5 ttest. FTS:Computed tomography (CT) scans of rats with AIACT images in the synovial joints from the front paws of rats had been acquired 25 days postimmunization. Briefly, rats anaesthetized by intraperitoneal ketamine (100 mg/kg) andStudent’s2013 British Society for Immunology, Clinical and Experimental Immunology, 175: 458E. Aizman et al.(a) 16 14 Clinical score 12 ten eight 6 4 2 0 11 12 13 14 15 16 Days postAIA induction 17 18 Control FTS Dexamethasone(b) Naive Front paws Control FTS Naive Hind paws Handle FTSFig. 1. Therapy with farnesylthiosalicylic acid (FTS) suppresses clinical and radiographic signs of adjuvantinduced arthritis (AIA). (a) AIA was induced in Lewis rats by injection of complete Freund’s adjuvant (CFA). Rats were treated daily, beginning from day three, with either oral FTS (80 mg/kg) or with 0 carboxy methyl cellulose (CMC) as a vehicle manage (n = 20 per group). A third group of AIAtreated rats was treated with dexamethasone (0 mg/kg) on days 1, two, three, five, eight and 11 (constructive handle). AIA severity was graded on a histological scale of 0, and on a clinical index with a maximal achievable score of 16 (see Supplies and approaches). The graph shows the mean clinical scores per group everyday, from days 11 to 18. P 05; P 001, Student’s ttest. (b) Photographs of the front and hind paws of AIAinduced rats (and naive rats where disease was not induced), 16 days after disease induction. (c) On day 16, front and hind paws of AIA and naive rats had been scanned (n = eight per group) by magnetic resonance imaging (MRI) making use of a 7T/30 spectrometer (Bruker Biospin, Ettlingen). T2weighted photos (TR = 3222 and TE = 67 ms) have been sequenced. Fourteen slices, 1 mm thick, had been acquired. Final image resolution was 056 056 mm3. Representative images are presented (upper panel). Statistical analysis in the outcomes is presented as implies regular error on the mean (decrease panel). P 001 compared with vehicletreated controls; Student’s ttest.(c) NaiveFront paws ControlFTSNaiveHind paws ControlFTS30 25 Infiltrate (104mm3) 20 15 ten five Manage FTS 0 Total Front paws Hind paws(eBioscience, San Diego, CA, USA), in accordance with the manufacturer’s instructions. FACScalibur flow cytometry and CBA computer software (BD Biosciences, San Jose, CA, USA) have been made use of for information collection and analysis.933708-92-0 web Cytokine measurementSerum levels on the cytokines interferon (IFN), TNF, interleukin (IL)four, IL6, IL10 and IL17 and transforming2013 British Society for Immunology, Clinical and Experimental Immunology, 175: 458FTS and arthritisgrowth aspect (TGF) have been determined by enzymelinked immunosorbent assay (ELISA) on day 18 (Bender MedSystems, Vienna, Austria).Price of 79208-84-7 Western blottingOn day 18, rat splenocytes and ILNs had been homogenized.PMID:24635174 The resulting lysates were subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE), followed by Western blotting, as described in Fig. 3. Protein bands were visualized making use of an enhanced chemiluminescence kit (ECL; Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA) and quantified by densitometry with Image EZQuantGel application. RasGTP in the lysates was measured by the Rasbinding domain GST pulldown assay (RBDGST), followed by Western blotting with antipanRas antibody [9]. The data had been normalized for the expression levels with the housekeeping protein tubulin, total Ras, total protein kinase B (AKT) and total extracellularregulated kinase (ERK).Differences appeared as early as day 11 after arthritis induction and have been maintained until.
