, Milano, Italy) and were uninfected or KRV infected, respectively. Fecal samples were collected on day 5 post-infection and frozen immediately at -80 degrees until use.
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Day 7 γδ T cells and DC were cultured either individually or together at a 1:1 ratio (106 cells/ml each). To some cultures the following reagents were added: a sonicate of Borrelia burgdorferi (10 μg/ml), zVAD-fmk (MP Biomedical, Santa Ana, CA) at the doses indicated, necrostatin (50 μM, R&D Systems, Minneapolis, MN), anti-TNF-α (10 μg/ml, Calbiochem, Darnstadt, Germany), anti-IL-1 Receptor antagonist, Anakinra (200 ng/ml, Amgen, Thousand Oaks, CA), anti-IL-12 (10 μg/ml, BioLegend San Diego, CA), anti-IL-18 (10 μg/ml, MBL, Woburn MA) or rat IgG (10 μg/ml Jackson Immunoresearch, West Grove PA). Transwell assays were performed using transparent collagen-treated microporous membranes (Corning cat. no. 3495, Corning, NY). 1×106 γδ T cells in 1 ml of complete medium + IL-2 placed in the lower chamber, with 5×105 DC in 100 μl placed in the upper chamber. Supernatants were collected after 20 h for cytokine analysis, and surface expression of CD25 by γδ T cells was determined by flow cytometry.
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Azacitidine (Sigma), cisplatin (Teva), cytarabine (Sigma), etoposide (Teva), fludarabine (Sagent), melphalan (Sigma), methotrexate (Hospira), paclitaxel (Hospira), vincristine (Hospira), 5-FU (Teva), doxorubicin (APP Pharmaceuticals), and anakinra (Amgen) were stored according to manufacturer’s recommendation. Lipopolysaccharide (LPS) from E. coli serotype 0111:B4 was purchased from Enzo Life Sciences. Nilotinib and sorafenib (LC Laboratories) and ponatinib (Tocris Bioscience) were dissolved in DMSO and stored at −80 °C. Insulin was purchased from Sigma. Trichloroacetic acid (TCA) was purchased from Fisher Scientific. Antibody against IL-1β (ab9722) was purchased from Abcam, phospho-JNK (9251) and phospho-p38 MAPK (9211) from Cell Signaling Technology, and p38 MAPK (sc-535) from Santa Cruz Biotechnology.
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Female Ldlr−/− mice were fed a western‐type diet (WTD; 22% fat, 0.15% cholesterol; catalogue #SF00‐219, Specialty Feeds) for 14 weeks. To induce lesion regression, the mice were then switched to a normal chow diet for 3 weeks and randomised into three groups (i. control, ii. K/BxN + vehicle, and iii. K/BxN + anakinra). After 1 week of a chow diet, inflammatory arthritis was induced using the K/BxN serum transfer model, as stated above. One day after clinical manifestations of arthritis (Day 5), anakinra was administered daily (10 mg kg−1; I.P; Amgen, CA, USA) until the end of the experiment. Control and control arthritic mice received vehicle (daily, saline). Treatments were administered in a blinded manner, and data analysis was also blinded. The primary outcome was blood monocyte levels.
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A single clinical isolate of NTHi [18 (link)] and a laboratory strain of rhinovirus, RV1B [19 (link)], were used for all experiments and prepared as previously described. PBMCs were stimulated with NTHi (multiplicity of infection (MOI) of 0.33) or RV1B (MOI 1) for 24 h, 72 h or 5 days, and 500 ng·mL−1 anakinra (Amgen, Thousand Oaks, CA) or 10 ng·mL−1 IL-1β was used to pretreat cells for 1 h before stimulation with NTHi or RV1B in relevant experiments.
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