Complete anti protease cocktail

All human studies were performed in accordance with an approved University of Michigan institutional review board protocol after legal consent. The tracheal aspirate samples were diluted 50:50 with PBS containing complete anti-protease cocktail (Sigma-Aldrich, St. Louis, MO) and 0.5% Triton X-100 nonionic detergent to dissociate the mucus. Samples were aliquoted in 75 μL and stored at −80°C until analysis. Tracheal aspirate samples were obtained from RSV infected children who were hospitalized and given mechanical ventilation in a pediatric intensive care unit. Control uninfected tracheal infant samples were obtained from children undergoing surgery for reasons unrelated to RSV infection, after parental consent. Uric acid levels were measured with a uric acid assay kit (Cayman Chemicals, USA), following manufacturer’s instructions.

All human studies were performed in accordance with an approved University of Michigan institutional review board protocol. Tracheal aspirate samples were obtained from RSV-infected infants hospitalized and mechanically ventilated in a pediatric intensive care unit; baseline clinical characteristics were collected from the medical record and are summarized in supplemental table 1. Infants’ parents or legal guardians provided informed consent. The samples were directly aspirated from the endotracheal tube. RSV infection was detected by clinical sputum PCR for initial diagnosis; this was confirmed subsequently by PCR in the lab (see below). Infants intubated for other non-infectious reasons provided control samples. Samples were divided for protein and cDNA analysis. Protein samples were diluted 1:1 with PBS-containing complete anti-protease cocktail (Sigma-Aldrich, St. Louis, MO) and 0.5% Triton X-100 nonionic detergent to dissociate mucus. TRIzol reagent was used for RNA extraction (Invitrogen, Carlsbad, CA). cDNA was synthesized using a murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). Pro-IL-1β mRNA was analyzed via commercial Taqman primers (Thermo Fisher Scientific, Waltham, MA). IL-1β and CCL5 protein were measured using a Bio-Plex 200 System (Bio-Rad Laboratories, Hercules, CA).