Pepinh myd

Proteinase K was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bay11-7082 and SP600125 were purchased from A.G. Scientific (San Diego, CA, USA). PD98059 and SB203580 were purchased from Selleck (Houston, TX, USA). Pepinh-MYD, OxPAPC, and CLI-095 were purchased from Invivogen (San Diego, CA, USA).

The following drugs and substances were used (abbreviations and suppliers in parentheses): serotonin (5-HT) from Sigma-Aldrich (St. Louis, MO). [³H]-5-HT (specific activity 28 Ci/mmol) was from PerkinElmer (Boston, MA). Goat polyclonal antibody anti-human SERT (ab130130), goat polyclonal anti-human TLR10 (ab53631), and rabbit monoclonal anti-human TLR2 (ab108998) were supplied by Abcam (Cambridge, UK). Goat polyclonal anti-human actin antibody (SC-1616r) and secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA), and Pepinh-MYD (an inhibitory peptide of MyD88) was from InvivoGen (San Diego, CA). All generic reagents were purchased from Sigma-Aldrich and Roche Applied Sciences (Sant Cugat del Vallés, Barcelona, Spain).

The lipopolysaccharide (LPS; from E. coli O26:B6), BzATP (ATP), nigericin, sodium orthovanadate, AC-YVAD-CMK and ethanol were all purchased from Sigma-Aldrich (Steinheim, Germany). The Pepinh-MYD (MyD88 inhibitory peptide, PI MyD88) was purchased from Invivogen (San Diego, CA, USA). The AZ10606120 dihydrochloride was acquired from Tocris Bioscience (Bristol, United Kingdom). The PYCARD (Apoptosis-associated speck-like protein containing a CARD) polyclonal antibody and Cellrox Green Reagent were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The Caspase-Glo^® 1 Inflammasome Assay was purchased from Promega (Madison, WI, USA). The Calcein AM Cell Viability Assay was purchased from R&D Systems (Wiesbaden, Germany).

Pepinh-MYD (RQIKIWFQNRRMKWKK-RDVLPGTCVNS-NH2) is a 26-amino acid peptide that blocks Myd88 signaling (InvivoGen, San Diego, CA, USA). Pepinh-Control (RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2) is a control peptide provided by the company. To examine the effect of Myd88 inactivation on poly(I:C)-induced antiviral activity, flounder were divided randomly into six equal-sized groups named A to F. Groups A and B, C and D, and E and F were administered via i.p. injection with 50 µM Pepinh-MYD, 50 µM Pepinh-Control, and PBS respectively. At 6 h post-administration, groups A, C, and E were injected i.m. with 20 µg poly(I:C), while the other three groups were injected with PBS. At 1 d post-poly(I:C) injection, the fish were challenged with megalocytivirus as described above. At 3 d, 5 d, and 7 d post-challenge, kidney and spleen from the fish (three fish/time point) were collected, and the viral amounts in the tissues were determined as above.

PBMCs from 13 TB-IRIS and non-IRIS patients (week 2, microarrayed) were defrosted and rested overnight at 37 °C in RPMI with 10% FCS. Total cell number was counted using a Coulter Counter (Beckman, Brea, CA) and cell viability was determined by Trypan blue staining in a TC-20 automatic cell counter (Bio-Rad, Hercules, CA). Lyophilized MyD88 peptide inhibitor (Pepinh-MYD; InvivoGen, San Diego, CA) and a control peptide (Pepinh-Control; InvivoGen) were dissolved in endotoxin-free water at 1 mM stock concentration. PBMCs were treated with MyD88 peptide inhibitor (50 μM), control peptide (50 μM) or RPMI mock for 5 h at 37 °C. Samples were either stimulated or unstimulated with heat-inactivated MTB (H37Rv; multiplicity of infection 1:1) overnight at 37 °C. TC supernatant was harvested and purified by centrifugation twice at 2,000 r.p.m. and stored at −80 °C until use. Cytokine concentrations in supernatants were quantified by Luminex analysis. Heat-inactivated H37Rv was used, because patients had undergone intensive antibiotic therapy at the time of sampling and any antigens present would probably be bacterial fragments rather than actively replicating bacilli.